U.S. patent application number 10/116788 was filed with the patent office on 2006-02-02 for genomic mammary amyloid a sequence.
Invention is credited to Marilynn A. Larson, Thomas L. McDonald, Annika Weber.
Application Number | 20060024804 10/116788 |
Document ID | / |
Family ID | 27396548 |
Filed Date | 2006-02-02 |
United States Patent
Application |
20060024804 |
Kind Code |
A9 |
McDonald; Thomas L. ; et
al. |
February 2, 2006 |
Genomic mammary amyloid a sequence
Abstract
A genomic nucleotide sequence encoding Serum Amyloid A (SAA),
isolated and purified from mammalian colostrum, is disclosed.
Methods of use for the same in transgenic protocols is also
disclosed.
Inventors: |
McDonald; Thomas L.;
(Lincoln, NE) ; Larson; Marilynn A.; (Lincoln,
NE) ; Weber; Annika; (Lincoln, NE) |
Correspondence
Address: |
MCKEE, VOORHEES & SEASE, P.L.C.;ATTN: UNIVERSITY OF NEBRASKA MEDICAL
CENTER
801 GRAND AVENUE, SUITE 3200
DES MOINES
IA
50309-2721
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20030170840 A1 |
September 11, 2003 |
|
|
Family ID: |
27396548 |
Appl. No.: |
10/116788 |
Filed: |
April 4, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US00/29065 |
Oct 20, 2000 |
|
|
|
10116788 |
Apr 4, 2002 |
|
|
|
09425679 |
Oct 22, 1999 |
6509444 |
|
|
10116788 |
Apr 4, 2002 |
|
|
|
60218611 |
Jul 17, 2000 |
|
|
|
PCT/US00/29065 |
|
|
|
|
60218482 |
Jul 14, 2000 |
|
|
|
PCT/US00/29065 |
|
|
|
|
Current U.S.
Class: |
435/183 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 13/12 20180101; C07K 14/4711 20130101; A61P 1/04 20180101;
A61P 1/00 20180101; A61P 13/02 20180101; A61P 1/12 20180101 |
Class at
Publication: |
435/183 ;
435/069.1; 435/320.1; 435/325; 536/023.2; 530/350 |
International
Class: |
C12N 9/00 20060101
C12N009/00; C07H 21/04 20060101 C07H021/04; C12P 21/02 20060101
C12P021/02; C12N 5/06 20060101 C12N005/06 |
Claims
1. A purified and isolated nucleic acid molecule that encodes
colostrum associated SAA wherein said protein encoded by said
nucleic acid molecule comprises a TFLK amino acid sequence present
in the N-terminal region of said protein said nucleic acid molecule
comprising one or more noncoding regions of approximately 50 or
more contiguous nucleotides from SEQ ID NO:31 and their
conservatively modified variants.
2. The nucleic acid molecule of claim 1 wherein said molecule is
purified and isolated from bovine species.
3. The nucleic acid molecule of claim 1 wherein said nucleic acid
encoding region encodes the peptide of SEQ ID NO: (SEQ ID NO:3) or
(SEQ ID NO:4) and one or more of a sequence selected from the group
consisting of (SEQ ID NO:5), (SEQ ID NO:6), (SEQ ID NO:7) and (SEQ
ID NO:8)
4. The nucleic acid molecule of claim 1 wherein said non coding
region is 5' of base 647 in SEQ ID NO:31.
5. The nucleic acid molecule of claim 1 wherein said non coding
region is selected from the group consisting of bases 738 to 2982
of SEQ ID NO: 31 and those regions that will hybridize under
conditions of high stringency to said bases.
6. The nucleic acid molecule of claim 1 wherein said non coding
region is selected from the group consisting of bases 3121 to 3510
of SEQ ID NO: 31 and those regions that will hybridize under
conditions of high stringency to said bases.
7. The nucleic acid molecule of claim 1 wherein said non coding
region is selected from the group consisting of bases 3674 to 4984
of SEQ ID NO: 31 and those regions that will hybridize under
conditions of high stringency to said bases.
8. A purified and isolated genomic bovine nucleotide sequence which
encodes colostrum associated SAA said sequence comprising a non
coding region selected from the group consisting of bases 1-647,
738-2982, 3121-3510, 3674-4984 of SEQ ID NO:31 and those regions
that will hybridize under conditions of high stringency to said
bases.
9. An expression cassette which comprises the nucleic acid molecule
of claim 1 operably linked to a promoter region.
10. A cloning or expression vector comprising the expression
cassette of claim 7.
11. A eucaryotic or procaryotic host cell transformed with the
vector of claim 8.
12. A purified and isolated nucleic acid molecule that encodes
colostrum associated SAA wherein said protein encoded by said
nucleic acid molecule comprises a TFLK amino acid sequence present
in the N-terminal region of said protein said nucleic acid molecule
comprising SEQ ID NO:31 and its conservatively modified
variants.
13. An expression cassette which comprises the nucleic acid
molecule of claim 10 operably linked to a promoter nucleotide.
14. A cloning or expression vector comprising the expression
cassette of claim 11.
15. A eucaryotic or procaryotic host cell transformed with the
vector of claim 12.
16. A method for producing MAA peptide comprising: administering a
MAA inducing agent to a bovine mammary epithelial cell line;
culturing said cell so that MAA is produced; and harvesting said
MAA from said mammary epithelial cell.
