Vaccines Comprising Heat-sensitive Transgenes

Abstract

The present disclosure provides temperature sensitive essential nucleic acid molecules from a psychrophilic bacterium, proteins encoded by the nucleic acid molecules, as well as recombinant cells into which have been introduced such nucleic acid molecules. The disclosed recombinant cells containing one or more essential nucleic acid molecules from a psychrophilic bacterium are thereby made temperature sensitive, and can be administered to a mammal to induce an immune response in the mammal.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation application of U.S. application Ser. No. 13/795,445 filed Mar. 12, 2013, pending, which is a continuation of U.S. application Ser. No. 13/496,723 filed Apr. 12, 2012, now U.S. Pat. No. 8,778,683, which is the U.S. National Stage of International Application No. PCT/CA2010/001561, filed Oct. 7, 2010, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Application No. 61/249,385 filed Oct. 7, 2009, and U.S. Provisional Application No. 61/322,634 filed on Apr. 9, 2010, all herein incorporated by reference.

FIELD

[0002] The technology relates to genes derived from psychrophilic bacteria, for use in the development of heat-sensitive vaccines. In one example, the technology relates to recombinant pathogens harboring the heat-sensitive gene ligA from Colwellia psychrerythraea, Pseudoalteromonas haloplanktis, and Shewanella frigidimarina and to genes ligA, pyrG, hemC, ftsZ, cmk, murG, fmt, and dnaK from C. psychrerythraea.

BACKGROUND

[0003] Vaccines against bacterial and viral diseases have played an important role in reducing infectious diseases in humans; however, there is still a need for innovative vaccines to reduce the current global burden of infectious diseases. Cold-adapted viruses have been used for decades as vaccines against human viral diseases. The best known example of such a vaccine is the Sabin polio virus vaccine. An alternate example is a cold adapted influenza vaccine called FluMist.RTM. (Medimmune LLC, Gaithersburg, Md., USA), which was introduced in the U.S. in 2003. FluMist.RTM. has been shown to be considerably more effective in certain demographic groups than influenza vaccines that practice the more common vaccination strategy of using inactivated virus to stimulate an immune response. Typically cold-adapted or "temperature-sensitive" (TS) viral strains have been developed by passing the virus repeatedly in eggs or cell culture at low temperatures and then testing the progeny for their inability to grow above about 37.degree. C., generally thought of as the "normal" human body temperature.

[0004] The concept of a "normal" human body temperature takes into consideration anatomical sites, individual variations, gender, physiological conditions and ambient temperature. Despite the number of variables, the human body can function only in a very narrow temperature range, which is generally about 36.degree. C.-39.degree. C. If the human body core temperature falls to about 35.degree. C., the body must be warmed or death will ensue. The skin temperature is always cooler than the body core regardless of the ambient temperature and clothing worn. At moderate temperatures (e.g., 21.degree. C.), the temperature of the skin is about 32.degree. C.-35.degree. C.

[0005] Those skilled in these arts are of the view that bacteria generally have a set of about 100 to 150 genes, called "essential genes" that are absolutely required for maintenance of bacterial viability. Identifying essential genes is difficult due to their nature, as knockouts of these genes results in death of the organism. Essential genes encode proteins composed of amino acid sequences that are highly conserved among almost all bacterial genera and species. This conservation presumably reflects their common function and structure among the different species. A select number of essential genes have been shown to be competent in substituting for a homologue in another bacterial species and in some cases these substitutions were from distantly related bacterial species. The conservation of amino acid sequences is widespread among bacteria, the deduced amino acid sequences of essential genes from psychrophiles and thermophiles shows high identity with their mesophilic counterparts. Microbiologists have generally used conditional lethal mutations, such as TS mutations, to identify essential genes.

[0006] Many bacterial species play significant roles in the global burden of infectious diseases. However, the causative agent of tuberculosis is probably the most significant contributor to human morbidity and mortality caused by an infectious bacterial disease. Although the Bacille Calmette-Guerin (BCG) vaccine has been used for several decades to protect against tuberculosis, its low efficacy has failed to lower the incidence of tuberculosis to acceptable levels.

SUMMARY

[0007] The present disclosure provides methods for engineering, producing and using heat-sensitive host microbial cells. In one example, recombinant pathogens contain heat-sensitive essential genes, for example inserted using homologous recombination. "Psychrophile" is a term that is applied to organisms that function optimally at cold temperatures e.g., <20.degree. C. Bacteria that live in cold ocean water, especially the Arctic and Antarctic oceans, are examples of psychrophilic bacteria. Enzymes and other proteins in psychrophilic bacteria function better in the cold than their homologous counterparts in mesophilic bacteria. Many of the enzymes from psychrophilic bacteria are also prone to denaturation at temperatures much lower than those that would affect their mesophilic counterpart. Presumably the pattern of temperature-sensitivity of psychrophilic enzymes extends to the products of essential genes.

[0008] Methods of identifying and manipulating psychrophilic essential genes with desired TS properties are provided. in vitro and in vivo recombinant technologies can be used. Francisella tularensis is the etiologic agent of the zoonotic disease, tularaemia. It can infect numerous animals by a variety of routes, and typically infects and grows in monocyte-derived cells in organs of the reticuloendothelial system. A closely related bacterium, Francisella novicida, has many of the properties of F. tularensis, and, in addition, is highly amenable to many genetic manipulations, including gene substitutions. The pathophysiology and genetic properties of F. novicida make it ideal for studying the effects of gene substitutions on a pathogenic bacterium. F. novicida is a mesophile with a maximal growth temperature of about 45.degree. C.

[0009] This disclosure also provides methods to determine maximal growth temperature of both bacterial strains and their growth properties at restrictive temperatures. The recombinant bacterial strains tested grew below the restrictive temperature but not above the restrictive temperature. When a psychrophilic essential allele encoding an essential gene is inserted into an area of a mammalian body that is colder than the human body core, e.g., the skin, the recombinant pathogenic bacteria will have the ability to thrive thereby inducing an immune response. When the pathogenic recombinant bacteria migrate to organs in the human body core where the temperature is higher, they die and are unable to harm the host.

[0010] The present disclosure provides isolated temperature-sensitive essential nucleic acid molecules from a psychrophilic bacterium comprising at least 80%, at least 90%, or at least 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 22, 23, or 24. In some examples, the psychrophilic bacteria are operable at a temperature of about -10.degree. C. to about 30.degree. C., but inoperable at a temperature greater than about 30.degree. C. Vectors and recombinant host cells (such as a recombinant bacterial host cell) that include such temperature-sensitive essential nucleic acid molecules from a psychrophilic bacterium are also provided. Immunogenic compositions that include such recombinant host bacteria (such as live or killed cells) are also disclosed. The disclosure also provides isolated proteins encoded by the disclosed isolated temperature-sensitive essential nucleic acid molecules, such as proteins having at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 26, 27, or 28.

[0011] Methods of making a temperature-sensitive microbial host cell, such as a recombinant host cell, are provided. In one example the method includes introducing (for example by inserting, substituting or replacing) a nucleic acid construct into the genome of a mesophilic bacterial strain, wherein the nucleic acid construct includes a temperature-sensitive essential nucleic acid molecule from a psychrophilic bacterial strain and one or more control sequences operably linked to the temperature-sensitive essential nucleic acid molecule, wherein the temperature-sensitive essential peptide encoded by the introduced temperature-sensitive essential nucleic acid molecule is operable (e.g., functional) at a temperature less than about 30.degree. C. and inoperable (e.g., non-functional) at a temperature greater than about 30.degree. C. In some examples the method also includes culturing the temperature-sensitive microbial host cell at a temperature wherein the temperature-sensitive peptide is operable, whereby said microbial host cell produces a plurality of peptides; increasing the culturing temperature to a temperature at which the temperature-sensitive peptide is inoperable; maintaining said culturing for a period of time sufficient to kill the temperature-sensitive microbial host cell; and harvesting the killed temperature-sensitive microbial host cells.

[0012] Methods for producing an immune response to a bacterium in a subject using the disclosed nucleic acid molecules, proteins, and recombinant host cells are provided. In one example the method includes administering to the subject a therapeutically effective amount of a temperature-sensitive bacterium, wherein the temperature-sensitive bacterium expresses a temperature-sensitive essential nucleic acid molecule from a psychrophilic bacterial strain, thereby inducing an immune response to the bacterium. Such methods can be used to prevent or treat a bacterial infection (such as a M. tuberculosis, Salmonella or Francisella infection).

[0013] The foregoing and other features of the disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1a is a flowchart illustrating an exemplary method using polymerase chain reaction (PCR), FIG. 1b is a schematic chart illustrating an exemplary method showing DNA integration-excision events that result in a gene substitution.

[0015] FIG. 2a is a schematic illustrating the sequence of the wild type (wt) F. novicida ligA gene as it exists normally in a chromosome, FIG. 2b is a schematic illustrating ligA.sub.Cp gene substitutions into the F. novicida chromosome according to an exemplary method of the present disclosure, FIG. 2c is a schematic illustrating ligA.sub.Sf gene substitution into the F. novicida chromosome according to an exemplary method of the present disclosure, FIG. 2d is a schematic illustrating ligA.sub.Ph gene substitutions into the F. novicida chromosome according to an exemplary method of the present disclosure, FIG. 2e is a schematic illustrating ligA.sub.Ph2 gene substitutions into the F. novicida chromosome according to an exemplary method of the present disclosure.

[0016] FIG. 3a is a graph illustrating the growth curve of wt F. novicida and F. novicida with the C. psychrerythraea ligA.sub.Cp gene substituted for the F. novicida homologue at 30.degree. C., FIG. 3b is a graph illustrating the growth curve of F. novicida with the C. psychrerythraea ligA.sub.Cp gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30.degree. C. to 33.degree. C. after 2 hours, FIG. 3c is a graph illustrating the growth curve of F. novicida with the C. psychrerythraea ligA.sub.Cp gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30.degree. C. to 34.degree. C. after 3.5 hours, FIG. 3d is a graph illustrating the growth curve of F. novicida with the C. psychrerythraea ligA.sub.Cp gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30.degree. C. to 35.degree. C. after 2 hours, FIG. 3e is a graph illustrating the growth curve of F. novicida with the C. psychrerythraea ligA.sub.Cp gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30.degree. C. to 37.degree. C. after 2 hours.

[0017] FIG. 4a is a graph illustrating the growth curve of wt F. novicida and F. novicida with the S. frigidimarina ligA.sub.Sf gene substituted for the F. novicida homologue at 30.degree. C., FIG. 4b is a graph illustrating the growth curve of F. novicida with the S. frigidimarina ligA.sub.Sf gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30.degree. C. to 33.degree. C. after 2 hours, FIG. 4c is a graph illustrating the growth curve of F. novicida with the S. frigidimarina ligA.sub.Sf gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30.degree. C. to 35.degree. C. after 2 hours, FIG. 4d is a graph illustrating the growth curve of F. novicida with the S. frigidimarina ligA.sub.Sf gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30.degree. C. to 37.degree. C. after 2 hours.

[0018] FIG. 5a is a graph illustrating the growth curve of wt F. novicida and F. novicida with the P. haloplanktis ligA.sub.Ph gene substituted for the F. novicida homologue at 30.degree. C., FIG. 5b is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligA.sub.Ph gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30.degree. C. to 33.degree. C. after 2 hours, FIG. 5c is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligA.sub.Ph gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30.degree. C. to 35.degree. C. after 2 hours, FIG. 5d is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligA.sub.Ph gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 30.degree. C. to 37.degree. C. after 2 hours.

[0019] FIG. 6a is a graph illustrating the growth curve of wt F. novicida and F. novicida with the P. haloplanktis ligA.sub.Ph2 gene substituted for the F. novicida homologue at 21.degree. C., FIG. 6b is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligA.sub.Ph2 gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 21.degree. C. to 26.degree. C. after 2 hours, FIG. 6c is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligA.sub.Ph2 gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 21.degree. C. to 28.degree. C. after 2 hours, FIG. 6d is a graph illustrating the growth curve of F. novicida with the P. haloplanktis ligA.sub.Ph2 gene substituted for the F. novicida homologue and wt F. novicida with a temperature shift from 21.degree. C. to 30.degree. C. after 2 hours.

[0020] FIG. 7 is a graph illustrating the decline in viability of wt F. novicida and F. novicida--ligA.sub.CP cultures at 37.degree. C. after being grown to late exponential phase at 33.degree. C.

[0021] FIG. 8 is a digital image illustrating the growth of S. ser. Typhimurium-ligA.sub.CP at 30.degree. C. and the lack of growth at 37.degree. C.

[0022] FIG. 9a is a graph illustrating the growth curve of wt Mycobacterium smegmatis and M. smegmatis-ligA.sub.CP at 30.degree. C., FIG. 9b is a graph illustrating the growth curve of M. smegmatis-ligA.sub.CP and wt M. smegmatis with a temperature shift from 30.degree. C. to 35.degree. C. after 4 hours, FIG. 9c is a graph illustrating the growth curve of M. smegmatis-ligA.sub.CP and wt M. smegmatis with a temperature shift from 30.degree. C. to 37.degree. C. after 4 hours.

[0023] FIGS. 10a-10d are a series of graphs showing the protective immunity induced by TS F. novicida strains.

[0024] FIGS. 11A-11L show sequences disclosed herein, with underlined portions being F. novicida sequence.

SEQUENCE LISTING

[0025] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.

[0026] SEQ ID NO: 1 is a full length nucleic acid coding sequence of the ligA.sub.Cp hybrid gene.

[0027] SEQ ID NO: 2 is the deduced 689 amino acid sequence of LigA.sub.Cp hybrid protein.

[0028] SEQ ID NO: 3 is a full length nucleic acid coding sequence of the ligA.sub.Ph hybrid gene.

[0029] SEQ ID NO: 4 is the deduced 673 amino acid sequence of LigA.sub.Ph hybrid protein.

[0030] SEQ ID NO: 5 is a full length nucleic acid coding sequence of the ligA.sub.Ph2 hybrid gene.

[0031] SEQ ID NO: 6 is the deduced 673 amino acid sequence of LigA.sub.Ph2 hybrid protein.

[0032] SEQ ID NO: 7 is a full length nucleic acid coding sequence of the ligA.sub.Sf hybrid gene.

[0033] SEQ ID NO: 8 is the deduced 670 amino acid sequence of LigA.sub.Sf hybrid protein.

[0034] SEQ ID NO: 9 is a full length nucleic acid coding sequence of the pyrG.sub.Cp hybrid gene.

[0035] SEQ ID NO: 10 is the deduced 545 amino acid sequence of PyrG.sub.Cp hybrid protein.

[0036] SEQ ID NO: 11 is a full length nucleic acid coding sequence of the hemC.sub.Cp hybrid gene.

[0037] SEQ ID NO: 12 is the deduced 317 amino acid sequence of HemC.sub.Cp hybrid protein.

[0038] SEQ ID NO: 13 is a full length nucleic acid coding sequence of the fmt.sub.Cp hybrid gene.

[0039] SEQ ID NO: 14 is the deduced 327 amino acid sequence of Fmt.sub.Cp hybrid protein.

[0040] SEQ ID NO: 15 is a full length nucleic acid coding sequence of the murG.sub.Cp hybrid gene.

[0041] SEQ ID NO: 16 is the deduced 387 amino acid sequence of MurG.sub.Cp hybrid protein.

[0042] SEQ ID NO: 17 is a full length nucleic acid coding sequence of codon optimized ligA.sub.Cp optimized for M. tuberculosis.

[0043] SEQ ID NO: 18 is the deduced 689 amino acid coding sequence of codon optimized LigA.sub.Cp hybrid protein with the first four codons changed to the M. tuberculosis form.

[0044] SEQ ID NO: 19 is a full length nucleic acid coding sequence of the dnaK.sub.Cp hybrid gene.

[0045] SEQ ID NO: 20 is the deduced 638 amino acid coding sequence of DnaK.sub.Cp hybrid protein.

[0046] SEQ ID NOS: 21 and 22 are a full length nucleic acid coding sequence of the essential gene tyrS from Colwellia psychrerythraea (normal font, uppercase), and the corresponding amino acid sequence, respectively.

[0047] SEQ ID NO: 23 and 24 are a full length nucleic acid coding sequence of the essential gene cmk from Colwellia psychrerythraea (normal font, uppercase) and the corresponding amino acid sequence, respectively. As shown in FIG. 11J, F. novicida sequence is underlined. The underlined regions correspond to the F. novicida sequence in both the nucleotide and amino acid sequence. The "non-underlined" is Colwellia psychrerythraea sequence. In the amino acid sequence there is no underlined amino acids at the end since the F. novicida sequence starts at the stop codon.

[0048] SEQ ID NO: 25 and 26 are a full length nucleic acid coding sequence of the essential gene dnaKsf from Shewanella frigidimarina (normal font, uppercase) and the corresponding amino acid sequence, respectively. As shown in FIG. 11K, Francisella novicida sequence is underlined. The underlined regions correspond to the F. novicida sequence in both the nucleotide and amino acid sequence. The "non-underlined" shows the Shewanella frigidimarina sequence. In the amino acid sequence at the beginning (MGK) is identical between Shewanella and Francisella, so it is double underlined. The single underline at the end of the amino acid sequence corresponds to the F. novicida sequence.

[0049] SEQ ID NO: 27 and 28 are a full length nucleic acid coding sequence of the essential gene ftsZ from Colwellia psychrerythraea (normal font, uppercase) and the corresponding amino acid sequence, respectively. As shown in FIG. 11L, Francisella novicida sequence is underlined. The underlined regions correspond to the F. novicida sequence in both the nucleotide and amino acid sequence. The "non-underlined" regions are Colwellia psychrerythraea sequence. There is extensive F. novicida region at the 5'-end (N-terminus).

DETAILED DESCRIPTION

[0050] The following explanations of terms and methods are provided to better describe the present disclosure. The singular forms "a," "an," and "the" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprising a nucleic acid molecule" includes single or plural nucleic acid molecules and is considered equivalent to the phrase "comprising at least one nucleic acid molecule." The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, "comprises" means "includes." Thus, "comprising A or B," means "including A, B, or A and B," without excluding additional elements.

[0051] Suitable methods and materials for the practice and/or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which a disclosed invention pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

[0052] The references cited herein are incorporated by reference.

[0053] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided. Unless otherwise noted, technical terms are used according to conventional usage by those skilled in the arts

[0054] Adjuvant: A vehicle used to enhance antigenicity, for example antigenicity of a recombinant host bacterium containing a TS essential psychrophilic bacteria sequence disclosed herein. Adjuvants include a suspension of minerals (e.g., alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example see U.S. Pat. No. 6,194,388; U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,214,806; U.S. Pat. No. 6,218,371; U.S. Pat. No. 6,239,116; U.S. Pat. No. 6,339,068; U.S. Pat. No. 6,406,705; and U.S. Pat. No. 6,429,199). Adjuvants include biological molecules (a "biological adjuvant"), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-.alpha., IFN-.gamma., G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

[0055] Administration: The introduction of a composition (such as an immunogenic composition) into a subject (such as a mammal, for example a human) by a selected route. Exemplary routes of administration include, but are not limited to, topical, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.

[0056] Ameliorate: The improvement of a disease or pathological condition (such as a bacterial infection) with respect to the effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known to those skilled in the arts specific to the particular disease.

[0057] Animal: Living multi-cellular vertebrate organisms, a category that includes mammals and birds. The term "mammal" includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects (such as mice, rats, rabbits, dogs, cats, horses, and cattle).

[0058] Antibody: A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V.sub.H) region and the variable light (V.sub.L) region. Together, the V.sub.H region and the V.sub.L region are responsible for binding the antigen recognized by the antibody.

[0059] Antibodies include intact immunoglobulins and the variants and portions of antibodies well known in the art, such as Fab fragments, Fab' fragments, F(ab)'.sub.2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.

[0060] Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (.lamda.) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

[0061] "Specifically binds" refers to the ability of individual antibodies to specifically immunoreact with an antigen, such as a bacterial antigen, relative to binding to unrelated proteins, such as non-bacterial proteins. The binding is a non-random binding reaction between an antibody molecule and an antigenic determinant of the T cell surface molecule. The desired binding specificity is typically determined from the reference point of the ability of the antibody to differentially bind the T cell surface molecule and an unrelated antigen, and therefore distinguish between two different antigens, particularly where the two antigens have unique epitopes. An antibody that specifically binds to a particular epitope is referred to as a "specific antibody".

[0062] In some examples, an antibody specifically binds to a target (such as a bacterial protein) with a binding constant that is at least 10.sup.3 M.sup.-1 greater, 10.sup.4 M.sup.-1 greater or 10.sup.5 M.sup.-1 greater than a binding constant for other molecules in a sample or subject. In some examples, an antibody or fragments thereof, has an equilibrium constant (Kd) of 1 nM or less. For example, an antibody binds to a target, such as a bacterial protein with a binding affinity of at least about 0.1.times.10.sup.-8 M, at least about 0.3.times.10.sup.-8 M, at least about 0.5.times.10.sup.-8 M, at least about 0.75.times.10.sup.-8 M, at least about 1.0.times.10.sup.-8 M, at least about 1.3.times.10.sup.-8 M at least about 1.5.times.10.sup.-8 M, or at least about 2.0.times.10.sup.-8 M. Kd values can, for example, be determined by competitive ELISA (enzyme-linked immunosorbent assay) or using a surface-plasmon resonance device such as the Biacore T100, which is available from Biacore, Inc., Piscataway, N.J.

[0063] Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes. "Epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells respond. In one embodiment, T cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Generally, T cells recognize epitopes of continuous amino acids. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.

[0064] Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants, such as those recognized by an immune cell. An antigen can be a tissue-specific antigen, or a disease-specific antigen. These terms are not exclusive, as a tissue-specific antigen can also be a disease specific antigen. A tissue-specific antigen is expressed in a limited number of tissues, such as a single tissue. A tissue specific antigen may be expressed by more than one related type of tissue, such as alveolar and bronchial tissue. A disease-specific antigen is expressed coincidentally with a disease process. Specific non-limiting examples of a disease-specific antigen are an antigen whose expression correlates with, or is predictive of, a bacterial infection, such as tuberculosis. A disease-specific antigen can be an antigen recognized by T cells or B cells.

[0065] CD4: Cluster of differentiation factor 4, a T cell surface protein that mediates interaction with the MHC Class II molecule. CD4 also serves as the primary receptor site for HIV on T cells during HIV infection. Cells that express CD4 are often helper T cells.

[0066] CD8: Cluster of differentiation factor 8, a T cell surface protein that mediates interaction with the MHC Class I molecule. Cells that express CD8 are often cytotoxic T cells. "CD8+ T cell mediated immunity" is an immune response implemented by presentation of antigens to CD8+ T cells.

[0067] Contacting: The process of incubating one agent in the presence of another. Thus, when a cell is contacted with an agent (such as an immunogenic composition), the cell is incubated with the agent for a sufficient period of time for the agent and the cell to interact.

[0068] Cool parts of the body: Regions of a human or other mammalian body that generally have a lower temperature than other parts of the body. The concept of natural human (or other mammal) body temperature variation due to anatomical sites, gender, physiological and ambient temperature. Despite the number of variables, the human (or other mammalian) body can function only in a very narrow temperature range, hence, for example the human body core remains at about 36.degree. C.-39.degree. C. Cool parts of the body include skin, mouth and rectum. Skin temperature, for example, is about 32.degree. C.-35.degree. C. Thus, in some examples, cool parts of the body have temperatures that are at least 1.degree. C. less, at least 2.degree. C. less, at least 3.degree. C. less, at least 4.degree. C. less, at least 4.degree. C. less, or at least 6.degree. C. less, such as 1.degree. C. to 8.degree. C. less, 1.degree. C. to 6.degree. C. less, 2.degree. C. to 6.degree. C. less, or 2.degree. C. to 4.degree. C. less, than other parts of the body, such as the core.

[0069] Cytokine: Proteins made by cells that affect the behavior of other cells, such as lymphocytes. In one embodiment, a cytokine is a chemokine, a molecule that affects cellular trafficking. Specific, non-limiting examples of cytokines include the interleukins (IL-2, IL-4, IL-6, IL-10, IL-21, etc.), and IFN-.gamma..

[0070] Degenerate variant: A TS essential psychrophilic bacteria nucleic acid sequence that encodes a TS essential psychrophilic bacteria protein that includes a nucleic acid sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in this disclosure as long as the amino acid sequence of the TS essential psychrophilic bacteria peptide encoded by the nucleotide sequence is unchanged.

[0071] Em.sup.R: Erythromycin resistance.

[0072] Essential gene: A gene that is necessary for the growth of the organism (such as a mesophilic bacterium) under all culturing conditions.

[0073] Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

[0074] A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters, are included (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences. In one embodiment, the promoter is a cytomegalovirus promoter.

[0075] Heat-sensitive: An inability to perform an essential biological function at temperatures above about 28.degree. C. Similarly, the term "heat-sensitive protein or polypeptide" refers to a non-functional mature protein resulting from heat-induced deactivation. An enzyme that does not catalyze its known reaction efficiently enough to support growth, development or life of the organism above about 28.degree. C. is an example of such a protein.

[0076] Heat-sensitive allele: An allele comprising a gene encoding a heat-sensitive protein. Similarly the term, "heat-sensitive gene" refers to a gene encoding a heat-sensitive protein.

[0077] Host cells: Cells into which a heterologous nucleic acid molecule has been introduced. For example, such cells may include a nucleic acid vector that is propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The cell can be prokaryotic, such as a bacterial cell. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.

[0078] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen or a particular TS recombinant microbial cell, such as mesophilic bacteria containing a psychrophile essential nucleic acid molecule provided herein. In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies. The development of an immune response following administration of mesophilic bacteria containing a psychrophile TS essential nucleic acid molecule can be measured using routine methods known in the art, for example by measuring cytokine production as an indication of a protective immune response.

[0079] Immunogenic composition: Compositions that include recombinant mesophilic bacteria containing a psychrophile TS essential nucleic acid molecule that induces a measurable CTL response against a recombinant mesophilic bacteria protein, or induces a measurable B cell response (such as production of antibodies that specifically bind a recombinant mesophilic bacteria-specific protein) against a recombinant mesophilic bacteria protein. For example, the immunogenic polypeptide or a nucleic acid encoding the immunogenic polypeptide can be present in a heat-sensitive mesophilic bacteria generated using the methods provided herein, wherein the bacteria is art of an immunogenic composition that can further include pharmaceutically acceptable carriers, and/or other therapeutic agents. An immunogenic composition can optionally include an adjuvant, a PD-1 antagonist, a co-stimulatory molecule, or a nucleic acid encoding a costimulatory molecule. An immunogenic composition can be readily tested for its ability to induce a CTL by art-recognized assays.

[0080] Immunogenic peptide: A peptide which comprises an allele-specific motif or other sequence such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response, or a B cell response (e.g. antibody production) against the antigen from which the immunogenic peptide is derived. Immunogenic peptides can also be identified by measuring their binding to a specific MHC protein and by their ability to stimulate CD4 and/or CD8 when presented in the context of the MHC protein.

[0081] Generally, immunogenic polypeptides can be used to induce an immune response in a subject, such as a B cell response or a T cell response. In one example, an immunogenic polypeptide, when bound to a MHC Class I molecule, activates cytotoxic T lymphocytes (CTLs) against the polypeptide. Induction of CTLs using synthetic peptides and CTL cytotoxicity assays are known in the art, see U.S. Pat. No. 5,662,907. In one example, an immunogenic peptide includes an allele-specific motif or other sequence such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response against the antigen from which the immunogenic peptide is derived.

[0082] Immunologically reactive conditions: Conditions that allow an antibody specific for a particular epitope to bind to that epitope to a greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. These conditions are dependent upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. The immunologically reactive conditions employed in the disclosed methods are "physiological conditions" which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organ and intracellular environment is generally about pH 7 (e.g., from pH 6.0 to pH 8.0, or pH 6.5 to pH 7.5, such as pH 7.2), contains water as the predominant solvent, and exists at a temperature above 0.degree. C. and below 50.degree. C. Osmolarity is within the range that is supportive of cell viability and proliferation. These conditions are well known to those skilled in these arts.

[0083] Interferon gamma (IFN-.gamma.): IFN-.gamma. is a dimeric protein with subunits of 146 amino acids. The protein is glycosylated at two sites, and the pI is 8.3-8.5. IFN-.gamma. is synthesized as a precursor protein of 166 amino acids including a secretory signal sequence of 23 amino acids. Two molecular forms of the biologically active protein of 20 and 25 kDa have been described. Both of them are glycosylated at position 25. The 25 kDa form is also glycosylated at position 97. The observed differences of natural IFN-.gamma. with respect to molecular mass and charge are due to variable glycosylation patterns. 40-60 kDa forms observed under non-denaturing conditions are dimers and tetramers of IFN-.gamma.. The human gene has a length of approximately 6 kb. It contains four exons and maps to chromosome 12q24.1.

[0084] IFN-.gamma. can be detected by sensitive immunoassays, such as an ELISPOT test that allows detection of individual cells producing IFN-.gamma.. Minute amounts of IFN-.gamma. can be detected indirectly by measuring IFN-induced proteins such as Mx protein. The induction of the synthesis of IP-10 has been used also to measure IFN-.gamma. concentrations. In addition, bioassays can be used to detect IFN-.gamma., such as an assay that employs induction of indoleamine 2,3-dioxygenase activity in 2D9 cells. The production of IFN-.gamma. can be used to assess T cell activation, such as activation of a T cell by bacterial antigen.

[0085] Isolated: A biological component (such as a nucleic acid molecule, protein or organelle) that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods. In another embodiment, "isolated" refers to nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0086] ligA: A wt allele of the gene encoding NAD-dependent DNA ligase found in mesophilic bacteria such as F. novicida, M. smegmatis or E. coli. Furthermore, ligA with a subscript, such as ligA.sub.Cp, ligA.sub.Sf, or ligA.sub.Ph, refers to a wt allele of the gene encoding NAD-dependent DNA ligase found in psychrophilic bacteria. For example ligA.sub.Cp refers to the wt allele of ligA found in the Arctic bacterium C. psychrerythraea strain 34H which has a maximal growth temperature below 18.degree. C. The ligA sequences from psychrophilic bacteria can be introduced into mesophilic bacteria, to confer temperature sensitivity to the mesophilic bacteria.

[0087] Mesophile: An organism naturally found in environments at temperatures between about 20.degree. C. and 50.degree. C. A bacterial mesophile refers to a bacterium that is normally associated with a mammal and thus is normally functioning at temperatures between about 32.degree. C. and 45.degree. C.

[0088] Psychrophile: An organism naturally found in environments that are permanently below 20.degree. C., often permanently below 10.degree. C. and sometimes below 0.degree. C. Such permanently cold environments include most ocean environments, permafrost soils, Arctic and Antarctic environments. Those skilled in these arts will understand that "psychrophile" and "psychrotroph" are commonly used to describe bacteria that grow in cold environments.

[0089] Psychrophilic: Features found in psychrophiles. For example, a "psychrophilic enzyme" is an enzyme isolated from a psychrophile.

[0090] Peptide modifications: Analogs (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences) and variants (homologs) of proteins that can be used in the methods and compositions provided herein. Peptides are comprised of amino acids, which may be either L- and/or D-amino acids, naturally occurring and otherwise. The peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. Modifications are well known to those skilled in these arts.

[0091] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic composition, such as an immunogenic composition.

[0092] The disclosed purified active compositions can be administered alone or combined with an acceptable carrier. Preparations can contain one type of therapeutic molecule, or can be composed of a combination of several types of therapeutic molecules. The nature of the carrier will depend on the particular mode of administration being utilized.

[0093] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0094] Preventing or treating a disease: "Preventing" a disease refers to inhibiting the full development of a disease, for example in a person who is known to be at risk of infection with M. tuberculosis, or M. leprae. An example of a person with a known predisposition is someone living with a person diagnosed with tuberculosis, health care professionals, or someone otherwise known to have been exposed to M. tuberculosis. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such as tuberculosis, after it has begun to develop.

[0095] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein is more pure than the protein in its originating environment within a cell. A preparation of a protein is typically purified such that the protein represents at least 50% of the total protein content of the preparation. However, more highly purified preparations may be required for certain applications. For example, for such applications, preparations in which the protein includes at least 75% or at least 90% of the total protein content may be employed.

