Phagemid vectors

Abstract

Phagemid vectors containing a sequence of features between a Col E1 origin and an f1 origin are useful for display of polypeptides or proteins, including antibody libraries.

Parent Case Text

This application claims priority under 35 USC 119(e) to provisional application 60/287,355, filed Apr. 27, 2001.

Description

BACKGROUND

1. Technical Field

This disclosure relates to cloning vectors. More specifically, phagemid vectors useful in the cloning and expression of foreign genetic information are disclosed.

2. Background of Related Art

Plasmids are extrachromosomal genetic elements capable of autonomous replication within their hosts. Bacterial plasmids range in size from 1 Kb to 200 Kb or more and encode a variety of useful properties. Plasmid encoded traits include resistance to antibiotics, production of antibiotics, degradation of complex organic molecules, production of bacteriocins, such as colicins, production of enterotoxins, and production of DNA restriction and modification enzymes.

Although plasmids have been studied for a number of years in their own right, particularly in terms of their replication, transmissibility, structure and evolution, with the advent of genetic engineering technology the focus of plasmid research has turned to the use of plasmids as vectors for the cloning and expression of foreign genetic information. In its application as a vector, the plasmid should possess one or more of the following properties. The plasmid DNA should be relatively small but capable of having relatively large amounts of foreign DNA incorporated into it. The size of the DNA insert is of concern in vectors based on bacteriophages where packing the nucleic acid into the phage particles can determine an upper limit. The plasmid should be under relaxed replication control. That is, where the replication of the plasmid molecule is not strictly coupled to the replication of the host DNA (stringent control), thereby resulting in multiple copies of plasmid DNA per host cell. The plasmid should express one or more selectable markers, such as the drug resistance markers, mentioned above, to permit the identification of host cells which contain the plasmid and also to provide a positive selection pressure for the maintenance of the plasmid in the host cell. Finally the plasmid should contain a single restriction site for one or more endonucleases in a region of plasmid which is not essential for plasmid replication. A vector as described above is useful, for example, for cloning genetic information, by which is meant integrating a segment of foreign DNA into the vector and reproducing identical copies of that information by virtue of the replication of the plasmid DNA.

The next step in the evolution of vector technology was the construction of so-called expression vectors. These vectors are characterized by their ability not only to replicate the inserted foreign genetic information but also to promote the transcription of the genetic information into mRNA and its subsequent translation into protein. This expression requires a variety of regulatory genetic sequences including but not necessarily limited to promoters, operators, transcription terminators, ribosomal binding sites and protein synthesis initiation and termination codons. These expression elements can be provided with the foreign DNA segment as parts thereof or can be integrated within the vector in a region adjacent to a restriction site so that when a foreign DNA segment is introduced into the vector it falls under the control of those elements to which it is now chemically joined.

Filamentous bacteriophage consist of a circular, single-stranded DNA molecule surrounded by a cylinder of coat proteins. There are about 2,700 molecules of the major coat proteins pVIII that envelope the phage. At one end of the phage particle, there are approximately five copies of each of gene III and VI proteins (pIII and pVI) that are involved in host cell binding and in the termination of the assembly process. The other end contains five copies of each of pVII and pIX that are required for the initiation of assembly and for maintenance of virion stability. In recent years, vectors have been developed and utilized for the display of foreign peptides and proteins on the surface of filamentous phage or phagemid particles.

The display of peptides and proteins on the surface of phage or phagemid particles represents a powerful methodology for selection of rare members in a complex library and for carrying out molecular evolution in the laboratory. The ability to construct libraries of enormous molecular diversity and to select for molecules with predetermined properties has made this technology applicable to a wide range of problems. A few of the many applications of such technology are: i) phage display of natural peptides including, mapping epitopes of monoclonal and polyclonal antibodies and generating immunogens; ii) phage display of random peptides, including mapping epitopes of monoclonal and polyclonal antibodies, identifying peptide ligands, and mapping substrate sites for proteases and kinases; and iii) phage display of protein and protein domains, including directed evolution of proteins, isolation of antibodies and cDNA expression screening.

Vectors have been developed which incorporate DNA from plasmids and bacteriophage. These phagemid vectors are derived by modifications of a plasmid genome containing an origin of replication from a bacteriophage, (e.g. f1, M13, fd) as well as the plasmid origin of replication. Phagemids are useful for the expression of foreign genetic information.

One known phagemid vector is .[.pBluescript.]. .Iadd.PBLUESCRIPT.TM. .Iaddend.II KS+ (pBS II KS+) (Stratagene, La Jolla, Calif.), which is a useful starting point for the construction of the present vector because of its small size and the fact that it contains the colE1 plasmid origin of replication and the phage f1 origin of replication in the desired orientation. The plasmid also carries an ampicillin resistance gene.

Vectors which due to their structures provide enhanced functionality would be desirable.

SUMMARY

Novel plasmid vectors capable of replication and expression of foreign genetic information in bacteria, such as, for example, cyanobacterium and E. coli are described herein. These new vectors contain a specific sequence of features after the ColE1 origin but before the f1 origin. Specifically, the present phagemid vector contains, after the ColE1 origin but before the f1 origin, a bacterial transcription terminator, a promoter, a first ribosomal binding site, a first leader sequence and a first cloning region, a second ribosomal binding site, a second leader sequence and a second cloning region. The second cloning region is adapted to receive a gene encoding a polypeptide to be displayed and a nucleotide sequence encoding at least a functional domain of a display protein.

The vectors described herein are constructed through a series of steps which convert a starting vector through a series of intermediate plasmids to the present novel vector which can be used for display of antibody libraries.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates the structure of pBS II KS+, a useful starting vector for making the novel vectors described herein;

FIG. 2 is a flow chart illustrating the method of making the novel vectors described herein;

FIG. 3 schematically illustrates the digestion of the starting vector and insertion of the promoter;

FIGS. 4A-C show the sequence (Seq. ID No. 19) of intermediate vector p110-81.6;

FIG. 5 schematically illustrates the insertion of the terminator;

FIGS. 6A-C show the sequence (Seq. ID No. 20) of intermediate vector p131-03.7;

FIG. 7 schematically illustrates the insertion of multiple cloning sites;

FIGS. 8A-C show the sequence (Seq. ID No. 21) of intermediate vector p131-39.1;

FIG. 9 schematically illustrates the insertion of the nucleotide sequence encoding the display protein and the two transcriptional control cassettes;

FIG. 10 is a map of plasmid pAX131; and

FIGS. 11A-D show the nucleic acid sequence (Seq. ID No. 18) of plasmid pAX131, including the domains corresponding to particular genes.

FIGS. 12A-G show the nucleic acid sequences of illustrative stuffer sequences.

FIGS. 13A-C show the nucleic acid sequence of plasmid pAX131 Xba/Not.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present novel phagemid vectors are useful for display of polypeptides such as, for example, antibody libraries. The vectors described herein can be prepared using any commercially available vector containing a ColE1 and an f1 origin of replication as the starting material. Such starting materials are known and are commercially available. One suitable starting material is the vector pBS II KS+ which is commercially available from Stratagene Corp., La Jolla, Calif. (Sec FIG. 1).