17. A bovine mammary epithelial cell line produced by the method of
claim 16.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of immunology and
mammalian immune systems. In particular, the invention provides
novel isoforms of serum amyloid A, which are found in
colostrum.
BACKGROUND OF THE INVENTION
[0002] Several scientific or patent publications are referenced in
this patent application to describe the state of the art to which
the invention pertains. Each of these publications is incorporated
by reference herein, in its entirety.
[0003] Mammals respond to tissue injury, trauma or infection by
executing a complex series of biological reactions in an effort to
prevent further tissue damage, to initiate repair of damaged
tissue, and to isolate and destroy infective organisms. This
process is referred to as the inflammatory response, the early and
intermediate stages of which are referred to as the acute phase
response.
[0004] The acute phase response involves a wide variety of
mediators, including cytokines, interleukins and tumor necrosis
factor. It also involves a radical alteration in the biosynthetic
profile of the liver. Under normal circumstances, the liver
synthesizes a range of plasma proteins at steady state
concentrations. Some of these proteins, the "acute phase" proteins
are induced in the inflammatory response to a level many times
greater than levels found under normal conditions. Acute phase
proteins are reviewed by Steel & Whitehead (Immunology Today
15: 81-87, 1994).
[0005] One of the massively induced acute phase proteins is Serum
Amyloid A (SAA). SAA actually comprises a family of polymorphic
proteins encoded by many genes in a number of mammalian species.
SAAs are small apolipoproteins that accumulate and associate
rapidly with high-density lipoprotein 3 (HDL3) during the acute
phase of the inflammatory response. Most SAA isoforms are induced
in response to inflammation; however, certain SAAs (e.g., human
SAA4) appear to be constitutively expressed or minimally induced in
the inflammatory response.
[0006] The liver has been considered the primary site of SAA
production. However, SAA production outside the liver has been
found, on a limited basis. For instance, expression of SAA mRNA has
been reported in human atherosclerotic lesions and in human
cultured smooth muscle cells and monocyte/macrophage cell lines
(Meek et al., 1994; Urieli-Shoval et al., 1994; Yamada et al.,
1996), and a unique isoform of SAA (SAA3) is produced by rabbit
synovial fibroblasts (Mitchell et al., J. Clin. Invest. 87:
1177-1185, 1991). More recently, it was discovered that SAA mRNA is
widely expressed in many histologically normal epithelial tissues,
including tissues of stomach, intestine, tonsil, breast, prostate,
thyroid, lung, pancreas, kidney, skin and brain neurons
(Urieli-Shoval et al., J. Histochem. Cytochem. 46: 1377-1384,
1998). The role of SAA in such tissues has not been elucidated, nor
has it been determined if the SAAs present in those tissues are the
same isoforms as those found in serum, or if they represent
additional isoforms of SAA.
SUMMARY OF THE INVENTION
[0007] According to one aspect of the invention, a Serum Amyloid A
(SAA) protein is provided, which is isolated and purified from
mammalian colostrum and is produced by ductal epithelial cells of
the mammary gland. In one embodiment, the SAA is isolated and
purified from horse colostrum. Preferably, the horse colostrum SAA
comprises (SEQ ID NO:3) or (SEQ ID NO:4) and one or more of a
sequence selected from the group consisting of (SEQ ID NO:5), (SEQ
ID NO:6), (SEQ ID NO:7) and (SEQ ID NO:8) See FIG. 1.
[0008] In another embodiment, the SAA is isolated and purified from
cow colostrum, and preferably comprises an N-terminal amino acid
sequence which is (SEQ ID NO:1).
[0009] Alternatively, the SAA is isolated and purified from sheep
colostrum and preferably comprises an N-terminal amino acid
sequence which is (SEQ ID NO:2). Further, an amino acid region from
the N-terminal sequence of colostrum SAA has been shown to be
preserved among most species and contains the active portion of the
molecule for several of the important properties of the
molecule.
[0010] According to another aspect of the invention, an isolated
nucleic acid molecule that encodes a mammalian colostrum SAA is
provided. The nucleic acid molecule may be a gene, cDNA or RNA and
may be single-stranded or double stranded. In a preferred
embodiment, the nucleic acid molecule comprises a sequence that
encodes one or more of (SEQ ID NO: 1-8), or their conservatively
modified variants. In a most preferred embodiment the nucleic acid
molecule comprises (SEQ ID NO:12) See FIG. 2 or its conservatively
modified variants, including other colostrum SAA sequences, a
similarly identified sequence, or any other nucleic acid sequence
which encodes a colostrum associated SAA as described in the
teachings herein.
[0011] Further according to the invention the genomic sequence for
mammalian bovine colostrum associated SAA has been determined
including the intron regions and the flanking region. SEQ ID NO:31.
Thus the invention also includes a nucleotide sequence which
encodes colostrum associated SAA which includes one or more of the
native noncoding or intron regions or their conservatively modified
variants. According to another aspect of the invention, a
population of synthetic oligonucleotides is provided, which
includes sequences obtained by back-translating one or more of
amino acid SEQ ID NOS: 1-8. One or more members of this population
of oligonucleotides specifically hybridizes to a gene or cDNA that
encodes a colostrum SAA.