[0096] Recombinant: A nucleic acid molecule that has a sequence not naturally occurring or a sequence that is made by an artificial combination of two naturally separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, by genetic engineering techniques, for example. Also refers to cells into which a non-native nucleic acid molecule has been introduced.

[0097] Resistant to infection: Animals (e.g., mammals) that demonstrate decreased symptoms of infection compared to non-resistant animals. Evidence of resistance to infection can appear as, for example, lower rates of mortality, increased life spans measured after exposure to the infective agent, fewer or less intense physiological symptoms, such as fewer lesions, or decreased cellular or tissue concentrations of the infective agent. In one embodiment, resistance to infection is demonstrated by a heightened immune response.

[0098] Restrictive temperature: The lowest temperature at which an organism is unable to grow. For example, in Table 1 "restrictive temperature" specifically refers to the lowest temperature at which the F. novicida strain with a psychrophilic gene integrated is unable to form an isolated colony on agar media. Due to the variation in the temperature of incubators, these temperatures are interpreted as being about .+-.1.degree. C.

[0099] sacB cassette: A modular DNA sequence encoding the enzyme levansucrase from Bacillus subtilus. Expression of this gene is lethal in the presence of sucrose to many bacteria and can thus be used as a counter-selection agent to help select for the loss of gene sequences.

[0100] Selective hybridization: Hybridization under moderately or highly stringent conditions that exclude non-related nucleotide sequences, the techniques of hybridization are known to those skilled in these arts.

[0101] Sequence identity: The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.

[0102] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0103] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

[0104] BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

[0105] Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1154 nucleotides is 75.0 percent identical to the test sequence (1166/1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 15-nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15/20*100=75).

[0106] For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). Homologs are typically characterized by possession of at least 30% sequence identity or more counted over the full-length alignment with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs use SEG. In addition, a manual alignment can be performed. Proteins with even greater similarity will show increasing percentage identities when assessed by this method, such as at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with a protein disclosed herein. Thus in one example, a protein that can be used in the disclosed methods and compositions has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 and retains the ability to confer TS (such as heat-sensitivity) to a mesophilic bacteria.

[0107] One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above. Nucleic acid sequences that do not show a high degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein. Such homologous nucleic acid sequences can, for example, possess at least about 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with a disclosed nucleic acid sequence as determined by this method. Thus in one example, a nucleic acid sequence that can be used in disclosed methods and compositions has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 and retains the ability to encode a protein that can confer TS (such as heat-sensitivity) to a mesophilic bacteria. An alternative (and not necessarily cumulative) indication that two nucleic acid sequences are substantially identical is that the peptide which the first nucleic acid encodes is immunologically cross reactive with the peptide encoded by the second nucleic acid.

[0108] Temperature-sensitive (TS)" or "heat-sensitive (HS): A bacterial component (such as a protein) or bacterium that is active up to about 30.degree. C. and inactivated at a temperature that is normally found in the human body, e.g., above about 30.degree. C.

[0109] Tester strain: A mesophilic bacterium that is amenable to gene replacement allowing the substitution of a psychrophilic essential gene for the homologue naturally found in the tester strain.

[0110] Therapeutically effective amount: An amount of a composition that alone, or together with an additional therapeutic agent(s) sufficient to achieve a desired effect in a subject, or in a cell, being treated with the agent. The effective amount of the agent (such as an immunogenic composition provided herein) can be dependent on several factors, including, but not limited to the subject or cells being treated, the particular therapeutic agent, and the manner of administration of the therapeutic composition. In one example, a therapeutically effective amount or concentration is one that is sufficient to prevent advancement, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by the disease, such as a bacterial infection (e.g., tuberculosis).

[0111] In one example, a desired response is to reduce or inhibit one or more symptoms associated with a bacterial infection. The one or more symptoms do not have to be completely eliminated for the composition to be effective. The effective amount of an agent that includes one of the disclosed immunogenic compositions that is administered to a human or veterinary subject will vary depending upon a number of factors associated with that subject, for example the overall health of the subject. An effective amount of an agent can be determined by varying the dosage of the product and measuring the resulting therapeutic response, such as the prevention of bacterial infection. Effective amounts also can be determined through various in vitro, in vivo or in situ immunoassays. The disclosed agents can be administered in a single dose, or in several doses, as needed to obtain the desired response.

[0112] In particular examples, a therapeutically effective dose of an immunogenic composition includes at least 10.sup.2 colony forming units (CFU), such as at least 10.sup.3, at least 10.sup.4, at least 10.sup.5, at least 10.sup.6, at least 10.sup.7, or at least 10.sup.8 CFU, for example 10.sup.2 to 10.sup.8 CFU. In one example, 10.sup.2 to 10.sup.8 CFU of live bacteria are administered intradermally or intranasally. However, one skilled in the art will recognize that higher or lower dosages also could be used, for example depending on the particular immunogenic composition. In particular examples, such daily dosages are administered in one or more divided doses (such as 2, 3, or 4 doses) or in a single formulation. The disclosed immunogenic composition can be administered alone, in the presence of a pharmaceutically acceptable carrier, in the presence of other therapeutic agents.

[0113] Treatment: A therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. In one example, the immunogenic compositions disclosed herein following administration to a mammal achieves a reduction in one or more signs of a bacterial infection.

[0114] Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transduced or transformed host cell, referred to herein as a recombinant cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker gene and other genetic elements known in the art. Vectors include plasmid vectors, including plasmids for expression in gram negative and gram positive bacterial cells. Exemplary vectors include those for expression in E. coli and Salmonella. Vectors also include viral vectors, such as, but are not limited to, retrovirus, orthopox, avipox, fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus, baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors.

[0115] Temperature-Sensitive Essential Genes from Psychrophilic Bacteria

[0116] It is disclosed herein that several nucleic acid molecules, and their corresponding peptides, can be introduced into a bacteria to confer temperature sensitivity (TS), such as heat-sensitivity, to the host bacteria. The resulting bacteria can be used to induce an immune response to the temperature-sensitive bacteria, such as a T cell response. Exemplary psychrophilic essential genes with desired temperature sensitivity, and their corresponding peptides, are provided herein. For example, host mesophilic bacteria can be transformed with one or more psychrophile TS essential nucleic acid molecules, thereby conferring TS to the mesophilic bacteria. The resulting recombinant mesophilic bacteria can be formulated into an immunogenic composition, to treat or prevent infection by the meosophilic bacteria. For example, recombinant mesophilic M. tuberculosis bacterium containing one or more psychrophile TS essential nucleic acid molecules can be used to treat or prevent tuberculosis. The same approach can be used to make TS forms of Bacillus anthracis, Brucella abortus, Burkholderia pseudomallei, Haemophilus influenzae, Mycobacterium bovis, Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus, Streptococcus pneumoniae, and Yersinia pestis which cause anthrax, brucellosis, melioidosis, meningitis, bovine tuberculosis, typhoid fever, dysentery, numerous types of nosocomial infections, pneumonia, and plague. Thus, such TS bacteria can be used to treat or prevent such conditions.

[0117] Temperature-sensitive essential proteins from a psychrophilic bacterium are provided herein, such as those from Colwellia sp., Pseudoalteromonas sp., or Shewanella sp. Exemplarily proteins include ligA, pyrG, hemC, ftsZ, cmk, murG, fmt, and dnaK. Exemplary sequences are provided in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28. However, one skilled in the art will appreciate that variant sequences can also be used. For example, a peptide having a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 is encompassed by the present disclosure, and can be used in the methods provided herein. Variant sequences retain the biological activity of the native temperature-sensitive essential protein from a psychrophilic bacterium, such as conferring the ability to make a bacterium TS (such as heat sensitivity), for example operable at a temperature of -10.degree. C. to about 30.degree. C. (such as 0.degree. C. to 30.degree. C.), but inoperable at a temperature greater than about 30.degree. C. (for example 4.degree. C. to 30.degree. C.), such as greater than 35.degree. C. Exemplary sequences can be obtained using computer programs that are readily available on the internet and the amino acid sequences set forth herein. In one example, the variant peptide retains a function of the native protein, such as the ability to confer temperature sensitivity to a bacterium.

[0118] A specific, non-limiting example of a variant protein is a conservative variant of the native protein (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28). Substitutions of the amino acids sequence shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 can be made based on this table, as long as the pathogenic mesophilic bacteria are rendered TS and are able to initiate an immune response to its pathogenic antigens. For example, protein sequences can be altered without significantly altering their biological properties, for example by introducing one or more conservative amino acid substitutions. Therefore, any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 24, 26, or 28 can be modified by making 1 to 20, 1 to 15, 1 to 12, 1 to 10, or 1 to 5 conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 50 conservative amino acid substitutions, while retaining the ability to render a mesophilic bacteria temperature sensitive (TS). Examples of conservative substitutions are shown below:

TABLE-US-00001 Original Conservative Residue Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

[0119] Minor modifications to the disclosed protein sequences can result in peptides which have substantially equivalent activity as compared to the unmodified counterpart protein described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the proteins produced by these modifications are included herein.

[0120] Temperature-sensitive essential proteins (and nucleic acid molecules) from a psychrophilic bacterium are disclosed herein that can be used to induce temperature sensitivity in a desired bacterial host, wherein the resulting recombinant bacteria can be used to induce an immune response (for example in a mammal). These peptides can include fragments of the full-length native protein, as long as the ability to confer temperature sensitivity in the host cell is retained. In these examples, the peptide does not include the full-length amino acid sequences set forth as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28. For example no more than 10%, no more than 5%, or no more than 1% of the amino acids can be deleted, such as 1% to 5% of the amino acids.

[0121] The isolated temperature-sensitive essential proteins can be part of a fusion protein. Thus, the fusion protein can include the temperature-sensitive essential protein (see above) and a second heterologous moiety, such as a myc protein, an enzyme or a carrier (such as a hepatitis carrier protein or bovine serum albumin) covalently linked to the temperature-sensitive essential protein. In additional examples, the temperature-sensitive essential protein includes six sequential histidine residues, a .beta.-galactosidase amino acid sequence, or an immunoglobulin amino acid sequence, for example at the C- or N-terminus of the temperature-sensitive essential protein. The temperature-sensitive essential protein can also be covalently linked to a carrier. Suitable carriers include, but are not limited to, a hepatitis B small envelope protein HBsAg.

[0122] The temperature-sensitive essential proteins disclosed herein can be chemically synthesized by standard methods, or can be produced recombinantly. An exemplary process for polypeptide production is described in Lu et al., Federation of European Biochemical Societies Letters. 429:31-35, 1998. Proteins can also be produced using molecular genetic techniques, such as by inserting a nucleic acid encoding a temperature-sensitive essential protein into an expression vector, introducing the expression vector into a host cell. They can also be isolated by methods including preparative chromatography and immunological separations.

[0123] Temperature-sensitive essential nucleic acid molecules from a psychrophilic bacterium are provided herein. Exemplary sequences are provided in the nucleic acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27. However, one skilled in the art will appreciate that variant sequences can also be used. For example, a nucleic acid molecule having a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleic acid sequence set forth in one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleic acid sequence set forth in nucleotides 10-2067 of SEQ ID NO: 1, nucleotides 10-2019 of SEQ ID NO: 3, nucleotides 10-2019 of SEQ ID NO: 5, nucleotides 10-2010 of SEQ ID NO: 7) is encompassed by the present disclosure, and can be used in the methods provided herein. In some examples, the codons of a nucleic acid molecule are optimized for the bacterium into which it is introduced. In some examples, such optimization does not alter the amino acid sequence encoded thereby. For example, the psychrophilic bacterium TS essential nucleic acid can be modified to optimize codon usage for the mesophilic bacterium (e.g., M. tuberculosis or F. novicida) into which the psychrophilic bacterium TS essential nucleic acid is introduced. Exemplary sequences can be obtained using computer programs that are readily available on the internet and the nucleic acid sequences set forth herein. In one example, the variant nucleic acid sequence retains the ability to encode a protein having the function of the native protein, such as the ability to confer temperature sensitivity (e.g., heat sensitivity) to a mesophilic bacterium.

[0124] The disclosed temperature-sensitive essential nucleic acid molecules from a psychrophilic bacterium include DNA, cDNA and RNA sequences which encode the temperature-sensitive essential peptide. Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue. Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (e.g., L. Stryer, 1988, Biochemistry, 3.sup.rd Edition, W.H. 5 Freeman and Co., NY).

[0125] A nucleic acid molecule encoding a temperature-sensitive essential peptide from a psychrophilic bacterium can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR) and the Q.beta. replicase amplification system (QB). For example, a polynucleotide encoding the protein can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule. A wide variety of cloning and in vitro amplification methodologies are well known to persons skilled in the art. PCR methods are described in, for example, U.S. Pat. No. 4,683,195; Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCR Technology, (Stockton Press, N Y, 1989). Polynucleotides also can be isolated by screening genomic or cDNA libraries with probes selected from the sequences of the desired polynucleotide under stringent hybridization conditions.

[0126] The nucleic acid molecules encoding a temperature-sensitive essential peptide from a psychrophilic bacterium include a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences. The nucleic acid molecules disclosed herein can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA.

[0127] The nucleic acid molecules encoding a temperature-sensitive essential peptide from a psychrophilic bacterium can be part of a vector, such as a plasmid or viral vector. Suitable vectors include retrovirus vectors, orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, adenoviral vectors, herpes virus vectors, alpha virus vectors, baculovirus vectors, Sindbis virus vectors, vaccinia virus vectors and poliovirus vectors. Specific exemplary vectors are poxvirus vectors such as vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus (MVA), adenovirus, baculovirus and the like. Other viral vectors that can be used include other DNA viruses such as herpes virus and adenoviruses, and RNA viruses such as retroviruses and polio.

[0128] The nucleic acid molecules encoding a temperature-sensitive essential peptide from a psychrophilic bacterium can be operably linked to at least one expression control element. The expression control elements are inserted in the vector or plasmid to control and regulate the expression of the nucleic acid sequence. For example, an expression control sequence operatively linked to a temperature-sensitive essential peptide coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. Specific examples of expression control elements include, but are not limited to, lac system, operator and promoter regions of phage lambda, yeast promoters and promoters derived from polyoma, adenovirus, retrovirus or SV40. Additional operational elements include, but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary for the appropriate transcription and subsequent translation of the nucleic acid sequence encoding the temperature-sensitive essential peptide from a psychrophilic bacterium in the host system. The expression vector can contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers. It will further be understood by one skilled in the art that such vectors are easily constructed using conventional methods (Ausubel et al., (1987) in "Current Protocols in Molecular Biology," John Wiley and Sons, New York, N.Y.) and are commercially available.

[0129] In one example, vector introduced into a host bacterium includes one or more of the following elements: (i) a prokaryotic origin of replication, so that the vector may be amplified in a prokaryotic host; (ii) a gene encoding a marker which allows selection of prokaryotic host cells that contain the vector (e.g., a gene encoding antibiotic resistance); (iii) at least one DNA sequence encoding one or more temperature-sensitive essential peptides from a psychrophilic bacterium located adjacent to a transcriptional promoter capable of directing the expression of the sequence; and (iv) DNA sequences homologous to the region of the parent virus genome where the foreign gene(s) will be inserted, flanking the construct of element (iii).

[0130] The vector can contain an additional gene that encodes a marker that will allow identification of recombinant cells containing inserted foreign DNA. These include genes that encode antibiotic or chemical resistance (e.g., see Spyropoulos et al., 1988, J. Virol. 62:1046; Falkner and Moss, 1988, J. Virol. 62:1849; Franke et al., 1985, Mol. Cell. Biol. 5:1918), as well as genes such as the E. coli lacZ gene, that permits identification of recombinant plaques by colorimetric assay.

[0131] Methods of introducing nucleic acid molecules, such as those that encode a temperature-sensitive essential peptide from a psychrophilic bacterium, are well known to those skilled in the art. Where the host is prokaryotic, such as, a bacterium, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl.sub.2 method using procedures well known in the art. Alternatively, MgCl.sub.2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation. Hosts cells can include bacterial cells, such as bacteria that cause disease. Examples of such bacteria that can be used as host cells for temperature-sensitive essential nucleic acids/peptides from a psychrophilic bacterium include without limitation any one or more of (or any combination of) Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum, Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetella sp. (such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi), Brucella sp. (such as Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis), Burkholderia sp. (such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenic E. coli) Enterococcus sp. (such as Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacter fennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiella pneumoniae, Klebsiella granulomatis and Klebsiella oxytoca), Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm sp. (such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica and Nocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides. Prevotella sp., Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii, Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) and Rickettsia typhi), Rhodococcus sp., Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium), Serratia sp. (such as Serratia marcesans and Serratia liquifaciens), Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcus pneumoniae (for example chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F Streptococcus pneumoniae), Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus pyogenes, Group B streptococci, Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus, Streptococcus equismilis, Group D streptococci, Streptococcus bovis, Group F streptococci, and Streptococcus anginosus Group G streptococci), Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela and Vibrio furnisii), Yersinia sp. (such as Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudotuberculosis) and Xanthomonas maltophilia among others.

[0132] Following transformation of bacterial cells, recombinant host cells can be identified by one of several techniques. For example, expression of a gene encoding a marker or indicator gene with the temperature-sensitive gene, as described above, can be used to identify recombinant progeny. One specific non-limiting example of an indicator gene is the E. coli lacZ gene. Recombinant bacterial cells expressing beta-galactosidase can be selected using a chromogenic substrate for the enzyme. Once a recombinant bacterium has been identified, it can be selected and amplified for use in an immunogenic composition provided herein.

Methods of Making Temperature-Sensitive Bacterial Strains

[0133] The exemplary embodiments relate to methods for generating recombinant TS bacteria for use in stimulating an immune response to the TS bacteria. In one aspect, an exemplary TS immunogenic composition is suitable for immunoprophylaxis to prevent infectious disease or alternatively immunotherapy to treat an infectious disease. Such TS bacteria are generated by the introduction of one or more TS essential genes from psychrophilic bacteria into a target bacteria (such as a mesophilic bacteria that causes a disease that one wants to treat or prevent). Thus, the disclosure provides safe immunogenic compositions based on live genetically altered bacterial microorganisms. This was accomplished by taking advantage of essential genes from psychrophilic bacteria, by creating a fusion of the psychrophilic structural genes with the transcriptional and translational control elements of the "host" genome or by making fusions between the host gene and the psychrophilic gene. The exemplary embodiments provide live vaccines and immunogenic compositions that mimic a number of cold adapted viral vaccines and are unable to grow at the normal body temperature.

[0134] According to another exemplary embodiment it is suitable for mass production purposes, specifically of antigen; due to the TS strain's non-virulent nature the aerosols produced are rendered harmless and therefore, this methods and compositions disclosed herein can significantly reduce or eliminate human risk of infection.

[0135] Another aspect, the methods and compositions provided herein has value as a research diagnostic, or as a research/educational tool because it allows for experimentation to be performed on organisms that are normally highly pathogenic in their viable state without posing threats to the researcher.

[0136] The methods and compositions provided herein can be employed to stimulate the immune system with TS organisms with the intention of prevention or treatment of a disease.

[0137] A large number of psychrophilic bacteria contain TS genes, which can be used to generate TS mesophilic bacteria of the present disclosure. For example, one or more TS essential genes from psychrophilic bacteria can be introduced into a mesophilic bacterium (for example into a chromosome of a mesophilic bacteria), thereby generating a TS strain that can be used to induce an immune response in a subject into whom it is administered. Recombinant methods for introducing a nucleic acid into bacteria are routine in the art. Appropriate TS essential genes from psychrophilic bacteria can be identified using the methods provided herein. As shown in Tables 1 and 2, nine of the twenty one essential genes from the psychrophilic C. psychrerhraea were introduced into F. novicida and substituted for an essential host gene to generate TS strains of F. novicida ("Group I"). Group I genes generated a range of TS phenotypes with the restrictive temperatures of about 33.degree. C. to 44.degree. C. Thus, the genes of Group I can be used to generate TS strains of the present disclosure. Group II in Table 1 consists of the C. psychrerhraea genes that either functioned poorly or not at all in the exemplary bacterial strain F. novicida. F. novicida strains carrying an integrate with the psychrophilic essential gene resolve the integrate under counter selection pressure generated by the presence of sacB and sucrose. However, the resolved strains retain copies of both the psychrophilic gene and the F. novicida homologue and the strains are not TS ("Group III" in Table 1); indicating that these psychrophilic essential genes do not function in the mesophilic host. Alleles of the same gene from different psychrophilic bacteria can be selected to identify those that generate hybrid strains with the same TS properties when substituted into the chromosomes of mesophilic bacteria. The ligA alleles from three different psychrophilic bacteria generated three different TS phenotypes when substituted into the mesophile F. novicida. The pyrG.sub.Cp allele from C. psychrerhraea created a TS strain when substituted into F. novicida but the pyrG.sub.Sf allele from S. frigidimarina (SF) did not. PH refers to P. haloplanktis.

TABLE-US-00002 TABLE 1 Restrictive Gene Temp.(.degree. C.) symbol Source Product Function Group ligA.sub.Ph2 28/PH NAD-dependent DNA ligase I ligA.sub.Sf 33/SF NAD-dependent DNA ligase I ligA.sub.Cp 34/CP NAD-dependent DNA ligase I ligA.sub.Ph 36.8/PH NAD-dependent DNA ligase I hemC.sub.Cp 36.8/CP Porphobilinogen deaminase (Hydroxymethylbilane I synthase) pyrG.sub.Cp 37.2/CP CTP synthetase I dnaK.sub.Cp 38.2/CP Molecular chaperone DnaK I murG.sub.Cp 38.2/CP UDP-N-acetylglucosamine-N-acetylmuramyl- I (pentapeptide) pyrophosphoryl-undecaprenol N- acetylglucosamine transferase fmt.sub.Cp 41/CP Methionyl-tRNA formyltransferase I ftsZ.sub.Cp 42/CP Cell division protein I cmk.sub.Cp 43/CP Cytidylate kinase I tyrS.sub.Cp 44/CP Aminoacyl tRNA synthetases for Tyr I adk.sub.Cp >44/CP Adenylate kinase (proved resolution) II accD.sub.Cp >44/CP AcetylCoA carboxylase. The F. novicida integrate II containing accD.sub.Cp fails to resolve. murI.sub.Cp >44/CP Glutamate racemase. The CP version of MurI II appears to function poorly at all temperatures in F. novicida. pyrG.sub.Sf >44/SF CTP synthetase III trxA.sub.Cp >44/CP Thioredoxin III glmS.sub.Cp >44/CP Glucosamine-fructose-6-phosphate III aminotransferase argS.sub.Cp >44/CP Aminoacyl tRNA synthetases for Arg III cds.sub.Cp >44/CP phosphatidate cytidylyltransferase III mur.sub.CpC >44/CP UDP-N-acetylmuramate-alanine ligase III valS.sub.Cp >44/CP Aminoacyl tRNA synthetases for Val III proS.sub.Cp >44/CP Aminoacyl tRNA synthetases for Pro III metK.sub.Cp .ltoreq.44/CP S-adenosylmethionine synthetase III ftsW.sub.Cp >44/CP Cell division protein III

TABLE-US-00003 TABLE 2 Mutation rate in F. novicida to Restricted Challenge temperature resistance Gene Temp (.degree. C.) Temp (.degree. C.) Trial #1 Trial #2 Trial #3 ligA.sub.Sf 33 37 .sup. 4.0 .times. 10.sup.-6 .sup. 3.3 .times. 10.sup.-7 .sup. 9.7 .times. 10.sup.-7 ligA.sub.Cp 34 37 <1.2 .times. 10.sup.-10 <7.93 .times. 10.sup.-11 <1.1 .times. 10.sup.-10 ligA.sub.Ph 36.8 39 <1.5 .times. 10.sup.-10 <7.8 .times. 10.sup.-11 <6.2 .times. 10.sup.-11 dnaK.sub.Cp 38.2 39.5 <3.2 .times. 10.sup.-10 <1.9 .times. 10.sup.-10 <3.2 .times. 10.sup.-10 hemC.sub.Cp 36.8 43 <2.5 .times. 10.sup.-10 <3.6 .times. 10.sup.-11 <3.7 .times. 10.sup.-11 pyrG.sub.Cp 37.2 40 .sup. 8.5 .times. 10.sup.-8 1.0 .times. 10.sup.-9 .sup. 6.5 .times. 10.sup.-8 murG.sub.Cp 38.2 43 .sup. 2.6 .times. 10.sup.-4 3.0 .times. 10.sup.-5 .sup. 8.5 .times. 10.sup.-5 dnaK.sub.Sf 39 42 3.1 .times. 10.sup.-10 8.5 .times. 10.sup.-10

[0138] To make a TS bacterial pathogen, an essential gene from an Arctic psychrophile bacterium was substituted into the genome of a mesophilic pathogenic bacterium. The Arctic bacterial essential gene ligA.sub.Sf rendered F. novicida unable to grow at a temperature of 33.degree. C. or higher. Table 2 outlines the restrictive temperature properties imposed on F. novicida following the replacement of the mesophilic essential gene for its psychrophilic counterpart. Any of the genes in Table 2 may be introduced into a pathogenic bacteria strain to create live heat-sensitive vaccines. Exemplary pathogenic bacteria include but are not limited to: Mycobacterium sp., Haemophilus sp., Vibrio sp., Escherichia sp., Salmonella sp., Streptococcus sp., Burkholderia sp., Campylobacter sp., Neisseria sp., and Francisella sp.

[0139] The disclosure relates to genes derived from psychrophilic bacteria for use in the development of heat-sensitive immunogenic compositions, and methods of using these compositions to stimulate an immune response in a subject. In a specific example, the disclosure provides recombinant pathogens (such as Mycobacterium sp., Haemophilus sp., Vibrio sp., Escherichia sp., Salmonella sp., Streptococcus sp., Burkholderia sp., Campylobacter sp., Neisseria sp., and Francisella sp.) containing one or more heat-sensitive genes, exemplified by ligA, pyrG, hemC, ftsZ, cmk, dnaK, and fmt, that can be administered to a subject to provide a prophylactic immune response against diseases caused by such bacteria.

[0140] Methods of making a recombinant temperature-sensitive (TS) bacterial cell are provided. In one example the method includes introducing into the genome of a mesophilic bacterial strain a nucleic acid construct that includes a TS essential nucleic acid molecule from a psychrophilic bacteria (such as one that encodes a peptide that is operable at a temperature of about -10.degree. C. to about 30.degree. C., and/or inoperable at a temperature greater than about 30.degree. C., for example Colwellia sp., Psuedoalteromonas sp., or Shewanella sp) and one or more control sequences operably linked to the TS essential nucleic acid molecule. The temperature-sensitive essential polynucleotide renders the mesophilic bacteria operable at a temperature less than about 30.degree. C. and inoperable at a temperature greater than about 30.degree. C. In some examples, the temperature-sensitive essential nucleic acid molecule includes a nucleotide sequence having at least 80%, at least 90%, or at least 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27. In some examples the method also includes isolating the TS essential nucleic acid molecule from the genome of the psychrophilic bacterial strain. The method can also include constructing or generating the nucleic acid construct comprising the TS essential nucleic acid molecule and one or more control sequences operably linked to the TS essential nucleic acid molecule.

[0141] In some examples, the method further includes culturing the recombinant TS bacterial host cell at a temperature wherein the temperature-sensitive peptide is operable, whereby said recombinant TS bacterial host cell produces a plurality of peptides; increasing the culturing temperature to a temperature at which the temperature-sensitive peptide is inoperable; maintaining said culturing for a period of time sufficient to kill the recombinant TS bacterial host cell; and harvesting the killed recombinant TS bacterial host cells.

[0142] Methods of making a recombinant TS bacterial host cell can also include the following. A psychrophilic microbial genome is screened for detection of a TS essential polynucleotide that encodes a peptide that is inactivated at about greater than 30.degree. C.; isolating said TS essential polynucleotide; constructing a nucleic acid construct comprising the TS essential polynucleotide and one or more control sequences operably linked to the TS polynucleotide; inserting the nucleic acid construct into the genome of a selected mesophilic bacterial host cell (such as Francisella novicida) thereby functionally replacing the host cell's homologue of the TS essential polynucleotide whereby the TS peptide (and thus the bacteria in which it is expressed) is operable at a temperature less than about 30.degree. C., and inoperable at a temperature greater than about 30.degree. C. and mimics the temperature sensitivity of the original designated host bacterium. The resulting recombinant mesophilic bacterial host cell comprising the TS polynucleotide is cultured or grown at a temperature less than about 30.degree. C. to confirm the viability of the recombinant mesophilic bacterial host cell; further culturing the recombinant mesophilic bacterial host cell comprising the TS polynucleotide at a temperature greater than about 30.degree. C. to determine if the mesophilic bacterial host cell is killed. If the mesophilic bacterial host cell is killed, the nucleic acid construct is introduced into the genome of a selected destination mesophilic bacterial host cell (such as Salmonella sp. or Mycobacterium sp.) thereby functionally replacing the host cell's homologue of the temperature-sensitive essential polynucleotide whereby the temperature-sensitive peptide (and thus the bacteria in which it is expressed) is operable at a temperature less than about 30.degree. C., and inoperable at a temperature greater than about 30.degree. C. and mimics the temperature sensitivity of the original tester host bacterium.

[0143] In some examples, the mesophilic bacteria is one that is operable at a temperature selected from the range of about 10.degree. C. to about 50.degree. C. prior to introduction of the TS essential nucleic acid molecule from a psychrophilic bacteria. Examples of such mesophilic bacteria include strains of fermentative bacteria or bioremediation bacteria. Other exemplary bacteria are provided above.

[0144] In some examples, the TS essential nucleic acid molecule expresses a peptide during a culturing of the recombinant TS bacteria, such as a peptide having at least 80%, at least 90%, or at least 95% sequence identity to an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28.

[0145] Recombinant TS bacteria generated by these methods, as well as compositions that include such bacteria, are also provided herein.

Temperature-Sensitive Bacterial Strain Compositions

[0146] Compositions are provided that include recombinant TS bacteria provided herein. In some examples, the compositions include more than one type of recombinant TS bacteria, such as 2, 3, 4 or 5 different recombinant TS bacteria. In some examples, the recombinant TS bacteria contain two or more different TS essential psychrophilic coding sequences (such as two or more of the Group I genes listed in Table 1, such as ligA and another Group I gene). In particular examples, the recombinant TS bacteria is a Francisella sp., Salmonella sp., or Mycobacterium sp. (other particular examples are provided above).

[0147] In some examples, such compositions are immunogenic, in that they can stimulate an immune response in a mammal. The compositions can include other components, such as pharmaceutically acceptable carriers (such as saline), adjuvants, preservatives, combinations thereof, and the like.

Methods of Stimulating an Immune Response Using Temperature-Sensitive Bacterial Strains

[0148] The TS recombinant bacteria disclosed herein can be used to generate an immune response in a subject. In some examples, the subject is infected with a bacterium, or as at risk of being infected with a bacterium (such as a health care worker), such as Mycobacterium tuberculosis. Thus, in several embodiments, the methods include administering to a subject a therapeutically effective amount of one or more of the TS recombinant bacteria disclosed herein in order to generate an immune response, such as, but not limited to, a protective immune response. For example, two or more different TS recombinant bacteria (such as those expressing different TS essential peptides from psychrophilic bacteria) can be used to generate an immune response in a subject. In some examples, the recombinant bacterium used to generate an immune response in a subject expresses two or more different temperature-sensitive essential peptides from a psychrophilic bacterium or the same temperature-sensitive essential peptide from two or more different psychrophilic bacteria.

[0149] The TS recombinant bacterium administered is selected based on the bacterial infection to be prevented or treated. For example, if the bacterial infection to be prevented or treated in the subject is tuberculosis, then the TS recombinant bacteria is Mycobacterium tuberculosis expressing at least one TS essential peptide from a psychrophilic bacterium. In another example, if the bacterial infection to be prevented or treated in the subject is tularemia, then the TS recombinant bacteria is F. tularensis expressing at least one TS essential peptide from a psychrophilic bacterium.

[0150] In exemplary applications, compositions are administered to a subject having in an amount sufficient to produce an immune response to the TS recombinant bacteria. These TS recombinant bacteria are of use to prevent a bacterial infection (such as Mycobacterium tuberculosis) prevent progression to disease in a subject having a latent bacterial infection, or to treat a disease resulting from the bacterial infection (such as tuberculosis). In several examples, administration of a therapeutically effective amount of a composition including the TS recombinant bacteria disclosed herein induces a sufficient immune response to decrease a symptom of a disease due to bacterial infection, to prevent the development of one or more symptoms of the disease associated with the infection, or to prevent infection with the bacteria.