FIG. 2 is a flow-chart showing one embodiment of the steps involved in converting a starting vector into one of the present novel vectors. Those skilled in the art will readily envision other schemes for preparing the present vectors. Accordingly, the present disclosure is not limited to the sequence of steps shown in FIG. 2.

In the first step, the starting vector is digested with restriction enzymes to remove a substantial portion of the vector between the ColE1 origin and the f1 origin of replication. Typically, the portion to be removed from the starting vector includes multiple cloning sites. Depending on the particular restriction sites present in the starting vector, suitable methods for digesting the starting vector are known to and readily selected by those skilled in the art.

Next, a promoter is inserted downstream of the ColE1 origin of the digested starting vector. Any promoter recognized by a host cell can be employed. Suitable promoters include, but are not limited to, ara, lac and trc promoters. The promoter drives expression of other sequences inserted into the vector, such as, for example expression of polypeptides. In particularly useful embodiments, a promoter sequence generated from the starting vector is employed as the promoter inserted downstream of the ColE1 origin as described in more detail below.

In the next step, a bacterial transcription terminator is inserted downstream of the ColE1 origin, and upstream of the promoter. Any terminator recognized by a host cell can be employed. Suitable terminators include, but are not limited to, the t.sub.HP terminator, the bgIG terminator, and the crp terminator. It should be noted that bioinformatics analysis has allowed the identification of over 100 rho-independent transcription terminators in the E. coli genome, all of which should be suitable for this purpose (Ermolaeva, et al, J. Mol Biol 301:27-33 (2000)).

In the next step, multiple restriction sites are inserted downstream of the promoter. The restriction site can be any known restriction site. Suitable restriction sites for insertion include, but are not limited to Nhe I, Hind III, Nco I, Xma I, Bgl II, Bst I, Pvu I, etc. The number of restriction sites inserted is not critical, provided a sufficient number of restriction sites are inserted to allow completion of the balance of the steps needed to create the present novel vectors. Thus as few as 2 to as many as 10 or more restriction sites can be inserted in this step. It should be understood that if one or more of the restriction sites selected for insertion is present in the starting vector, it may be desirable to remove or disable the native restriction site to avoid unwanted digestion during further processing. The restriction site can be inserted using any technique known to those skilled in the art. A particularly preferred combination of restriction sites inserted in this step is Not I, Sfi I, Spe I, Xho I, Xba I and EcoR I.

The next step involves inserting a nucleotide sequence encoding a product that enables display of a polypeptide on the surface of a phagemid particle. The product encoded can thus be considered at least a functional domain of a display protein. The display protein can be any natural or synthetic polypeptide to which a polypeptide to be displayed can be fused and which can present the polypeptide to be displayed for screening processes. Suitable display polypeptides include proteins that can be incorporated into the coat of a phage particle. As those skilled in the art will appreciate, filamentous bacteriophage consist of a circular, single-stranded DNA molecule a surrounded by a cylinder of coat proteins. There are about 2,700 molecules of the major coat protein pVIII that encapsidate the phage. At one end of the phage particle, there are approximately five copies each of gene III and VI proteins (pIII and pVI) that are involved in host-cell binding and in the termination of the assembly process. The other end contains five copies each of pVII and pIX that are required for the initiation of assembly and for maintenance of virion stability. A nucleotide sequence encoding any of these coat proteins can be employed in making the novel vectors herein. Particularly preferred are nucleotide sequences encoding at least a functional domain of pIII. The nucleotide sequence encoding at least a functional domain of pill can be natural or synthetic. The nucleotide sequence inserted can encode a truncated pIII provided the display function of the protein is maintained. An example of a synthetic or artificial coat protein useful herein is that disclosed in Weiss et al., Mol Biol., 300(1), 213-219 (2000), the disclosure of which is incorporated herein by reference.

In the next step, two transcriptional control cassettes are inserted, an upstream transcriptional control cassette and a downstream transcriptional control cassette. Each of the transcriptional control cassettes include a ribosomal binding site, a leader sequence and a cloning site for receiving a gene encoding a polypeptide to be expressed. Any known ribosomal binding site (RBS) and leader sequence recognized by the host cell can be employed. Preferably, the RBS and leader sequence employed is optimized for expression in E. coli. The cloning site is a region of the nucleic acid between two restriction sites, typically with a nonessential region of nucleotide sequence (commonly referred to as a "stuffer" sequence) positioned therebetween. Alternatively, the stuffer sequence may contain a non-essential region and a portion of an antibody constant domain. Suitable stuffer sequences include, for example, those shown in FIGS. 12A-G.

The downstream transcriptional control cassette is inserted adjacent to the nucleotide sequence encoding at least the functional domain of the display protein. In this manner, a fusion protein will be expressed when a gene encoding a polypeptide to be displayed is inserted at the cloning site of the downstream transcriptional control cassette. As those skilled in the art will appreciate, a suppressible stop codon could be positioned between the gene encoding the polypeptide to be displayed and the nucleotide sequence encoding at least a functional domain of the display protein such that fusion display is obtained in a suppressing host (as long as the gene is inserted in-frame) and a secreted protein without the display protein is obtained in a non-suppressing host.

The upstream transcriptional control cassette is inserted upstream of the downstream transcriptional control cassette. The upstream transcriptional control cassette provides a second cloning region for receiving a second gene encoding a polypeptide that can dimerize with the polypeptide to be displayed. For example, where the vector expresses a heavy chain Fd fused to a display protein, the second gene preferably encodes an antibody light chain. As with the cloning site of the downstream transcriptional control cassette, the cloning site of the upstream transcriptional control cassette is a region of the vector between two restriction sites, typically with a stuffer positioned therebetween. It should of course be understood that where a polypeptide other than an antibody is to be displayed (such as, for example, where monomeric display of a single polypeptide or protein is intended) a second gene need not be cloned into the vector at the cloning site of the upstream transcriptional control cassette. In such cases the second cloning site can simply remain unused. As those skilled in the art will also appreciate, where a single chain antibody is encoded by the gene inserted at the cloning site of the downstream transcriptional control cassette, there is no need to insert a second gene into the vector at the cloning site of the upstream transcriptional control cassette.

Thus, the phagmid vector produced by the process illustrated in FIG. 2 will contain, after the ColE1 origin but before the f1 origin, a terminator, a promoter, a first ribosomal binding site, a first leader sequence and a first cloning region, a second ribosomal binding site, a second leader sequence and, a second cloning region for receiving a gene encoding a polypeptide to be displayed and a nucleotide sequence encoding at least a functional domain of a display protein.