[0012] According to another aspect of the invention, antibodies
immunologically specific for one or more epitopes of colostrum SAA
are provided. Preferably, the antibodies are immunologically
specific for at least one epitope of the colostrum SAA that
distinguishes colostrum SAA from serum SAA.
[0013] According to another aspect of the invention, a process is
provided for obtaining SAA from a mammal. The process comprises the
steps of: (1) providing a sample of colostrum from the mammal, and
(2) separating SAA contained in the sample from other materials
contained in the sample, thereby obtaining the SAA from the mammal.
SAA produced by this process is also provided.
[0014] According to the invention the novel colostrum associated
SAA particularly the highly conserved region, (TFLK) also has an
ability to stimulate mucin 3 (MUC3) production. Thus colostrum
associated SAA may be used to treat and prevent enteric infections
or other mucin inhibited disease states, such as traveler's
diarrhea, infant diarrhea, necrotizing enterocolitis, urinary
infections, and provide veterinary medicine for prevention of
diarrhea in herd animals. The invention thus includes
pharmaceutical compositions comprising a pharmaceutically effective
amount of colostrum associated SAA peptide and a carrier to treat
these and other diseases with similar pathology. Finally, other
epithelial cell linings of mucosal surfaces such as nasopharynx,
bladder etc., which produce mucins may also be treated with the
pharmaceutical compositions of the invention to stimulate mucin
production to prevent or treat infections associated therewith.
[0015] In yet another embodiment, the specificity of the promoter
can be used to stimulate SAA production to aid in treatment of
diseases associated with the teats or other mammary tissue of
animals. For example, the colostrum associated SAA promoter is
induced by prolactin. Thus one could administer prolactin or other
colostrum SAA inducing agent to stimulate its production and cause
increased MAA at the mammary tissue of said animal. Other features
and advantages of the present invention will be better understood
by reference to the drawings, detailed descriptions and examples
that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. N-terminal amino acid sequence alignment of tryptic
fragments of cow (SEQ ID NO:1), sheep (SEQ ID NO:2) and horse (SEQ
ID NO:3) colostrum SAA, tryptic digest fragments of horse colostrum
SAA (SEQ ID NOS: 4-8), rabbit synovial fibroblast SAA3 (SEQ ID
NO:9); horse serum SAA, (SEQ ID NO:10); and mink serum SAA1, (SEQ
ID NO:11).
[0017] FIG. 2. Nucleotide and complete deduced amino acid sequences
of bovine colostrum associated SAA cDNA. The. nucleotide position
is indicated on the right. The predicted amino acid sequence is
shown in single-letter code above the coding sequence, the stop
codon is indicated with an asterisk, and the presumed signal
peptide cleavage site to remove the leader sequence is denoted by
an inverted triangle. Sequences determined by Edman degradation of
the purified colostrum associated SAA protein are double
underlined. Residues back-translated for the initial degenerate
oligonucleotide primers used in the PCR amplification of the
colostrum associated SAA 300 bp cDNA sequence are italicized.
Oligonucleotide primers M5GW2 and M3GW2 used in the PCR
amplification of the 5' and 3' regions of the colostrum associated
SAA cDNA sequence, respectively, are underlined.
[0018] FIG. 3. This graph shows the production of colostrum-SAA by
the MAC-T bovine epithelial cells over a period of eight days after
they were stimulated with prolactin. Measurable quantities of
colostrum-SAA could be detected by day two.
[0019] FIG. 4. SDS-PAGE 12% gel of the different fractions
generated in the affinity purification of colostrum-SAA. The
fractions are as follows: original culture fluid (lane 1),
non-bound (lane 2), wash fractions (lanes 3-8) and eluted fractions
(lanes 9-15). Blot (A.) is stained with CCB and (B.) the identical
blot stained with a biotinylated anti-SAA monoclonal antibody. Lane
11 contains the colostrum-SAA as identified with the monoclonal
antibody. This is the fraction that was used for amino acid
sequence analysis and for IEF.
[0020] FIG. 5. The listing of the amino acids as determined by
sequence analysis. The major sequence contains the N-terminal
sequence for colostrum-SAA.
[0021] FIG. 6. These blots represent 2-D gels from the isoelectric
focusing of MAC-T cell fluid (A. and B.), bovine serum (C.) and
bovine colostrum (D.). FIG. 6A. represents the blot of the 2-D gel
which was obtained as a second dimension from the pH 3-10 IPG strip
which was used for the isoelectric focusing of the affinity
purified MAC-T cell fluid along with the standards. The blot has
been stained with CBB so that all of the proteins are stained. The
isoelectric point (pI) of some of the commercial standards as well
as the approximate apparent pI of the colostrum SAA (pI range
9.4-9.6) have been noted on the figure. This agrees with the
predicted pI of 9.6 for colostrum SAA using the compute pI/Mw tool
of ExPASy Proteomic Server of the Swiss Institute of
Bioinformatics. FIG. 6B. is the identical blot, but this has been
stained with the monoclonal antibody with specificity to SAA. The
only spot that stains is the one corresponding to the colostrum SAA
and this spot matches the spot observed on the CBB stained blot.