[0151] In some examples, the compositions are of use in preventing a future bacterial infection. Thus, a therapeutically effective amount of the composition is administered to a subject at risk of becoming infected with a bacterium, such as Mycobacterium tuberculosis. For example the disclosed compositions can be used to prevent the development of tuberculosis, such as latent or active tuberculosis in the subject upon subsequent exposure to Mycobacterium tuberculosis. In one example, the compositions are administered to a subject with a latent Mycobacterium tuberculosis infection, and prevent the development of symptoms of tuberculosis. Thus the compositions are of use in treating a subject with latent tuberculosis, such that the subject does not develop active tuberculosis.

[0152] Amounts effective for these uses will depend upon the severity of the disease, the general state of the patient's health, and the robustness of the patient's immune system. In one example, a therapeutically effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. In other examples, a therapeutically effective amount is an amount sufficient to prevent an infection with the bacterium in a subject upon subsequent exposure of the subject to the bacterium. In additional examples, a therapeutically effective amount is an amount sufficient to prevent development of symptom in a subject infected with a bacterium.

[0153] The TS recombinant bacteria-containing composition can be administered by any means known to one of skill in the art either locally or systemically, such as by intramuscular injection, subcutaneous injection, intraperitoneal infection, intravenous injection, oral administration, nasal administration, transdermal administration or even anal administration. In one embodiment, administration is by oral, subcutaneous injection or intramuscular injection. To extend the time during which the TS recombinant bacteria is available to stimulate a response, the TS recombinant bacteria can be provided as an implant, an oily injection, or as a particulate system. The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle. A particulate carrier based on a synthetic polymer has been shown to act as an adjuvant to enhance the immune response, in addition to providing a controlled release. Aluminum salts can also be used as adjuvants to produce an immune response.

[0154] In one specific, non-limiting example, the TS recombinant bacteria are administered in a manner to direct the immune response to a cellular response (that is, a cytotoxic T lymphocyte (CTL) response), rather than a humoral (antibody) response.

[0155] Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES, GM-CSF, TNF-.alpha., or IFN-.gamma., one or more growth factors, such as GM-CSF or G-CSF; one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72, B7-1, B7-2, or other B7 related molecules; one or more molecules such as OX-40L or 41 BBL, or combinations of these molecules, can be used as biological adjuvants (see, for example, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can be administered systemically (or locally) to the subject. In some examples, IL-2, RANTES, GM-CSF, TNF-.alpha., IFN-.gamma., G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1 B7-2, OX-40L, 41 BBL and ICAM-1 are administered. In various embodiments, the nucleic acid encoding the biological adjuvant can be cloned into same vector as the psychrophilic TS essential peptide coding sequence, or the nucleic acid can be cloned into one or more separate vectors for co-administration into the bacteria.

[0156] A pharmaceutical composition including TS recombinant bacteria is thus provided. These compositions are of use to promote an immune response to a particular bacterium. In one embodiment, TS recombinant bacteria are mixed with an adjuvant containing two or more of a stabilizing detergent, a micelle-forming agent, and an oil. Suitable stabilizing detergents, micelle-forming agents, and oils are detailed in U.S. Pat. No. 5,585,103; U.S. Pat. No. 5,709,860; U.S. Pat. No. 5,270,202; and U.S. Pat. No. 5,695,770, all of which are incorporated by reference. A stabilizing detergent is any detergent that allows the components of the emulsion to remain as a stable emulsion. Such detergents include polysorbate, 80 (TWEEN) (Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured by ICI Americas, Wilmington, Del.), TWEEN 40.TM., TWEEN 20.TM., TWEEN 60.TM., ZWITTERGENT.TM. 3-12, TEEPOL HB7.TM., and SPAN 85.TM.. These detergents are usually provided in an amount of approximately 0.05 to 0.5%, such as at about 0.2%. A micelle forming agent is an agent which is able to stabilize the emulsion formed with the other components such that a micelle-like structure is formed. Such agents generally cause some irritation at the site of injection in order to recruit macrophages to enhance the cellular response. Examples of such agents include polymer surfactants described by BASF Wyandotte publications, e.g., Schmolka, J. Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al., J. Immunol 129:1244, 1981, PLURONIC.TM. L62LF, L101, and L64, PEG1000, and TETRONIC.TM. 1501, 150R1, 701, 901, 1301, and 130R1. The chemical structures of such agents are well known in the art. In one embodiment, the agent is chosen to have a hydrophile-lipophile balance (HLB) of between 0 and 2, as defined by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent can be provided in an effective amount, for example between 0.5 and 10%, or in an amount between 1.25 and 5%.

[0157] In one example oil is included in the composition. Examples of such oils include squalene, Squalane, EICOSANE.TM., tetratetracontane, glycerol, and peanut oil or other vegetable oils. In one specific, non-limiting example, the oil is provided in an amount between 1 and 10%, or between 2.5 and 5%. The oil should be both biodegradable and biocompatible so that the body can break down the oil over time, and so that no adverse affects, such as granulomas, are evident upon use of the oil.

[0158] In one embodiment, the adjuvant in the composition is a mixture of stabilizing detergents, micelle-forming agent, and oil available under the name PROVAX.RTM. (IDEC Pharmaceuticals, San Diego, Calif.). An adjuvant can also be an immunostimulatory nucleic acid, such as a nucleic acid including a CpG motif, or a biological adjuvant (see above).

[0159] Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems, see Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, Pa., 1995. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 .mu.m are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 .mu.m so that only nanoparticles are administered intravenously. Microparticles are typically around 100 .mu.m in diameter and are administered subcutaneously or intramuscularly (see Kreuter, Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339, 1992).

[0160] In particular examples, at least 10.sup.2 CFU of the TS bacteria disclosed herein are administered per dose, such as at least 10.sup.3 CFU, at least 10.sup.4 CFU, at least 10.sup.5 CFU, at least 10.sup.6 CFU, at least 10.sup.7 CFU, at least 10.sup.8 CFU, such as 10.sup.2 to 10.sup.8 CFU or 10.sup.4 to 10.sup.8 CFU. In particular examples, such dosages are administered intradermal or intranasal.

[0161] Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the subject. In one embodiment, the dosage is administered once as a bolus, but in another embodiment can be applied periodically until a therapeutic result is achieved. In one embodiment, the dose is sufficient to treat or ameliorate symptoms or signs of bacterial infection without producing unacceptable toxicity to the subject. In another embodiment, the dose is sufficient to prevent infection with a bacterium upon subsequent exposure to the bacterium (such as M. tuberculosis). In a further embodiment, the dose is sufficient to prevent a symptom of bacterial infection (e.g., tuberculosis) in a subject with a latent bacterial infection. Systemic or local administration can be utilized.

[0162] Thus the disclosure provides methods for producing an immune response to a bacterium in a subject. The method can include administering to the subject a therapeutically effective amount of a TS bacterium, wherein the temperature-sensitive bacterium expresses a psychrophilic TS essential protein or nucleic acid molecule provided herein (such as a nucleic acid coding sequence in a vector), thereby inducing an immune response to the bacterium. The method can further include administering other agents, such as an adjuvant or antimicrobial agent (such as an antibiotic). In some examples, the immune response is a protective immune response. The subject may have a bacterial infection, be at risk for acquiring a bacterial infection, or have a latent bacterial infection. Exemplary bacterial infections include infections with is M. tuberculosis, Salmonella or Francisella.

[0163] Methods of measuring an immune response following stimulation with a bacterial antigen, such as a cytokine response, are known in the art. In some examples, the method further includes measuring an immune response following administration of the therapeutic compositions provided herein. In one example, a cytokine response is increased following administration of the composition provided herein, such as an increase relative to the absence of administration of the composition. In one example, cytokine production increases by at least 20%, such as at least 40%, at least 50%, at least 75%, at least 90%, or at least 95% following administration of the composition, relative to the cytokine response in the absence of administration of the composition.

[0164] The disclosure is illustrated by the following non-limiting Examples.

Example 1

[0165] This example pertains to an exemplary method to create recombinant psychrophilic genes joined to flanking DNA of a mesophilic host.

[0166] FIG. 1a exemplifies the fusion PCR (also known as, "extension overlap PCR", "overlap PCR" or "splice overlap PCR") strategy used to incorporate the C. psychrerythraea essential gene (C2) into the wt F. novicida genome. The C. psychrerythraea genes were engineered with overlap PCR to contain the ribosome binding site (RBS) and the initial three codons and stop codon of the surrounding F. novicida genes (F1 and F3) to promote translation of the C2 gene at normal levels for F. novicida. The fusion PCR product was ligated to an erythromycin resistant sacB cassette (Em.sup.R-sacB) prior to its transformation into F. novicida. Em.sup.R colonies containing the fusion PCR product were grown in the presence of sucrose and colonies were screened for the loss of Em.sup.R, the F. novicida essential gene (F2), and the presence of C2.

[0167] FIG. 1b illustrates the introduction of the psychrophilic gene fusion construct into the target organism's chromosome via a single crossover event. Furthermore, it illustrates that the excision can be enhanced using the counter-selectable sacB marker. For genes that are not a part of a multi-cistronic operon the upstream pathogen genomic region was fused to the psychrophilic structural gene from codon 4 through to the stop codon. A similar approach was used when substituting a psychrophilic allele into the middle of an operon. However, as one skilled in the art can appreciate, depending on the nature of the operon, some of the codons at the C terminus of the host homologue remained if they were important for translation of the downstream cistron.

Example 2

[0168] This example pertains to an exemplary method to insert the psychrophilic allele into the mesophilic bacteria.

[0169] FIG. 2a illustrates the substitution region for the psychrophilic ligA gene, corresponding to SEQ ID NO: 1. Additionally, it illustrates its incorporation into the wt F. novicida chromosome. FIGS. 2b-e illustrate the integration point for the psychrophilic ligA genes of C. psychrerythraea, S. frigidmarina, P. haloplanktis I, and P. haloplanktis 2 respectively. The first three codons for F. novicida were retained in order to maximize the potential for ligA expression levels. FIG. 2a-e illustrates that in most cases the integration and excision events result in a simple substitution of the psychrophilic gene for the mesophilic host homologue. However, the integration and excision events may also lead to the formation of a hybrid gene as illustrated in FIG. 1b.

Example 3

[0170] This example pertains to an exemplary method to determine the maximal growth temperature of each bacterial strain and to show its growth properties at restrictive temperatures.

[0171] Each bacterial strain was tested on agar plates placed in a highly stable (.+-.1.degree. C.) incubator; the restrictive temperatures were defined as the lowest temperatures that did not permit the formation of isolated colonies on an agar streak plate. The growth properties at different temperatures of four different transgenic strains of F. novicida carrying psychrophilic ligA substitutions and the growth properties of wt F. novicida are shown in FIGS. 3-6. The psychrophilic ligA genes are ligA.sub.Cp, ligA.sub.Sf, ligA.sub.Ph and ligA.sub.Ph2, as represented by SEQ ID NOs: 1, 7, 3, and 5 respectively. In the first panels in FIGS. 3-6, growth is shown at a permissive temperature, i.e., a temperature below the restrictive temperature. In subsequent panels, growth of both the transgenic and wt strains is shown before and after a shift to the restrictive temperature or higher.

[0172] Extended growth curves of both the F. novicida transgenic and wt strains are shown as inserts in select panels of FIG. 3-6. These curves were generated by taking a fully grown culture, diluting it, and monitoring its growth in fresh growth media. More specifically, the wt and TS transgenic F. novicida cultures were grown at restrictive temperatures until they reached stationary phase at which point, they were diluted and re-incubated for growth at the restrictive temperatures again. The additional growth curves demonstrate that the cessation of growth exhibited by the transgenic strain is a real phenomenon, as opposed to a temporary adjustment to the temperature shift.

Example 4

[0173] This pertains to an exemplary method used to determine the frequency of mutations that permit bacterial growth at temperatures higher than the restrictive temperature of TS F. novicida transgenic strains.

[0174] Cultures were grown to late logarithmic phase at the permissive growth temperature, they were then diluted in a series of 10.sup.9-10.sup.5 cells/plate on agar and incubated at temperatures about 3.degree. C. above the restrictive temperature, as well as at temperatures about 3.degree. C. below the restrictive temperature. From this dilution series the rate of mutations that allow for growth at higher temperatures were calculated, Table 2 exemplifies the frequency of mutation to temperature resistance in F. novicida. Remarkably, some of the psychrophilic genes are unable to mutate to forms that will function above their restrictive temperature. One skilled in the art may hypothesize that the millions of years required to adapt to a cold climate renders some of the psychrophilic essential gene products unable to adopt simple changes allowing them to function in temperatures typical to their mesophilic counterparts. These include ligACp, ligA.sub.Ph, hemC.sub.Cp, dnaK.sub.Cp, fmt.sub.Cp, and dnaK.sub.Sf.

Example 5

[0175] This example pertains to an exemplary method to determine the duration of viability of the recombinant TS bacterial strains at the restrictive temperature.

[0176] An exemplary culture of a TS transgenic strain that has a maximal growth temperature of about 33.degree. C., was grown at about 30.degree. C. and a sample of the culture was incubated at about 37.degree. C. to mimic the typical temperature of human body core tissues. Samples were taken at varying time points between 0-24 hours, and the individual samples were re-diluted, plated on to growth media, and then cultured at about 30.degree. C. to determine the death rate above the restrictive temperature. As a control, the same experiment was carried out with the wt bacterium.

[0177] The persistence of F. tularensis strains carrying the psychrophilic essential genes within their macrophages was determined. Transgenic strains were cultured at about 30.degree. C. and used to infect macrophages at about 37.degree. C. in 24 well tissue culture plates using standard methods known to those skilled in these arts. For several days monitoring the infected macrophages a subset of cells were lysed and the bacteria were plated onto agar medium and incubated at about 30.degree. C. The data generated in these experiments showed the lifespan of transgene strains during an infection with macrophages at a restrictive temperature and helped to predict the persistence of TS strains during infections.

[0178] This example can be extrapolated to provide an in vitro correlation for what can occur in a mammal. A TS transgenic strain will grow in a cool part of the body such as the skin. Replication of the strain at and about this cool site will constantly cause the TS transgenic strain progeny to be moved into the draining lymph nodes. Depending on the locations of the lymph nodes and the restrictive temperature of the TS transgenic strain, the TS progeny will die over a period of several hours. The presence of the TS transgenic strain both in its live and dead states will stimulate an immune response.

Example 6

[0179] This pertains to an exemplary method to determine the ability of a TS essential gene from a psychrophile to impart its TS phenotype on a mesophilic bacterium. Specifically, it provides a method for transferring a psychrophilic essential gene encoding a TS product to a variety of bacteria as well as the transfer of the TS essential gene between mesophiles.

[0180] Several psychrophilic essential genes were substituted into the genome of the mesophilic bacterium F. novicida. Multiple approaches can be used to inserting a psychrophilic essential gene into a given bacterium in place of its mesophilic homologue. Furthermore, it can be appreciated that one can substitute a given psychrophilic essential gene into many different bacteria. The following three methods exemplify various ways of substituting ligA.sub.Cp into three different bacteria. A common approach to gene substitution is illustrated in FIG. 1b, and involves the integration of a foreign gene in a bacterium that is in close proximity to the hosts' homologous gene through PCR. Following integration, a counter selective marker, such as sacB, can be used to help identify the results of the integration and excision events. Specifically this approach was used to replace the F. novicida ligA gene with the psychrophilic ligA.sub.Cp gene.

[0181] An alternate approach was used to replace the S. enterica ligA. The strain of S. enterica used had a bacteriophage Mu insertion in the chromosomal copy of ligA (Park et al., 1989. J. Bacteriol. 171: 2173-80.). A wt copy of the bacteriophage T4 DNA ligase was carried on the ampicillin resistant plasmid, pBR313. The ligA.sub.Cp gene was introduced on the compatible chloramphenicol resistant plasmid, pSUP2716, and the recombinant S. enterica strain was cultured in the absence of ampicillin and the presence of chloramphenicol. These growth conditions allow the pBR313:T4 DNA ligase recombinant plasmid to be lost. S. enterica strains that had lost the plasmid encoding the T4 DNA ligase, rendering them ampicillin sensitive, were dependant on the ligA.sub.Cp for viability and were TS.

[0182] Another alternate approach can be employed when introducing a psychrophilic essential gene into Gram-positive bacteria. The method of insertion of ligA.sub.Cp into M. smegmatis described herein exemplifies this method. A version of ligA.sub.Cp (SEQ ID NOS: 17 and 18) designed with optimal codons was cloned into the mycobacterial plasmid, pSM1; this a precautionary step due to the low G+C content in the ligA.sub.Cp gene when compared to that of the M. smegmatis and M. tuberculosis ligA genes. The recombinant pSMT3:ligA.sub.Cp was electroporated into M. smegmatis. Subsequently a large fragment of the M. smegmatis ligA gene was deleted resulting in a strain dependent on ligA.sub.Cp for viability. This strain was TS at about 34.degree. C. This temperature is reflective of the TS nature of the F. novicida transgene strain encoding ligA.sub.Cp.

[0183] This example illustrates the use of a mesophilic tester strain which contains a psychrophilic essential gene to predict the TS phenotype when said psychrophilic essential gene is used to construct a transgene strain of another mesophilic bacterium. In this example, the tester strain was F. novicida. The substitution of ligA.sub.Cp for the F. novicida ligA homologue showed that ligA.sub.Cp functioned in the mesophile and imparted a TS phenotype having a restrictive temperature of about 34.degree. C. The phenotype of the transgenic strain of F. novicida carrying ligA.sub.Cp predicted that substitution of the ligA.sub.Cp gene into other mesophiles (destination hosts) would results in viable bacteria that had a restrictive temperature of 34.degree. C. The phenotype of the Salmonella and Mycobacteria transgene strains carrying ligA.sub.Cp showed that the inter-genus transfer of a TS psychrophilic essential gene could result in a phenotype seen in the tester strain.

Example 7

[0184] This example describes an exemplary method to combine psychrophilic genes or fragments thereof (as represented by SEQ ID NO 1-24) or mutant essential psychrophilic genes to create gene products with desired TS properties.

[0185] Combining about 30%, at the 5'-end, of the novicida pyrG gene with about 2/3 of the 3'-end of the C. psychrerythraea pyrG gene (pyrG.sub.Cp) in the region of codon 157-159 created a recombinant gene that was TS at 37.degree. C. The F. novicida and C. psychrerythraea pyrG genes are identical at codons 157-159 inclusive. Additionally, the single point mutation at amino acid residue 149 in ligA.sub.Ph from an asparagine ("N") residue to a lysine ("K") residue changes the restrictive temperature from 37.degree. C. to 28.degree. C.

[0186] This approach could be applied to different psychrophilic genes by using either in vitro or in vivo recombinant technologies to combine two or more homologues of the same gene.

Example 8

[0187] This example pertains to an exemplary method to determine the distribution of a transgenic strain from a site of infection in a mammal.

[0188] F. novicida (a.k.a. F. tularensis subspecies novicida) carrying a psychrophilic transgene was used. One skilled in the art will appreciate that similar methods can be used to generate and examine TS strains of F. tularensis. F. novicida is highly virulent in mice. The infection of mice by F. novicida serves as a model for the infection of larger mammals with F. tularensis. Most strains of F. tularensis are highly virulent in most mammals.

[0189] The distribution of F. novicida transgenic strains from the site of infection was assessed either by injecting the recombinant strains through the skin, or by introduction via the nose, and measuring the amount of viable F. novicida cells in internal organs such as the lung, liver and spleen about three to ten days after the inoculation. It was found that TS F. novicida transgenic strains did not spread significantly from the site of inoculation. A direct correlation between the inactivation temperature of the psychrophilic essential gene and the level of distribution throughout the system was observed; the dissemination of TS F. novicida strains is Lewis Rats is outlined in Table 3.

TABLE-US-00004 TABLE 3 F. novicida Restrictive CFU/Tail strain Temp. (.degree. C.) injection site CFU/Spleen wt.sup.- 45 9.7 .times. 10.sup.3/7.1 .times. 10.sup.3 3.7 .times. 10.sup.6/2.2 .times. 10.sup.6 ligA.sub.Cp 34 5 .times. 10.sup.2/3 .times. 10.sup.2 0/0 ligA.sub.Ph 36.8 3 .times. 10.sup.2/2 .times. 10.sup.2 0/0 dnaK.sub.Cp 38.2 1.5 .times. 10.sup.4/7.6 .times. 10.sup.4 5.0 .times. 10.sup.2/0 .sup. fmt.sub.Cp 41 5.2 .times. 10.sup.3/2.4 .times. 10.sup.3 3.5 .times. 10.sup.5/2.1 .times. 10.sup.5

[0190] As a further example, one of the psychrophilic essential genes (ligA.sub.Cp) was substituted into the genome of M. tuberculosis to create a transgenic strain. Some psychrophilic essential genes originate in bacteria with DNA with low G+C content. Thus the genes were optimized with codons for M. tuberculosis prior to inserting the psychrophilic genes into the pathogenic bacteria (SEQ ID NOS: 17 and 18 provide the optimized sequences). Codon optimization is a method well known to those skilled in these arts and can be accomplished using freely available bioinformatic tools. The codon optimized psychrophilic essential genes were inserted into M. tuberculosis by methods that are well described in Examples 1 and 2. M. tuberculosis, like M. smegmatis, are Gram-positive bacteria.

[0191] Another exemplary method pertains to an exemplary method the distribution of a Gram-negative pathogenic strain. A psychrophilic essential gene was introduced into S. enterica. Upon introduction of the ligA.sub.Cp psychrophilic essential gene into S. enterica, the result was a transgenic strain that was unable to grow at 37.degree. C., as illustrated in FIG. 8. Furthermore, this strain was unable to disperse from the site of inoculation in infected mice, as evidenced by the inability of the strain to migrate to the lungs, liver or spleen.

Example 9

[0192] This example pertains to an exemplary method to determine the level of protective immune response generated from the inoculation of a mammal with a TS transgenic bacterial strain. Methods of inoculation are known in the art, and can include i.v., i.m., s.c., or i.p injection, as well as inhalation, oral, and transdermal routes of delivery. One skilled in the art will appreciate that methods similar to those described in this example can be used to test any transgenic TS bacterial strain that includes one or more psychrophilic essential nucleic acid sequences.

[0193] Inoculation of mice with a TS F. novicida transgenic strain (Fn-ligA.sub.Ph, Fn-ligA.sub.Cp or Fn-dnaK.sub.Cp) caused the cells of their immune systems to be stimulated (as measured by reduced bacterial organ burdens) resulting in protection against infection with wt F. novicida (FIGS. 10a-d). Mice were initially inoculated with the TS transgenic strain and then challenged with an inoculation three weeks later of the wt F. novicida strain. This resulted in reduced growth in the livers and spleens of mice infected with the wt strains as compared to mice that had not been inoculated with recombinant F. novicida. Furthermore, decreases in the morbidity and mortalities were observed among the inoculated group of mice resulting in the conclusion that immune protection was achieved.

[0194] Similarly, mice vaccinated with M. tuberculosis and S. enterica transgenic strains (ligAPh) were shown to be more resistant to infections with the wt pathogens than were un-vaccinated mice.

Example 10

[0195] This example pertains to an exemplary method of discovering novel psychrophilic essential genes.

[0196] Psychrophilic bacterium can be isolated from a cold environment, for example ocean waters near the Earth's poles. Essential genes can be identified by using degenerate PCR or other standard techniques to find highly conserved genes, such as bacterial essential genes. Once these genes have been identified, they can be substituted into the genome of a mesophile using the methods provided herein or known in the art, displacing the host homologue of the gene. The resulting strain can then be tested for temperature sensitivity as described herein.

Example 11

[0197] This example pertains to an exemplary method of using TS transgene strains in drug discovery research. Although a TS F. tularensis strain is exemplified, one skilled in the art will appreciate that similar methods can be used for other TS strains generated using the methods provided herein.

[0198] A TS transgenic strain of F. tularensis (ligA.sub.Ph) that was inoperable above about 37.degree. C. was used to infect cell line macrophages grown in microtiter plates at 34.degree. C. A library of antimicrobial drug candidates was introduced to individual wells that contained the infected macrophages, and the effect of the drug candidates on the killing of F. tularensis was measured by lysing the macrophages at various time points and determining the number of viable TS transgenic F. tularensis by plating on agar plates. Wt F. tularensis is extremely infectious and causes a deadly disease. The use of the TS transgenic F. tularensis strain allowed one to use greatly relaxed biological containment conditions because the strain is incapable of causing disease in humans.

Example 12

[0199] This example pertains to an exemplary method of generating and using TS strains of Mycobacterium containing temperature-sensitive essential nucleic acid molecules from psychrophilic bacteria to develop an immunogenic composition, which for example can be used to stimulate an immune response in a mammal, to protect or treat an M. tuberculosis infection in the mammal.

[0200] The ligA.sub.Ph and pryG.sub.Cp genes will separately be introduced into M. tuberculosis H37Rv using an integration/excision approach. The counter-selectable marker sacB will be used to enhance the generation of excision events that can be detected. C57BL/6 mice will be vaccinated by introducing 10,000 bacteria subcutaneously at the base of the tail. Negative controls mice injected with PBS and positive control mice injected with the BCG strain will processed at the same time. The mice will be rested for 30 days. Following this period all of the mice will be exposed to an aerosol of M. tuberculosis H37Rv that deposits 150 bacteria into the lungs. At weeks 0, 4, 8 16 and 32 following exposure to M. tuberculosis H37Rv, the mice will be euthanized and the number of M. tuberculosis H37Rv in the lungs and spleens determined. If the transgenes TS M. tuberculosis strains are successful at inducing a protective immune response, the number of bacteria in the mice organs will be less than that of the negative control. Subsequent experiments will be performed in a guinea pig model of tuberculosis.

[0201] In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that illustrated embodiments are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Sequence CWU 1