The present vectors also include a selectable marker. Either an ampicillin resistant or a CAT resistant vector can be produced in accordance with the present disclosure. The ampicillin or CAT resistance can be provided by simply choosing a starting vector having the desired resistance. Alternatively, if the starting vector is ampicillin resistant to produce a CAT resistant vector, the ampicillin resistant gene is removed and replaced with the chloramphenicol transferase gene. Techniques for providing either ampicillin or CAT resistance in the present vectors will be readily apparent to those skilled in the art. Other suitable selectable markers include, but are not limited to, tetracycline or kanamycin resistance.

The vectors described herein can be transformed into a host cell using known techniques (e.g., electroporation) and amplified. The vectors described herein can also be digested and have a first gene and optionally a second gene ligated therein in accordance with this disclosure. The vector so engineered can be transformed into a host cell using known techniques and amplified or to effect expression of polypeptides and/or proteins encoded thereby to produce phage particles displaying single polypeptides or dimeric species. Those skilled in the art will readily envision other uses for the novel vectors described herein.

The following examples illustrate the present invention without limiting its scope. The steps involved in constructing the vectors described herein are discussed in detail in the Examples. Those skilled in the art possess knowledge of suitable techniques to accomplish the steps described below without the need for undue experimentation, such techniques being well known to those skilled in the art.

EXAMPLE 1

This example illustrates methods and compositions for the construction of one embodiment of a phagemid vector according to the present disclosure. The starting phagemid selected for construction was pBS II KS+ which contains an ampicillin resistant gene which results in a final vector, pAX131, which is ampicillin resistant.

Digestion of Starting Vector and Insertion of Promoter

The commercially available vector pBS II KS+ (Stratagene, LaJolla, Calif.) was digested with Pvu I and Sap I to generate a 2424 bp pBS II KS+ fragment which lacks the bases at positions 500 to 1037 corresponding to the multiple cloning region. The resulting fragment contains the Ampicillin resistant gene (AmpR), phage f1 origin, and the Col E1origin. (See FIG. 3.) Next, two mutagenic primers were used with the pBS II KS+ fragment in a PCR reaction followed by digestion with EcoR I and Sap I to generate a 209 bp fragment containing the lac promoter. The primers used were as follows: 5' AAC CGT ATT ACC GCC TTT GAG TG 3' (SEQ. ID. NO. 1): and 5' CCT GAA TTC AAT TGT TAT CCG CTC ACA ATT CCA C 3' (SEQ. ID. NO. 2).

The 2424 bp fragment and the 209 bp fragment were combined in a three-way ligation reaction with two overlapping oligonucleotides which contain a Not I, EcoR I and Pvu I sites to form a first intermediate plasmid (designated p110-81.6). (See FIG. 3.) The oligonucleotides used for this reaction were: 5' CGG TAA TGC GGC CGC TAC ATG 3' (SEQ. ID. NO. 3); and 5' AAT TCA TGT AGC GGC CGC ATT ACC GAT 3' (SEQ. ID. NO. 4).

The resulting plasmid p110-81.6 was digested and sequenced in the altered region to identify a clone with the correct incorporation of the lac promoter, Pvu I, Sap I, EcoR, and Not I sites. The sequencing of p110-81.6 revealed a nucleic acid change at position 875 within the lac promoter. The published sequence of pBS II KS+ had an adenine at position 875. However, sequencing of p110-81.6 and the original pBS II KS+ revealed a guanine at position 875. The sequence (Seq. ID No. 19) of intermediate plasmid p110-81.6 is shown in FIGS. 4A-C.

Insertion of Terminator

A transcription termination sequence was inserted into the first intermediate plasmid (p110-81.6) upstream of the lac promoter at the Sap I site. (See FIG. 5.)

Plasmid 110-81.6 was digested with Sap I to create an insertion point for the oligonucleotides which contained a t.sub.HP terminator (Nohno et al., Molecular and General Genetics, Vol. 205, pages 260-269 (1986). The oligonucleotides used in this ligation were: 5' AGC GTA CCC GAT AAA AGC GGC TTC CTG ACA GGA GGC CGT TTT GTT TTG CAG CCC ACC T 3'; (SEQ. ID. No. 5); and 5' GCT AGG TGG GCT GCA AAA CAA AAC GGC CTC CTG TCA GGA AGC CGC TTT TAT CGG GTA C 3' (SEQ. ID. NO. 6).

The resulting intermediate vector (designated p131-03.7) was digested and sequenced in the altered region to determine its identity. The sequence (Seq. ID No. 20) of intermediate vector p131-03.7 is shown in FIGS. 6A-C.

Insertion of Multiple Restriction Sites

Oligonucleotides containing the Xba I, XhoI, SpeI and Sfi sites were then inserted into intermediate plasmid p131-03.7.(See FIG. 7.)

Intermediate vector p131-03.7 was digested with EcoR I and Not I and then gel purified. Then overlapping oligonucleotides containing the Xba I, Xho I, Spe I and Sfi I sites were ligated into the p131-03.7 backbone. The oligonucleotides inserted were: 5' AAT TCA CAT CTA GAT ATC TCG AGT CAA TAC TAG TGG CCA GGC CGG CCA GC 3' (SEQ. ID. NO. 7); and 5' GGC CGC TGG CCG GCC TGG CCA CTA GTA TTG ACT CGA GAT ATC TAG ATG TG 3' (SEQ. ID. NO. 8).

The resulting intermediate plasmid (designated p131-39.1) was sequenced and analyzed to determine its identity. The sequence (Seq. ID No. 21) of intermediate plasmid p131-39.1 is shown in FIGS. 8A-C.

Construction of Nucleotide Sequence Encoding Display Protein

Single stranded DNA from phage f1 (ATCC #15766-B2) was used as a template for the cloning of gene III. (Sec FIG. 9.)

The primers used were: 5' AGT GGC CAG GCC GGC CTT GAA ACT GTT GAA AGT TGT TTA GCA AA 3' (SEQ. ID. NO. 9) which contains the Sfi I site, bases to maintain the coding frame and a portion of gene III; and 5 TCT GCG GCC GCT TAG CTA GCT TAA GAC TCT TTA TTA CGC AGT ATG TTA GCA 3' (SEQ. ID. NO. 10); which contains the end of gene III in which an internal ribosome binding site ordinarily used for the next downstream gene has been removed by changing a silent third base position in the corresponding codon. This oligonucleotide also contains a stop codon, Nhe I site for potential use in removal of the fusion, a second stop codon for use with the fusion, and the Not I site for cloning. The PCR fragment was digested with Sfi I and Not I and inserted into p131-39.1 digested with Sfi I and Not I to create intermediate vector p131-44.2. The integrity of the gene III region and flanking sequences was confirmed by sequence analysis. Creation of the Upstream Transcriptional Control Cassette

Plasmid 131-39.1 was utilized as a shuttle vector for cloning the oligonucleotides containing the ompA signal peptide coding sequence. The upstream transcriptional control cassette was generated within intermediate plasmid 131-39.1 by inserting a pair of oligonucleotides which contain EcoR I, the ompA signal peptide leader, followed by a Sac I site, a small stuffer region, and a ribosome binding site. (See FIG. 9.) The oligonucleotides used were:

Eco Xba: 5' AAT TCA AGG AGT TAA TTA TGA AAA AAA CCG CGA TTG CGA TTG CGG TGG CGC TGG CGG GCT TTG CGA CCG TGG CCC AGG CGG CCG AGC TCA TCT T 3' (SEQ. ID. NO. 11); and Xba Eco: 5' CTA GAA GAT GAG CTC GGC CGC CTG GGC CAC GGT CGC AAA GCC CGC CAG CGC CAC CGC AAT CGC AAT CGC GGT TTT TTT CAT AAT TAA CTC CTT G 3' (SEQ. ID. NO. 12). The RBS and leader sequences included in the upstream transcriptional control cassette are optimized for use in E. coli. These novel sequences are: 5' AAG GAG 3' (Seq. ID No.13) for the RBS; and 5' ATG AAA AAA ACC GCG ATT GCG ATT GCG GTG GCG CTG GCG GGC TTT GCG ACC GTG GCC CAG GCG GCC 3' (Seq. ID No. 14) for the ompA leader. The resulting plasmid was sequenced to confirm the identity of the insert and digested at the EcoRI and Xbal sites to generate a 94 bp fragment which is the upstream transcriptional control cassette. Creation of the Downstream Transcriptional Control Cassette

Intermediate plasmid 131-39.1 was utilized as a shuttle vector for cloning the oligonucleotides containing the pelB signal peptide coding sequence. The downstream transcriptional control cassette was generated within intermediate plasmid 131-39.1 by inserting a pair of oligonucleotides containing the pelB signal peptide, Xba I, site, and a ribosome binding site. The oligonucleotides used were:

XbaXho: 5' CTA GAT ATA ATT AAG GAG ATA AAT ATG AAA TAT CTG CTG CCG ACC GCG GCG GCG GGC CTG CTG CTG CTG GCG GCG CAG CCG GCG ATG GCGC 3' (SEQ. ID. NO. 15); and XhoXba: 5' TCG AGC GCC ATC GCC GGC TGC GCC GCC AGC AGC AGC AGG CCC GCC GCC GCG GTC GGC AGC AGA TAT TTC ATA TTT ATC TCC TTA ATT ATA T 3' (SEQ. ID. NO. 16).

The novel pelB leader sequence was optimized for use in E. coli and had the sequence 5' TAT GAA ATA TCT GCT GCC GAC CGC GGC GGC GGG CCT GCT GCT GCT GGC GGC GCA GCC GGC GAT GGC G 3' (Seq. ID No. 17). The resulting plasmid was sequenced to confirm the identity of the insert and digested at the Xbal and XhoI sites to generate a 91 bp fragment which is the downstream transcriptional control cassette. Construction of pAx131 Vector

The upstream transcriptional control cassette and the downstream transcriptional control cassette were combined with intermediate plasmid p131-44.2 digested with EcoRI and XhoI in a 3-way ligation reaction to produce pAX131 (See FIG. 9). FIG. 10 is a map of the resulting pAX131 vector. The pAX131 was analyzed to determine its nucleic acid sequence (SEQ. ID. NO. 18) which is shown in FIGS. 11A-D.

EXAMPLE 2

Insertion of an alternate upstream transcriptional control cassette

PAX131 vector was digested with Not I restriction enzyme. The resulting DNA overhangs were then filled in with Klenow fragment Polymerase to blunt end the DNA followed by ligation. This was performed to remove the existing Not I site. The Not I deleted PAX131 vector was digested with EcoR I/Xba I, and ligated with a duplexed oligo containing EcoR I and Spe I overhangs (Xba I, and Spe I have compatible ends).

Eco/Spe oligo: 5' AAT TCA AGG AGT TAA TTA TGA AAA AAA CCG CGA TTG CGA TTG CGG TGG CGC TGG CGG GCT TTG CGA CCG TGG CCC AGG CGG CCT CTA GAA TCT GCG GCC GCA 3' (SEQ. ID NO. 22) Spe/Eco oligo: 5' CTA GTG CGG CCG CAG ATT CTA GAG GCC GCC TGG GCC ACG GTC GCA AAG CCC GCC AGC GCC ACC GCA ATC GCA ATC GCG GTT TTT TTC ATA ATT AAC TCC TTG 3' (SEQ. ID NO. 23)

The resulting vector (pAX131 Xba/Not) had Xba I, and Not I sites for cloning of a gene, such as light chains, rather than Sac I and Xba I. FIGS. 13A-C show the nucleic acid sequence for vector (pAX131 Xba/Not.

It is contemplated that the present novel vectors can be used in connection with the production and screening of libraries made in accordance with conventional phage display technologies. Both natural and synthetic antibody repertoires have been generated as phage displayed libraries. Natural antibodies can be cloned from B-cell mRNA isolated from peripheral blood lymphocytes, bone marrow, spleen, or other lymphatic tissue of a human or non-human donor. Donors with an immune response to the antigen(s) of interest can be used to create immune antibody libraries. Alternatively, non-immune libraries may be generated from donors by isolating naive antibody B cell genes. PCR using antibody specific primers on the 18.sup.st strand cDNA allows the isolation of light chain and heavy chain antibody fragments which can then be cloned into the display vector.

Synthetic antibodies or antibody libraries can be made up in part or entirely with regions of synthetically derived sequence. Library diversity can be engineered within variable regions, particularly within CDRs, through the use of degenerate oligonucleotides. For example, a single Fab gene may be modified at the heavy chain CDR3 position to contain random nucleotide sequences. The random sequence can be introduced into the heavy chain gene using an oligonucleotide which contains the degenerate coding region in an overlap PCR approach. Alternatively, degenerate oligo cassettes can be cloned into restriction sites that flank the CDR(s) to create diversity. The resulting library generated by this or other approaches can then be cloned into a display vector in accordance with this disclosure.

Upon introduction of the display library into bacteria, phage particles will be generated that have antibody displayed on the surface. The resulting collection of phage-displayed antibodies can be selected for those with the ability to bind to the antigen of interest using techniques known to those skilled in the art. Antibodies identified by this system can be used therapeutically, as diagnostic reagents, or as research tools.

It is contemplated that single and double stranded versions of the vectors described herein are within the scope of the present invention. It is well within the purview of those skilled in the art to prepare either single or double stranded vectors having the features described herein.

It will be understood that various modifications may be made to the embodiments described herein. For example, as those skilled in the art will appreciate, a first gene encoding a fusion protein having an antibody light chain to be fused to and displayed by pVIII and a second gene encoding a heavy chain Fd can be inserted into the vector at the newly created restriction site to provide effective antibody display. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto.