FIG. 6C. is the monoclonal antibody stained blot from the analysis
of the semi-pure bovine serum. Three isoforms with the approximate
apparent pI values of 7.0, 5.8 and 5.5 stain with the monoclonal
antibody with specificity to SAA. FIG. D. is the monoclonal
antibody stained blot of the 2-D gel from affinity purified bovine
colostrum. Only the spot that corresponds to colostrum-SAA at the
approximate apparent pI in the range of 9.4-9.6 stains with the
monoclonal antibody. MAC-T generated SAA has pI of 9.4-9.6. Thus
both the MAC-T culture fluid and the colostrum contain
colostrum-SAA since the spots identified with the monoclonal
antibody have the identical pI and molecular weight (12 Kda) and
this value is significantly different from the pI of any of the
isoforms found in the serum.
[0022] FIG. 7. (N-Terminal Peptide Titration Mucin 3 (MUC3)). FIG.
7 depicts the results of an assay measuring MUC3 specific mRNA. It
shows that the N-Terminal 10 amino acid, bovine Colostrum-SAA
peptide containing TFLK stimulated the production of MUC3 mRNA up
to 1-1/2 times that of base line control levels (significance of
P<0.0002). The ideal concentration was 50 .mu.g/ml medium.
[0023] FIG. 8. (Mucin 3 (MUC3) Stimulation). FIG. 8 is a graph
showing a comparison of MUC3 stimulating activity of the N-terminal
10 amino acid bovine colostrum-SAA peptide was compared activity of
the "Limited Scramble", the "Total Scramble" and "C-Terminal
peptides" on cells. FIG. 8 shows that the original N-Terminal
peptide was the only peptide that stimulated MUC3 mRNA
statistically significantly over the control values
(p<0.008).
[0024] FIG. 9. (Mucin 2 (MUC2) Stimulation). FIG. 9 is a graph of
MUC2 production under culture conditions favoring MUC2 expression,
none of the peptides stimulated the production of MUC2 mRNA
including the N-Terminal 10 amino acid peptide that stimulated MUC3
production. None of the experimental values were significantly
different from control values.
[0025] FIG. 10. FIG. 10 is the genomic bovine colostrum associated
SAA sequence comprising the introns, and flanking region of the
gene. (SEQ ID NO:13). The TATA box is double underlined in the
promoter region for bovine mammary-associated serum amyloid A 3
(M-SAA3, MAA). The additional three upstream nucleotides (single
underlined) from the TATA box are also conserved in most "milk
protein" TATA boxes. Putative consensus STAT5 DNA-binding sites at
-1741 to -1749 and at -2184 to -2193 are italicized in bold type
and denoted above the sequence. Other possible transcription factor
DNA-binding sites are underlined and the corresponding
transcription factor for this consensus sequence is identified
above the sequence (MatInspector V2.2/TRANSFAC 4.0, Quandt et al.,
1995). The transcriptional start site is underlined and indicated
above the nucleotide with +1. The beginning and ending of the three
introns are denoted with an arrow above these regions. The start
and stop codons are underlined and indicated above the nucleotides.
The encoded amino acids are denoted under the double-stranded DNA
sequence. The presumed signal peptide cleavage site to remove the
leader sequence predicted by the SignalP program (Version 1.1)
(Nielsen et al., 1997) with 100% certainty is denoted by an
inverted triangle. The polyadenylation signal (-6307 to -6312) is
underlined and the probable site for cleavage and polyadenylation
is indicated with a double arrow. sequence data.
[0026] FIG. 11. is a graph showing the production of colostrum-SAA
by the MAC T bovine epithelial cells over a periods of 13 days of
stimulation with different levels of lipopolysaccharide (LPS).
Measurable quantities of colostrum-SAA could be detected by day 1
when the cells were stimulated with LPS at 5 .mu.g/ml.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0027] Various terms relating to the compositions and methods of
the present invention are used herein above and also throughout the
specification and claims.
[0028] Units, prefixes, and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxy orientation,
respectively. Numeric ranges are inclusive of the numbers defining
the range and include each integer within the defined range. Amino
acids may be referred to herein by either their commonly known
three letter symbols or by the one-letter symbols recommended by
the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted
single-letter codes. Unless otherwise provided for, software,
electrical, and electronics terms as used herein are as defined in
The New IEEE Standard Dictionary of Electrical and Electronics
Terms (5.sup.th edition, 1993). The terms defined below are more
fully defined by reference to the specification as a whole.
[0029] By "amplified" is meant the construction of multiple copies
of a nucleic acid sequence or multiple copies complementary to the
nucleic acid sequence using at least one of the nucleic acid
sequences as a template. Amplification systems include the
polymerase chain reaction (PCR) system, ligase chain reaction (LCR)
system, nucleic acid sequence based amplification (NASBA, Canteen,
Mississauga, Ontario), Q-Beta Replicase systems,
transcription-based amplification system (TAS), and strand
displacement amplification (SDA). See, e.g., Diagnostic Molecular
Microbiology: Principles and Applications, D. H. Persing et al.,
Ed., American Society for Microbiology, Washington, D.C. (1993).
The product of amplification is termed an amplicon.
[0030] The term "antibody" includes reference to antigen binding
forms of antibodies (e.g., Fab, F(ab).sub.2). The term "antibody"
frequently refers to a polypeptide substantially encoded by an
immunoglobulin gene or immunoglobulin genes, or fragments thereof
which specifically bind and recognize an analyte (antigen).