1

2812070DNAArtificial SequenceligACp hybrid 1atg act cca gtc gaa aag aaa att agc caa ctg caa cag cag ctt aat 48Met Thr Pro Val Glu Lys Lys Ile Ser Gln Leu Gln Gln Gln Leu Asn 1 5 10 15 caa tat aat cat gaa tat tat gta tta gac caa cct agt gtg cct gat 96Gln Tyr Asn His Glu Tyr Tyr Val Leu Asp Gln Pro Ser Val Pro Asp 20 25 30 gca gaa tat gac cga tta atg aca gca tta atc gat tta gaa aag act 144Ala Glu Tyr Asp Arg Leu Met Thr Ala Leu Ile Asp Leu Glu Lys Thr 35 40 45 aat cct gag ctt aag act att gac tca cct agc caa aaa gtt ggc ggt 192Asn Pro Glu Leu Lys Thr Ile Asp Ser Pro Ser Gln Lys Val Gly Gly 50 55 60 cag gca tta aaa tct ttc act caa gta act cat cag ctg ccg atg ctt 240Gln Ala Leu Lys Ser Phe Thr Gln Val Thr His Gln Leu Pro Met Leu 65 70 75 80 tct ctt gat aat gtt ttt tct tta gat gat ttt cac gca ttt gtt aaa 288Ser Leu Asp Asn Val Phe Ser Leu Asp Asp Phe His Ala Phe Val Lys 85 90 95 cgc gta aaa gat agg tta aat gac aat caa gcg ata gtc ttt tgt gcc 336Arg Val Lys Asp Arg Leu Asn Asp Asn Gln Ala Ile Val Phe Cys Ala 100 105 110 gag cct aaa tta gac ggt tta gca gtg agt tta cgt tat gag cac ggg 384Glu Pro Lys Leu Asp Gly Leu Ala Val Ser Leu Arg Tyr Glu His Gly 115 120 125 cag tta ata caa gcg gct aca cgt ggc gat ggt agt gta ggg gag aat 432Gln Leu Ile Gln Ala Ala Thr Arg Gly Asp Gly Ser Val Gly Glu Asn 130 135 140 att acg act aac att cgt aca ata aaa tct att ccg ctt aag tta atg 480Ile Thr Thr Asn Ile Arg Thr Ile Lys Ser Ile Pro Leu Lys Leu Met 145 150 155 160 ggc aca cca ggt aaa gat ttt cct gat atc gtt gaa gtc cgc ggt gaa 528Gly Thr Pro Gly Lys Asp Phe Pro Asp Ile Val Glu Val Arg Gly Glu 165 170 175 gtt ttt atg cct aag gca agt ttt gac gca tta aat aca tkg gct aaa 576Val Phe Met Pro Lys Ala Ser Phe Asp Ala Leu Asn Thr Xaa Ala Lys 180 185 190 aaa cgt ggc gag aaa ggt ttt gca aat cca cgt aat gca gcg gcg gga 624Lys Arg Gly Glu Lys Gly Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly 195 200 205 agt tta cga caa ctt gat tct aaa atc acc gct aaa cgt aat tta gct 672Ser Leu Arg Gln Leu Asp Ser Lys Ile Thr Ala Lys Arg Asn Leu Ala 210 215 220 ttt tac gct tat agc ctt gga ttt gta ggg aaa ctg tct gat gga ggc 720Phe Tyr Ala Tyr Ser Leu Gly Phe Val Gly Lys Leu Ser Asp Gly Gly 225 230 235 240 gct gaa agt acc gat tta acc aat gac ttt ttt gct aac tct cat cat 768Ala Glu Ser Thr Asp Leu Thr Asn Asp Phe Phe Ala Asn Ser His His 245 250 255 gaa aga cta tgt cag ctt aaa agg ttg ggt ttg cct atg tgt cca gaa 816Glu Arg Leu Cys Gln Leu Lys Arg Leu Gly Leu Pro Met Cys Pro Glu 260 265 270 gta cgc tta ctt gaa agt gag caa gcc tgt gat gcg ttt tat caa gat 864Val Arg Leu Leu Glu Ser Glu Gln Ala Cys Asp Ala Phe Tyr Gln Asp 275 280 285 atc tta gca aag cgt agt gcc ttg agt tat gaa att gat ggc act gta 912Ile Leu Ala Lys Arg Ser Ala Leu Ser Tyr Glu Ile Asp Gly Thr Val 290 295 300 tta aaa gtt gat gaa atc tct ttg cag aaa cgt tta ggg ttt gtc gca 960Leu Lys Val Asp Glu Ile Ser Leu Gln Lys Arg Leu Gly Phe Val Ala 305 310 315 320 cgt gcc cca cgt tgg gct att gct tat aaa ttc cct gcg gaa gaa gaa 1008Arg Ala Pro Arg Trp Ala Ile Ala Tyr Lys Phe Pro Ala Glu Glu Glu 325 330 335 tta acc tgt gtt gaa gat gtc gag ttt caa gta ggg cgt acc ggc gcg 1056Leu Thr Cys Val Glu Asp Val Glu Phe Gln Val Gly Arg Thr Gly Ala 340 345 350 att act ccc gta gca cgt ttg aaa ccg gta ttt gtt ggt ggc gta aca 1104Ile Thr Pro Val Ala Arg Leu Lys Pro Val Phe Val Gly Gly Val Thr 355 360 365 gtt tct aat gcc aca tta cat aac caa gat gaa ata acc cga tta ggg 1152Val Ser Asn Ala Thr Leu His Asn Gln Asp Glu Ile Thr Arg Leu Gly 370 375 380 ctg aaa gtg aat gat ttc gtg gtt atc cgc cgt gcc ggt gat gtt att 1200Leu Lys Val Asn Asp Phe Val Val Ile Arg Arg Ala Gly Asp Val Ile 385 390 395 400 cct caa att gtt agc gta gta ctt gat aaa cga cca gat aat gcc gtc 1248Pro Gln Ile Val Ser Val Val Leu Asp Lys Arg Pro Asp Asn Ala Val 405 410 415 gat ata gtc ttt cct acc agt tgc cct gtt tgt gac tct gca gtg gct 1296Asp Ile Val Phe Pro Thr Ser Cys Pro Val Cys Asp Ser Ala Val Ala 420 425 430 aaa cct gaa ggt gaa gcc gta ctg aga tgt acc gcc gga ctt ttc tgt 1344Lys Pro Glu Gly Glu Ala Val Leu Arg Cys Thr Ala Gly Leu Phe Cys 435 440 445 gcg gcg caa aga aaa gaa gct att aaa cat ttt gct tct cga aaa gca 1392Ala Ala Gln Arg Lys Glu Ala Ile Lys His Phe Ala Ser Arg Lys Ala 450 455 460 cat gat gtt gat ggt tta ggt gac aaa cta gta gag caa ctt gta gat 1440His Asp Val Asp Gly Leu Gly Asp Lys Leu Val Glu Gln Leu Val Asp 465 470 475 480 gaa aag tta att aat acg cyc gct gat tta ttc aaa tta acc gaa ata 1488Glu Lys Leu Ile Asn Thr Xaa Ala Asp Leu Phe Lys Leu Thr Glu Ile 485 490 495 caa gtt agt act ata gat cgt atg ggt aaa aaa tca gcg acc aat tta 1536Gln Val Ser Thr Ile Asp Arg Met Gly Lys Lys Ser Ala Thr Asn Leu 500 505 510 att aat gga ctt gag cag gct aaa agt acc aca cta gca aaa ttt att 1584Ile Asn Gly Leu Glu Gln Ala Lys Ser Thr Thr Leu Ala Lys Phe Ile 515 520 525 tat ggt ctg ggc ata cgc gaa gtc ggt gaa gca act gct gct aat cta 1632Tyr Gly Leu Gly Ile Arg Glu Val Gly Glu Ala Thr Ala Ala Asn Leu 530 535 540 gca aat cat ttt tat acc tta gcg gca att gaa agt gct tct ctt gaa 1680Ala Asn His Phe Tyr Thr Leu Ala Ala Ile Glu Ser Ala Ser Leu Glu 545 550 555 560 gac tta caa aat gtt tca gat gtt ggc gaa gtc gtt gcc aaa aat att 1728Asp Leu Gln Asn Val Ser Asp Val Gly Glu Val Val Ala Lys Asn Ile 565 570 575 att aat ttc ttt aaa gaa gag cat aac tta gcg atc gtt tct gga cta 1776Ile Asn Phe Phe Lys Glu Glu His Asn Leu Ala Ile Val Ser Gly Leu 580 585 590 agt gaa gta atg cac tgg cca act att gaa ata aag tca gct gag gag 1824Ser Glu Val Met His Trp Pro Thr Ile Glu Ile Lys Ser Ala Glu Glu 595 600 605 tta ccg ctt gca gag cag ata ttt gtt tta aca ggc aca tta acc caa 1872Leu Pro Leu Ala Glu Gln Ile Phe Val Leu Thr Gly Thr Leu Thr Gln 610 615 620 atg gga aga act gaa gct aaa aca gcc tta cag tcc ttg gga gct aaa 1920Met Gly Arg Thr Glu Ala Lys Thr Ala Leu Gln Ser Leu Gly Ala Lys 625 630 635 640 gta tca ggt agt gtc tcg aag aat aca cac ttc gtt gtt gca ggt gat 1968Val Ser Gly Ser Val Ser Lys Asn Thr His Phe Val Val Ala Gly Asp 645 650 655 aaa gcg gga tct aaa ctg act aag gct cag gat tta ggt atc tca gtg 2016Lys Ala Gly Ser Lys Leu Thr Lys Ala Gln Asp Leu Gly Ile Ser Val 660 665 670 ctt acc gaa gat ggg tta gta gcg tta ctt gcc gaa cat ggc ata act 2064Leu Thr Glu Asp Gly Leu Val Ala Leu Leu Ala Glu His Gly Ile Thr 675 680 685 att tga 2070Ile 2689PRTArtificial Sequencemisc_feature(190)..(190)The 'Xaa' at location 190 stands for Trp, or Leu. 2Met Thr Pro Val Glu Lys Lys Ile Ser Gln Leu Gln Gln Gln Leu Asn 1 5 10 15 Gln Tyr Asn His Glu Tyr Tyr Val Leu Asp Gln Pro Ser Val Pro Asp 20 25 30 Ala Glu Tyr Asp Arg Leu Met Thr Ala Leu Ile Asp Leu Glu Lys Thr 35 40 45 Asn Pro Glu Leu Lys Thr Ile Asp Ser Pro Ser Gln Lys Val Gly Gly 50 55 60 Gln Ala Leu Lys Ser Phe Thr Gln Val Thr His Gln Leu Pro Met Leu 65 70 75 80 Ser Leu Asp Asn Val Phe Ser Leu Asp Asp Phe His Ala Phe Val Lys 85 90 95 Arg Val Lys Asp Arg Leu Asn Asp Asn Gln Ala Ile Val Phe Cys Ala 100 105 110 Glu Pro Lys Leu Asp Gly Leu Ala Val Ser Leu Arg Tyr Glu His Gly 115 120 125 Gln Leu Ile Gln Ala Ala Thr Arg Gly Asp Gly Ser Val Gly Glu Asn 130 135 140 Ile Thr Thr Asn Ile Arg Thr Ile Lys Ser Ile Pro Leu Lys Leu Met 145 150 155 160 Gly Thr Pro Gly Lys Asp Phe Pro Asp Ile Val Glu Val Arg Gly Glu 165 170 175 Val Phe Met Pro Lys Ala Ser Phe Asp Ala Leu Asn Thr Xaa Ala Lys 180 185 190 Lys Arg Gly Glu Lys Gly Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly 195 200 205 Ser Leu Arg Gln Leu Asp Ser Lys Ile Thr Ala Lys Arg Asn Leu Ala 210 215 220 Phe Tyr Ala Tyr Ser Leu Gly Phe Val Gly Lys Leu Ser Asp Gly Gly 225 230 235 240 Ala Glu Ser Thr Asp Leu Thr Asn Asp Phe Phe Ala Asn Ser His His 245 250 255 Glu Arg Leu Cys Gln Leu Lys Arg Leu Gly Leu Pro Met Cys Pro Glu 260 265 270 Val Arg Leu Leu Glu Ser Glu Gln Ala Cys Asp Ala Phe Tyr Gln Asp 275 280 285 Ile Leu Ala Lys Arg Ser Ala Leu Ser Tyr Glu Ile Asp Gly Thr Val 290 295 300 Leu Lys Val Asp Glu Ile Ser Leu Gln Lys Arg Leu Gly Phe Val Ala 305 310 315 320 Arg Ala Pro Arg Trp Ala Ile Ala Tyr Lys Phe Pro Ala Glu Glu Glu 325 330 335 Leu Thr Cys Val Glu Asp Val Glu Phe Gln Val Gly Arg Thr Gly Ala 340 345 350 Ile Thr Pro Val Ala Arg Leu Lys Pro Val Phe Val Gly Gly Val Thr 355 360 365 Val Ser Asn Ala Thr Leu His Asn Gln Asp Glu Ile Thr Arg Leu Gly 370 375 380 Leu Lys Val Asn Asp Phe Val Val Ile Arg Arg Ala Gly Asp Val Ile 385 390 395 400 Pro Gln Ile Val Ser Val Val Leu Asp Lys Arg Pro Asp Asn Ala Val 405 410 415 Asp Ile Val Phe Pro Thr Ser Cys Pro Val Cys Asp Ser Ala Val Ala 420 425 430 Lys Pro Glu Gly Glu Ala Val Leu Arg Cys Thr Ala Gly Leu Phe Cys 435 440 445 Ala Ala Gln Arg Lys Glu Ala Ile Lys His Phe Ala Ser Arg Lys Ala 450 455 460 His Asp Val Asp Gly Leu Gly Asp Lys Leu Val Glu Gln Leu Val Asp 465 470 475 480 Glu Lys Leu Ile Asn Thr Xaa Ala Asp Leu Phe Lys Leu Thr Glu Ile 485 490 495 Gln Val Ser Thr Ile Asp Arg Met Gly Lys Lys Ser Ala Thr Asn Leu 500 505 510 Ile Asn Gly Leu Glu Gln Ala Lys Ser Thr Thr Leu Ala Lys Phe Ile 515 520 525 Tyr Gly Leu Gly Ile Arg Glu Val Gly Glu Ala Thr Ala Ala Asn Leu 530 535 540 Ala Asn His Phe Tyr Thr Leu Ala Ala Ile Glu Ser Ala Ser Leu Glu 545 550 555 560 Asp Leu Gln Asn Val Ser Asp Val Gly Glu Val Val Ala Lys Asn Ile 565 570 575 Ile Asn Phe Phe Lys Glu Glu His Asn Leu Ala Ile Val Ser Gly Leu 580 585 590 Ser Glu Val Met His Trp Pro Thr Ile Glu Ile Lys Ser Ala Glu Glu 595 600 605 Leu Pro Leu Ala Glu Gln Ile Phe Val Leu Thr Gly Thr Leu Thr Gln 610 615 620 Met Gly Arg Thr Glu Ala Lys Thr Ala Leu Gln Ser Leu Gly Ala Lys 625 630 635 640 Val Ser Gly Ser Val Ser Lys Asn Thr His Phe Val Val Ala Gly Asp 645 650 655 Lys Ala Gly Ser Lys Leu Thr Lys Ala Gln Asp Leu Gly Ile Ser Val 660 665 670 Leu Thr Glu Asp Gly Leu Val Ala Leu Leu Ala Glu His Gly Ile Thr 675 680 685 Ile 32022DNAArtificial SequenceligAPh hybrid 3atg act cca agc att agt gag caa ata aac cat ctt cgt agt acg ctt 48Met Thr Pro Ser Ile Ser Glu Gln Ile Asn His Leu Arg Ser Thr Leu 1 5 10 15 gaa cag cac agt tac aat tat tat gta ctt gat acc ccc agt att cct 96Glu Gln His Ser Tyr Asn Tyr Tyr Val Leu Asp Thr Pro Ser Ile Pro 20 25 30 gat gct gaa tac gac cgt tta tta caa caa ctc agc gca cta gaa act 144Asp Ala Glu Tyr Asp Arg Leu Leu Gln Gln Leu Ser Ala Leu Glu Thr 35 40 45 cag cac cca gaa tta ata act gcc gac tca cca acc caa aaa gtg ggc 192Gln His Pro Glu Leu Ile Thr Ala Asp Ser Pro Thr Gln Lys Val Gly 50 55 60 ggt gct gcg cta agt aaa ttt gag caa gta gcg cac caa gtg cct atg 240Gly Ala Ala Leu Ser Lys Phe Glu Gln Val Ala His Gln Val Pro Met 65 70 75 80 tta tcg ctt gat aac gcc ttt agc gaa gat gag ttt att gcc ttt aat 288Leu Ser Leu Asp Asn Ala Phe Ser Glu Asp Glu Phe Ile Ala Phe Asn 85 90 95 cgc cgt ata aaa gag cgt tta atg agt acc gaa gag ctt act ttt tgt 336Arg Arg Ile Lys Glu Arg Leu Met Ser Thr Glu Glu Leu Thr Phe Cys 100 105 110 tgt gag cca aaa cta gat ggc tta gct gtg tcg att att tat cgt gat 384Cys Glu Pro Lys Leu Asp Gly Leu Ala Val Ser Ile Ile Tyr Arg Asp 115 120 125 ggc gta cta gtg caa gcc gcg acc cga ggt gat ggg ttg acg gga gaa 432Gly Val Leu Val Gln Ala Ala Thr Arg Gly Asp Gly Leu Thr Gly Glu 130 135 140 aat gta act caa aac gtt aaa aca att cgt aat gtg cca ctt aaa tta 480Asn Val Thr Gln Asn Val Lys Thr Ile Arg Asn Val Pro Leu Lys Leu 145 150 155 160 cga ggt agc gat tat cct gct gaa cta gaa gtg cgc ggc gaa gtg ttt 528Arg Gly Ser Asp Tyr Pro Ala Glu Leu Glu Val Arg Gly Glu Val Phe 165 170 175 atg gat aat gca ggc ttt gaa aag ttt aac att gaa gct gaa aaa cgt 576Met Asp Asn Ala Gly Phe Glu Lys Phe Asn Ile Glu Ala Glu Lys Arg 180 185 190 ggt gaa aaa gta ttt gta aac cca cgc aac gcc gcc gca ggt agc ctg 624Gly Glu Lys Val Phe Val Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu 195 200 205 cgc cag ctt gac tct aaa att acg gct aaa cgc cca ctg atg ttt tat 672Arg Gln Leu Asp Ser Lys Ile Thr Ala Lys Arg Pro Leu Met Phe Tyr 210 215 220 gcc tac agc aca ggt ctt gta gcc gac ggt agc att gca gag gat cat

720Ala Tyr Ser Thr Gly Leu Val Ala Asp Gly Ser Ile Ala Glu Asp His 225 230 235 240 tat cag caa tta gaa aaa ttg act gat tgg ggg tta cca ctt tgc cct 768Tyr Gln Gln Leu Glu Lys Leu Thr Asp Trp Gly Leu Pro Leu Cys Pro 245 250 255 gaa aca aaa tta gta gaa ggc cca caa gct gca ctg gct tat tat act 816Glu Thr Lys Leu Val Glu Gly Pro Gln Ala Ala Leu Ala Tyr Tyr Thr 260 265 270 gac att tta acg cgc cgt ggc gag ctt aaa tat gaa ata gat ggc gtg 864Asp Ile Leu Thr Arg Arg Gly Glu Leu Lys Tyr Glu Ile Asp Gly Val 275 280 285 gta ata aaa ata aat caa aaa gcc tta caa gag cgt tta ggc ttt gta 912Val Ile Lys Ile Asn Gln Lys Ala Leu Gln Glu Arg Leu Gly Phe Val 290 295 300 gca cgc gct ccg cgt tgg gct att gct tat aag ttc ccg gcc caa gaa 960Ala Arg Ala Pro Arg Trp Ala Ile Ala Tyr Lys Phe Pro Ala Gln Glu 305 310 315 320 gaa ata acc aaa tta ctc gat gta gag ttt cag gtg ggg cgt acg gga 1008Glu Ile Thr Lys Leu Leu Asp Val Glu Phe Gln Val Gly Arg Thr Gly 325 330 335 gca att aca ccg gtt gca cgc tta gag ccg gta ttt gtt ggt ggt gtt 1056Ala Ile Thr Pro Val Ala Arg Leu Glu Pro Val Phe Val Gly Gly Val 340 345 350 act gta tca aac gct acc ttg cac aat ggc gat gaa ata gcg cgc tta 1104Thr Val Ser Asn Ala Thr Leu His Asn Gly Asp Glu Ile Ala Arg Leu 355 360 365 ggc gta aaa gtg ggc gac acg gta att att cgc cgt gca ggg gac gta 1152Gly Val Lys Val Gly Asp Thr Val Ile Ile Arg Arg Ala Gly Asp Val 370 375 380 att cca caa ata acg caa gta gta ctt gag cgc cgc cct gat gat gcc 1200Ile Pro Gln Ile Thr Gln Val Val Leu Glu Arg Arg Pro Asp Asp Ala 385 390 395 400 cgc gat att gag ttt ccg gta act tgc cca att tgt gac tcc cat gta 1248Arg Asp Ile Glu Phe Pro Val Thr Cys Pro Ile Cys Asp Ser His Val 405 410 415 gaa aaa gta gaa ggt gaa gcc gta gcg cgt tgt act ggt ggt tta gtg 1296Glu Lys Val Glu Gly Glu Ala Val Ala Arg Cys Thr Gly Gly Leu Val 420 425 430 tgc ccg gcg caa cgt aaa caa gcg att aaa cac ttt gca tcg cgc aaa 1344Cys Pro Ala Gln Arg Lys Gln Ala Ile Lys His Phe Ala Ser Arg Lys 435 440 445 gca ctc gat att gac ggc ctt ggc gat aaa att gtt gat caa ctc gtc 1392Ala Leu Asp Ile Asp Gly Leu Gly Asp Lys Ile Val Asp Gln Leu Val 450 455 460 gac aga gag ctg att aaa acc cct gca gat ttg ttt att tta aag caa 1440Asp Arg Glu Leu Ile Lys Thr Pro Ala Asp Leu Phe Ile Leu Lys Gln 465 470 475 480 gga cat ttt gaa tcg ctt gag cgt atg ggg cca aag tcg gct aaa aat 1488Gly His Phe Glu Ser Leu Glu Arg Met Gly Pro Lys Ser Ala Lys Asn 485 490 495 ttg gtt act gcg ctt caa gac gct aaa gca aca act ttg gct aag ttt 1536Leu Val Thr Ala Leu Gln Asp Ala Lys Ala Thr Thr Leu Ala Lys Phe 500 505 510 tta tac tca ttg ggt att cgt gaa gcg ggt gag gca acc aca caa aat 1584Leu Tyr Ser Leu Gly Ile Arg Glu Ala Gly Glu Ala Thr Thr Gln Asn 515 520 525 tta gct aat cat ttc tta acc ctt gaa aac gta ata aat gcc agc att 1632Leu Ala Asn His Phe Leu Thr Leu Glu Asn Val Ile Asn Ala Ser Ile 530 535 540 gat agt tta act caa gta agt gat gtg ggc gaa ata gta gca acc cat 1680Asp Ser Leu Thr Gln Val Ser Asp Val Gly Glu Ile Val Ala Thr His 545 550 555 560 gta cgt agc ttt ttt gcc gaa cag cat aat tta gat gtt gta aat gcg 1728Val Arg Ser Phe Phe Ala Glu Gln His Asn Leu Asp Val Val Asn Ala 565 570 575 ctg gta gag caa ggt att aat tgg cct gaa ctt act cca cct tca gcg 1776Leu Val Glu Gln Gly Ile Asn Trp Pro Glu Leu Thr Pro Pro Ser Ala 580 585 590 caa gag cag cca tta gct ggc ctt gtt tat gtg ctt acc ggt acc tta 1824Gln Glu Gln Pro Leu Ala Gly Leu Val Tyr Val Leu Thr Gly Thr Leu 595 600 605 aac aca tta aac cgt aat gac gcc aaa gca cgt ttg caa cag tta ggt 1872Asn Thr Leu Asn Arg Asn Asp Ala Lys Ala Arg Leu Gln Gln Leu Gly 610 615 620 gct aaa gtg tcg ggt agt gtg tcg gct aaa acc gat gcg tta gta gca 1920Ala Lys Val Ser Gly Ser Val Ser Ala Lys Thr Asp Ala Leu Val Ala 625 630 635 640 ggc gaa aag gcc ggc tct aaa cta act aag gca caa gac tta ggt ata 1968Gly Glu Lys Ala Gly Ser Lys Leu Thr Lys Ala Gln Asp Leu Gly Ile 645 650 655 gat gta ctg aca gaa gaa gat tta att aat tta tta gag caa cat aat 2016Asp Val Leu Thr Glu Glu Asp Leu Ile Asn Leu Leu Glu Gln His Asn 660 665 670 ggc tga 2022Gly 4673PRTArtificial SequenceSynthetic Construct 4Met Thr Pro Ser Ile Ser Glu Gln Ile Asn His Leu Arg Ser Thr Leu 1 5 10 15 Glu Gln His Ser Tyr Asn Tyr Tyr Val Leu Asp Thr Pro Ser Ile Pro 20 25 30 Asp Ala Glu Tyr Asp Arg Leu Leu Gln Gln Leu Ser Ala Leu Glu Thr 35 40 45 Gln His Pro Glu Leu Ile Thr Ala Asp Ser Pro Thr Gln Lys Val Gly 50 55 60 Gly Ala Ala Leu Ser Lys Phe Glu Gln Val Ala His Gln Val Pro Met 65 70 75 80 Leu Ser Leu Asp Asn Ala Phe Ser Glu Asp Glu Phe Ile Ala Phe Asn 85 90 95 Arg Arg Ile Lys Glu Arg Leu Met Ser Thr Glu Glu Leu Thr Phe Cys 100 105 110 Cys Glu Pro Lys Leu Asp Gly Leu Ala Val Ser Ile Ile Tyr Arg Asp 115 120 125 Gly Val Leu Val Gln Ala Ala Thr Arg Gly Asp Gly Leu Thr Gly Glu 130 135 140 Asn Val Thr Gln Asn Val Lys Thr Ile Arg Asn Val Pro Leu Lys Leu 145 150 155 160 Arg Gly Ser Asp Tyr Pro Ala Glu Leu Glu Val Arg Gly Glu Val Phe 165 170 175 Met Asp Asn Ala Gly Phe Glu Lys Phe Asn Ile Glu Ala Glu Lys Arg 180 185 190 Gly Glu Lys Val Phe Val Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu 195 200 205 Arg Gln Leu Asp Ser Lys Ile Thr Ala Lys Arg Pro Leu Met Phe Tyr 210 215 220 Ala Tyr Ser Thr Gly Leu Val Ala Asp Gly Ser Ile Ala Glu Asp His 225 230 235 240 Tyr Gln Gln Leu Glu Lys Leu Thr Asp Trp Gly Leu Pro Leu Cys Pro 245 250 255 Glu Thr Lys Leu Val Glu Gly Pro Gln Ala Ala Leu Ala Tyr Tyr Thr 260 265 270 Asp Ile Leu Thr Arg Arg Gly Glu Leu Lys Tyr Glu Ile Asp Gly Val 275 280 285 Val Ile Lys Ile Asn Gln Lys Ala Leu Gln Glu Arg Leu Gly Phe Val 290 295 300 Ala Arg Ala Pro Arg Trp Ala Ile Ala Tyr Lys Phe Pro Ala Gln Glu 305 310 315 320 Glu Ile Thr Lys Leu Leu Asp Val Glu Phe Gln Val Gly Arg Thr Gly 325 330 335 Ala Ile Thr Pro Val Ala Arg Leu Glu Pro Val Phe Val Gly Gly Val 340 345 350 Thr Val Ser Asn Ala Thr Leu His Asn Gly Asp Glu Ile Ala Arg Leu 355 360 365 Gly Val Lys Val Gly Asp Thr Val Ile Ile Arg Arg Ala Gly Asp Val 370 375 380 Ile Pro Gln Ile Thr Gln Val Val Leu Glu Arg Arg Pro Asp Asp Ala 385 390 395 400 Arg Asp Ile Glu Phe Pro Val Thr Cys Pro Ile Cys Asp Ser His Val 405 410 415 Glu Lys Val Glu Gly Glu Ala Val Ala Arg Cys Thr Gly Gly Leu Val 420 425 430 Cys Pro Ala Gln Arg Lys Gln Ala Ile Lys His Phe Ala Ser Arg Lys 435 440 445 Ala Leu Asp Ile Asp Gly Leu Gly Asp Lys Ile Val Asp Gln Leu Val 450 455 460 Asp Arg Glu Leu Ile Lys Thr Pro Ala Asp Leu Phe Ile Leu Lys Gln 465 470 475 480 Gly His Phe Glu Ser Leu Glu Arg Met Gly Pro Lys Ser Ala Lys Asn 485 490 495 Leu Val Thr Ala Leu Gln Asp Ala Lys Ala Thr Thr Leu Ala Lys Phe 500 505 510 Leu Tyr Ser Leu Gly Ile Arg Glu Ala Gly Glu Ala Thr Thr Gln Asn 515 520 525 Leu Ala Asn His Phe Leu Thr Leu Glu Asn Val Ile Asn Ala Ser Ile 530 535 540 Asp Ser Leu Thr Gln Val Ser Asp Val Gly Glu Ile Val Ala Thr His 545 550 555 560 Val Arg Ser Phe Phe Ala Glu Gln His Asn Leu Asp Val Val Asn Ala 565 570 575 Leu Val Glu Gln Gly Ile Asn Trp Pro Glu Leu Thr Pro Pro Ser Ala 580 585 590 Gln Glu Gln Pro Leu Ala Gly Leu Val Tyr Val Leu Thr Gly Thr Leu 595 600 605 Asn Thr Leu Asn Arg Asn Asp Ala Lys Ala Arg Leu Gln Gln Leu Gly 610 615 620 Ala Lys Val Ser Gly Ser Val Ser Ala Lys Thr Asp Ala Leu Val Ala 625 630 635 640 Gly Glu Lys Ala Gly Ser Lys Leu Thr Lys Ala Gln Asp Leu Gly Ile 645 650 655 Asp Val Leu Thr Glu Glu Asp Leu Ile Asn Leu Leu Glu Gln His Asn 660 665 670 Gly 52022DNAArtificial SequenceLigAPh2 hybrid 5atg act cca agc att agt gag caa ata aac cat ctt cgt agt acg ctt 48Met Thr Pro Ser Ile Ser Glu Gln Ile Asn His Leu Arg Ser Thr Leu 1 5 10 15 gaa cag cac agt tac aat tat tat gta ctt gat acc ccc agt att cct 96Glu Gln His Ser Tyr Asn Tyr Tyr Val Leu Asp Thr Pro Ser Ile Pro 20 25 30 gat gct gaa tac gac cgt tta tta caa caa ctc agc gca cta gaa act 144Asp Ala Glu Tyr Asp Arg Leu Leu Gln Gln Leu Ser Ala Leu Glu Thr 35 40 45 cag cac cca gaa tta ata act gcc gac tca cca acc caa aaa gtg ggc 192Gln His Pro Glu Leu Ile Thr Ala Asp Ser Pro Thr Gln Lys Val Gly 50 55 60 ggt gct gcg cta agt aaa ttt gag caa gta gcg cac caa gtg cct atg 240Gly Ala Ala Leu Ser Lys Phe Glu Gln Val Ala His Gln Val Pro Met 65 70 75 80 tta tcg ctt gat aac gcc ttt agc gaa gat gag ttt att gcc ttt aat 288Leu Ser Leu Asp Asn Ala Phe Ser Glu Asp Glu Phe Ile Ala Phe Asn 85 90 95 cgc cgt ata aaa gag cgt tta atg agt acc gaa gag ctt act ttt tgt 336Arg Arg Ile Lys Glu Arg Leu Met Ser Thr Glu Glu Leu Thr Phe Cys 100 105 110 tgt gag cca aaa cta gat ggc tta gct gtg tcg att att tat cgt gat 384Cys Glu Pro Lys Leu Asp Gly Leu Ala Val Ser Ile Ile Tyr Arg Asp 115 120 125 ggc gta cta gtg caa gcc gcg acc cga ggt gat ggg ttg acg gga gaa 432Gly Val Leu Val Gln Ala Ala Thr Arg Gly Asp Gly Leu Thr Gly Glu 130 135 140 aat gta act caa aaa gtt aaa aca att cgt aat gtg cca ctt aaa tta 480Asn Val Thr Gln Lys Val Lys Thr Ile Arg Asn Val Pro Leu Lys Leu 145 150 155 160 cga ggt agc gat tat cct gct gaa cta gaa gtg cgc ggc gaa gtg ttt 528Arg Gly Ser Asp Tyr Pro Ala Glu Leu Glu Val Arg Gly Glu Val Phe 165 170 175 atg gat aat gca ggc ttt gaa aag ttt aac att gaa gct gaa aaa cgt 576Met Asp Asn Ala Gly Phe Glu Lys Phe Asn Ile Glu Ala Glu Lys Arg 180 185 190 ggt gaa aaa gta ttt gta aac cca cgc aac gcc gcc gca ggt agc ctg 624Gly Glu Lys Val Phe Val Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu 195 200 205 cgc cag ctt gac tct aaa att acg gct aaa cgc cca ctg atg ttt tat 672Arg Gln Leu Asp Ser Lys Ile Thr Ala Lys Arg Pro Leu Met Phe Tyr 210 215 220 gcc tac agc aca ggt ctt gta gcc gac ggt agc att gca gag gat cat 720Ala Tyr Ser Thr Gly Leu Val Ala Asp Gly Ser Ile Ala Glu Asp His 225 230 235 240 tat cag caa tta gaa aaa ttg act gat tgg ggg tta cca ctt tgc cct 768Tyr Gln Gln Leu Glu Lys Leu Thr Asp Trp Gly Leu Pro Leu Cys Pro 245 250 255 gaa aca aaa tta gta gaa ggc cca caa gct gca ctg gct tat tat act 816Glu Thr Lys Leu Val Glu Gly Pro Gln Ala Ala Leu Ala Tyr Tyr Thr 260 265 270 gac att tta acg cgc cgt ggc gag ctt aaa tat gaa ata gat ggc gtg 864Asp Ile Leu Thr Arg Arg Gly Glu Leu Lys Tyr Glu Ile Asp Gly Val 275 280 285 gta ata aaa ata aat caa aaa gcc tta caa gag cgt tta ggc ttt gta 912Val Ile Lys Ile Asn Gln Lys Ala Leu Gln Glu Arg Leu Gly Phe Val 290 295 300 gca cgc gct ccg cgt tgg gct att gct tat aag ttc ccg gcc caa gaa 960Ala Arg Ala Pro Arg Trp Ala Ile Ala Tyr Lys Phe Pro Ala Gln Glu 305 310 315 320 gaa ata acc aaa tta ctc gat gta gag ttt cag gtg ggg cgt acg gga 1008Glu Ile Thr Lys Leu Leu Asp Val Glu Phe Gln Val Gly Arg Thr Gly 325 330 335 gca att aca ccg gtt gca cgc tta gag ccg gta ttt gtt ggt ggt gtt 1056Ala Ile Thr Pro Val Ala Arg Leu Glu Pro Val Phe Val Gly Gly Val 340 345 350 act gta tca aac gct acc ttg cac aat ggc gat gaa ata gcg cgc tta 1104Thr Val Ser Asn Ala Thr Leu His Asn Gly Asp Glu Ile Ala Arg Leu 355 360 365 ggc gta aaa gtg ggc gac acg gta att att cgc cgt gca ggg gac gta 1152Gly Val Lys Val Gly Asp Thr Val Ile Ile Arg Arg Ala Gly Asp Val 370 375 380 att cca caa ata acg caa gta gta ctt gag cgc cgc cct gat gat gcc 1200Ile Pro Gln Ile Thr Gln Val Val Leu Glu Arg Arg Pro Asp Asp Ala 385 390 395 400 cgc gat att gag ttt ccg gta act tgc cca att tgt gac tcc cat gta 1248Arg Asp Ile Glu Phe Pro Val Thr Cys Pro Ile Cys Asp Ser His Val 405 410 415 gaa aaa gta gaa ggt gaa gcc gta gcg cgt tgt act ggt ggt tta gtg 1296Glu Lys Val Glu Gly Glu Ala Val Ala Arg Cys Thr Gly Gly Leu Val 420 425 430 tgc ccg gcg caa cgt aaa caa gcg att aaa cac ttt gca tcg cgc aaa 1344Cys Pro Ala Gln Arg Lys Gln Ala Ile Lys His Phe Ala Ser Arg Lys 435 440 445 gca ctc gat att gac ggc ctt ggc gat aaa att gtt gat caa ctc gtc 1392Ala Leu Asp Ile Asp Gly Leu Gly Asp Lys Ile Val Asp Gln Leu Val 450 455 460 gac aga gag ctg att aaa acc cct gca gat ttg ttt att tta aag caa 1440Asp Arg Glu Leu Ile Lys Thr Pro Ala Asp Leu Phe Ile Leu Lys Gln 465 470 475 480 gga cat ttt gaa tcg ctt gag cgt atg ggg cca aag tcg gct aaa aat 1488Gly His Phe Glu Ser Leu Glu Arg Met Gly Pro Lys Ser Ala Lys Asn