SEQUENCE LISTINGS

1

31123DNAartificial sequenceprimer 1 aaccgtatta ccgcctttga gtg 23 234DNAartificial sequenceprimer 2 cctgaattca attgttatcc gctcacaatt ccac 34 321DNAartificial sequenceoligonucleotide 3 cggtaatgcg gccgctacat g 21 427DNAartificial sequenceoligonucleotide 4 aattcatgta gcggccgcat taccgat 27 558DNAartificial sequenceoligonucleotide 5 agcgtacccg ataaaagcgg cttcctgaca ggaggccgtt ttgttttgca g cccacct 58 658DNAartificial sequenceoligonucleotide 6 gctaggtggg ctgcaaaaca aaacggcctc ctgtcaggaa gccgctttta t cgggtac 58 750DNAartificial sequenceoligonucleotide 7 aattcacatc tagatatctc gagtcaatac tagtggccag gccggccagc 50 850DNAartificial sequenceoligonucleotide 8 ggccgctggc cggcctggcc actagtattg actcgagata tctagatgtg 50 944DNAartificial sequenceprimer 9 agtggccagg ccggccttga aactgttgaa agttgtttag caaa 44 1051DNAartificial sequenceprimer 10 tctgcggccg cttagctagc ttaagactct ttattacgca gtatgttagc a 51 1194DNAartificial sequenceoligonucleotide 11 aattcaagga gttaattatg aaaaaaaccg cgattgcgat tgcggtggcg c tggcgggct 60 ttgcgaccgt ggcccaggcg gccgagctca tctt 94 1294DNAartificial sequenceoligonucleotide 12 ctagaagatg agctcggccg cctgggccac ggtcgcaaag cccgccagcg c caccgcaat 60 cgcaatcgcg gtttttttca taattaactc cttg 94 136DNAartificial sequenceRBS 13 aaggag 6 1466DNAartificial sequenceompA leader 14 atgaaaaaaa ccgcgattgc gattgcggtg gcgctggcgg gctttgcgac c gtggcccag 60 gcggcc 66 1591DNAartificial sequenceoligonucleotide 15 ctagatataa ttaaggagat aaatatgaaa tatctgctgc cgaccgcggc g gcgggcctg 60 ctgctgctgg cggcgcagcc ggcgatggcg c 91 1691DNAartificial sequenceoligonucleotide 16 tcgagcgcca tcgccggctg cgccgccagc agcagcaggc ccgccgccgc g gtcggcagc 60 agatatttca tatttatctc cttaattata t 91 1767DNAartificial sequencepelB leader 17 tatgaaatat ctgctgccga ccgcggcggc gggcctgctg ctgctggcgg c gcagccggc 60 gatggcg 67 184154DNAartificial sequenceplasmid vector 18 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc t aaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa t attgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt g cggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct g aagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc c ttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta t gtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac t attctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc a tgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac t tacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg g atcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac g agcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc g aactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt g caggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga g ccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc c gtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag a tcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca t atatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc c tttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca g accccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc t gcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta c caactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt c tagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc g ctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg t tggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg t gcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag c tatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc a gggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat a gtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg g ggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc t ggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt a ccgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca g tgagcgagg 1920 aagcgtaccc gataaaagcg gcttcctgac aggaggccgt tttgttttgc a gcccaccta 1980 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat t cattaatgc 2040 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc a attaatgtg 2100 agttagctca ctcattaggc accccaggct ttacacttta tgctcccggc t cgtatgttg 2160 tgtggaattg tgagcggata acaattgaat tcaaggagtt aattatgaaa a aaaccgcga 2220 ttgcgattgc ggtggcgctg gcgggctttg cgaccgtggc ccaggcggcc g agctcatct 2280 tctagatata attaaggaga taaatatgaa atatctgctg ccgaccgcgg c ggcgggcct 2340 gctgctgctg gcggcgcagc cggcgatggc gctcgagtca atactagtgg c caggccggc 2400 cttgaaactg ttgaaagttg tttagcaaaa ccccatacag aaaattcatt t actaacgtc 2460 tggaaagacg acaaaacttt agatcgttac gctaactatg agggctgtct g tggaatgct 2520 acaggcgttg tagtttgtac tggtgacgaa actcagtgtt acggtacatg g gttcctatt 2580 gggcttgcta tccctgaaaa tgagggtggt ggctctgagg gtggcggttc t gagggtggc 2640 ggctctgagg gtggcggtac taaacctcct gagtacggtg atacacctat t ccgggctat 2700 acttatatca accctctcga cggcacttat ccgcctggta ctgagcaaaa c cccgctaat 2760 cctaatcctt ctcttgagga gtctcagcct cttaatactt tcatgtttca g aataatagg 2820 ttccgaaata ggcagggggc attaactgtt tatacgggca ctgttactca a ggcactgac 2880 cccgttaaaa cttattacca gtacactcct gtatcatcaa aagccatgta t gacgcttac 2940 tggaacggta aattcagaga ctgcgctttc cattctggct ttaatgagga t ccattcgtt 3000 tgtgaatatc aaggccaatc gtctgacctg cctcaacctc ctgttaatgc t ggcggcggc 3060 tctggtggtg gttctggtgg cggctctgag ggtggtggct ctgagggtgg c ggttctgag 3120 ggtggcggct ctgagggtgg cggttccggt ggtggctctg gttccggtga t tttgattat 3180 gaaaagatgg caaacgctaa taagggggct atgaccgaaa atgccgatga a aacgatgaa 3240 aacgcgctac agtctgacgc taaaggcaaa cttgattctg tcgctactga t tacggtgct 3300 gctatcgacg gtttcattgg tgacgtttcc ggccttgcta atggtaatgg t gctactggt 3360 gattttgctg gctctaattc ccaaatggct caagtcggtg acggtgataa t tcaccttta 3420 atgaataatt tccgtcaata tttaccttcc ctccctcaat cggttgaatg t cgccctttt 3480 gtctttggcg ctggtaaacc atatgaattt tctattgatt gtgacaaaat a aacttattc 3540 cgtggtgtct ttgcgtttct tttatatgtt gccaccttta tgtatgtatt t tcgacgttt 3600 gctaacatac tgcgtaataa agagtcttaa gctagctaag cggccgcatt a ccgatcgcc 3660 cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc g gcgcattaa 3720 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc g ccctagcgc 3780 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt c cccgtcaag 3840 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac c tcgacccca 3900 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag a cggtttttc 3960 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa a ctggaacaa 4020 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg a tttcggcct 4080 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac a aaatattaa 4140 cgcttacaat ttag 4154 192654DNAartificial sequenceplasmid vector 19 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt a aatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag a atagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga a cgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg a accatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc c taaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg a agggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc g cgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat t caggctgcg 480 caactgttgg gaagggcgat cggtaatgcg gccgctacat gaattcaatt g ttatccgct 540 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg g tgcctaatg 600 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt c gggaaacct 660 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt t gcgtattgg 720 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc t gcggcgagc 780 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg a taacgcagg 840 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg c cgcgttgct 900 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac g ctcaagtca 960 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg g aagctccct 1020 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct t tctcccttc 1080 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg t gtaggtcgt 1140 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct g cgccttatc 1200 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac t ggcagcagc 1260 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt t cttgaagtg 1320 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc t gctgaagcc 1380 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca c cgctggtag 1440 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat c tcaagaaga 1500 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac g ttaagggat 1560 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt a aaaatgaag 1620 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc a atgcttaat 1680 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg c ctgactccc 1740 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg c tgcaatgat 1800 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc c agccggaag 1860 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta t taattgttg 1920 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg t tgccattgc 1980 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct c cggttccca 2040 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta g ctccttcgg 2100 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg t tatggcagc 2160 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga c tggtgagta 2220 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt g cccggcgtc 2280 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca t tggaaaacg 2340 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt c gatgtaacc 2400 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt c tgggtgagc 2460 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga a atgttgaat 2520 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt g tctcatgag 2580 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc g cacatttcc 2640 ccgaaaagtg ccac 2654 202712DNAartificial sequenceplasmid vector 20 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt a aatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag a atagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga a cgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg a accatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc c taaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg a agggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc g cgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat t caggctgcg 480 caactgttgg gaagggcgat cggtaatgcg gccgctacat gaattcaatt g ttatccgct 540 cacaattcca cacaacatac gagccgggag cataaagtgt aaagcctggg g tgcctaatg 600 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt c gggaaacct 660 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt t gcgtattgg 720 gcgctcttcc gctaggtggg ctgcaaaaca aaacggcctc ctgtcaggaa g ccgctttta 780 tcgggtacgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg c ggcgagcgg 840 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat a acgcaggaa 900 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc g cgttgctgg 960 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc t caagtcaga 1020 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga a gctccctcg 1080 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt c tcccttcgg 1140 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg t aggtcgttc 1200 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc g ccttatccg 1260 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg g cagcagcca 1320 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc t tgaagtggt 1380 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg c tgaagccag 1440 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc g ctggtagcg 1500 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct c aagaagatc 1560 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt t aagggattt 1620 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa a aatgaagtt 1680 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa t gcttaatca 1740 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc t gactccccg 1800 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct g caatgatac 1860 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca g ccggaaggg 1920 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt a attgttgcc 1980 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt g ccattgcta 2040 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc g gttcccaac 2100 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc t ccttcggtc 2160 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt a tggcagcac 2220 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact g gtgagtact 2280 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc c cggcgtcaa 2340 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt g gaaaacgtt 2400 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg a tgtaaccca 2460 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct g ggtgagcaa 2520 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa t gttgaatac 2580 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt c tcatgagcg 2640 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc a catttcccc 2700 gaaaagtgcc ac 2712 212750DNAartificial sequenceplasmid vector 21 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc t aaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa t attgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt g cggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct g aagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc c ttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta t gtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac t attctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc a tgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac t tacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg g atcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac g agcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc g aactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt g caggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga g ccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc c gtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag a tcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca t atatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc c tttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca g accccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc t gcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta c caactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt c tagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc g ctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg t tggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg t gcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag c tatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc a gggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat a gtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg g ggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc t ggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt a ccgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca g tgagcgagg 1920 aagcgtaccc gataaaagcg gcttcctgac aggaggccgt tttgttttgc a gcccaccta 1980 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat t cattaatgc 2040

agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc a attaatgtg 2100 agttagctca ctcattaggc accccaggct ttacacttta tgctcccggc t cgtatgttg 2160 tgtggaattg tgagcggata acaattgaat tcacatctag atatctcgag t caatactag 2220 tggccaggcc ggccagcggc cgcattaccg atcgcccttc ccaacagttg c gcagcctga 2280 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg g tggttacgc 2340 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct t tcttccctt 2400 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg c tccctttag 2460 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag g gtgatggtt 2520 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg g agtccacgt 2580 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc t cggtctatt 2640 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat g agctgattt 2700 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag 2750 22102DNAartificial sequenceoligonucleotide 22 aattcaagga gttaattatg aaaaaaaccg cgattgcgat tgcggtggcg c tggcgggct 60 ttgcgaccgt ggcccaggcg gcctctagaa tctgcggccg ca 102 23102DNAartificial sequenceoligonucleotide 23 ctagtgcggc cgcagattct agaggccgcc tgggccacgg tcgcaaagcc c gccagcgcc 60 accgcaatcg caatcgcggt ttttttcata attaactcct tg 102 24565DNAartificial sequencestuffer sequence 24 tctagataac tgtggctgca ccatctgtct tcatcttccc gccatctgat g agcagttga 60 aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga g aggccaaag 120 tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt g tcacagagc 180 aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc a aagcagact 240 acgagaaaca caaagtctac gcctgcgaag tcacccatca gggcctgagc t ccggaggtg 300 cctcagtcgt gtgcttcttg aacaacttct accccaaaga catcaatgtc a agtggaaga 360 ttgatggcag tgaacgacaa aatggcgtcc tgaacagttg gactgatcag g acagcaaag 420 acagcaccta cagcatgagc agcaccctca cgttgaccaa ggacgagtat g aacgacata 480 acagctatac ctgtgaggcc actcacaaga catcaacttc acccattgtc a agagcttca 540 acaggaatga gtgttaagcg gccgc 565 251131DNAartificial sequencestuffer sequence 25 ctcgagctga tgagccatgg aagctgtgtc gcctgcacca ggctcccacg g ctcgtggtg 60 cggtgcgctt ctggtgttcg ctgcctacag ccgacacgtc gagcttcgtg c ccctagagt 120 tgcgcgtcac agcagcctcc ggcgctccgc gatatcaccg tgtcatccac a tcaatgaag 180 tagtgctcct agacgccccc gtggggctgg tggcgcggtt ggctgacgag a gcggccacg 240 tagtgttgcg ctggctcccg ccgcctgaga cacccatgac gtctcacatc c gctacgagg 300 tggacgtctc ggccggcaac ggcgcaggga gcgtacagag ggtggagatc c tggagggcc 360 gcaccgagtg tgtgctgagc aacctgcggg gccggacgcg ctacaccttc g ccgtccgcg 420 cgcgtatggc tgagccgagc ttcggcggct tctggagcgc ctggtcggag c ctgtgtcgc 480 tgctgacgcc tagcgacctg gaccccctca tcctgacgct ctccctcatc c tcgtggtca 540 tcctggtgct gctgaccgtg ctcgcgctgc tctcccaccg ccgggctctg a agcagaaga 600 tctggcctgg catcccgagc ccagagagcg agtttgaagg cctcttcacc a cccacaagg 660 gtaacttcca gctgtggctg taccagaatg atggctgcct gtggtggagc c cctgcaccc 720 ccttcacgga ggacccacct gcttccctgg aagtcctctc agagcgctgc t gggggacga 780 tgcaggcagt ggagccgggg acagatgatg agggcccttt tccccctcgt c tcctgtgag 840 aattccccgt cggatacgag cagcgtggcc gttggctgcc tcgcacagga c ttccttccc 900 gactccatca ctttctcctg gaaatacaag aacaactctg acatcagcag c acccggggc 960 ttcccatcag tcctgagagg gggcaagtac gcagccacct cacaggtgct g ctgccttcc 1020 aaggacgtca tgcagggcac agacgaacac gtggtgtgca aagtccagca c cccaacggc 1080 aacaaagaaa agaacgtgcc tcttccagtg attgctgagc tgcctactag t 1131 261121DNAartificial sequencestuffer sequence 26 ctcgagctga tgagccatgg aagctgtgtc gcctgcacca ggctcccacg g ctcgtggtg 60 cggtgcgctt ctggtgttcg ctgcctacag ccgacacgtc gagcttcgtg c ccctagagt 120 tgcgcgtcac agcagcctcc ggcgctccgc gatatcaccg tgtcatccac a tcaatgaag 180 tagtgctcct agacgccccc gtggggctgg tggcgcggtt ggctgacgag a gcggccacg 240 tagtgttgcg ctggctcccg ccgcctgaga cacccatgac gtctcacatc c gctacgagg 300 tggacgtctc ggccggcaac ggcgcaggga gcgtacagag ggtggagatc c tggagggcc 360 gcaccgagtg tgtgctgagc aacctgcggg gccggacgcg ctacaccttc g ccgtccgcg 420 cgcgtatggc tgagccgagc ttcggcggct tctggagcgc ctggtcggag c ctgtgtcgc 480 tgctgacgcc tagcgacctg gaccccctca tcctgacgct ctccctcatc c tcgtggtca 540 tcctggtgct gctgaccgtg ctcgcgctgc tctcccaccg ccgggctctg a agcagaaga 600 tctggcctgg catcccgagc ccagagagcg agtttgaagg cctcttcacc a cccacaagg 660 gtaacttcca gctgtggctg taccagaatg atggctgcct gtggtggagc c cctgcaccc 720 ccttcacgga ggacccacct gcttccctgg aagtcctctc agagcgctgc t gggggacga 780 tgcaggcagt ggagccgggg acagatgatg agggcccatc ggtcttcccc c tggcaccct 840 cctccaagag cacctctggc ggcacagcgg ccctgggctg cctggtcaag g actacttcc 900 ccgaaccggt gacggtgtcg tggaactcag gcgctctgac cagcggcgtg c acaccttcc 960 cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc g tgccatcca 1020 gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc a acaccaagg 1080 tggacaagaa agttgagccc aaatcttgtg acaaaactag t 1121 27337DNAartificial sequencestuffer sequence 27 tctagataac tgtggctgca ccatctgtct tcatcttccc gccatctgat g agcagttga 60 aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga g aggccaaag 120 tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt g tcacagagc 180 aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc a aagcagact 240 acgagaaaca caaagtctac gcctgcgaag tcacccatca gggcctgagc t cgcccgtca 300 caaagagctt caacagggga gagtgttaag cggccgc 337 28509DNAartificial sequencestuffer sequence 28 tctagataac tgtggctgca ccatctgtct tcatcttccc gccatctgat g agcagttga 60 aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga g aggccaaag 120 tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt g tcacagagc 180 aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc a aagcagact 240 acgagaaaca caaagtctac gcctgcgaag tcacccatca gggcctgagc t ctgacagtg 300 gcttggaaag cagatagcag ccccgtcaag gcgggagtgg agaccaccac a ccctccaaa 360 caaagcaaca acaagtacgc ggccagcagc tatctgagcc tgacgcctga g cagtggaag 420 tcccacagaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga g aagacagtg 480 gcccctacag aatgttcata agcggccgc 509 291059DNAartificial sequencestuffer sequence 29 ctcgagctga tgagccatgg aagctgtgtc gcctgcacca ggctcccacg g ctcgtggtg 60 cggtgcgctt ctggtgttcg ctgcctacag ccgacacgtc gagcttcgtg c ccctagagt 120 tgcgcgtcac agcagcctcc ggcgctccgc gatatcaccg tgtcatccac a tcaatgaag 180 tagtgctcct agacgccccc gtggggctgg tggcgcggtt ggctgacgag a gcggccacg 240 tagtgttgcg ctggctcccg ccgcctgaga cacccatgac gtctcacatc c gctacgagg 300 tggacgtctc ggccggcaac ggcgcaggga gcgtacagag ggtggagatc c tggagggcc 360 gcaccgagtg tgtgctgagc aacctgcggg gccggacgcg ctacaccttc g ccgtccgcg 420 cgcgtatggc tgagccgagc ttcggcggct tctggagcgc ctggtcggag c ctgtgtcgc 480 tgctgacgcc tagcgacctg gaccccctca tcctgacgct ctccctcatc c tcgtggtca 540 tcctggtgct gctgaccgtg ctcgcgctgc tctcccaccg ccgggctctg