However, while various antibody fragments can be defined in terms
of the digestion of an intact antibody, one of skill will
appreciate that such fragments may be synthesized de novo either
chemically or by utilizing recombinant DNA methodology. Thus, the
term antibody, as used herein, also includes antibody fragments
such as single chain Fv, chimeric antibodies (i.e., comprising
constant and variable regions from different species), humanized
antibodies (i.e., comprising a complementarity determining region
(CDR) from a non-human source) and heteroconjugate antibodies
(e.g., bispecific antibodies).
[0031] As used herein, "antisense orientation" includes reference
to a duplex polynucleotide sequence that is operably linked to a
promoter in an orientation where the antisense strand is
transcribed. The antisense strand is sufficiently complementary to
an endogenous transcription product such that translation of the
endogenous transcription product is often inhibited.
[0032] As used herein, "colostrum associated serum amyloid A",
"colostrum associated SAA" and/or "colostrum SAA" are used
interchangeably and include but is not limited to the sequences
disclosed herein, their conservatively modified variants,
regardless of source and any other variants which retain the
biological properties of the colostrum SAA and as demonstrated by
the assays disclosed herein.
[0033] As used herein, "chromosomal region" includes reference to a
length of a chromosome that may be measured by reference to the
linear segment of DNA that it comprises. The chromosomal region can
be defined by reference to two unique DNA sequences, i.e.,
markers.
[0034] The term "conservatively modified variants" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refers to
those nucleic acids which encode identical or conservatively
modified variants of the amino acid sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations" and represent one species
of conservatively modified variation. Every nucleic acid sequence
herein that encodes a polypeptide also, by reference to the genetic
code, describes every possible silent variation of the nucleic
acid. One of ordinary skill will recognize that each codon in a
nucleic acid (except AUG, which is ordinarily the only codon for
methionine; and UGG, which is ordinarily the only codon for
tryptophan) can be modified to yield a functionally identical
molecule. Accordingly, each silent variation of a nucleic acid
which encodes a polypeptide of the present invention is implicit in
each described polypeptide sequence and is within the scope of the
present invention.
[0035] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Thus, any number of amino acid
residues selected from the group of integers consisting of from 1
to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10
alterations can be made. Conservatively modified variants typically
provide similar biological activity as the unmodified polypeptide
sequence from which they are derived. For example, substrate
specificity, enzyme activity, or ligand/receptor binding is
generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the
native protein for its native substrate. Conservative substitution
tables providing functionally similar amino acids are well known in
the art.
[0036] The following six groups each contain amino acids that are
conservative substitutions for one another: [0037] 1) Alanine (A),
Serine (S), Threonine (T); [0038] 2) Aspartic acid (D), Glutamic
acid (E); [0039] 3) Asparagine (N), Glutamine (Q); [0040] 4)
Arginine (R), Lysine (K); [0041] 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (V); and [0042] 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W). See also, Creighton (1984) Proteins
W. H. Freeman and Company.
[0043] By "encoding" or "encoded", with respect to a specified
nucleic acid, is meant comprising the information for translation
into the specified protein. A nucleic acid encoding a protein may
comprise non-translated sequences (e.g., introns) within translated
regions of the nucleic acid, or may lack such intervening
non-translated sequences (e.g., as in cDNA). The information by
which a protein is encoded is specified by the use of codons.
Typically, the amino acid sequence is encoded by the nucleic acid
using the "universal" genetic code. However, variants of the
universal code, such as are present in some plant, animal, and
fungal mitochondria, the bacterium Mycoplasma capricolum, or the
ciliate Macronucleus, may be used when the nucleic acid is
expressed therein.
[0044] When the nucleic acid is prepared or altered synthetically,
advantage can be taken of known codon preferences of the intended
host where the nucleic acid is to be expressed. For example,
although nucleic acid sequences of the present invention may be
expressed in both monocotyledonous and dicotyledonous plant
species, sequences can be modified to account for the specific
codon preferences and GC content preferences of monocotyledons or
dicotyledons as these preferences have been shown to differ (Murray
et al. Nucl. Acids Res. 17:477-498 (1989)). Thus, the maize
preferred codon for a particular amino acid may be derived from
known gene sequences from maize. Maize codon usage for 28 genes
from maize plants are listed in Table 4 of Murray et al.,
supra.
[0045] As used herein "full-length sequence" in reference to a
specified polynucleotide or its encoded protein means having the
entire amino acid sequence of, a native (non-synthetic),
endogenous, biologically active form of the specified protein.
Methods to determine whether a sequence is full-length are well
known in the art including such exemplary techniques as northern or
western blots, primer extensions, S1 protection, and ribonuclease
protection. See, e.g., Plant Molecular Biology: A Laboratory
Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to
known full-length homologous (orthologous and/or paralogous)
sequences can also be used to identify full-length sequences of the
present invention. Additionally, consensus sequences typically
present at the 5' and 3' untranslated regions of mRNA aid in the
identification of a polynucleotide as full-length. For example, the
consensus sequence ANNNNAUGG, where the underlined codon represents
the N-terminal methionine, aids in determining whether the
polynucleotide has a complete 5' end. Consensus sequences at the 3'
end, such as polyadenylation sequences, aid in determining whether
the polynucleotide has a complete 3' end.