485 490 495 ttg gtt act gcg ctt caa gac gct aaa gca aca act ttg gct aag ttt 1536Leu Val Thr Ala Leu Gln Asp Ala Lys Ala Thr Thr Leu Ala Lys Phe 500 505 510 tta tac tca ttg ggt att cgt gaa gcg ggt gag gca acc aca caa aat 1584Leu Tyr Ser Leu Gly Ile Arg Glu Ala Gly Glu Ala Thr Thr Gln Asn 515 520 525 tta gct aat cat ttc tta acc ctt gaa aac gta ata aat gcc agc att 1632Leu Ala Asn His Phe Leu Thr Leu Glu Asn Val Ile Asn Ala Ser Ile 530 535 540 gat agt tta act caa gta agt gat gtg ggc gaa ata gta gca acc cat 1680Asp Ser Leu Thr Gln Val Ser Asp Val Gly Glu Ile Val Ala Thr His 545 550 555 560 gta cgt agc ttt ttt gcc gaa cag cat aat tta gat gtt gta aat gcg 1728Val Arg Ser Phe Phe Ala Glu Gln His Asn Leu Asp Val Val Asn Ala 565 570 575 ctg gta gag caa ggt att aat tgg cct gaa ctt act cca cct tca gcg 1776Leu Val Glu Gln Gly Ile Asn Trp Pro Glu Leu Thr Pro Pro Ser Ala 580 585 590 caa gag cag cca tta gct ggc ctt gtt tat gtg ctt acc ggt acc tta 1824Gln Glu Gln Pro Leu Ala Gly Leu Val Tyr Val Leu Thr Gly Thr Leu 595 600 605 aac aca tta aac cgt aat gac gcc aaa gca cgt ttg caa cag tta ggt 1872Asn Thr Leu Asn Arg Asn Asp Ala Lys Ala Arg Leu Gln Gln Leu Gly 610 615 620 gct aaa gtg tcg ggt agt gtg tcg gct aaa acc gat gcg tta gta gca 1920Ala Lys Val Ser Gly Ser Val Ser Ala Lys Thr Asp Ala Leu Val Ala 625 630 635 640 ggc gaa aag gcc ggc tct aaa cta act aag gca caa gac tta ggt ata 1968Gly Glu Lys Ala Gly Ser Lys Leu Thr Lys Ala Gln Asp Leu Gly Ile 645 650 655 gat gta ctg aca gaa gaa gat tta att aat tta tta gag caa cat aat 2016Asp Val Leu Thr Glu Glu Asp Leu Ile Asn Leu Leu Glu Gln His Asn 660 665 670 ggc tga 2022Gly 6673PRTArtificial SequenceSynthetic Construct 6Met Thr Pro Ser Ile Ser Glu Gln Ile Asn His Leu Arg Ser Thr Leu 1 5 10 15 Glu Gln His Ser Tyr Asn Tyr Tyr Val Leu Asp Thr Pro Ser Ile Pro 20 25 30 Asp Ala Glu Tyr Asp Arg Leu Leu Gln Gln Leu Ser Ala Leu Glu Thr 35 40 45 Gln His Pro Glu Leu Ile Thr Ala Asp Ser Pro Thr Gln Lys Val Gly 50 55 60 Gly Ala Ala Leu Ser Lys Phe Glu Gln Val Ala His Gln Val Pro Met 65 70 75 80 Leu Ser Leu Asp Asn Ala Phe Ser Glu Asp Glu Phe Ile Ala Phe Asn 85 90 95 Arg Arg Ile Lys Glu Arg Leu Met Ser Thr Glu Glu Leu Thr Phe Cys 100 105 110 Cys Glu Pro Lys Leu Asp Gly Leu Ala Val Ser Ile Ile Tyr Arg Asp 115 120 125 Gly Val Leu Val Gln Ala Ala Thr Arg Gly Asp Gly Leu Thr Gly Glu 130 135 140 Asn Val Thr Gln Lys Val Lys Thr Ile Arg Asn Val Pro Leu Lys Leu 145 150 155 160 Arg Gly Ser Asp Tyr Pro Ala Glu Leu Glu Val Arg Gly Glu Val Phe 165 170 175 Met Asp Asn Ala Gly Phe Glu Lys Phe Asn Ile Glu Ala Glu Lys Arg 180 185 190 Gly Glu Lys Val Phe Val Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu 195 200 205 Arg Gln Leu Asp Ser Lys Ile Thr Ala Lys Arg Pro Leu Met Phe Tyr 210 215 220 Ala Tyr Ser Thr Gly Leu Val Ala Asp Gly Ser Ile Ala Glu Asp His 225 230 235 240 Tyr Gln Gln Leu Glu Lys Leu Thr Asp Trp Gly Leu Pro Leu Cys Pro 245 250 255 Glu Thr Lys Leu Val Glu Gly Pro Gln Ala Ala Leu Ala Tyr Tyr Thr 260 265 270 Asp Ile Leu Thr Arg Arg Gly Glu Leu Lys Tyr Glu Ile Asp Gly Val 275 280 285 Val Ile Lys Ile Asn Gln Lys Ala Leu Gln Glu Arg Leu Gly Phe Val 290 295 300 Ala Arg Ala Pro Arg Trp Ala Ile Ala Tyr Lys Phe Pro Ala Gln Glu 305 310 315 320 Glu Ile Thr Lys Leu Leu Asp Val Glu Phe Gln Val Gly Arg Thr Gly 325 330 335 Ala Ile Thr Pro Val Ala Arg Leu Glu Pro Val Phe Val Gly Gly Val 340 345 350 Thr Val Ser Asn Ala Thr Leu His Asn Gly Asp Glu Ile Ala Arg Leu 355 360 365 Gly Val Lys Val Gly Asp Thr Val Ile Ile Arg Arg Ala Gly Asp Val 370 375 380 Ile Pro Gln Ile Thr Gln Val Val Leu Glu Arg Arg Pro Asp Asp Ala 385 390 395 400 Arg Asp Ile Glu Phe Pro Val Thr Cys Pro Ile Cys Asp Ser His Val 405 410 415 Glu Lys Val Glu Gly Glu Ala Val Ala Arg Cys Thr Gly Gly Leu Val 420 425 430 Cys Pro Ala Gln Arg Lys Gln Ala Ile Lys His Phe Ala Ser Arg Lys 435 440 445 Ala Leu Asp Ile Asp Gly Leu Gly Asp Lys Ile Val Asp Gln Leu Val 450 455 460 Asp Arg Glu Leu Ile Lys Thr Pro Ala Asp Leu Phe Ile Leu Lys Gln 465 470 475 480 Gly His Phe Glu Ser Leu Glu Arg Met Gly Pro Lys Ser Ala Lys Asn 485 490 495 Leu Val Thr Ala Leu Gln Asp Ala Lys Ala Thr Thr Leu Ala Lys Phe 500 505 510 Leu Tyr Ser Leu Gly Ile Arg Glu Ala Gly Glu Ala Thr Thr Gln Asn 515 520 525 Leu Ala Asn His Phe Leu Thr Leu Glu Asn Val Ile Asn Ala Ser Ile 530 535 540 Asp Ser Leu Thr Gln Val Ser Asp Val Gly Glu Ile Val Ala Thr His 545 550 555 560 Val Arg Ser Phe Phe Ala Glu Gln His Asn Leu Asp Val Val Asn Ala 565 570 575 Leu Val Glu Gln Gly Ile Asn Trp Pro Glu Leu Thr Pro Pro Ser Ala 580 585 590 Gln Glu Gln Pro Leu Ala Gly Leu Val Tyr Val Leu Thr Gly Thr Leu 595 600 605 Asn Thr Leu Asn Arg Asn Asp Ala Lys Ala Arg Leu Gln Gln Leu Gly 610 615 620 Ala Lys Val Ser Gly Ser Val Ser Ala Lys Thr Asp Ala Leu Val Ala 625 630 635 640 Gly Glu Lys Ala Gly Ser Lys Leu Thr Lys Ala Gln Asp Leu Gly Ile 645 650 655 Asp Val Leu Thr Glu Glu Asp Leu Ile Asn Leu Leu Glu Gln His Asn 660 665 670 Gly 72013DNAArtificial SequenceLigASf hybrid 7atg act cca att caa act gaa atg gat caa ctt act cac acc att aac 48Met Thr Pro Ile Gln Thr Glu Met Asp Gln Leu Thr His Thr Ile Asn 1 5 10 15 caa cat aat att cgt tat tac gtt gat gat gct ccg tca ata ccc gat 96Gln His Asn Ile Arg Tyr Tyr Val Asp Asp Ala Pro Ser Ile Pro Asp 20 25 30 gct gaa tac gac aga tta att aag cgc tta act gag tta gaa cgt gac 144Ala Glu Tyr Asp Arg Leu Ile Lys Arg Leu Thr Glu Leu Glu Arg Asp 35 40 45 tat ccg caa ttt aaa tcg gta gat tca ccg aca caa cgc gtc ggt ggt 192Tyr Pro Gln Phe Lys Ser Val Asp Ser Pro Thr Gln Arg Val Gly Gly 50 55 60 ata gca tta caa aaa ttt gct caa att acc cac ctt aaa ccg atg tta 240Ile Ala Leu Gln Lys Phe Ala Gln Ile Thr His Leu Lys Pro Met Leu 65 70 75 80 agt ctc gac aat gcg ttt gaa caa gcc gat ttt gca gca ttt aat aag 288Ser Leu Asp Asn Ala Phe Glu Gln Ala Asp Phe Ala Ala Phe Asn Lys 85 90 95 cgt ata act gat aaa gtc gat agc gtc gat tat gtt tgc gaa cca aaa 336Arg Ile Thr Asp Lys Val Asp Ser Val Asp Tyr Val Cys Glu Pro Lys 100 105 110 cta gac gga ttg gcc gtg agt att act tat cgt ttt ggc gtt ctt gaa 384Leu Asp Gly Leu Ala Val Ser Ile Thr Tyr Arg Phe Gly Val Leu Glu 115 120 125 cgc gcc gca acg cga ggt gat ggc agt gtc ggc gaa gat att acc gct 432Arg Ala Ala Thr Arg Gly Asp Gly Ser Val Gly Glu Asp Ile Thr Ala 130 135 140 aat gtg cgt act att cgt tca att cct ctt aag tta cgc ggt gaa gga 480Asn Val Arg Thr Ile Arg Ser Ile Pro Leu Lys Leu Arg Gly Glu Gly 145 150 155 160 ttt cca gat tta gtt gaa gta cgt ggc gaa gtg ttt atg cct aaa gcg 528Phe Pro Asp Leu Val Glu Val Arg Gly Glu Val Phe Met Pro Lys Ala 165 170 175 gca ttt gag gca tta aac cag cgt caa atc agc aaa ggt gac aaa gtc 576Ala Phe Glu Ala Leu Asn Gln Arg Gln Ile Ser Lys Gly Asp Lys Val 180 185 190 ttt gtt aat cct cgc aac gca gct gcc ggc agt ttg cgc caa tta gac 624Phe Val Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp 195 200 205 agt aaa att acc gct tca agg gct ctt ggg ttt tat gct tat gca tta 672Ser Lys Ile Thr Ala Ser Arg Ala Leu Gly Phe Tyr Ala Tyr Ala Leu 210 215 220 ggt gta gtc gaa ggc gag tca caa ccg atg caa aca agc cac tat ggc 720Gly Val Val Glu Gly Glu Ser Gln Pro Met Gln Thr Ser His Tyr Gly 225 230 235 240 caa cta aca cag ctg caa caa tgg ggt att ccc gtt agt agt gaa gtg 768Gln Leu Thr Gln Leu Gln Gln Trp Gly Ile Pro Val Ser Ser Glu Val 245 250 255 aaa gtg act gat tta tta gaa aaa gtc tat gca tat tac gcc gat att 816Lys Val Thr Asp Leu Leu Glu Lys Val Tyr Ala Tyr Tyr Ala Asp Ile 260 265 270 atg gcc aga cga agt gcg ctt gaa tat gaa att gac ggc gtc gtc ata 864Met Ala Arg Arg Ser Ala Leu Glu Tyr Glu Ile Asp Gly Val Val Ile 275 280 285 aag gtt aat gac att gcc aag caa caa aca ctt ggt ttt gtg gct aaa 912Lys Val Asn Asp Ile Ala Lys Gln Gln Thr Leu Gly Phe Val Ala Lys 290 295 300 gct cct cga tgg gcc ata gcc tat aaa ttt cca gcc cag gaa gaa atg 960Ala Pro Arg Trp Ala Ile Ala Tyr Lys Phe Pro Ala Gln Glu Glu Met 305 310 315 320 acc ttg tta gag tct gtt gac ttt cag gtt ggc cga acg ggt gct gtt 1008Thr Leu Leu Glu Ser Val Asp Phe Gln Val Gly Arg Thr Gly Ala Val 325 330 335 acc cct gtc gct cgc ctc aaa ccg ata ttt gtc ggt ggc gtg act gtg 1056Thr Pro Val Ala Arg Leu Lys Pro Ile Phe Val Gly Gly Val Thr Val 340 345 350 tcg aat gcg acc ttg cac aat gct gat gaa att gcc cgt ctt ggg gtg 1104Ser Asn Ala Thr Leu His Asn Ala Asp Glu Ile Ala Arg Leu Gly Val 355 360 365 aaa ata ggc gat aca gtg att att cgc cgc gca ggt gac gtt atc ccg 1152Lys Ile Gly Asp Thr Val Ile Ile Arg Arg Ala Gly Asp Val Ile Pro 370 375 380 caa att gtt gct atc gtg cca gaa aag cgc cct gat gat gca caa gat 1200Gln Ile Val Ala Ile Val Pro Glu Lys Arg Pro Asp Asp Ala Gln Asp 385 390 395 400 att atc ttt cca ctg cat tgt cct gtg tgc caa agc att gtt gag cgt 1248Ile Ile Phe Pro Leu His Cys Pro Val Cys Gln Ser Ile Val Glu Arg 405 410 415 tta gaa ggt gaa gct gta gcg cgt tgt agt ggt gga ctt ttt tgt gaa 1296Leu Glu Gly Glu Ala Val Ala Arg Cys Ser Gly Gly Leu Phe Cys Glu 420 425 430 gcg caa cgt aaa gag gcg att aaa cat ttt gca tcc cgt aaa gca tta 1344Ala Gln Arg Lys Glu Ala Ile Lys His Phe Ala Ser Arg Lys Ala Leu 435 440 445 aat att gat ggc atg ggc gat aaa atc gtt gag caa tta att gat aaa 1392Asn Ile Asp Gly Met Gly Asp Lys Ile Val Glu Gln Leu Ile Asp Lys 450 455 460 gaa cta gtc aaa acg cca gca gac ttg ttt tcc ctt acc gct tct agc 1440Glu Leu Val Lys Thr Pro Ala Asp Leu Phe Ser Leu Thr Ala Ser Ser 465 470 475 480 atc acg atg tta gat cgc atg gcg atg aag tca gcc aca aat att gtc 1488Ile Thr Met Leu Asp Arg Met Ala Met Lys Ser Ala Thr Asn Ile Val 485 490 495 gcg gcg att aaa cac gct aaa gcc act aca tta gcg cgt ttt tta tat 1536Ala Ala Ile Lys His Ala Lys Ala Thr Thr Leu Ala Arg Phe Leu Tyr 500 505 510 agt ctt ggg atc cgc gaa gtc ggc gaa gct acc gcc gct aat tta gcc 1584Ser Leu Gly Ile Arg Glu Val Gly Glu Ala Thr Ala Ala Asn Leu Ala 515 520 525 caa cac ttt gcc gaa ttt gag cgt att cga act gct agc gtt gaa caa 1632Gln His Phe Ala Glu Phe Glu Arg Ile Arg Thr Ala Ser Val Glu Gln 530 535 540 ctg ctc gaa gtc gct gat gtt ggt gac att gta gca aaa cac att cga 1680Leu Leu Glu Val Ala Asp Val Gly Asp Ile Val Ala Lys His Ile Arg 545 550 555 560 caa ttt ttt gca cag cca cat aac att gaa gta ata gag caa ttg ctt 1728Gln Phe Phe Ala Gln Pro His Asn Ile Glu Val Ile Glu Gln Leu Leu 565 570 575 gaa gcc ggc att act tgg cct gtt att gaa caa gct gac gaa tcg cag 1776Glu Ala Gly Ile Thr Trp Pro Val Ile Glu Gln Ala Asp Glu Ser Gln 580 585 590 ctt agt ctt aaa ggg caa acg tgg gtg tta act ggt acg cta act caa 1824Leu Ser Leu Lys Gly Gln Thr Trp Val Leu Thr Gly Thr Leu Thr Gln 595 600 605 ctt aat cgt aac gat gcc aaa gcc caa tta cag gct ttg ggc gcc aaa 1872Leu Asn Arg Asn Asp Ala Lys Ala Gln Leu Gln Ala Leu Gly Ala Lys 610 615 620 gtg gct ggc agt gtt tcg aaa aat act gat tgc ctt gtt gct ggt gaa 1920Val Ala Gly Ser Val Ser Lys Asn Thr Asp Cys Leu Val Ala Gly Glu 625 630 635 640 gca gcg ggt tct aaa tta gca aaa gct gaa gaa ttg ggc gtt aag gtg 1968Ala Ala Gly Ser Lys Leu Ala Lys Ala Glu Glu Leu Gly Val Lys Val 645 650 655 ata gat gaa caa gct ctg atg gat tta ttg aat gcg gct aac tga 2013Ile Asp Glu Gln Ala Leu Met Asp Leu Leu Asn Ala Ala Asn 660 665 670 8670PRTArtificial SequenceSynthetic Construct 8Met Thr Pro Ile Gln Thr Glu Met Asp Gln Leu Thr His Thr Ile Asn 1 5 10 15 Gln His Asn Ile Arg Tyr Tyr Val Asp Asp Ala Pro Ser Ile Pro Asp 20 25 30 Ala Glu Tyr Asp Arg Leu Ile Lys Arg Leu Thr Glu Leu Glu Arg Asp 35 40 45 Tyr Pro Gln Phe Lys Ser Val Asp Ser Pro Thr Gln Arg Val Gly Gly 50 55 60 Ile Ala Leu Gln Lys Phe Ala Gln Ile Thr His Leu Lys Pro Met Leu 65 70 75 80 Ser Leu Asp Asn Ala Phe Glu Gln Ala Asp Phe Ala Ala Phe Asn Lys 85 90 95 Arg Ile Thr Asp Lys Val Asp Ser Val Asp Tyr Val Cys Glu Pro Lys 100 105 110 Leu Asp Gly Leu Ala Val Ser Ile Thr Tyr Arg Phe Gly Val Leu Glu 115 120 125 Arg Ala Ala Thr Arg Gly Asp

Gly Ser Val Gly Glu Asp Ile Thr Ala 130 135 140 Asn Val Arg Thr Ile Arg Ser Ile Pro Leu Lys Leu Arg Gly Glu Gly 145 150 155 160 Phe Pro Asp Leu Val Glu Val Arg Gly Glu Val Phe Met Pro Lys Ala 165 170 175 Ala Phe Glu Ala Leu Asn Gln Arg Gln Ile Ser Lys Gly Asp Lys Val 180 185 190 Phe Val Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp 195 200 205 Ser Lys Ile Thr Ala Ser Arg Ala Leu Gly Phe Tyr Ala Tyr Ala Leu 210 215 220 Gly Val Val Glu Gly Glu Ser Gln Pro Met Gln Thr Ser His Tyr Gly 225 230 235 240 Gln Leu Thr Gln Leu Gln Gln Trp Gly Ile Pro Val Ser Ser Glu Val 245 250 255 Lys Val Thr Asp Leu Leu Glu Lys Val Tyr Ala Tyr Tyr Ala Asp Ile 260 265 270 Met Ala Arg Arg Ser Ala Leu Glu Tyr Glu Ile Asp Gly Val Val Ile 275 280 285 Lys Val Asn Asp Ile Ala Lys Gln Gln Thr Leu Gly Phe Val Ala Lys 290 295 300 Ala Pro Arg Trp Ala Ile Ala Tyr Lys Phe Pro Ala Gln Glu Glu Met 305 310 315 320 Thr Leu Leu Glu Ser Val Asp Phe Gln Val Gly Arg Thr Gly Ala Val 325 330 335 Thr Pro Val Ala Arg Leu Lys Pro Ile Phe Val Gly Gly Val Thr Val 340 345 350 Ser Asn Ala Thr Leu His Asn Ala Asp Glu Ile Ala Arg Leu Gly Val 355 360 365 Lys Ile Gly Asp Thr Val Ile Ile Arg Arg Ala Gly Asp Val Ile Pro 370 375 380 Gln Ile Val Ala Ile Val Pro Glu Lys Arg Pro Asp Asp Ala Gln Asp 385 390 395 400 Ile Ile Phe Pro Leu His Cys Pro Val Cys Gln Ser Ile Val Glu Arg 405 410 415 Leu Glu Gly Glu Ala Val Ala Arg Cys Ser Gly Gly Leu Phe Cys Glu 420 425 430 Ala Gln Arg Lys Glu Ala Ile Lys His Phe Ala Ser Arg Lys Ala Leu 435 440 445 Asn Ile Asp Gly Met Gly Asp Lys Ile Val Glu Gln Leu Ile Asp Lys 450 455 460 Glu Leu Val Lys Thr Pro Ala Asp Leu Phe Ser Leu Thr Ala Ser Ser 465 470 475 480 Ile Thr Met Leu Asp Arg Met Ala Met Lys Ser Ala Thr Asn Ile Val 485 490 495 Ala Ala Ile Lys His Ala Lys Ala Thr Thr Leu Ala Arg Phe Leu Tyr 500 505 510 Ser Leu Gly Ile Arg Glu Val Gly Glu Ala Thr Ala Ala Asn Leu Ala 515 520 525 Gln His Phe Ala Glu Phe Glu Arg Ile Arg Thr Ala Ser Val Glu Gln 530 535 540 Leu Leu Glu Val Ala Asp Val Gly Asp Ile Val Ala Lys His Ile Arg 545 550 555 560 Gln Phe Phe Ala Gln Pro His Asn Ile Glu Val Ile Glu Gln Leu Leu 565 570 575 Glu Ala Gly Ile Thr Trp Pro Val Ile Glu Gln Ala Asp Glu Ser Gln 580 585 590 Leu Ser Leu Lys Gly Gln Thr Trp Val Leu Thr Gly Thr Leu Thr Gln 595 600 605 Leu Asn Arg Asn Asp Ala Lys Ala Gln Leu Gln Ala Leu Gly Ala Lys 610 615 620 Val Ala Gly Ser Val Ser Lys Asn Thr Asp Cys Leu Val Ala Gly Glu 625 630 635 640 Ala Ala Gly Ser Lys Leu Ala Lys Ala Glu Glu Leu Gly Val Lys Val 645 650 655 Ile Asp Glu Gln Ala Leu Met Asp Leu Leu Asn Ala Ala Asn 660 665 670 91638DNAArtificial SequencePyrGCp hybrid 9atg aat tct aac act aaa att att ttc gtc aca ggt ggg gta gta tca 48Met Asn Ser Asn Thr Lys Ile Ile Phe Val Thr Gly Gly Val Val Ser 1 5 10 15 tca ctt ggt aag ggt gta act gcg gca tct ttg gct act ctc tta gaa 96Ser Leu Gly Lys Gly Val Thr Ala Ala Ser Leu Ala Thr Leu Leu Glu 20 25 30 agt cgt ggt ctt aat gta aca atg atg aag ctt gat cca tac atc aat 144Ser Arg Gly Leu Asn Val Thr Met Met Lys Leu Asp Pro Tyr Ile Asn 35 40 45 gtt gat cca ggg act atg agt cca ttg caa cat ggt gaa gtt ttt gta 192Val Asp Pro Gly Thr Met Ser Pro Leu Gln His Gly Glu Val Phe Val 50 55 60 acc gaa gat ggc gca gag act gat ctt gat tta ggt cat tat gag cgc 240Thr Glu Asp Gly Ala Glu Thr Asp Leu Asp Leu Gly His Tyr Glu Arg 65 70 75 80 ttt atc cgc aat aag atg act caa gca aat aac ttc aca acc ggt aaa 288Phe Ile Arg Asn Lys Met Thr Gln Ala Asn Asn Phe Thr Thr Gly Lys 85 90 95 gta tac cag agt gtg tta aga aga gag cgt aag ggt gat tat cta ggt 336Val Tyr Gln Ser Val Leu Arg Arg Glu Arg Lys Gly Asp Tyr Leu Gly 100 105 110 gct act atc cag gtg att cca cat atc att gat gag atc aaa agg cgt 384Ala Thr Ile Gln Val Ile Pro His Ile Ile Asp Glu Ile Lys Arg Arg 115 120 125 att tgt agt ggt att gct gat gat gtt gat gtt gcg att gtt gag att 432Ile Cys Ser Gly Ile Ala Asp Asp Val Asp Val Ala Ile Val Glu Ile 130 135 140 ggt ggt act gtt ggt gat atc gag tca caa cca ttt tta gaa gct att 480Gly Gly Thr Val Gly Asp Ile Glu Ser Gln Pro Phe Leu Glu Ala Ile 145 150 155 160 cgt caa ttg gca tta gag gta ggt cgt gat cgt gct atg ttt atg cat 528Arg Gln Leu Ala Leu Glu Val Gly Arg Asp Arg Ala Met Phe Met His 165 170 175 ttg acc tta gtg cca tat tta gca gca gca ggt gaa atc aaa act aaa 576Leu Thr Leu Val Pro Tyr Leu Ala Ala Ala Gly Glu Ile Lys Thr Lys 180 185 190 cca aca cag cac tca gta aaa gat tta cgc tct atc ggt att ttt cct 624Pro Thr Gln His Ser Val Lys Asp Leu Arg Ser Ile Gly Ile Phe Pro 195 200 205 gac att tta gta tgt cgt tca gac cgc gct att cct aac gcc gaa cgc 672Asp Ile Leu Val Cys Arg Ser Asp Arg Ala Ile Pro Asn Ala Glu Arg 210 215 220 gct aaa ata tct ctc ttc act aat gtt gaa gag aaa gcg gtt gta tca 720Ala Lys Ile Ser Leu Phe Thr Asn Val Glu Glu Lys Ala Val Val Ser 225 230 235 240 atg cgt gat gta gac agt att tat aag att cct gct tta tta aaa gct 768Met Arg Asp Val Asp Ser Ile Tyr Lys Ile Pro Ala Leu Leu Lys Ala 245 250 255 caa ggt acc gat gaa ata gtt gtt aag cga ttt ggt tta gat gta cct 816Gln Gly Thr Asp Glu Ile Val Val Lys Arg Phe Gly Leu Asp Val Pro 260 265 270 gaa gcc gac tta act gaa tgg gaa gaa gtg ctt tac cat gaa gca aat 864Glu Ala Asp Leu Thr Glu Trp Glu Glu Val Leu Tyr His Glu Ala Asn 275 280 285 cct atc ggt gaa gtg act att ggt atg gtt ggt aaa tac act gaa tta 912Pro Ile Gly Glu Val Thr Ile Gly Met Val Gly Lys Tyr Thr Glu Leu 290 295 300 cct gat gcg tac aaa tca gta aac gaa gcg tta aaa cat gca ggt ctt 960Pro Asp Ala Tyr Lys Ser Val Asn Glu Ala Leu Lys His Ala Gly Leu 305 310 315 320 aaa aac caa gtc act gta aat att aaa tac att gac tcg caa gat gta 1008Lys Asn Gln Val Thr Val Asn Ile Lys Tyr Ile Asp Ser Gln Asp Val 325 330 335 gaa gtc aaa ggt gtt gaa atc tta gct aac ttg gat gct att tta gtt 1056Glu Val Lys Gly Val Glu Ile Leu Ala Asn Leu Asp Ala Ile Leu Val 340 345 350 cct ggt ggt ttc ggt gaa cgt ggt gtt gaa ggt aaa att tta acg gca 1104Pro Gly Gly Phe Gly Glu Arg Gly Val Glu Gly Lys Ile Leu Thr Ala 355 360 365 caa tat gcg cgt gaa aac aaa gta cct tat tta ggt att tgt tta ggt 1152Gln Tyr Ala Arg Glu Asn Lys Val Pro Tyr Leu Gly Ile Cys Leu Gly 370 375 380 atg caa gta gcc tta att gaa ttt gct cgt aat gtt gcc ggt tta act 1200Met Gln Val Ala Leu Ile Glu Phe Ala Arg Asn Val Ala Gly Leu Thr 385 390 395 400 gat gcg cac agt act gaa ttt aat agc gaa act cca cac cca gtg gtt 1248Asp Ala His Ser Thr Glu Phe Asn Ser Glu Thr Pro His Pro Val Val 405 410 415 ggt tta atc agt gaa tgg tta gac gaa gaa ggc caa gtt gag tac cga 1296Gly Leu Ile Ser Glu Trp Leu Asp Glu Glu Gly Gln Val Glu Tyr Arg 420 425 430 aat gag caa tca gat tta ggt ggt act atg cgt tta ggt tca caa ttg 1344Asn Glu Gln Ser Asp Leu Gly Gly Thr Met Arg Leu Gly Ser Gln Leu 435 440 445 tgc cac ttg gtg aaa ggt acc aag gct tgc gac gta tat ggt agt gaa 1392Cys His Leu Val Lys Gly Thr Lys Ala Cys Asp Val Tyr Gly Ser Glu 450 455 460 aca atc aat gag aga cac cgt cat cgt ttt gag gta aat aat aac tac 1440Thr Ile Asn Glu Arg His Arg His Arg Phe Glu Val Asn Asn Asn Tyr 465 470 475 480 cga gaa caa tta agc aaa gca ggt ttg att ttc tcg ggt tta tcg tca 1488Arg Glu Gln Leu Ser Lys Ala Gly Leu Ile Phe Ser Gly Leu Ser Ser 485 490 495 gat aaa agt tta gtt gag gtg att gaa ata gcg gat cat cca tgg ttt 1536Asp Lys Ser Leu Val Glu Val Ile Glu Ile Ala Asp His Pro Trp Phe 500 505 510 att gcg ggt caa ttc cat cct gag ttt aat tct act cca cgt gat ggt 1584Ile Ala Gly Gln Phe His Pro Glu Phe Asn Ser Thr Pro Arg Asp Gly 515 520 525 cac ccg tta ttc gaa agc ttt gtt gca gcg agt ttt aaa ctg caa aat 1632His Pro Leu Phe Glu Ser Phe Val Ala Ala Ser Phe Lys Leu Gln Asn 530 535 540 aat tag 1638Asn 545 10545PRTArtificial SequenceSynthetic Construct 10Met Asn Ser Asn Thr Lys Ile Ile Phe Val Thr Gly Gly Val Val Ser 1 5 10 15 Ser Leu Gly Lys Gly Val Thr Ala Ala Ser Leu Ala Thr Leu Leu Glu 20 25 30 Ser Arg Gly Leu Asn Val Thr Met Met Lys Leu Asp Pro Tyr Ile Asn 35 40 45 Val Asp Pro Gly Thr Met Ser Pro Leu Gln His Gly Glu Val Phe Val 50 55 60 Thr Glu Asp Gly Ala Glu Thr Asp Leu Asp Leu Gly His Tyr Glu Arg 65 70 75 80 Phe Ile Arg Asn Lys Met Thr Gln Ala Asn Asn Phe Thr Thr Gly Lys 85 90 95 Val Tyr Gln Ser Val Leu Arg Arg Glu Arg Lys Gly Asp Tyr Leu Gly 100 105 110 Ala Thr Ile Gln Val Ile Pro His Ile Ile Asp Glu Ile Lys Arg Arg 115 120 125 Ile Cys Ser Gly Ile Ala Asp Asp Val Asp Val Ala Ile Val Glu Ile 130 135 140 Gly Gly Thr Val Gly Asp Ile Glu Ser Gln Pro Phe Leu Glu Ala Ile 145 150 155 160 Arg Gln Leu Ala Leu Glu Val Gly Arg Asp Arg Ala Met Phe Met His 165 170 175 Leu Thr Leu Val Pro Tyr Leu Ala Ala Ala Gly Glu Ile Lys Thr Lys 180 185 190 Pro Thr Gln His Ser Val Lys Asp Leu Arg Ser Ile Gly Ile Phe Pro 195 200 205 Asp Ile Leu Val Cys Arg Ser Asp Arg Ala Ile Pro Asn Ala Glu Arg 210 215 220 Ala Lys Ile Ser Leu Phe Thr Asn Val Glu Glu Lys Ala Val Val Ser 225 230 235 240 Met Arg Asp Val Asp Ser Ile Tyr Lys Ile Pro Ala Leu Leu Lys Ala 245 250 255 Gln Gly Thr Asp Glu Ile Val Val Lys Arg Phe Gly Leu Asp Val Pro 260 265 270 Glu Ala Asp Leu Thr Glu Trp Glu Glu Val Leu Tyr His Glu Ala Asn 275 280 285 Pro Ile Gly Glu Val Thr Ile Gly Met Val Gly Lys Tyr Thr Glu Leu 290 295 300 Pro Asp Ala Tyr Lys Ser Val Asn Glu Ala Leu Lys His Ala Gly Leu 305 310 315 320 Lys Asn Gln Val Thr Val Asn Ile Lys Tyr Ile Asp Ser Gln Asp Val 325 330 335 Glu Val Lys Gly Val Glu Ile Leu Ala Asn Leu Asp Ala Ile Leu Val 340 345 350 Pro Gly Gly Phe Gly Glu Arg Gly Val Glu Gly Lys Ile Leu Thr Ala 355 360 365 Gln Tyr Ala Arg Glu Asn Lys Val Pro Tyr Leu Gly Ile Cys Leu Gly 370 375 380 Met Gln Val Ala Leu Ile Glu Phe Ala Arg Asn Val Ala Gly Leu Thr 385 390 395 400 Asp Ala His Ser Thr Glu Phe Asn Ser Glu Thr Pro His Pro Val Val 405 410 415 Gly Leu Ile Ser Glu Trp Leu Asp Glu Glu Gly Gln Val Glu Tyr Arg 420 425 430 Asn Glu Gln Ser Asp Leu Gly Gly Thr Met Arg Leu Gly Ser Gln Leu 435 440 445 Cys His Leu Val Lys Gly Thr Lys Ala Cys Asp Val Tyr Gly Ser Glu 450 455 460 Thr Ile Asn Glu Arg His Arg His Arg Phe Glu Val Asn Asn Asn Tyr 465 470 475 480 Arg Glu Gln Leu Ser Lys Ala Gly Leu Ile Phe Ser Gly Leu Ser Ser 485 490 495 Asp Lys Ser Leu Val Glu Val Ile Glu Ile Ala Asp His Pro Trp Phe 500 505 510 Ile Ala Gly Gln Phe His Pro Glu Phe Asn Ser Thr Pro Arg Asp Gly 515 520 525 His Pro Leu Phe Glu Ser Phe Val Ala Ala Ser Phe Lys Leu Gln Asn 530 535 540 Asn 545 11954DNAArtificial SequencehemCCp hybrid 11atg aaa caa act aca gta cga att gcc acg cgt aaa agc gcc ctc gcc 48Met Lys Gln Thr Thr Val Arg Ile Ala Thr Arg Lys Ser Ala Leu Ala 1 5 10 15 tta tgg caa gca gaa tat gtt aaa gcg caa ctt gag cat ttt cat gac 96Leu Trp Gln Ala Glu Tyr Val Lys Ala Gln Leu Glu His Phe His Asp 20 25 30 ggt att aat gtt gaa tta gtg cct atg aca acg aaa ggc gac atc att 144Gly Ile Asn Val Glu Leu Val Pro Met Thr Thr Lys Gly Asp Ile Ile 35 40 45 tta gac acg cct tta gcc aaa gtc ggc ggt aaa ggt tta ttt gtt aaa 192Leu Asp Thr Pro Leu Ala Lys Val Gly Gly Lys Gly Leu Phe Val Lys 50 55 60 gag ctt gaa gta gca atg ctt gaa gac cgt gct gat att gct gtt cat 240Glu Leu Glu Val Ala Met Leu Glu Asp Arg Ala Asp Ile Ala Val His 65 70 75 80 tca atg aaa gat gtt cct gtc gat ttt cca gaa ggc tta gga tta gaa 288Ser Met Lys Asp Val Pro Val Asp Phe Pro Glu Gly Leu Gly Leu Glu 85 90 95 gtc att tgt cct cgt gaa gat ccc cgt gat gct ttt gtt tct aat acc 336Val Ile Cys Pro Arg Glu Asp Pro Arg Asp Ala Phe Val Ser Asn Thr 100 105 110 atc aaa tca tta agt gat tta cca caa ggc tct att gtt ggc acc tca 384Ile Lys Ser Leu Ser Asp Leu Pro Gln Gly Ser Ile Val Gly Thr Ser 115 120 125 agc tta cgc cgt cag tgt caa tta aaa gca agc cgc cct gat tta gat 432Ser Leu Arg Arg Gln Cys Gln Leu Lys Ala Ser Arg Pro Asp Leu Asp 130 135 140