a agcagaaga 600 tctggcctgg catcccgagc ccagagagcg agtttgaagg cctcttcacc a cccacaagg 660 gtaacttcca gctgtggctg taccagaatg atggctgcct gtggtggagc c cctgcaccc 720 ccttcacgga ggacccacct gcttccctgg aagtcctctc agagcgctgc t gggggacga 780 tgcaggcagt ggagccgggg acagatgatg agggccctag gatgcctggt c aagggttat 840 ttccctgagc cagtgacctt gacctggaac tctggatccc tgtccagtgg t gtgcacacc 900 ttcccagctg tcctgcagtc tgacctctac accctcagca gctcagtgac t gtaacctcc 960 agcacctggc ccagccagtc catcacctgc aatgtggccc acccggcaag c agcaccaag 1020 gtggacaaga aaattgagcc cagagtgccc acaactagt 1059 301056DNAartificial sequencestuffer sequence 30 ctcgagctga tgagccatgg aagctgtgtc gcctgcacca ggctcccacg g ctcgtggtg 60 cggtgcgctt ctggtgttcg ctgcctacag ccgacacgtc gagcttcgtg c ccctagagt 120 tgcgcgtcac agcagcctcc ggcgctccgc gatatcaccg tgtcatccac a tcaatgaag 180 tagtgctcct agacgccccc gtggggctgg tggcgcggtt ggctgacgag a gcggccacg 240 tagtgttgcg ctggctcccg ccgcctgaga cacccatgac gtctcacatc c gctacgagg 300 tggacgtctc ggccggcaac ggcgcaggga gcgtacagag ggtggagatc c tggagggcc 360 gcaccgagtg tgtgctgagc aacctgcggg gccggacgcg ctacaccttc g ccgtccgcg 420 cgcgtatggc tgagccgagc ttcggcggct tctggagcgc ctggtcggag c ctgtgtcgc 480 tgctgacgcc tagcgacctg gaccccctca tcctgacgct ctccctcatc c tcgtggtca 540 tcctggtgct gctgaccgtg ctcgcgctgc tctcccaccg ccgggctctg a agcagaaga 600 tctggcctgg catcccgagc ccagagagcg agtttgaagg cctcttcacc a cccacaagg 660 gtaacttcca gctgtggctg taccagaatg atggctgcct gtggtggagc c cctgcaccc 720 ccttcacgga ggacccacct gcttccctgg aagtcctctc agagcgctgc t gggggacga 780 tgcaggcagt ggagccgggg acagatgatg agggccctag gatgcctggt c aagggctat 840 ttccctgagc cagtgacagt gacctggaac tctggatccc tgtccagcgg t gtgcacacc 900 ttcccagctg tcctgcagtc tgacctctac actctgagca gctcagtgac t gtcccctcc 960 agcacctggc ccagcgagac cgtcacctgc aacgttgccc acccggccag c agcaccaag 1020 gtggacaaga aaattgtgcc cagggattgt actagt 1056 314153DNAartificial sequenceplasmid vector 31 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc t aaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa t attgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt g cggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct g aagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc c ttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta t gtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac t attctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc a tgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac t tacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg g atcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac g agcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc g aactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt g caggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga g ccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc c gtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag a tcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca t atatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc c tttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca g accccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc t gcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta c caactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt c tagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc g ctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg t tggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg t gcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag c tatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc a gggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat a gtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg g ggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc t ggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt a ccgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca g tgagcgagg 1920 aagcgtaccc gataaaagcg gcttcctgac aggaggccgt tttgttttgc a gcccaccta 1980 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat t cattaatgc 2040 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc a attaatgtg 2100 agttagctca ctcattaggc accccaggct ttacacttta tgctcccggc t cgtatgttg 2160 tgtggaattg tgagcggata acaattgaat tcaaggagtt aattatgaaa a aaaccgcga 2220 ttgcgattgc ggtggcgctg gcgggctttg cgaccgtggc ccaggcggcc t ctagaatct 2280 gcggccgcac tagatataat taaggagata aatatgaaat atctgctgcc g accgcggcg 2340 gcgggcctgc tgctgctggc ggcgcagccg gcgatggcgc tcgagtcaat a ctagtggcc 2400 aggccggcct tgaaactgtt gaaagttgtt tagcaaaacc ccatacagaa a attcattta 2460 ctaacgtctg gaaagacgac aaaactttag atcgttacgc taactatgag g gctgtctgt 2520 ggaatgctac aggcgttgta gtttgtactg gtgacgaaac tcagtgttac g gtacatggg 2580 ttcctattgg gcttgctatc cctgaaaatg agggtggtgg ctctgagggt g gcggttctg 2640 agggtggcgg ctctgagggt ggcggtacta aacctcctga gtacggtgat a cacctattc 2700 cgggctatac ttatatcaac cctctcgacg gcacttatcc gcctggtact g agcaaaacc 2760 ccgctaatcc taatccttct cttgaggagt ctcagcctct taatactttc a tgtttcaga 2820 ataataggtt ccgaaatagg cagggggcat taactgttta tacgggcact g ttactcaag 2880 gcactgaccc cgttaaaact tattaccagt acactcctgt atcatcaaaa g ccatgtatg 2940 acgcttactg gaacggtaaa ttcagagact gcgctttcca ttctggcttt a atgaggatc 3000 cattcgtttg tgaatatcaa ggccaatcgt ctgacctgcc tcaacctcct g ttaatgctg 3060 gcggcggctc tggtggtggt tctggtggcg gctctgaggg tggtggctct g agggtggcg 3120 gttctgaggg tggcggctct gagggtggcg gttccggtgg tggctctggt t ccggtgatt 3180 ttgattatga aaagatggca aacgctaata agggggctat gaccgaaaat g ccgatgaaa 3240 acgcgctaca gtctgacgct aaaggcaaac ttgattctgt cgctactgat t acggtgctg 3300 ctatcgacgg tttcattggt gacgtttccg gccttgctaa tggtaatggt g ctactggtg 3360 attttgctgg ctctaattcc caaatggctc aagtcggtga cggtgataat t cacctttaa 3420 tgaataattt ccgtcaatat ttaccttccc tccctcaatc ggttgaatgt c gcccttttg 3480 tctttggcgc tggtaaacca tatgaatttt ctattgattg tgacaaaata a acttattcc 3540 gtggtgtctt tgcgtttctt ttatatgttg ccacctttat gtatgtattt t cgacgtttg 3600 ctaacatact gcgtaataaa gagtcttaag ctagctaagc ggccgcatta c cgatcgccc 3660 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg g cgcattaag 3720 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg c cctagcgcc 3780 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc c ccgtcaagc 3840 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc t cgaccccaa 3900 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga c ggtttttcg 3960 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa c tggaacaac 4020 actcaaccct atctcggtct attcttttga tttataaggg attttgccga t ttcggccta 4080 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca a aatattaac 4140 gcttacaatt tag 4153