[0046] With respect to proteins or peptides, the term "isolated
protein (or peptide)" or "isolated and purified protein (or
peptide)" is sometimes used herein. This term may refer to a
protein that has been sufficiently separated from other proteins
with which it would naturally be associated, so as to exist in
"substantially pure" form. Alternatively, this term may refer to a
protein produced by expression of an isolated nucleic acid
molecule.
[0047] With reference to nucleic acid molecules, the term "isolated
nucleic acid" is sometimes used. This term, when applied to DNA,
refers to a DNA molecule that is separated from sequences with
which it is immediately contiguous (in the 5' and 3' directions) in
the naturally occurring genome of the organism from which it was
derived. For example, the "isolated nucleic acid" may comprise a
DNA molecule inserted into a vector, such as a plasmid or virus
vector, or integrated into the genomic DNA of a procaryote or
eucaryote. An "isolated nucleic acid molecule" may also comprise a
cDNA molecule.
[0048] With respect to RNA molecules, the term "isolated nucleic
acid" primarily refers to an RNA molecule encoded by an isolated
DNA molecule as defined above. Alternatively, the term may refer to
an RNA molecule that has been sufficiently separated from RNA
molecules with which it would be associated in its natural state
(i.e., in cells or tissues), such that it exists in a
"substantially pure" form (the term "substantially pure" is defined
below).
[0049] As used herein, "heterologous" in reference to a nucleic
acid is a nucleic acid that originates from a foreign species, or,
if from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. For example, a promoter operably linked to a
heterologous structural gene is from a species different from that
from which the structural gene was derived, or, if from the same
species, one or both are substantially modified from their original
form. A heterologous protein may originate from a foreign species
or, if from the same species, is substantially modified from its
original form by deliberate human intervention.
[0050] By "host cell" is meant a cell which contains a vector and
supports the replication and/or expression of the vector. Host
cells may be prokaryotic cells such as E. coli, or eukaryotic cells
such as yeast, insect, amphibian, or mammalian cells. Preferably,
host cells are monocotyledonous or dicotyledonous plant cells. A
particularly preferred monocotyledonous host cell is a maize host
cell.
[0051] The term "hybridization complex" includes reference to a
duplex nucleic acid structure formed by two single-stranded nucleic
acid sequences selectively hybridized with each other.
[0052] The term "introduced" in the context of inserting a nucleic
acid into a cell, means "transfection" or "transformation" or
"transduction" and includes reference to the incorporation of a
nucleic acid into a eukaryotic or prokaryotic cell where the
nucleic acid may be incorporated into the genome of the cell (e.g.,
chromosome, plasmid, plastid or mitochondrial DNA), converted into
an autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0053] Unless otherwise stated, the term "colostrum associated SAA
encoding nucleic acid" is a nucleic acid of the present invention
and means a nucleic acid comprising a polynucleotide of the present
invention encoding a colostrum associated SAA. A "colostrum
associated SAA gene" is a gene of the present invention and refers
to a heterologous genomic form of a full-length colostrum
associated SAA polynucleotide.
[0054] As used herein, "localized within the chromosomal region
defined by and including" with respect to particular markers
includes reference to a contiguous length of a chromosome delimited
by and including the stated markers.
[0055] As used herein, "marker" includes reference to a locus on a
chromosome that serves to identify a unique position on the
chromosome. A "polymorphic marker" includes reference to a marker
which appears in multiple forms (alleles) such that different forms
of the marker, when they are present in a homologous pair, allow
transmission of each of the chromosomes of that pair to be
followed. A genotype may be defined by use of one or a plurality of
markers.
[0056] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues having the essential nature of natural nucleotides
in that they hybridize to single-stranded nucleic acids in a manner
similar to naturally occurring nucleotides (e.g., peptide nucleic
acids).
[0057] By "nucleic acid library" is meant a collection of isolated
DNA or RNA molecules which comprise and substantially represent the
entire transcribed fraction of a genome of a specified organism.
Construction of exemplary nucleic acid libraries, such as genomic
and cDNA libraries, is taught in standard molecular biology
references such as Berger and Kimmel, Guide to Molecular Cloning
Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc.,
San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning--A
Laboratory Manual, 2.sup.nd ed., Vol. 1-3 (1989); and Current
Protocols in Molecular Biology, F. M. Ausubel et al., Eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc. (1994).
[0058] As used herein, "polynucleotide" includes reference to a
deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof
that have the essential nature of a natural ribonucleotide in that
they hybridize, under stringent hybridization conditions, to
substantially the same nucleotide sequence as naturally occurring
nucleotides and/or allow translation into the same amino acid(s) as
the naturally occurring nucleotide(s). A polynucleotide can be
full-length or a subsequence of a native or heterologous structural
or regulatory gene. Unless otherwise indicated, the term includes
reference to the specified sequence as well as the complementary
sequence thereof. Thus, DNAs or RNAs with backbones modified for
stability or for other reasons as "polynucleotides" as that term is
intended herein. Moreover, DNAs or RNAs comprising unusual bases,
such as inosine, or modified bases, such as tritylated bases, to
name just two examples, are polynucleotides as the term is used
herein. It will be appreciated that a great variety of
modifications have been made to DNA and RNA that serve many useful
purposes known to those of skill in the art. The term
polynucleotide as it is employed herein embraces such chemically,
enzymatically or metabolically modified forms of polynucleotides,
as well as the chemical forms of DNA and RNA characteristic of
viruses and cells, including among other things, simple and complex
cells.