att cgt gat tta cgt ggc aat gta aat acc cgc cta aga aaa tta gat 480Ile Arg Asp Leu Arg Gly Asn Val Asn Thr Arg Leu Arg Lys Leu Asp 145 150 155 160 gaa ggt cag tac gac gct att ata tta gcc gct gca ggc cta att cgc 528Glu Gly Gln Tyr Asp Ala Ile Ile Leu Ala Ala Ala Gly Leu Ile Arg 165 170 175 tta gaa atg agc gag cgt att gca cag ttt atc gaa cca gaa gaa atg 576Leu Glu Met Ser Glu Arg Ile Ala Gln Phe Ile Glu Pro Glu Glu Met 180 185 190 ctt cct gca aat ggc caa ggc gct gtt ggc att gaa tgt cgt aat gat 624Leu Pro Ala Asn Gly Gln Gly Ala Val Gly Ile Glu Cys Arg Asn Asp 195 200 205 gat gcg aca att aaa gcc tta tta gca cca tta gaa tgt gct acc acc 672Asp Ala Thr Ile Lys Ala Leu Leu Ala Pro Leu Glu Cys Ala Thr Thr 210 215 220 cgt att cgt gtt ctt gca gaa cgt gca atg aat aga gca tta caa ggc 720Arg Ile Arg Val Leu Ala Glu Arg Ala Met Asn Arg Ala Leu Gln Gly 225 230 235 240 ggt tgc cag gtt cct atc ggt agc tat ggt gtt att tct gct gat ggt 768Gly Cys Gln Val Pro Ile Gly Ser Tyr Gly Val Ile Ser Ala Asp Gly 245 250 255 aaa aat atc cac tta cgt ggc tta gtt ggc tct gtc gat ggt agt gaa 816Lys Asn Ile His Leu Arg Gly Leu Val Gly Ser Val Asp Gly Ser Glu 260 265 270 atg ata gaa agt gaa atc acc ggc cct gtt gaa gaa ggt gaa gcg ctc 864Met Ile Glu Ser Glu Ile Thr Gly Pro Val Glu Glu Gly Glu Ala Leu 275 280 285 ggc aat aaa ctc gcg caa gag tta cta agc cga ggt gca gat aaa att 912Gly Asn Lys Leu Ala Gln Glu Leu Leu Ser Arg Gly Ala Asp Lys Ile 290 295 300 tta cag caa gtt tat tca gaa aat gat atc aaa gag agt taa 954Leu Gln Gln Val Tyr Ser Glu Asn Asp Ile Lys Glu Ser 305 310 315 12317PRTArtificial SequenceSynthetic Construct 12Met Lys Gln Thr Thr Val Arg Ile Ala Thr Arg Lys Ser Ala Leu Ala 1 5 10 15 Leu Trp Gln Ala Glu Tyr Val Lys Ala Gln Leu Glu His Phe His Asp 20 25 30 Gly Ile Asn Val Glu Leu Val Pro Met Thr Thr Lys Gly Asp Ile Ile 35 40 45 Leu Asp Thr Pro Leu Ala Lys Val Gly Gly Lys Gly Leu Phe Val Lys 50 55 60 Glu Leu Glu Val Ala Met Leu Glu Asp Arg Ala Asp Ile Ala Val His 65 70 75 80 Ser Met Lys Asp Val Pro Val Asp Phe Pro Glu Gly Leu Gly Leu Glu 85 90 95 Val Ile Cys Pro Arg Glu Asp Pro Arg Asp Ala Phe Val Ser Asn Thr 100 105 110 Ile Lys Ser Leu Ser Asp Leu Pro Gln Gly Ser Ile Val Gly Thr Ser 115 120 125 Ser Leu Arg Arg Gln Cys Gln Leu Lys Ala Ser Arg Pro Asp Leu Asp 130 135 140 Ile Arg Asp Leu Arg Gly Asn Val Asn Thr Arg Leu Arg Lys Leu Asp 145 150 155 160 Glu Gly Gln Tyr Asp Ala Ile Ile Leu Ala Ala Ala Gly Leu Ile Arg 165 170 175 Leu Glu Met Ser Glu Arg Ile Ala Gln Phe Ile Glu Pro Glu Glu Met 180 185 190 Leu Pro Ala Asn Gly Gln Gly Ala Val Gly Ile Glu Cys Arg Asn Asp 195 200 205 Asp Ala Thr Ile Lys Ala Leu Leu Ala Pro Leu Glu Cys Ala Thr Thr 210 215 220 Arg Ile Arg Val Leu Ala Glu Arg Ala Met Asn Arg Ala Leu Gln Gly 225 230 235 240 Gly Cys Gln Val Pro Ile Gly Ser Tyr Gly Val Ile Ser Ala Asp Gly 245 250 255 Lys Asn Ile His Leu Arg Gly Leu Val Gly Ser Val Asp Gly Ser Glu 260 265 270 Met Ile Glu Ser Glu Ile Thr Gly Pro Val Glu Glu Gly Glu Ala Leu 275 280 285 Gly Asn Lys Leu Ala Gln Glu Leu Leu Ser Arg Gly Ala Asp Lys Ile 290 295 300 Leu Gln Gln Val Tyr Ser Glu Asn Asp Ile Lys Glu Ser 305 310 315 13984DNAArtificial SequencefmtCp hybrid 13atg aaa aaa cca cta aat atc att ttt gca ggt act cct gaa ttc gct 48Met Lys Lys Pro Leu Asn Ile Ile Phe Ala Gly Thr Pro Glu Phe Ala 1 5 10 15 gcc caa cat tta gca gcg tta att aat tct gaa cat aat att gtc gcc 96Ala Gln His Leu Ala Ala Leu Ile Asn Ser Glu His Asn Ile Val Ala 20 25 30 gtt tat tgt ccc cct gat aaa cca gct ggc cgc ggt aaa aaa cta aca 144Val Tyr Cys Pro Pro Asp Lys Pro Ala Gly Arg Gly Lys Lys Leu Thr 35 40 45 gct tgt gca aca aag tta ctc gca ata gag cac gac att att gtt gag 192Ala Cys Ala Thr Lys Leu Leu Ala Ile Glu His Asp Ile Ile Val Glu 50 55 60 caa cct att aac ttt aaa aat gag gaa gac caa caa caa tta gcg aaa 240Gln Pro Ile Asn Phe Lys Asn Glu Glu Asp Gln Gln Gln Leu Ala Lys 65 70 75 80 tat aac gct gat atc atg gtt gtt gtt gct tat ggt ctg cta tta cct 288Tyr Asn Ala Asp Ile Met Val Val Val Ala Tyr Gly Leu Leu Leu Pro 85 90 95 gaa gtc att tta aac tct cca cgt tta ggc tgc att aac gta cat ggc 336Glu Val Ile Leu Asn Ser Pro Arg Leu Gly Cys Ile Asn Val His Gly 100 105 110 tca att cta cca aaa tgg cgt ggt gca gca cct att caa cgt tct ctt 384Ser Ile Leu Pro Lys Trp Arg Gly Ala Ala Pro Ile Gln Arg Ser Leu 115 120 125 gaa gct gga gat aag aaa acc ggt gtc acc att atg caa atg gat aaa 432Glu Ala Gly Asp Lys Lys Thr Gly Val Thr Ile Met Gln Met Asp Lys 130 135 140 ggg tta gac acg gga gac atg att cta tcc gct gag tgc gaa ata gaa 480Gly Leu Asp Thr Gly Asp Met Ile Leu Ser Ala Glu Cys Glu Ile Glu 145 150 155 160 aat aca gat acc agt gca agt ctt tat gaa aaa ctt gcc aac tta ggg 528Asn Thr Asp Thr Ser Ala Ser Leu Tyr Glu Lys Leu Ala Asn Leu Gly 165 170 175 cca act gcc tta gtt aat aca tta act att atg gct gaa cct gat tat 576Pro Thr Ala Leu Val Asn Thr Leu Thr Ile Met Ala Glu Pro Asp Tyr 180 185 190 caa gcc agt aat cat aat atc gct caa gat gat gaa tta gcg act tat 624Gln Ala Ser Asn His Asn Ile Ala Gln Asp Asp Glu Leu Ala Thr Tyr 195 200 205 gcc aag aaa ctt gat aaa act gaa gca gag ctt aac tgg caa ttc agt 672Ala Lys Lys Leu Asp Lys Thr Glu Ala Glu Leu Asn Trp Gln Phe Ser 210 215 220 gct gat gaa cta cat cga aaa att cgt gct tat att cct tgg cca gtt 720Ala Asp Glu Leu His Arg Lys Ile Arg Ala Tyr Ile Pro Trp Pro Val 225 230 235 240 gct caa ttt acc ttt aca gaa tct gaa ggt aag cag cat agg tta cgc 768Ala Gln Phe Thr Phe Thr Glu Ser Glu Gly Lys Gln His Arg Leu Arg 245 250 255 ata tgg caa gca tcc gtg caa gaa tat cga ggc aat gct gat cca ggc 816Ile Trp Gln Ala Ser Val Gln Glu Tyr Arg Gly Asn Ala Asp Pro Gly 260 265 270 acg ata ata aag gca gac aaa gaa ggg ata gaa gta gca aca acc agt 864Thr Ile Ile Lys Ala Asp Lys Glu Gly Ile Glu Val Ala Thr Thr Ser 275 280 285 ggt tcg tta cga cta gaa gtc att caa ctt cca ggg aaa aaa gca tta 912Gly Ser Leu Arg Leu Glu Val Ile Gln Leu Pro Gly Lys Lys Ala Leu 290 295 300 gcc gta aaa gac atc cta aat ggt cgc agc gat tgg ttc gtt gtt ggc 960Ala Val Lys Asp Ile Leu Asn Gly Arg Ser Asp Trp Phe Val Val Gly 305 310 315 320 agc act att aac aag cta gga taa 984Ser Thr Ile Asn Lys Leu Gly 325 14327PRTArtificial SequenceSynthetic Construct 14Met Lys Lys Pro Leu Asn Ile Ile Phe Ala Gly Thr Pro Glu Phe Ala 1 5 10 15 Ala Gln His Leu Ala Ala Leu Ile Asn Ser Glu His Asn Ile Val Ala 20 25 30 Val Tyr Cys Pro Pro Asp Lys Pro Ala Gly Arg Gly Lys Lys Leu Thr 35 40 45 Ala Cys Ala Thr Lys Leu Leu Ala Ile Glu His Asp Ile Ile Val Glu 50 55 60 Gln Pro Ile Asn Phe Lys Asn Glu Glu Asp Gln Gln Gln Leu Ala Lys 65 70 75 80 Tyr Asn Ala Asp Ile Met Val Val Val Ala Tyr Gly Leu Leu Leu Pro 85 90 95 Glu Val Ile Leu Asn Ser Pro Arg Leu Gly Cys Ile Asn Val His Gly 100 105 110 Ser Ile Leu Pro Lys Trp Arg Gly Ala Ala Pro Ile Gln Arg Ser Leu 115 120 125 Glu Ala Gly Asp Lys Lys Thr Gly Val Thr Ile Met Gln Met Asp Lys 130 135 140 Gly Leu Asp Thr Gly Asp Met Ile Leu Ser Ala Glu Cys Glu Ile Glu 145 150 155 160 Asn Thr Asp Thr Ser Ala Ser Leu Tyr Glu Lys Leu Ala Asn Leu Gly 165 170 175 Pro Thr Ala Leu Val Asn Thr Leu Thr Ile Met Ala Glu Pro Asp Tyr 180 185 190 Gln Ala Ser Asn His Asn Ile Ala Gln Asp Asp Glu Leu Ala Thr Tyr 195 200 205 Ala Lys Lys Leu Asp Lys Thr Glu Ala Glu Leu Asn Trp Gln Phe Ser 210 215 220 Ala Asp Glu Leu His Arg Lys Ile Arg Ala Tyr Ile Pro Trp Pro Val 225 230 235 240 Ala Gln Phe Thr Phe Thr Glu Ser Glu Gly Lys Gln His Arg Leu Arg 245 250 255 Ile Trp Gln Ala Ser Val Gln Glu Tyr Arg Gly Asn Ala Asp Pro Gly 260 265 270 Thr Ile Ile Lys Ala Asp Lys Glu Gly Ile Glu Val Ala Thr Thr Ser 275 280 285 Gly Ser Leu Arg Leu Glu Val Ile Gln Leu Pro Gly Lys Lys Ala Leu 290 295 300 Ala Val Lys Asp Ile Leu Asn Gly Arg Ser Asp Trp Phe Val Val Gly 305 310 315 320 Ser Thr Ile Asn Lys Leu Gly 325 151164DNAArtificial SequencemurGCp hybrid 15atg agt cta aat cat ggc caa ggt aat aaa gat tta gca aaa act ttg 48Met Ser Leu Asn His Gly Gln Gly Asn Lys Asp Leu Ala Lys Thr Leu 1 5 10 15 tta gtc atg gct ggt ggc acc ggt gga cat ata ttc cct ggt att gcg 96Leu Val Met Ala Gly Gly Thr Gly Gly His Ile Phe Pro Gly Ile Ala 20 25 30 gtc gcc gat gag ctg aaa gcg caa gga tgg aaa atc cat tgg ttg gga 144Val Ala Asp Glu Leu Lys Ala Gln Gly Trp Lys Ile His Trp Leu Gly 35 40 45 act gcc gat cgt atg gaa gct caa att gta cct atg cat ggt tat gat 192Thr Ala Asp Arg Met Glu Ala Gln Ile Val Pro Met His Gly Tyr Asp 50 55 60 att tcg ttt atc aat ata agt ggt ctg cgt ggt aaa aat cta tta aca 240Ile Ser Phe Ile Asn Ile Ser Gly Leu Arg Gly Lys Asn Leu Leu Thr 65 70 75 80 acg ctt gtt atg cct ttt aaa ttg tta agg tcg ctt ttt caa gcg aga 288Thr Leu Val Met Pro Phe Lys Leu Leu Arg Ser Leu Phe Gln Ala Arg 85 90 95 cgc gtg att aaa aca gtg aaa cct gat gtt gtt ata ggc atg ggt ggc 336Arg Val Ile Lys Thr Val Lys Pro Asp Val Val Ile Gly Met Gly Gly 100 105 110 tat gca agt gct ccg ggt ggt ttg gcc gct tgg cta agt aaa ata ccg 384Tyr Ala Ser Ala Pro Gly Gly Leu Ala Ala Trp Leu Ser Lys Ile Pro 115 120 125 cta atc gtt cat gaa caa aat gct gct gcc gga tta agt aat cgc ttg 432Leu Ile Val His Glu Gln Asn Ala Ala Ala Gly Leu Ser Asn Arg Leu 130 135 140 tta gcg cgt atc gcc aat aaa gta tgc tgc gcc ttt cct aat gca ttt 480Leu Ala Arg Ile Ala Asn Lys Val Cys Cys Ala Phe Pro Asn Ala Phe 145 150 155 160 gtt agc gga att gat gtt gaa gtg gtt ggt aat cct tta cgc gcg tca 528Val Ser Gly Ile Asp Val Glu Val Val Gly Asn Pro Leu Arg Ala Ser 165 170 175 atc ggt cag caa gca ctg gtt tca gaa aat ata gat caa agc cac gaa 576Ile Gly Gln Gln Ala Leu Val Ser Glu Asn Ile Asp Gln Ser His Glu 180 185 190 ggt agt aaa aat att cta gtg gta ggt ggt agt tta ggc gct caa gtc 624Gly Ser Lys Asn Ile Leu Val Val Gly Gly Ser Leu Gly Ala Gln Val 195 200 205 tta aat aag gtg atg ccg gat agc ttt aag gat tta tca gaa agt gat 672Leu Asn Lys Val Met Pro Asp Ser Phe Lys Asp Leu Ser Glu Ser Asp 210 215 220 gag aaa tat tgt ata tgg cac caa acg ggc gac aat aac caa gca cta 720Glu Lys Tyr Cys Ile Trp His Gln Thr Gly Asp Asn Asn Gln Ala Leu 225 230 235 240 gtc acc gca tct tat aaa cag gaa tat att gat act gga aaa gtg aga 768Val Thr Ala Ser Tyr Lys Gln Glu Tyr Ile Asp Thr Gly Lys Val Arg 245 250 255 gtt acc gaa ttt att act gat att gct gct gca tat cag tgg gct gat 816Val Thr Glu Phe Ile Thr Asp Ile Ala Ala Ala Tyr Gln Trp Ala Asp 260 265 270 ata gtg att tgt cgt gcg gga gcg cta acc gtt tca gaa tta gcc atg 864Ile Val Ile Cys Arg Ala Gly Ala Leu Thr Val Ser Glu Leu Ala Met 275 280 285 gca gca aca cca gcc att ttt gta cca cta ccg cat gca gta gat gat 912Ala Ala Thr Pro Ala Ile Phe Val Pro Leu Pro His Ala Val Asp Asp 290 295 300 cat caa aca aaa aat gcg ttg tac ctc gta aag cga gat gca gca aag 960His Gln Thr Lys Asn Ala Leu Tyr Leu Val Lys Arg Asp Ala Ala Lys 305 310 315 320 tta ttg cca cag gca gaa cta aat aat gag agt atc acg tcg tta ata 1008Leu Leu Pro Gln Ala Glu Leu Asn Asn Glu Ser Ile Thr Ser Leu Ile 325 330 335 atc gag ctg ttt gat cag cct caa act tta gct gac atg gct aaa gct 1056Ile Glu Leu Phe Asp Gln Pro Gln Thr Leu Ala Asp Met Ala Lys Ala 340 345 350 tct ttg agt gct gca act agt gat gca agt cag aaa gta gca aaa ttg 1104Ser Leu Ser Ala Ala Thr Ser Asp Ala Ser Gln Lys Val Ala Lys Leu 355 360 365 tgc caa cag ctt tca ata tcg aat ggc gca aaa ctt aga aat aat gaa 1152Cys Gln Gln Leu Ser Ile Ser Asn Gly Ala Lys Leu Arg Asn Asn Glu 370 375 380 gag aac aaa taa 1164Glu Asn Lys 385 16387PRTArtificial SequenceSynthetic Construct 16Met Ser Leu Asn His Gly Gln Gly Asn Lys Asp Leu Ala Lys Thr Leu 1 5 10 15 Leu Val Met Ala Gly Gly Thr Gly Gly His Ile Phe Pro Gly Ile Ala 20 25 30 Val Ala Asp Glu Leu Lys Ala Gln Gly Trp Lys Ile His Trp Leu Gly 35 40 45 Thr Ala Asp Arg Met Glu Ala Gln Ile Val Pro Met His Gly Tyr Asp 50 55 60 Ile Ser Phe Ile Asn Ile Ser Gly Leu Arg Gly Lys Asn Leu Leu Thr 65 70

75 80 Thr Leu Val Met Pro Phe Lys Leu Leu Arg Ser Leu Phe Gln Ala Arg 85 90 95 Arg Val Ile Lys Thr Val Lys Pro Asp Val Val Ile Gly Met Gly Gly 100 105 110 Tyr Ala Ser Ala Pro Gly Gly Leu Ala Ala Trp Leu Ser Lys Ile Pro 115 120 125 Leu Ile Val His Glu Gln Asn Ala Ala Ala Gly Leu Ser Asn Arg Leu 130 135 140 Leu Ala Arg Ile Ala Asn Lys Val Cys Cys Ala Phe Pro Asn Ala Phe 145 150 155 160 Val Ser Gly Ile Asp Val Glu Val Val Gly Asn Pro Leu Arg Ala Ser 165 170 175 Ile Gly Gln Gln Ala Leu Val Ser Glu Asn Ile Asp Gln Ser His Glu 180 185 190 Gly Ser Lys Asn Ile Leu Val Val Gly Gly Ser Leu Gly Ala Gln Val 195 200 205 Leu Asn Lys Val Met Pro Asp Ser Phe Lys Asp Leu Ser Glu Ser Asp 210 215 220 Glu Lys Tyr Cys Ile Trp His Gln Thr Gly Asp Asn Asn Gln Ala Leu 225 230 235 240 Val Thr Ala Ser Tyr Lys Gln Glu Tyr Ile Asp Thr Gly Lys Val Arg 245 250 255 Val Thr Glu Phe Ile Thr Asp Ile Ala Ala Ala Tyr Gln Trp Ala Asp 260 265 270 Ile Val Ile Cys Arg Ala Gly Ala Leu Thr Val Ser Glu Leu Ala Met 275 280 285 Ala Ala Thr Pro Ala Ile Phe Val Pro Leu Pro His Ala Val Asp Asp 290 295 300 His Gln Thr Lys Asn Ala Leu Tyr Leu Val Lys Arg Asp Ala Ala Lys 305 310 315 320 Leu Leu Pro Gln Ala Glu Leu Asn Asn Glu Ser Ile Thr Ser Leu Ile 325 330 335 Ile Glu Leu Phe Asp Gln Pro Gln Thr Leu Ala Asp Met Ala Lys Ala 340 345 350 Ser Leu Ser Ala Ala Thr Ser Asp Ala Ser Gln Lys Val Ala Lys Leu 355 360 365 Cys Gln Gln Leu Ser Ile Ser Asn Gly Ala Lys Leu Arg Asn Asn Glu 370 375 380 Glu Asn Lys 385 172067DNAArtificial SequenceCodon optimized ligACp optimized for M. tuberculosis 17gtg agc gag aag gag aag aaa ata tcc cag ctg caa cag caa ctg aac 48Val Ser Glu Lys Glu Lys Lys Ile Ser Gln Leu Gln Gln Gln Leu Asn 1 5 10 15 caa tat aac cat gag tac tat gtc ctc gac cag cca tcg gtc ccc gat 96Gln Tyr Asn His Glu Tyr Tyr Val Leu Asp Gln Pro Ser Val Pro Asp 20 25 30 gcg gag tac gat cgc ctg atg acc gcg tta atc gat ctg gaa aag acc 144Ala Glu Tyr Asp Arg Leu Met Thr Ala Leu Ile Asp Leu Glu Lys Thr 35 40 45 aac ccg gag ttg aag acg atc gac agt ccg tcg cag aag gtg ggc ggt 192Asn Pro Glu Leu Lys Thr Ile Asp Ser Pro Ser Gln Lys Val Gly Gly 50 55 60 cag gcc ctg aag agc ttc acc caa gtg acg cat cag ctg ccc atg ctc 240Gln Ala Leu Lys Ser Phe Thr Gln Val Thr His Gln Leu Pro Met Leu 65 70 75 80 tcg ctt gac aac gtc ttt tcg ctg gat gac ttc cac gcc ttc gtc aag 288Ser Leu Asp Asn Val Phe Ser Leu Asp Asp Phe His Ala Phe Val Lys 85 90 95 agg gtc aaa gac cgt ctc aat gac aac cag gcg atc gtg ttc tgt gcc 336Arg Val Lys Asp Arg Leu Asn Asp Asn Gln Ala Ile Val Phe Cys Ala 100 105 110 gag ccg aag ctg gac ggc ctc gcg gta tcg ctc cgc tac gag cat ggc 384Glu Pro Lys Leu Asp Gly Leu Ala Val Ser Leu Arg Tyr Glu His Gly 115 120 125 cag ctc atc cag gcg gcc acg cgg ggc gac ggc tca gtc ggg gag aat 432Gln Leu Ile Gln Ala Ala Thr Arg Gly Asp Gly Ser Val Gly Glu Asn 130 135 140 atc acc acg aac atc cgg acg atc aag tcc atc ccc ctg aag ctc atg 480Ile Thr Thr Asn Ile Arg Thr Ile Lys Ser Ile Pro Leu Lys Leu Met 145 150 155 160 ggc act ccc ggc aaa gac ttt cca gac att gtg gaa gtc cgg ggc gaa 528Gly Thr Pro Gly Lys Asp Phe Pro Asp Ile Val Glu Val Arg Gly Glu 165 170 175 gtg ttc atg ccg aag gcc tcg ttc gac gcg ctg aac acc ctg gct aag 576Val Phe Met Pro Lys Ala Ser Phe Asp Ala Leu Asn Thr Leu Ala Lys 180 185 190 aaa cgg ggg gag aag ggc ttc gct aac ccg cgg aac gcg gca gcc ggc 624Lys Arg Gly Glu Lys Gly Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly 195 200 205 agt ctg cgt cag ctg gac agc aag atc acg gcc aag cgc aac ctg gcg 672Ser Leu Arg Gln Leu Asp Ser Lys Ile Thr Ala Lys Arg Asn Leu Ala 210 215 220 ttc tat gcc tac agc cta ggt ttc gtg ggg aaa ctg agc gac ggg ggc 720Phe Tyr Ala Tyr Ser Leu Gly Phe Val Gly Lys Leu Ser Asp Gly Gly 225 230 235 240 gcg gaa agc acc gac ttg acg aac gac ttt ttc gcg aac tcg cac cat 768Ala Glu Ser Thr Asp Leu Thr Asn Asp Phe Phe Ala Asn Ser His His 245 250 255 gag cga ttg tgt caa ttg aag cga ctg ggt ttg ccg atg tgt ccg gag 816Glu Arg Leu Cys Gln Leu Lys Arg Leu Gly Leu Pro Met Cys Pro Glu 260 265 270 gtg cgg ctg cta gaa tcg gag cag gct tgc gac gcg ttc tac cag gac 864Val Arg Leu Leu Glu Ser Glu Gln Ala Cys Asp Ala Phe Tyr Gln Asp 275 280 285 atc ctc gcg aag cgc tcg gct ctt tca tac gaa atc gac ggt acc gtt 912Ile Leu Ala Lys Arg Ser Ala Leu Ser Tyr Glu Ile Asp Gly Thr Val 290 295 300 ttg aag gtt gac gag atc tcc ctc cag aag cgc ctg ggt ttc gtg gcg 960Leu Lys Val Asp Glu Ile Ser Leu Gln Lys Arg Leu Gly Phe Val Ala 305 310 315 320 cgg gca ccg cgc tgg gcc atc gcc tac aag ttc cca gca gag gaa gag 1008Arg Ala Pro Arg Trp Ala Ile Ala Tyr Lys Phe Pro Ala Glu Glu Glu 325 330 335 ctg acc tgc gtg gag gac gta gaa ttt caa gtg ggc cgc acc ggt gcc 1056Leu Thr Cys Val Glu Asp Val Glu Phe Gln Val Gly Arg Thr Gly Ala 340 345 350 atc acc ccg gtt gcc cgc ctg aag cct gtc ttc gtg ggc ggt gtc acc 1104Ile Thr Pro Val Ala Arg Leu Lys Pro Val Phe Val Gly Gly Val Thr 355 360 365 gtg agc aac gct acc ctt cat aac cag gac gag atc aca cgt ctg ggg 1152Val Ser Asn Ala Thr Leu His Asn Gln Asp Glu Ile Thr Arg Leu Gly 370 375 380 ctg aag gtc aac gat ttc gtc gtg att cgc cgg gca ggc gac gtt att 1200Leu Lys Val Asn Asp Phe Val Val Ile Arg Arg Ala Gly Asp Val Ile 385 390 395 400 ccg cag atc gtg tcg gtg gtc ctg gac aaa agg ccg gat aac gcc gtc 1248Pro Gln Ile Val Ser Val Val Leu Asp Lys Arg Pro Asp Asn Ala Val 405 410 415 gat atc gtc ttc ccc acg tcg tgc ccg gtg tgc gac tcg gcc gtg gcc 1296Asp Ile Val Phe Pro Thr Ser Cys Pro Val Cys Asp Ser Ala Val Ala 420 425 430 aag ccc gaa ggc gag gca gtc ctg cgg tgc aca gcc ggg ctc ttc tgt 1344Lys Pro Glu Gly Glu Ala Val Leu Arg Cys Thr Ala Gly Leu Phe Cys 435 440 445 gcg gcc cag cgc aag gaa gcc atc aag cac ttc gcc tcc cgc aag gcc 1392Ala Ala Gln Arg Lys Glu Ala Ile Lys His Phe Ala Ser Arg Lys Ala 450 455 460 cac gac gtc gac gga ctg ggc gac aag ctc gtc gag cag ctt gta gac 1440His Asp Val Asp Gly Leu Gly Asp Lys Leu Val Glu Gln Leu Val Asp 465 470 475 480 gag aag ctg atc aac acc ccc gcg gat ctg ttc aag ctc acc gaa atc 1488Glu Lys Leu Ile Asn Thr Pro Ala Asp Leu Phe Lys Leu Thr Glu Ile 485 490 495 cag gtg agt acc att gac aga atg gga aag aag tct gcc acc aac ctg 1536Gln Val Ser Thr Ile Asp Arg Met Gly Lys Lys Ser Ala Thr Asn Leu 500 505 510 ata aat ggt ctg gag cag gcg aag agc act acg ctg gcg aag ttc att 1584Ile Asn Gly Leu Glu Gln Ala Lys Ser Thr Thr Leu Ala Lys Phe Ile 515 520 525 tac ggc ctg ggg atc cgg gaa gtg gga gag gcc acg gcc gcg aac ctg 1632Tyr Gly Leu Gly Ile Arg Glu Val Gly Glu Ala Thr Ala Ala Asn Leu 530 535 540 gcc aac cac ttc tac acc ctc gcc gcg atc gag agc gcc agc ttg gag 1680Ala Asn His Phe Tyr Thr Leu Ala Ala Ile Glu Ser Ala Ser Leu Glu 545 550 555 560 gat ctg cag aac gta tcc gac gtg ggt gag gtc gtg gca aag aac atc 1728Asp Leu Gln Asn Val Ser Asp Val Gly Glu Val Val Ala Lys Asn Ile 565 570 575 att aat ttc ttc aag gaa gag cac aac ctg gcg atc gtc agc ggt ttg 1776Ile Asn Phe Phe Lys Glu Glu His Asn Leu Ala Ile Val Ser Gly Leu 580 585 590 agc gaa gtg atg cac tgg ccc acc atc gag atc aag tcg gcc gag gag 1824Ser Glu Val Met His Trp Pro Thr Ile Glu Ile Lys Ser Ala Glu Glu 595 600 605 ctt cct ctg gcg gag cag atc ttc gtc ctc acc gga act ctc acc cag 1872Leu Pro Leu Ala Glu Gln Ile Phe Val Leu Thr Gly Thr Leu Thr Gln 610 615 620 atg ggc cgc acg gag gcg aag acc gcc ttg caa tcc ctg ggc gct aag 1920Met Gly Arg Thr Glu Ala Lys Thr Ala Leu Gln Ser Leu Gly Ala Lys 625 630 635 640 gtc tcg ggc tcc gtc tcc aag aac acc cac ttc gtg gtt gcg ggc gac 1968Val Ser Gly Ser Val Ser Lys Asn Thr His Phe Val Val Ala Gly Asp 645 650 655 aag gct ggc agc aag ctg acg aag gcg cag gac ctc ggc atc tca gtc 2016Lys Ala Gly Ser Lys Leu Thr Lys Ala Gln Asp Leu Gly Ile Ser Val 660 665 670 ctg aca gag gat ggc ctg gtc gcc ctg ctg gca gag cac ggc atc acc 2064Leu Thr Glu Asp Gly Leu Val Ala Leu Leu Ala Glu His Gly Ile Thr 675 680 685 att 2067Ile 18689PRTArtificial SequenceSynthetic Construct 18Val Ser Glu Lys Glu Lys Lys Ile Ser Gln Leu Gln Gln Gln Leu Asn 1 5 10 15 Gln Tyr Asn His Glu Tyr Tyr Val Leu Asp Gln Pro Ser Val Pro Asp 20 25 30 Ala Glu Tyr Asp Arg Leu Met Thr Ala Leu Ile Asp Leu Glu Lys Thr 35 40 45 Asn Pro Glu Leu Lys Thr Ile Asp Ser Pro Ser Gln Lys Val Gly Gly 50 55 60 Gln Ala Leu Lys Ser Phe Thr Gln Val Thr His Gln Leu Pro Met Leu 65 70 75 80 Ser Leu Asp Asn Val Phe Ser Leu Asp Asp Phe His Ala Phe Val Lys 85 90 95 Arg Val Lys Asp Arg Leu Asn Asp Asn Gln Ala Ile Val Phe Cys Ala 100 105 110 Glu Pro Lys Leu Asp Gly Leu Ala Val Ser Leu Arg Tyr Glu His Gly 115 120 125 Gln Leu Ile Gln Ala Ala Thr Arg Gly Asp Gly Ser Val Gly Glu Asn 130 135 140 Ile Thr Thr Asn Ile Arg Thr Ile Lys Ser Ile Pro Leu Lys Leu Met 145 150 155 160 Gly Thr Pro Gly Lys Asp Phe Pro Asp Ile Val Glu Val Arg Gly Glu 165 170 175 Val Phe Met Pro Lys Ala Ser Phe Asp Ala Leu Asn Thr Leu Ala Lys 180 185 190 Lys Arg Gly Glu Lys Gly Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly 195 200 205 Ser Leu Arg Gln Leu Asp Ser Lys Ile Thr Ala Lys Arg Asn Leu Ala 210 215 220 Phe Tyr Ala Tyr Ser Leu Gly Phe Val Gly Lys Leu Ser Asp Gly Gly 225 230 235 240 Ala Glu Ser Thr Asp Leu Thr Asn Asp Phe Phe Ala Asn Ser His His 245 250 255 Glu Arg Leu Cys Gln Leu Lys Arg Leu Gly Leu Pro Met Cys Pro Glu 260 265 270 Val Arg Leu Leu Glu Ser Glu Gln Ala Cys Asp Ala Phe Tyr Gln Asp 275 280 285 Ile Leu Ala Lys Arg Ser Ala Leu Ser Tyr Glu Ile Asp Gly Thr Val 290 295 300 Leu Lys Val Asp Glu Ile Ser Leu Gln Lys Arg Leu Gly Phe Val Ala 305 310 315 320 Arg Ala Pro Arg Trp Ala Ile Ala Tyr Lys Phe Pro Ala Glu Glu Glu 325 330 335 Leu Thr Cys Val Glu Asp Val Glu Phe Gln Val Gly Arg Thr Gly Ala 340 345 350 Ile Thr Pro Val Ala Arg Leu Lys Pro Val Phe Val Gly Gly Val Thr 355 360 365 Val Ser Asn Ala Thr Leu His Asn Gln Asp Glu Ile Thr Arg Leu Gly 370 375 380 Leu Lys Val Asn Asp Phe Val Val Ile Arg Arg Ala Gly Asp Val Ile 385 390 395 400 Pro Gln Ile Val Ser Val Val Leu Asp Lys Arg Pro Asp Asn Ala Val 405 410 415 Asp Ile Val Phe Pro Thr Ser Cys Pro Val Cys Asp Ser Ala Val Ala 420 425 430 Lys Pro Glu Gly Glu Ala Val Leu Arg Cys Thr Ala Gly Leu Phe Cys 435 440 445 Ala Ala Gln Arg Lys Glu Ala Ile Lys His Phe Ala Ser Arg Lys Ala 450 455 460 His Asp Val Asp Gly Leu Gly Asp Lys Leu Val Glu Gln Leu Val Asp 465 470 475 480 Glu Lys Leu Ile Asn Thr Pro Ala Asp Leu Phe Lys Leu Thr Glu Ile 485 490 495 Gln Val Ser Thr Ile Asp Arg Met Gly Lys Lys Ser Ala Thr Asn Leu 500 505 510 Ile Asn Gly Leu Glu Gln Ala Lys Ser Thr Thr Leu Ala Lys Phe Ile 515 520 525 Tyr Gly Leu Gly Ile Arg Glu Val Gly Glu Ala Thr Ala Ala Asn Leu 530 535 540 Ala Asn His Phe Tyr Thr Leu Ala Ala Ile Glu Ser Ala Ser Leu Glu 545 550 555 560 Asp Leu Gln Asn Val Ser Asp Val Gly Glu Val Val Ala Lys Asn Ile 565 570 575 Ile Asn Phe Phe Lys Glu Glu His Asn Leu Ala Ile Val Ser Gly Leu 580 585 590 Ser Glu Val Met His Trp Pro Thr Ile Glu Ile Lys Ser Ala Glu Glu 595 600 605 Leu Pro Leu Ala Glu Gln Ile Phe Val Leu Thr Gly Thr Leu Thr Gln 610 615 620 Met Gly Arg Thr Glu Ala Lys Thr Ala Leu Gln Ser Leu Gly Ala Lys 625 630 635 640 Val Ser Gly Ser Val Ser Lys Asn Thr His Phe Val Val Ala Gly Asp 645 650 655 Lys Ala Gly Ser Lys Leu Thr Lys Ala Gln Asp Leu Gly Ile Ser Val 660 665 670 Leu Thr Glu Asp Gly Leu Val Ala Leu Leu Ala Glu His Gly Ile Thr 675 680 685 Ile 191917DNAColwellia psychroerythrusCDS(1)..(1917)misc_feature(556)..(556)s at location 556 is g or c 19atg gga aaa att att ggt att gac cta gga aca act aac tca tgt gtt 48Met Gly Lys Ile Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser Cys Val 1 5 10 15 gct gtt tta gat ggc gac agt gta cgt gtt att gaa aat gca gaa ggc 96Ala Val Leu Asp Gly Asp Ser Val Arg Val Ile Glu Asn Ala Glu Gly 20 25 30 gat cgt aca act cct tct att att ggt tat aca gcc gaa ggc gaa aca 144Asp Arg Thr Thr Pro Ser Ile Ile Gly Tyr Thr Ala Glu Gly Glu Thr 35 40 45