Claims

We claim:

.[.1. A phagemid vector comprising: a selectable marker; a ColE1 origin; an f1 origin; and after the ColE1 origin but before the f1 origin, further comprising the following features: a bacterial transcription terminator; a promoter, a first ribosomal binding site; a first leader sequence; a first cloning region; a second ribosomal binding site; a second leader sequence; a second cloning region for receiving a gene encoding a polypeptide to be displayed; and a nucleotide sequence encoding a product that enables display of a polypeptide on the surface of a phagemid particle..].

.[.2. A phagemid vector as in claim 1 wherein at least one of the first or second ribosomal binding sites comprises Seq. ID No. 13..].

3. .[.A phagemid vector as in claim 1.]. .Iadd.A phagemid vector comprising: a selectable marker; a ColE1 origin; an f1 origin; and after the ColE1 origin but before the f1 origin, further comprising the following features: a bacterial transcription terminator and a promoter wherein the bacterial transcription terminator is upstream of the promoter; a first ribosomal binding site; a first leader sequence; a first cloning region; a second ribosomal binding site; a second leader sequence; a second cloning region for receiving a gene encoding a polypeptide to be displayed; and a nucleotide sequence encoding a product that enables display of a polypeptide on the surface of a phagemid particle, .Iaddend. wherein at least one of the first or second leader sequences comprises a sequence selected from the group consisting of Seq. ID No. 14 and Seq. ID No. 17.

.[.4. A phagemid vector as in claim 1 wherein the nucleotide sequence encoding a product encodes a protein selected from the group consisting of pIII and pVIII..].

.[.5. A phagemid vector as in claim 1 wherein the nucleotide sequence encoding a product encodes a truncated pIII..].

.[.6. A phagemid vector as in claim 1 wherein the nucleotide sequence encoding a product encodes a synthetic pIII..].

.[.7. A phagemid vector as in claim 1 wherein the selectable marker is selected from the group consisting of ampicillin resistance, chloramphenicol transferase resistance, tetracycline resistance and kanamycin resistance..].

8. A phagemid vector comprising Seq. ID No. 18.

9. A vector comprising a sequence selected from the group consisting of Seq. ID Nos. 19, 20 and 21.