[0059] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers. The essential nature of
such analogues of naturally occurring amino acids is that, when
incorporated into a protein, that protein is specifically reactive
to antibodies elicited to the same protein but consisting entirely
of naturally occurring amino acids. The terms "polypeptide",
"peptide" and "protein" are also inclusive of modifications
including, but not limited to, phosphorylation, glycosylation,
lipid attachment, sulfation, gamma-carboxylation of glutamic acid
residues, hydroxylation and ADP-ribosylation. It will be
appreciated, as is well known and as noted above, that polypeptides
are not entirely linear. For instance, polypeptides may be branched
as a result of ubiquitination, and they may be circular, with or
without branching, generally as a result of posttranslation events,
including natural processing event and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translation natural process and by entirely synthetic methods,
as well. Further, this invention contemplates the use of both the
methionine-containing and the methionine-less amino terminal
variants of the protein of the invention. With respect to a
protein, the term "N-terminal region" shall include approximately
50 amino acids adjacent to the amino terminal end of a protein.
[0060] As used herein "TFLK motif" shall include any formulation
whether by amino acids or otherwise that would maintain the
structural integrity and biological activity of the TFLK active
site of colostrum SAA.
[0061] As used herein "recombinant" includes reference to a cell or
vector, that has been modified by the introduction of a
heterologous nucleic acid or that the cell is derived from a cell
so modified. Thus, for example, recombinant cells express genes
that are not found in identical form within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under-expressed or not expressed at
all as a result of deliberate human intervention. The term
"recombinant" as used herein does not encompass the alteration of
the cell or vector by naturally occurring events (e.g., spontaneous
mutation, natural transformation/transduction/transposition) such
as those occurring without deliberate human intervention.
[0062] As used herein, a "recombinant expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically,
with a series of specified nucleic acid elements which permit
transcription of a particular nucleic acid in a host cell. The
recombinant expression cassette can be incorporated into a plasmid,
chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid
fragment. Typically, the recombinant expression cassette portion of
an expression vector includes, among other sequences, a nucleic
acid to be transcribed, and a promoter.
[0063] The term "residue" or "amino acid residue" or "amino acid"
are used interchangeably herein to refer to an amino acid that is
incorporated into a protein, polypeptide, or peptide (collectively
"protein"). The amino acid may be a naturally occurring amino acid
and, unless otherwise limited, may encompass non-natural analogs of
natural amino acids that can function in a similar manner as
naturally occurring amino acids.
[0064] The term "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a probe
will hybridize to its target sequence, to a detectably greater
degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and may be
different in different circumstances. By controlling the stringency
of the hybridization and/or washing conditions, target sequences
can be identified which are 100% complementary to the probe
(homologous probing). Alternatively, stringency conditions can be
adjusted to allow some mismatching in sequences so that lower
degrees of similarity are detected (heterologous probing).
Generally, a probe is less than about 1000 nucleotides in length,
optionally less than 500 nucleotides in length.
[0065] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.5.times. to 1.times.SSC at 55 to 50.degree. C. Exemplary high
stringency conditions include hybridization in 50% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to
65.degree. C.
[0066] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
T.sub.m can be approximated from the equation of Meinkoth and Wahl,
Anal. Biochem., 138:267-284 (1984): T.sub.m=81.5.degree.
C.+16.6(log M)+0.41(%GC)-0.61(%form)-500/L; where M is the molarity
of monovalent cations, %GC is the percentage of guanosine and
cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the complementary target
sequence hybridizes to a perfectly matched probe. T.sub.m is
reduced by about 1.degree. C. for each 1% of mismatching; thus,
T.sub.m, hybridization and/or wash conditions can be adjusted to
hybridize to sequences of the desired identity. For example, if
sequences with .gtoreq.90% identity are sought, the T.sub.m can be
decreased 10.degree. C. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence and its complement at a
defined ionic strength and pH. However, severely stringent
conditions can utilize a hybridization and/or wash at 1, 2, 3, or
4.degree. C. lower than the thermal melting point (T.sub.m);
moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting
point (T.sub.m); low stringency conditions can utilize a
hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C.
lower than the thermal melting point (T.sub.m). Using the equation,
hybridization and wash compositions, and desired T.sub.m, those of
ordinary skill will understand that variations in the stringency of
hybridization and/or wash solutions are inherently described. If
the desired degree of mismatching results in a T.sub.m of less than
45.degree. C. (aqueous solution) or 32.degree. C. (formamide
solution) it is preferred to increase the SSC concentration so that
a higher temperature can be used. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Laboratory
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Acids Probes, Part I, Chapter 2, Ausubel, et al.,
Eds., Greene Publishing and Wiley-Interscience, New York
(1995).
[0067] The term "substantially pure" refers to a preparation
comprising at least 50-60% by weight the compound of interest
(e.g., nucleic acid, oligonucleotide, protein, etc.). More
preferably, the preparation comprises at least 75% by weight, and
most preferably 90-99% by weight, the compound of interest. Purity
is measured by methods appropriate for the compound of interest
(e.g. chromatographic methods, agarose or polyacrylamide gel
electrophoresis, HPLC analysis, and the like).