tta gta ggt caa cct gct aag cgt caa tct gta act aac cca gaa aac 192Leu Val Gly Gln Pro Ala Lys Arg Gln Ser Val Thr Asn Pro Glu Asn 50 55 60 act tta tat gca att aaa cgc tta atc ggt cgt cgt ttc gaa gat aaa 240Thr Leu Tyr Ala Ile Lys Arg Leu Ile Gly Arg Arg Phe Glu Asp Lys 65 70 75 80 gaa aca caa cgt gac atc gat att atg cca ttt ggt att gtt aaa gcg 288Glu Thr Gln Arg Asp Ile Asp Ile Met Pro Phe Gly Ile Val Lys Ala 85 90 95 gat aac ggt gat gct tgg gtt caa gta aaa ggc gag aaa att gct ccg 336Asp Asn Gly Asp Ala Trp Val Gln Val Lys Gly Glu Lys Ile Ala Pro 100 105 110 cca caa gtt tca gct gaa gtt ctt aag aaa atg aaa aag act gct gaa 384Pro Gln Val Ser Ala Glu Val Leu Lys Lys Met Lys Lys Thr Ala Glu 115 120 125 gac ttc tta ggt gaa acc gta act gaa gct gtt att act gta cct gct 432Asp Phe Leu Gly Glu Thr Val Thr Glu Ala Val Ile Thr Val Pro Ala 130 135 140 tac ttt aac gat tca caa cgc caa gca acg aaa gat gct ggt cgt att 480Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp Ala Gly Arg Ile 145 150 155 160 gct ggt ctt gaa gtc aaa cgt att atc aac gaa cct act gct gct gcc 528Ala Gly Leu Glu Val Lys Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala 165 170 175 ctt gct tac ggc atg gac aaa caa gaa sgt gac aaa gtt gtt gca gtt 576Leu Ala Tyr Gly Met Asp Lys Gln Glu Xaa Asp Lys Val Val Ala Val 180 185 190 tac gat tta ggt ggt ggt aca ttc gat att tca atc att gaa att gat 624Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Ile Glu Ile Asp 195 200 205 gaa atg gat ggc gaa cac act ttt gaa gta tta gcg act aac ggt gat 672Glu Met Asp Gly Glu His Thr Phe Glu Val Leu Ala Thr Asn Gly Asp 210 215 220 act cac tta ggt ggt gaa gat ttt gat aac cgt tta atc aac tac ctt 720Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Leu Ile Asn Tyr Leu 225 230 235 240 gta gct gaa ttc aaa aaa gac caa ggc atg gac tta acg tct gat cct 768Val Ala Glu Phe Lys Lys Asp Gln Gly Met Asp Leu Thr Ser Asp Pro 245 250 255 tta gca atg cag cgt tta aaa gaa gca gca gaa aaa gct aaa tgt gaa 816Leu Ala Met Gln Arg Leu Lys Glu Ala Ala Glu Lys Ala Lys Cys Glu 260 265 270 ctt tct tca gca caa caa aca gat gta aac tta cct tac atc act gct 864Leu Ser Ser Ala Gln Gln Thr Asp Val Asn Leu Pro Tyr Ile Thr Ala 275 280 285 gat ggt tca ggt cct aag cac atg aac atc aaa gtg act cgt gct aag 912Asp Gly Ser Gly Pro Lys His Met Asn Ile Lys Val Thr Arg Ala Lys 290 295 300 tta gaa tca cta gtt gaa gat atg gtt aaa gca aca tta gag ccg ctt 960Leu Glu Ser Leu Val Glu Asp Met Val Lys Ala Thr Leu Glu Pro Leu 305 310 315 320 aaa caa gcg ctt aaa gat gca gac tta tca gta agc aag att gat gat 1008Lys Gln Ala Leu Lys Asp Ala Asp Leu Ser Val Ser Lys Ile Asp Asp 325 330 335 gtt att tta gtt ggt ggt caa tct cgt atg cca cta gtt caa aaa act 1056Val Ile Leu Val Gly Gly Gln Ser Arg Met Pro Leu Val Gln Lys Thr 340 345 350 gtt act gat ttc ttc ggt aaa gag cca cgt aaa gat gtt aac cct gat 1104Val Thr Asp Phe Phe Gly Lys Glu Pro Arg Lys Asp Val Asn Pro Asp 355 360 365 gaa gca gta gct tct ggt gcg gcg att caa gcg ggt gtt ctt tct ggt 1152Glu Ala Val Ala Ser Gly Ala Ala Ile Gln Ala Gly Val Leu Ser Gly 370 375 380 gat gtg act gac gtt ctt tta tta gac gtt aca cca cta tca tta ggt 1200Asp Val Thr Asp Val Leu Leu Leu Asp Val Thr Pro Leu Ser Leu Gly 385 390 395 400 atc gaa act atg ggc ggt gtg atg act aag gtt atc gac aaa aac act 1248Ile Glu Thr Met Gly Gly Val Met Thr Lys Val Ile Asp Lys Asn Thr 405 410 415 act atc cca act aag caa tca caa act ttc tct aca gct gat gat aac 1296Thr Ile Pro Thr Lys Gln Ser Gln Thr Phe Ser Thr Ala Asp Asp Asn 420 425 430 caa gct gca gta act gtt cat gtt tgt cag ggt gag cgt aag caa gct 1344Gln Ala Ala Val Thr Val His Val Cys Gln Gly Glu Arg Lys Gln Ala 435 440 445 tca gca aac aaa tct tta ggt caa ttt aac ctt gaa ggt att gaa cca 1392Ser Ala Asn Lys Ser Leu Gly Gln Phe Asn Leu Glu Gly Ile Glu Pro 450 455 460 gca caa cgt ggt aca cca caa atc gaa gta act ttt gat att gat gct 1440Ala Gln Arg Gly Thr Pro Gln Ile Glu Val Thr Phe Asp Ile Asp Ala 465 470 475 480 gat ggt atc ttg cac gtt acg gct aaa gat aag aat act ggt aaa gag 1488Asp Gly Ile Leu His Val Thr Ala Lys Asp Lys Asn Thr Gly Lys Glu 485 490 495 caa aaa atc act atc aaa gcc tct tct ggt tta tct gat gaa gaa gta 1536Gln Lys Ile Thr Ile Lys Ala Ser Ser Gly Leu Ser Asp Glu Glu Val 500 505 510 gag cag atg gta cgt gat gca gaa gct aac gct gat gct gat gct aaa 1584Glu Gln Met Val Arg Asp Ala Glu Ala Asn Ala Asp Ala Asp Ala Lys 515 520 525 ttt gaa gag cta gta act gca cgt aat caa gct gat ggc atg att cac 1632Phe Glu Glu Leu Val Thr Ala Arg Asn Gln Ala Asp Gly Met Ile His 530 535 540 gcg act cgc aag caa gtt gaa gaa gca ggc gaa gag tta cca agc gaa 1680Ala Thr Arg Lys Gln Val Glu Glu Ala Gly Glu Glu Leu Pro Ser Glu 545 550 555 560 gat aaa gaa aaa att gaa gca gca tta act gag ctt gaa gaa gca gtt 1728Asp Lys Glu Lys Ile Glu Ala Ala Leu Thr Glu Leu Glu Glu Ala Val 565 570 575 aaa ggt gat gat aaa gaa gta att gaa gct aaa act caa gca ctt atg 1776Lys Gly Asp Asp Lys Glu Val Ile Glu Ala Lys Thr Gln Ala Leu Met 580 585 590 gaa gca tca gct aag tta atg gaa att gct caa gct aaa gaa caa gct 1824Glu Ala Ser Ala Lys Leu Met Glu Ile Ala Gln Ala Lys Glu Gln Ala 595 600 605 caa agc gct cct gaa ggt gct caa gaa gct gac gca gct cct gca gac 1872Gln Ser Ala Pro Glu Gly Ala Gln Glu Ala Asp Ala Ala Pro Ala Asp 610 615 620 gat gtt gtt gat gct gag ttt gaa gaa gtt aaa gac aaa aaa taa 1917Asp Val Val Asp Ala Glu Phe Glu Glu Val Lys Asp Lys Lys 625 630 635 20638PRTColwellia psychroerythrusmisc_feature(186)..(186)The 'Xaa' at location 186 stands for Gly, or Arg. 20Met Gly Lys Ile Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser Cys Val 1 5 10 15 Ala Val Leu Asp Gly Asp Ser Val Arg Val Ile Glu Asn Ala Glu Gly 20 25 30 Asp Arg Thr Thr Pro Ser Ile Ile Gly Tyr Thr Ala Glu Gly Glu Thr 35 40 45 Leu Val Gly Gln Pro Ala Lys Arg Gln Ser Val Thr Asn Pro Glu Asn 50 55 60 Thr Leu Tyr Ala Ile Lys Arg Leu Ile Gly Arg Arg Phe Glu Asp Lys 65 70 75 80 Glu Thr Gln Arg Asp Ile Asp Ile Met Pro Phe Gly Ile Val Lys Ala 85 90 95 Asp Asn Gly Asp Ala Trp Val Gln Val Lys Gly Glu Lys Ile Ala Pro 100 105 110 Pro Gln Val Ser Ala Glu Val Leu Lys Lys Met Lys Lys Thr Ala Glu 115 120 125 Asp Phe Leu Gly Glu Thr Val Thr Glu Ala Val Ile Thr Val Pro Ala 130 135 140 Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp Ala Gly Arg Ile 145 150 155 160 Ala Gly Leu Glu Val Lys Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala 165 170 175 Leu Ala Tyr Gly Met Asp Lys Gln Glu Xaa Asp Lys Val Val Ala Val 180 185 190 Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Ile Glu Ile Asp 195 200 205 Glu Met Asp Gly Glu His Thr Phe Glu Val Leu Ala Thr Asn Gly Asp 210 215 220 Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Leu Ile Asn Tyr Leu 225 230 235 240 Val Ala Glu Phe Lys Lys Asp Gln Gly Met Asp Leu Thr Ser Asp Pro 245 250 255 Leu Ala Met Gln Arg Leu Lys Glu Ala Ala Glu Lys Ala Lys Cys Glu 260 265 270 Leu Ser Ser Ala Gln Gln Thr Asp Val Asn Leu Pro Tyr Ile Thr Ala 275 280 285 Asp Gly Ser Gly Pro Lys His Met Asn Ile Lys Val Thr Arg Ala Lys 290 295 300 Leu Glu Ser Leu Val Glu Asp Met Val Lys Ala Thr Leu Glu Pro Leu 305 310 315 320 Lys Gln Ala Leu Lys Asp Ala Asp Leu Ser Val Ser Lys Ile Asp Asp 325 330 335 Val Ile Leu Val Gly Gly Gln Ser Arg Met Pro Leu Val Gln Lys Thr 340 345 350 Val Thr Asp Phe Phe Gly Lys Glu Pro Arg Lys Asp Val Asn Pro Asp 355 360 365 Glu Ala Val Ala Ser Gly Ala Ala Ile Gln Ala Gly Val Leu Ser Gly 370 375 380 Asp Val Thr Asp Val Leu Leu Leu Asp Val Thr Pro Leu Ser Leu Gly 385 390 395 400 Ile Glu Thr Met Gly Gly Val Met Thr Lys Val Ile Asp Lys Asn Thr 405 410 415 Thr Ile Pro Thr Lys Gln Ser Gln Thr Phe Ser Thr Ala Asp Asp Asn 420 425 430 Gln Ala Ala Val Thr Val His Val Cys Gln Gly Glu Arg Lys Gln Ala 435 440 445 Ser Ala Asn Lys Ser Leu Gly Gln Phe Asn Leu Glu Gly Ile Glu Pro 450 455 460 Ala Gln Arg Gly Thr Pro Gln Ile Glu Val Thr Phe Asp Ile Asp Ala 465 470 475 480 Asp Gly Ile Leu His Val Thr Ala Lys Asp Lys Asn Thr Gly Lys Glu 485 490 495 Gln Lys Ile Thr Ile Lys Ala Ser Ser Gly Leu Ser Asp Glu Glu Val 500 505 510 Glu Gln Met Val Arg Asp Ala Glu Ala Asn Ala Asp Ala Asp Ala Lys 515 520 525 Phe Glu Glu Leu Val Thr Ala Arg Asn Gln Ala Asp Gly Met Ile His 530 535 540 Ala Thr Arg Lys Gln Val Glu Glu Ala Gly Glu Glu Leu Pro Ser Glu 545 550 555 560 Asp Lys Glu Lys Ile Glu Ala Ala Leu Thr Glu Leu Glu Glu Ala Val 565 570 575 Lys Gly Asp Asp Lys Glu Val Ile Glu Ala Lys Thr Gln Ala Leu Met 580 585 590 Glu Ala Ser Ala Lys Leu Met Glu Ile Ala Gln Ala Lys Glu Gln Ala 595 600 605 Gln Ser Ala Pro Glu Gly Ala Gln Glu Ala Asp Ala Ala Pro Ala Asp 610 615 620 Asp Val Val Asp Ala Glu Phe Glu Glu Val Lys Asp Lys Lys 625 630 635 211220DNAArtificial SequencetyrS hybrid 21tataaatata atg tcg agc ttt aac caa gca ttc gcc gaa cta aaa cgc 49 Met Ser Ser Phe Asn Gln Ala Phe Ala Glu Leu Lys Arg 1 5 10 gga gca gaa gaa ata tta gta gaa gaa gaa tta tta aca aag ctt aag 97Gly Ala Glu Glu Ile Leu Val Glu Glu Glu Leu Leu Thr Lys Leu Lys 15 20 25 aca ggt aag ccg cta aaa atc aaa gcg ggt ttt gat cct act gcg cct 145Thr Gly Lys Pro Leu Lys Ile Lys Ala Gly Phe Asp Pro Thr Ala Pro 30 35 40 45 gac tta cat tta ggc cac acg gta tta att aac aag ctt cgt caa ttc 193Asp Leu His Leu Gly His Thr Val Leu Ile Asn Lys Leu Arg Gln Phe 50 55 60 caa caa tta ggt cat gaa gtt att ttc ttg att ggt gac ttc acc gga 241Gln Gln Leu Gly His Glu Val Ile Phe Leu Ile Gly Asp Phe Thr Gly 65 70 75 atg att ggt gat cca acg ggt aaa aat gtg acg cgt aag gca ctc act 289Met Ile Gly Asp Pro Thr Gly Lys Asn Val Thr Arg Lys Ala Leu Thr 80 85 90 aaa gaa gac gta tta gcc aat gct gaa acg tat aaa gag caa gtc ttt 337Lys Glu Asp Val Leu Ala Asn Ala Glu Thr Tyr Lys Glu Gln Val Phe 95 100 105 aaa ata tta gat ccc gct aaa aca acc gtt gcc ttt aac tct act tgg 385Lys Ile Leu Asp Pro Ala Lys Thr Thr Val Ala Phe Asn Ser Thr Trp 110 115 120 125 atg gat aaa tta ggc gcg gca ggt atg tta caa ctt gcc tct cgt caa 433Met Asp Lys Leu Gly Ala Ala Gly Met Leu Gln Leu Ala Ser Arg Gln 130 135 140 acg gtt gcc cgt atg atg gag cgt gac gac ttt aaa aaa cgt tat gct 481Thr Val Ala Arg Met Met Glu Arg Asp Asp Phe Lys Lys Arg Tyr Ala 145 150 155 aac ggc cag gcc att gct att cat gag ttt atg tac cct tta gta caa 529Asn Gly Gln Ala Ile Ala Ile His Glu Phe Met Tyr Pro Leu Val Gln 160 165 170 ggt tgg gat tca gtt gcg ctt gag gct gat gtt gag ctg ggt ggt acc 577Gly Trp Asp Ser Val Ala Leu Glu Ala Asp Val Glu Leu Gly Gly Thr 175 180 185 gac caa aag ttt aat tta tta atg ggt cgt gag tta caa aaa tct gaa 625Asp Gln Lys Phe Asn Leu Leu Met Gly Arg Glu Leu Gln Lys Ser Glu 190 195 200 205 ggc cag cgt cca caa aca gta tta atg atg cca tta ctt gaa ggc cta 673Gly Gln Arg Pro Gln Thr Val Leu Met Met Pro Leu Leu Glu Gly Leu 210 215 220 gat ggc gtt cag aaa atg tct aag tca tta ggc aac tac att ggc att 721Asp Gly Val Gln Lys Met Ser Lys Ser Leu Gly Asn Tyr Ile Gly Ile 225 230 235 act gat acg cct acc gac atg ttt ggc aaa ata atg tca att tca gat 769Thr Asp Thr Pro Thr Asp Met Phe Gly Lys Ile Met Ser Ile Ser Asp 240 245 250 gta tta atg tgg cgt tac tac gag tta ctt agc ttt aaa ccg ctt gaa 817Val Leu Met Trp Arg Tyr Tyr Glu Leu Leu Ser Phe Lys Pro Leu Glu 255 260 265 gaa att gaa ggt tat aaa acc gag ata gaa aat ggc aaa aat cct cgt 865Glu Ile Glu Gly Tyr Lys Thr Glu Ile Glu Asn Gly Lys Asn Pro Arg 270 275 280 285 gat gtt aaa att gat tta gcc aaa gaa ttg att gct cgt ttt cat gat 913Asp Val Lys Ile Asp Leu Ala Lys Glu Leu Ile Ala Arg Phe His Asp 290 295 300 gaa gct gct gca caa gct gcc cat gat gaa ttc atc aat cgt ttc caa 961Glu Ala Ala Ala Gln Ala Ala His Asp Glu Phe Ile Asn Arg Phe Gln 305 310 315 aaa ggt gcg tta cct gat gat atg ccg gaa tta acg att acc act gaa 1009Lys Gly Ala Leu Pro Asp Asp Met Pro Glu Leu Thr Ile Thr Thr Glu 320 325 330 aat ggt gaa ata gcc att gct aac ttg ctt aaa gat gca gga tta gtc 1057Asn Gly Glu Ile Ala Ile Ala Asn Leu Leu Lys Asp Ala Gly Leu Val 335 340 345 ggt agt act tct gat gcc ttt aga atg atc aaa caa ggg gcg gct aaa 1105Gly Ser Thr Ser Asp Ala Phe Arg Met Ile Lys Gln Gly Ala Ala Lys 350 355 360 365

att gat agc gaa aaa gta act gac cgt agc tta gtt att agc gct ggc 1153Ile Asp Ser Glu Lys Val Thr Asp Arg Ser Leu Val Ile Ser Ala Gly 370 375 380 acg acg gca gtt tat caa gtc ggc aaa cgt aaa ttt gct cgt att acc 1201Thr Thr Ala Val Tyr Gln Val Gly Lys Arg Lys Phe Ala Arg Ile Thr 385 390 395 ata aaa taaggggttg taa 1220Ile Lys 22399PRTArtificial SequenceSynthetic Construct 22Met Ser Ser Phe Asn Gln Ala Phe Ala Glu Leu Lys Arg Gly Ala Glu 1 5 10 15 Glu Ile Leu Val Glu Glu Glu Leu Leu Thr Lys Leu Lys Thr Gly Lys 20 25 30 Pro Leu Lys Ile Lys Ala Gly Phe Asp Pro Thr Ala Pro Asp Leu His 35 40 45 Leu Gly His Thr Val Leu Ile Asn Lys Leu Arg Gln Phe Gln Gln Leu 50 55 60 Gly His Glu Val Ile Phe Leu Ile Gly Asp Phe Thr Gly Met Ile Gly 65 70 75 80 Asp Pro Thr Gly Lys Asn Val Thr Arg Lys Ala Leu Thr Lys Glu Asp 85 90 95 Val Leu Ala Asn Ala Glu Thr Tyr Lys Glu Gln Val Phe Lys Ile Leu 100 105 110 Asp Pro Ala Lys Thr Thr Val Ala Phe Asn Ser Thr Trp Met Asp Lys 115 120 125 Leu Gly Ala Ala Gly Met Leu Gln Leu Ala Ser Arg Gln Thr Val Ala 130 135 140 Arg Met Met Glu Arg Asp Asp Phe Lys Lys Arg Tyr Ala Asn Gly Gln 145 150 155 160 Ala Ile Ala Ile His Glu Phe Met Tyr Pro Leu Val Gln Gly Trp Asp 165 170 175 Ser Val Ala Leu Glu Ala Asp Val Glu Leu Gly Gly Thr Asp Gln Lys 180 185 190 Phe Asn Leu Leu Met Gly Arg Glu Leu Gln Lys Ser Glu Gly Gln Arg 195 200 205 Pro Gln Thr Val Leu Met Met Pro Leu Leu Glu Gly Leu Asp Gly Val 210 215 220 Gln Lys Met Ser Lys Ser Leu Gly Asn Tyr Ile Gly Ile Thr Asp Thr 225 230 235 240 Pro Thr Asp Met Phe Gly Lys Ile Met Ser Ile Ser Asp Val Leu Met 245 250 255 Trp Arg Tyr Tyr Glu Leu Leu Ser Phe Lys Pro Leu Glu Glu Ile Glu 260 265 270 Gly Tyr Lys Thr Glu Ile Glu Asn Gly Lys Asn Pro Arg Asp Val Lys 275 280 285 Ile Asp Leu Ala Lys Glu Leu Ile Ala Arg Phe His Asp Glu Ala Ala 290 295 300 Ala Gln Ala Ala His Asp Glu Phe Ile Asn Arg Phe Gln Lys Gly Ala 305 310 315 320 Leu Pro Asp Asp Met Pro Glu Leu Thr Ile Thr Thr Glu Asn Gly Glu 325 330 335 Ile Ala Ile Ala Asn Leu Leu Lys Asp Ala Gly Leu Val Gly Ser Thr 340 345 350 Ser Asp Ala Phe Arg Met Ile Lys Gln Gly Ala Ala Lys Ile Asp Ser 355 360 365 Glu Lys Val Thr Asp Arg Ser Leu Val Ile Ser Ala Gly Thr Thr Ala 370 375 380 Val Tyr Gln Val Gly Lys Arg Lys Phe Ala Arg Ile Thr Ile Lys 385 390 395 23741DNAArtificial Sequencecmk hybrid 23aagtctagaa gatgtcgaaa aacttgtaca gtttatgcag gaatttgaga atg aac 56 Met Asn 1 aat agc aca cca gtt ata acc att gat ggc cca agt ggg gct ggt aaa 104Asn Ser Thr Pro Val Ile Thr Ile Asp Gly Pro Ser Gly Ala Gly Lys 5 10 15 gga acc gtt gca agg ata gtt gcg gac caa tta ggt tgg cac ctt ctt 152Gly Thr Val Ala Arg Ile Val Ala Asp Gln Leu Gly Trp His Leu Leu 20 25 30 gac agt ggg gct att tac cgc gtc tta gct gtt gcc att caa cat cac 200Asp Ser Gly Ala Ile Tyr Arg Val Leu Ala Val Ala Ile Gln His His 35 40 45 50 caa ctt tca tta gat gat gaa gag cct ctt atc cct atg gct gca cat 248Gln Leu Ser Leu Asp Asp Glu Glu Pro Leu Ile Pro Met Ala Ala His 55 60 65 tta gat gtt caa ttt gaa att aat agt caa ggt gaa gct aaa gtt att 296Leu Asp Val Gln Phe Glu Ile Asn Ser Gln Gly Glu Ala Lys Val Ile 70 75 80 tta gaa ggt gaa aat gtt act gaa att att cgt act gaa gaa gtt ggc 344Leu Glu Gly Glu Asn Val Thr Glu Ile Ile Arg Thr Glu Glu Val Gly 85 90 95 gga tta gca tcg aaa gta gca gca ttt cca cgt gtt aga gaa gcg cta 392Gly Leu Ala Ser Lys Val Ala Ala Phe Pro Arg Val Arg Glu Ala Leu 100 105 110 tta cga aga caa cgt gca ttt agc gtt agc cct ggc tta att gca gat 440Leu Arg Arg Gln Arg Ala Phe Ser Val Ser Pro Gly Leu Ile Ala Asp 115 120 125 130 ggt cgc gac atg gga acc gtt gtt ttt ccg aaa gct cca gta aaa ata 488Gly Arg Asp Met Gly Thr Val Val Phe Pro Lys Ala Pro Val Lys Ile 135 140 145 ttt tta act gct agt gct gaa gaa cga gct gac cga aga ttt aat cag 536Phe Leu Thr Ala Ser Ala Glu Glu Arg Ala Asp Arg Arg Phe Asn Gln 150 155 160 ttg aaa gaa aaa gga att gat gtt aac atc ggg cgc ctt ttg gat gac 584Leu Lys Glu Lys Gly Ile Asp Val Asn Ile Gly Arg Leu Leu Asp Asp 165 170 175 ata cgt caa cga gat gag cga gat caa aac cgc aag gta gct cct ctt 632Ile Arg Gln Arg Asp Glu Arg Asp Gln Asn Arg Lys Val Ala Pro Leu 180 185 190 atc ccg gca gaa gga gcg tta act att gat tct act gat att tct att 680Ile Pro Ala Glu Gly Ala Leu Thr Ile Asp Ser Thr Asp Ile Ser Ile 195 200 205 210 aca gaa gtc gtc aat aaa atc ctt atg ttt gcc aat ggc aaa tta acg 728Thr Glu Val Val Asn Lys Ile Leu Met Phe Ala Asn Gly Lys Leu Thr 215 220 225 tag atattttagc 74124226PRTArtificial SequenceSynthetic Construct 24Met Asn Asn Ser Thr Pro Val Ile Thr Ile Asp Gly Pro Ser Gly Ala 1 5 10 15 Gly Lys Gly Thr Val Ala Arg Ile Val Ala Asp Gln Leu Gly Trp His 20 25 30 Leu Leu Asp Ser Gly Ala Ile Tyr Arg Val Leu Ala Val Ala Ile Gln 35 40 45 His His Gln Leu Ser Leu Asp Asp Glu Glu Pro Leu Ile Pro Met Ala 50 55 60 Ala His Leu Asp Val Gln Phe Glu Ile Asn Ser Gln Gly Glu Ala Lys 65 70 75 80 Val Ile Leu Glu Gly Glu Asn Val Thr Glu Ile Ile Arg Thr Glu Glu 85 90 95 Val Gly Gly Leu Ala Ser Lys Val Ala Ala Phe Pro Arg Val Arg Glu 100 105 110 Ala Leu Leu Arg Arg Gln Arg Ala Phe Ser Val Ser Pro Gly Leu Ile 115 120 125 Ala Asp Gly Arg Asp Met Gly Thr Val Val Phe Pro Lys Ala Pro Val 130 135 140 Lys Ile Phe Leu Thr Ala Ser Ala Glu Glu Arg Ala Asp Arg Arg Phe 145 150 155 160 Asn Gln Leu Lys Glu Lys Gly Ile Asp Val Asn Ile Gly Arg Leu Leu 165 170 175 Asp Asp Ile Arg Gln Arg Asp Glu Arg Asp Gln Asn Arg Lys Val Ala 180 185 190 Pro Leu Ile Pro Ala Glu Gly Ala Leu Thr Ile Asp Ser Thr Asp Ile 195 200 205 Ser Ile Thr Glu Val Val Asn Lys Ile Leu Met Phe Ala Asn Gly Lys 210 215 220 Leu Thr 225 251937DNAArtificial SequencednaKsf hybrid 25ggagaatcaa atg gga aaa att att ggt atc gat tta ggc aca aca aac 49 Met Gly Lys Ile Ile Gly Ile Asp Leu Gly Thr Thr Asn 1 5 10 tcg tgt gta gca gtc ctt gat ggc ggc aaa gca cgc gta att gaa aac 97Ser Cys Val Ala Val Leu Asp Gly Gly Lys Ala Arg Val Ile Glu Asn 15 20 25 gca gag ggt gat cgc aca acc cca tca att atc gct tat acc gat gat 145Ala Glu Gly Asp Arg Thr Thr Pro Ser Ile Ile Ala Tyr Thr Asp Asp 30 35 40 45 gaa att att gta ggc cag cca gca aag cgt cag gct gta acc aac cca 193Glu Ile Ile Val Gly Gln Pro Ala Lys Arg Gln Ala Val Thr Asn Pro 50 55 60 aca aac aca ttc ttt gcc atc aag cgt tta atc ggt cgt cgt ttt aaa 241Thr Asn Thr Phe Phe Ala Ile Lys Arg Leu Ile Gly Arg Arg Phe Lys 65 70 75 gat gac gaa gtt caa cgt gat gtg aac atc atg cca ttc aaa att atc 289Asp Asp Glu Val Gln Arg Asp Val Asn Ile Met Pro Phe Lys Ile Ile 80 85 90 gca gct gat aat ggt gat gca tgg gtt gag tca cgt ggt aac aaa atg 337Ala Ala Asp Asn Gly Asp Ala Trp Val Glu Ser Arg Gly Asn Lys Met 95 100 105 gca cca cca caa gtt tca gct gaa atc ttg aaa aag atg aaa aag act 385Ala Pro Pro Gln Val Ser Ala Glu Ile Leu Lys Lys Met Lys Lys Thr 110 115 120 125 gct gaa gac ttt tta ggt gaa gaa gtg act gaa gcg gtt att acc gtt 433Ala Glu Asp Phe Leu Gly Glu Glu Val Thr Glu Ala Val Ile Thr Val 130 135 140 cct gct tac ttt aac gat tca caa cgt caa gcc act aaa gat gct ggt 481Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp Ala Gly 145 150 155 cgt atc gca ggt ctt gat gtt aag cgt att atc aac gaa cct act gct 529Arg Ile Ala Gly Leu Asp Val Lys Arg Ile Ile Asn Glu Pro Thr Ala 160 165 170 gct gca ctt gca tac ggt atc gac aag aaa caa ggc gac aac att gtt 577Ala Ala Leu Ala Tyr Gly Ile Asp Lys Lys Gln Gly Asp Asn Ile Val 175 180 185 gct gta tac gat tta ggt ggt ggt aca ttc gat atc tct atc atc gaa 625Ala Val Tyr Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Ile Glu 190 195 200 205 att gac agc aac gat ggt gac caa aca ttt gaa gta cta gca acc aat 673Ile Asp Ser Asn Asp Gly Asp Gln Thr Phe Glu Val Leu Ala Thr Asn 210 215 220 ggt gat act cac tta ggt ggt gaa gac ttt gat aac cgt atg att aac 721Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Met Ile Asn 225 230 235 tat tta gct gat gaa ttc aaa aaa gac caa ggc tta gat ctt cgt aga 769Tyr Leu Ala Asp Glu Phe Lys Lys Asp Gln Gly Leu Asp Leu Arg Arg 240 245 250 gat cct tta gca atg caa cgt ttg aaa gaa gcc gct gaa aaa gca aaa 817Asp Pro Leu Ala Met Gln Arg Leu Lys Glu Ala Ala Glu Lys Ala Lys 255 260 265 atc gag ctt tca agc act aac cac act gaa gtt aac ttg cct tac atc 865Ile Glu Leu Ser Ser Thr Asn His Thr Glu Val Asn Leu Pro Tyr Ile 270 275 280 285 act gct gat gca tca ggt cct aag cat tta gtg gtt aaa att act cgt 913Thr Ala Asp Ala Ser Gly Pro Lys His Leu Val Val Lys Ile Thr Arg 290 295 300 gct aag tta gag tca tta gtt gaa gat tta att caa cgt act cta gag 961Ala Lys Leu Glu Ser Leu Val Glu Asp Leu Ile Gln Arg Thr Leu Glu 305 310 315 ccg ctt aaa gtt gca cta gct gat gct gat tta tca ata tca gat atc 1009Pro Leu Lys Val Ala Leu Ala Asp Ala Asp Leu Ser Ile Ser Asp Ile 320 325 330 aat gaa gtg att ctt gtg ggt ggt cag act cgt atg cct aaa gta caa 1057Asn Glu Val Ile Leu Val Gly Gly Gln Thr Arg Met Pro Lys Val Gln 335 340 345 gaa gca gtc act aac ttc ttt ggc aaa gag cct cgt aaa gat gtt aac 1105Glu Ala Val Thr Asn Phe Phe Gly Lys Glu Pro Arg Lys Asp Val Asn 350 355 360 365 cct gat gaa gcg gtt gct gtt ggt gcg gcg att cag gct ggc gta ctt 1153Pro Asp Glu Ala Val Ala Val Gly Ala Ala Ile Gln Ala Gly Val Leu 370 375 380 tct ggt gaa gtg aaa gac gta ctt cta ctt gac gtt acc cca cta tct 1201Ser Gly Glu Val Lys Asp Val Leu Leu Leu Asp Val Thr Pro Leu Ser 385 390 395 ctt ggt att gaa acc atg ggc agt gtg atg aca aag ctt atc gag aag 1249Leu Gly Ile Glu Thr Met Gly Ser Val Met Thr Lys Leu Ile Glu Lys 400 405 410 aac acc act atc ccg act aaa gct cag caa gta ttc tca aca gct gac 1297Asn Thr Thr Ile Pro Thr Lys Ala Gln Gln Val Phe Ser Thr Ala Asp 415 420 425 gac aac caa agt gcc gtg act att cac gta ctt caa ggt gaa cgt aag 1345Asp Asn Gln Ser Ala Val Thr Ile His Val Leu Gln Gly Glu Arg Lys 430 435 440 445 caa gcg agt gct aac aag tca tta ggt caa ttt aac ctt gaa ggt att 1393Gln Ala Ser Ala Asn Lys Ser Leu Gly Gln Phe Asn Leu Glu Gly Ile 450 455 460 gag cca gca cca cgt ggc caa cca cag gtt gaa gtg atg ttc gac att 1441Glu Pro Ala Pro Arg Gly Gln Pro Gln Val Glu Val Met Phe Asp Ile 465 470 475 gat gct gat ggt atc tta cat gtg tct gca aca gac aag aaa aca ggt 1489Asp Ala Asp Gly Ile Leu His Val Ser Ala Thr Asp Lys Lys Thr Gly 480 485 490 aag aaa caa aac att act atc aaa gcc tct tca ggt tta tct gat gaa 1537Lys Lys Gln Asn Ile Thr Ile Lys Ala Ser Ser Gly Leu Ser Asp Glu 495 500 505 gaa gtt gaa caa atg gta cgt gat gca gaa gct cat gct gat gaa gat 1585Glu Val Glu Gln Met Val Arg Asp Ala Glu Ala His Ala Asp Glu Asp 510 515 520 525 gct aaa ttt gaa gag tta gtt aaa gcg cgt aat caa gca gat ggt tta 1633Ala Lys Phe Glu Glu Leu Val Lys Ala Arg Asn Gln Ala Asp Gly Leu 530 535 540 gct cat tca act aaa aaa caa gtt gaa gaa gct ggc gat gca cta gct 1681Ala His Ser Thr Lys Lys Gln Val Glu Glu Ala Gly Asp Ala Leu Ala 545 550 555 agt gac gaa aaa gaa aag att gaa gca gca atc gca act tta gaa act 1729Ser Asp Glu Lys Glu Lys Ile Glu Ala Ala Ile Ala Thr Leu Glu Thr 560 565 570 gcc ata aaa ggc aaa gat aaa gaa gcc att gat aca gca act caa gcg 1777Ala Ile Lys Gly Lys Asp Lys Glu Ala Ile Asp Thr Ala Thr Gln Ala 575 580 585 cta atc gaa gcg tct gct aag tta atg gaa att gct caa gct aaa gct 1825Leu Ile Glu Ala Ser Ala Lys Leu Met Glu Ile Ala Gln Ala Lys Ala 590 595 600 605 caa ggt gaa gca gaa ggt caa gcg cac gat gct ggc caa gaa aag cct 1873Gln Gly Glu Ala Glu Gly Gln Ala His Asp Ala Gly Gln Glu Lys Pro 610 615 620 gct gat gat gtt gtt gat gct gag ttc gaa gaa gtt aaa gac gac aaa 1921Ala Asp Asp Val Val Asp Ala Glu Phe Glu Glu Val Lys Asp Asp Lys 625 630 635 aaa taa ataatctttt 1937Lys 26638PRTArtificial SequenceSynthetic Construct 26Met Gly Lys Ile Ile Gly Ile Asp Leu Gly Thr Thr Asn Ser Cys Val 1 5 10 15 Ala Val Leu Asp Gly Gly Lys Ala Arg Val Ile Glu Asn Ala Glu Gly 20 25 30 Asp Arg Thr Thr Pro Ser Ile Ile Ala Tyr Thr Asp Asp Glu Ile

Ile 35 40 45 Val Gly Gln Pro Ala Lys Arg Gln Ala Val Thr Asn Pro Thr Asn Thr 50 55 60 Phe Phe Ala Ile Lys Arg Leu Ile Gly Arg Arg Phe Lys Asp Asp Glu 65 70 75 80 Val Gln Arg Asp Val Asn Ile Met Pro Phe Lys Ile Ile Ala Ala Asp 85 90 95 Asn Gly Asp Ala Trp Val Glu Ser Arg Gly Asn Lys Met Ala Pro Pro 100 105 110 Gln Val Ser Ala Glu Ile Leu Lys Lys Met Lys Lys Thr Ala Glu Asp 115 120 125 Phe Leu Gly Glu Glu Val Thr Glu Ala Val Ile Thr Val Pro Ala Tyr 130 135 140 Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp Ala Gly Arg Ile Ala 145 150 155 160 Gly Leu Asp Val Lys Arg Ile Ile Asn Glu Pro Thr Ala Ala Ala Leu 165 170 175 Ala Tyr Gly Ile Asp Lys Lys Gln Gly Asp Asn Ile Val Ala Val Tyr 180 185 190 Asp Leu Gly Gly Gly Thr Phe Asp Ile Ser Ile Ile Glu Ile Asp Ser 195 200 205 Asn Asp Gly Asp Gln Thr Phe Glu Val Leu Ala Thr Asn Gly Asp Thr 210 215 220 His Leu Gly Gly Glu Asp Phe Asp Asn Arg Met Ile Asn Tyr Leu Ala 225 230 235 240 Asp Glu Phe Lys Lys Asp Gln Gly Leu Asp Leu Arg Arg Asp Pro Leu 245 250 255 Ala Met Gln Arg Leu Lys Glu Ala Ala Glu Lys Ala Lys Ile Glu Leu 260 265 270 Ser Ser Thr Asn His Thr Glu Val Asn Leu Pro Tyr Ile Thr Ala Asp 275 280 285 Ala Ser Gly Pro Lys His Leu Val Val Lys Ile Thr Arg Ala Lys Leu 290 295 300 Glu Ser Leu Val Glu Asp Leu Ile Gln Arg Thr Leu Glu Pro Leu Lys 305 310 315 320 Val Ala Leu Ala Asp Ala Asp Leu Ser Ile Ser Asp Ile Asn Glu Val 325 330 335 Ile Leu Val Gly Gly Gln Thr Arg Met Pro Lys Val Gln Glu Ala Val 340 345 350 Thr Asn Phe Phe Gly Lys Glu Pro Arg Lys Asp Val Asn Pro Asp Glu 355 360 365 Ala Val Ala Val Gly Ala Ala Ile Gln Ala Gly Val Leu Ser Gly Glu 370 375 380 Val Lys Asp Val Leu Leu Leu Asp Val Thr Pro Leu Ser Leu Gly Ile 385 390 395 400 Glu Thr Met Gly Ser Val Met Thr Lys Leu Ile Glu Lys Asn Thr Thr 405 410 415 Ile Pro Thr Lys Ala Gln Gln Val Phe Ser Thr Ala Asp Asp Asn Gln 420 425 430 Ser Ala Val Thr Ile His Val Leu Gln Gly Glu Arg Lys Gln Ala Ser 435 440 445 Ala Asn Lys Ser Leu Gly Gln Phe Asn Leu Glu Gly Ile Glu Pro Ala 450 455 460 Pro Arg Gly Gln Pro Gln Val Glu Val Met Phe Asp Ile Asp Ala Asp 465 470 475 480 Gly Ile Leu His Val Ser Ala Thr Asp Lys Lys Thr Gly Lys Lys Gln 485 490 495 Asn Ile Thr Ile Lys Ala Ser Ser Gly Leu Ser Asp Glu Glu Val Glu 500 505 510 Gln Met Val Arg Asp Ala Glu Ala His Ala Asp Glu Asp Ala Lys Phe 515 520 525 Glu Glu Leu Val Lys Ala Arg Asn Gln Ala Asp Gly Leu Ala His Ser 530 535 540 Thr Lys Lys Gln Val Glu Glu Ala Gly Asp Ala Leu Ala Ser Asp Glu 545 550 555 560 Lys Glu Lys Ile Glu Ala Ala Ile Ala Thr Leu Glu Thr Ala Ile Lys 565 570 575 Gly Lys Asp Lys Glu Ala Ile Asp Thr Ala Thr Gln Ala Leu Ile Glu 580 585 590 Ala Ser Ala Lys Leu Met Glu Ile Ala Gln Ala Lys Ala Gln Gly Glu 595 600 605 Ala Glu Gly Gln Ala His Asp Ala Gly Gln Glu Lys Pro Ala Asp Asp 610 615 620 Val Val Asp Ala Glu Phe Glu Glu Val Lys Asp Asp Lys Lys 625 630 635 271175DNAArtificial SequenceftsZ hybrid 27atg ttt gat ttt aac gat tca atg gtt tca aat gcc ata att aaa gtt 48Met Phe Asp Phe Asn Asp Ser Met Val Ser Asn Ala Ile Ile Lys Val 1 5 10 15 gtc ggt gtt ggt ggc ggt ggc ggt aat gct gta caa cat atg tgt gaa 96Val Gly Val Gly Gly Gly Gly Gly Asn Ala Val Gln His Met Cys Glu 20 25 30 gaa gtt tct gat gtt gag ttt ttt gcc cta aat aca gat ggt caa gca 144Glu Val Ser Asp Val Glu Phe Phe Ala Leu Asn Thr Asp Gly Gln Ala 35 40 45 tta tca aaa tca aaa gtt caa aat ata tta caa att ggt aca aac cta 192Leu Ser Lys Ser Lys Val Gln Asn Ile Leu Gln Ile Gly Thr Asn Leu 50 55 60 aca aaa ggt tta ggt gct ggt gcg aat cct gaa att ggt aag aga gct 240Thr Lys Gly Leu Gly Ala Gly Ala Asn Pro Glu Ile Gly Lys Arg Ala 65 70 75 80 gca act gaa gat aga gcg aaa atc gag caa ctt tta gag ggt gct gat 288Ala Thr Glu Asp Arg Ala Lys Ile Glu Gln Leu Leu Glu Gly Ala Asp 85 90 95 atg gtt ttc atc act gct ggt atg ggt ggt ggt aca ggt aca ggt gga 336Met Val Phe Ile Thr Ala Gly Met Gly Gly Gly Thr Gly Thr Gly Gly 100 105 110 gct cct gta gtt gca gaa gtt gca aaa gag atg ggt ata ctt aca gta 384Ala Pro Val Val Ala Glu Val Ala Lys Glu Met Gly Ile Leu Thr Val 115 120 125 gct gta gtt act aag cct ttc cct ttt gaa gga cca aga aga atg aaa 432Ala Val Val Thr Lys Pro Phe Pro Phe Glu Gly Pro Arg Arg Met Lys 130 135 140 gca gca gag caa ggt att gag ttt tta tct aaa agt gtt gat tca ctg 480Ala Ala Glu Gln Gly Ile Glu Phe Leu Ser Lys Ser Val Asp Ser Leu 145 150 155 160 att act att cct aac gaa aag tta ctg aaa gta ctt ggc cct gga aca 528Ile Thr Ile Pro Asn Glu Lys Leu Leu Lys Val Leu Gly Pro Gly Thr 165 170 175 agc tta tta gat gcc ttt aaa gca gca aat aac gtg cta ctt ggc gcc 576Ser Leu Leu Asp Ala Phe Lys Ala Ala Asn Asn Val Leu Leu Gly Ala 180 185 190 gtt cag ggt att gca gaa tta att act cgt cct ggt ttg ata aat gtc 624Val Gln Gly Ile Ala Glu Leu Ile Thr Arg Pro Gly Leu Ile Asn Val 195 200 205 gat ttt gct gat gta cgt acc gtt atg tct gag atg ggt act gcc atg 672Asp Phe Ala Asp Val Arg Thr Val Met Ser Glu Met Gly Thr Ala Met 210 215 220 atg ggt tct ggt act gct tct ggc gat gat aga gca caa gaa gct gct 720Met Gly Ser Gly Thr Ala Ser Gly Asp Asp Arg Ala Gln Glu Ala Ala 225 230 235 240 gat gct gct att tca agt cct tta tta gag gat gtg gat tta gct ggt 768Asp Ala Ala Ile Ser Ser Pro Leu Leu Glu Asp Val Asp Leu Ala Gly 245 250 255 gca cgc ggg atc tta gtt aat att acc gca ggt atg gat att agt atc 816Ala Arg Gly Ile Leu Val Asn Ile Thr Ala Gly Met Asp Ile Ser Ile 260 265 270 gat gag ttt gaa act gtt ggt aat gcc gtt aaa gct ttc gct tct gaa 864Asp Glu Phe Glu Thr Val Gly Asn Ala Val Lys Ala Phe Ala Ser Glu 275 280 285 aat gcg act gtt gtt gtt ggt gct gtt att gat atg gat atg aca gat 912Asn Ala Thr Val Val Val Gly Ala Val Ile Asp Met Asp Met Thr Asp 290 295 300 gag ctt cgt gtg act gtt gtt gct acg ggt att ggc gct gaa agt aag 960Glu Leu Arg Val Thr Val Val Ala Thr Gly Ile Gly Ala Glu Ser Lys 305 310 315 320 cct gat att acg tta gta aat cct atg cca atg gct gaa gca aaa gtt 1008Pro Asp Ile Thr Leu Val Asn Pro Met Pro Met Ala Glu Ala Lys Val 325 330 335 gtc ggt ggg gat tat aca cca gct gca cca cag gca aat tta gcg act 1056Val Gly Gly Asp Tyr Thr Pro Ala Ala Pro Gln Ala Asn Leu Ala Thr 340 345 350 gaa gca ata gct atg act gat agc aat gcg cag aaa gca gca gca acc 1104Glu Ala Ile Ala Met Thr Asp Ser Asn Ala Gln Lys Ala Ala Ala Thr 355 360 365 gac tta gat act tat tta gat att cct gct ttt tta cgt aag caa gcg 1152Asp Leu Asp Thr Tyr Leu Asp Ile Pro Ala Phe Leu Arg Lys Gln Ala 370 375 380 gat taataaaaac caaaattaag 1175Asp 385 28385PRTArtificial SequenceSynthetic Construct 28Met Phe Asp Phe Asn Asp Ser Met Val Ser Asn Ala Ile Ile Lys Val 1 5 10 15 Val Gly Val Gly Gly Gly Gly Gly Asn Ala Val Gln His Met Cys Glu 20 25 30 Glu Val Ser Asp Val Glu Phe Phe Ala Leu Asn Thr Asp Gly Gln Ala 35 40 45 Leu Ser Lys Ser Lys Val Gln Asn Ile Leu Gln Ile Gly Thr Asn Leu 50 55 60 Thr Lys Gly Leu Gly Ala Gly Ala Asn Pro Glu Ile Gly Lys Arg Ala 65 70 75 80 Ala Thr Glu Asp Arg Ala Lys Ile Glu Gln Leu Leu Glu Gly Ala Asp 85 90 95 Met Val Phe Ile Thr Ala Gly Met Gly Gly Gly Thr Gly Thr Gly Gly 100 105 110 Ala Pro Val Val Ala Glu Val Ala Lys Glu Met Gly Ile Leu Thr Val 115 120 125 Ala Val Val Thr Lys Pro Phe Pro Phe Glu Gly Pro Arg Arg Met Lys 130 135 140 Ala Ala Glu Gln Gly Ile Glu Phe Leu Ser Lys Ser Val Asp Ser Leu 145 150 155 160 Ile Thr Ile Pro Asn Glu Lys Leu Leu Lys Val Leu Gly Pro Gly Thr 165 170 175 Ser Leu Leu Asp Ala Phe Lys Ala Ala Asn Asn Val Leu Leu Gly Ala 180 185 190 Val Gln Gly Ile Ala Glu Leu Ile Thr Arg Pro Gly Leu Ile Asn Val 195 200 205 Asp Phe Ala Asp Val Arg Thr Val Met Ser Glu Met Gly Thr Ala Met 210 215 220 Met Gly Ser Gly Thr Ala Ser Gly Asp Asp Arg Ala Gln Glu Ala Ala 225 230 235 240 Asp Ala Ala Ile Ser Ser Pro Leu Leu Glu Asp Val Asp Leu Ala Gly 245 250 255 Ala Arg Gly Ile Leu Val Asn Ile Thr Ala Gly Met Asp Ile Ser Ile 260 265 270 Asp Glu Phe Glu Thr Val Gly Asn Ala Val Lys Ala Phe Ala Ser Glu 275 280 285 Asn Ala Thr Val Val Val Gly Ala Val Ile Asp Met Asp Met Thr Asp 290 295 300 Glu Leu Arg Val Thr Val Val Ala Thr Gly Ile Gly Ala Glu Ser Lys 305 310 315 320 Pro Asp Ile Thr Leu Val Asn Pro Met Pro Met Ala Glu Ala Lys Val 325 330 335 Val Gly Gly Asp Tyr Thr Pro Ala Ala Pro Gln Ala Asn Leu Ala Thr 340 345 350 Glu Ala Ile Ala Met Thr Asp Ser Asn Ala Gln Lys Ala Ala Ala Thr 355 360 365 Asp Leu Asp Thr Tyr Leu Asp Ile Pro Ala Phe Leu Arg Lys Gln Ala 370 375 380 Asp 385

Claims

1. A method of stimulating an immune response in an animal, comprising: administering to the animal a therapeutically effective amount of a recombinant mesophilic bacterium that is temperature-sensitive (TS), wherein the recombinant mesophilic bacterium that is TS is made by a method comprising: introducing into the genome of a mesophilic bacterium a nucleic acid construct comprising a TS essential nucleic acid molecule from a psychrophilic bacterium, and functionally replacing the mesophilic bacterium's homolog of the TS essential nucleic acid molecule, thereby making the recombinant mesophilic bacterium that is TS, wherein a protein encoded by the TS essential nucleic acid molecule is operable at a temperature less than 30.degree. C. and inoperable at a temperature greater than 30.degree. C., and wherein the recombinant mesophilic bacterium that is TS has a restrictive temperature between 33.degree. C. and 44.degree. C., thereby stimulating the immune response in the animal.

2. The method of claim 1, wherein the psychrophilic bacterium is Colwellia sp., Pseudoalteromonas sp., or Shewanella sp.

3. The method of claim 1, wherein the recombinant mesophilic bacterium that is TS is Salmonella sp., Mycobacterium sp. or Escherichia sp.

4. The method of claim 1, wherein the TS essential nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, wherein expression of the TS essential nucleic acid molecule confers temperature sensitivity.

5. The method of claim 1, wherein the protein encoded by the TS essential nucleic acid molecule comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28.

6. The method of claim 1, wherein the nucleic acid construct comprising the TS essential nucleic acid molecule comprises a vector.

7. The method of claim 1, wherein the recombinant mesophilic bacterium that is TS is present in a composition comprising an adjuvant.

8. The method of claim 1, wherein the recombinant mesophilic bacterium that is TS is present in a composition comprising a pharmaceutically acceptable carrier.

9. The method of claim 1, wherein the animal is a bird.

10. The method of claim 1, wherein the animal is a mammal.

11. The method of claim 1, wherein at least 10.sup.5 CFU of the recombinant mesophilic bacterium that is TS are administered to the animal.

12. The method of claim 1, wherein administering comprises intramuscular injection, subcutaneous injection, oral administration, intranasal administration or inhalation.

13. The method of claim 1, wherein the immune response comprises a cytotoxic T lymphocyte (CTL) response.

14. The method of claim 1, wherein the immune response comprises a B cell response.

15. The method of claim 1, wherein the method treats a Francisella novicida, Mycobacterium tuberculosis, Salmonella enteritidis, or Escherichia coli infection in the animal.

16. The method of claim 1, wherein the method prevents a Francisella novicida, Mycobacterium tuberculosis, Salmonella enteritidis, or Escherichia coli infection in the animal.

17. The method of claim 1, wherein more than one type of recombinant mesophilic bacterium that is TS is administered to the animal.

18. The method of claim 1, further comprising: culturing the recombinant mesophilic bacterium that is TS at a temperature wherein a protein encoded by the TS essential nucleic acid molecule is operable, whereby the recombinant mesophilic bacterium that is TS produces a plurality of peptides; increasing the culturing temperature to a temperature at which a protein encoded by the TS nucleic acid molecule is inoperable; maintaining said culturing for a period of time sufficient to kill the recombinant mesophilic bacterium that is TS; and harvesting the killed recombinant mesophilic bacterium that is TS.

19. A method of stimulating an immune response in an animal, comprising: administering to the animal a therapeutically effective amount of a recombinant mesophilic bacterium that is temperature-sensitive (TS), wherein the recombinant mesophilic bacterium that is TS is made by a method comprising: introducing into the genome of a mesophilic bacterium by homologous recombination a nucleic acid construct comprising a TS essential nucleic acid molecule from a psychrophilic bacterium flanked on both sides by a nucleic acid molecule homologous to a region of the mesophilic bacterium genome where the TS essential nucleic acid molecule from the psychrophilic bacterium will be inserted into the mesophilic bacterium genome, and functionally replacing the mesophilic bacterium's homolog of the TS essential nucleic acid molecule, thereby making a recombinant mesophilic bacterium that is TS, wherein a protein encoded by the TS essential nucleic acid molecule is operable at a temperature less than 30.degree. C. and inoperable at a temperature greater than 30.degree. C., and wherein the recombinant mesophilic bacterium that is TS has a restrictive temperature between 33.degree. C. and 44.degree. C., thereby stimulating the immune response in the animal.

20. A method of stimulating an immune response in an animal, comprising: administering to the animal a therapeutically effective amount of a recombinant mesophilic bacterium that is temperature-sensitive (TS), wherein the recombinant mesophilic bacterium that is TS is made by a method comprising: introducing into the genome of a mesophilic bacterium a nucleic acid construct comprising a TS essential nucleic acid molecule from a psychrophilic bacterium flanked on both sides by a nucleic acid molecule homologous to a region of the mesophilic bacterium genome where the TS essential nucleic acid molecule from the psychrophilic bacterium will be inserted into the mesophilic bacterium genome, wherein a protein encoded by the TS essential nucleic acid molecule is operable at a temperature less than 30.degree. C. and inoperable at a temperature greater than 30.degree. C., and wherein the recombinant mesophilic bacterium that is TS is viable at a temperature of 0.degree. C. to 30.degree. C., nonviable at a temperature greater than 30.degree. C., and has a restrictive temperature between 33.degree. C. and 44.degree. C.; culturing the recombinant mesophilic bacterium that is TS at a temperature wherein a protein encoded by the TS essential nucleic acid molecule is operable, whereby the recombinant mesophilic bacterium that is TS produces a plurality of peptides; increasing the culturing temperature to a temperature at which a protein encoded by the TS nucleic acid molecule is inoperable; maintaining said culturing for a period of time sufficient to kill the recombinant mesophilic bacterium that is TS; and harvesting the killed recombinant mesophilic bacterium that is TS, thereby stimulating the immune response in the animal.