[0068] Nucleic acid sequences and amino acid sequences can be
compared using computer programs that align the similar sequences
of the nucleic or amino acids thus define the differences. The
BLAST programs (NCBI) and parameters used therein are used by many
practitioners to align amino acid sequence fragments. However,
equivalent alignments and similarity/identity assessments can be
obtained through the use of any standard alignment software. For
instance, the GCG Wisconsin Package version 9.1, available from the
Genetics Computer Group in Madison, Wis., and the default
parameters used (gap creation penalty=12, gap extension penalty=4)
by Best-Fit program may also be used to compare sequence identity
and similarity.
[0069] The term "substantially the same" refers to nucleic acid or
amino acid sequences having sequence variation that do not
materially affect the nature of the protein (i.e. the structure,
stability characteristics, substrate specificity and/or biological
activity of the protein). With particular reference to nucleic acid
sequences, the term "substantially the same" is intended to refer
to the coding region and to conserved sequences governing
expression, and refers primarily to degenerate codons encoding the
same amino acid, or alternate codons encoding conservative
substitute amino acids in the encoded polypeptide. With reference
to amino acid sequences, the term "substantially the same" refers
generally to conservative substitutions and/or variations in
regions of the polypeptide not involved in determination of
structure or function.
[0070] The terms "percent identical" and "percent similar" are also
used herein in comparisons among amino acid and nucleic acid
sequences. When referring to amino acid sequences, "percent
identical" refers to the percent of the amino acids of the subject
amino acid sequence that have been matched to identical amino acids
in the compared amino acid sequence by a sequence analysis program.
"Percent similar" refers to the percent of the amino acids of the
subject amino acid sequence that have been matched to identical or
conserved amino acids. Conserved amino acids are those which differ
in structure but are similar in physical properties such that the
exchange of one for another would not appreciably change the
tertiary structure of the resulting protein. Conservative
substitutions are defined in Taylor (1986, J. Theor. Biol.
119:205). When referring to nucleic acid molecules, "percent
identical" refers to the percent of the nucleotides of the subject
nucleic acid sequence that have been matched to identical
nucleotides by a sequence analysis program.
[0071] With respect to antibodies, the term "immunologically
specific" refers to antibodies that bind to one or more epitopes of
a protein of interest, but which do not substantially recognize and
bind other molecules in a sample containing a mixed population of
antigenic biological molecules.
[0072] With respect to oligonucleotides or other single-stranded
nucleic acid molecules, the term "specifically hybridizing" refers
to the association between two single-stranded nucleic acid
molecules of sufficiently complementary sequence to permit such
hybridization under pre-determined conditions generally used in the
art i.e., conditions of stringency (sometimes termed "substantially
complementary"). In particular, the term refers to hybridization of
an oligonucleotide with a substantially complementary sequence
contained within a single-stranded DNA or RNA molecule, to the
substantial exclusion of hybridization of the oligonucleotide with
single-stranded nucleic acids of non-complementary sequence.
[0073] A "coding sequence" or "coding region" refers to a nucleic
acid molecule having sequence information necessary to produce a
gene product, when the sequence is expressed.
[0074] The term "operably linked" or "operably inserted" means that
the regulatory sequences necessary for expression of the coding
sequence are placed in a nucleic acid molecule in the appropriate
positions relative to the coding sequence so as to enable
expression of the coding sequence. This same definition is
sometimes applied to the arrangement other transcription control
elements (e.g. enhancers) in an expression vector.
[0075] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell.
[0076] The terms "promoter", "promoter region" or "promoter
sequence" refer generally to transcriptional regulatory regions of
a gene, which may be found at the 5' or 3' side of the coding
region, or within the coding region, or within introns. Typically,
a promoter is a DNA regulatory region capable of binding RNA
polymerase in a cell and initiating transcription of a downstream
(3' direction) coding sequence. The typical 5' promoter sequence is
bounded at its 3' terminus by the transcription initiation site and
extends upstream (5' direction) to include the minimum number of
bases or elements necessary to initiate transcription at levels
detectable above background. Within the promoter sequence is a
transcription initiation site (conveniently defined by mapping with
nuclease S1), as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase.
[0077] A "vector" is a replicon, such as plasmid, phage, cosmid, or
virus to which another nucleic acid segment may be operably
inserted so as to bring about the replication or expression of the
segment.
[0078] The term "nucleic acid construct" or "DNA construct" is
sometimes used to refer to a coding sequence or sequences operably
linked to appropriate regulatory sequences and inserted into a
vector for transforming a cell. This term may be used
interchangeably with the term "transforming DNA". Such a nucleic
acid construct may contain a coding sequence for a gene product of
interest, along with a selectable marker gene and/or a reporter
gene.
[0079] The term "selectable marker gene" refers to a gene encoding
a product that, when expressed, confers a selectable phenotype such
as antibiotic resistance on a transformed cell.
[0080] The term "reporter gene" refers to a gene that encodes a
product which is easily detectable by standard methods, either
directly or indirectly.
[0081] A "heterologous" region of a nucleic acid construct is an
identifiable segment (or segments) of the nucleic acid molecule
within a larger molecule that is not found in association with the
larger molecule in nature. Thus, when the heterologous region
encodes a mammalian gene, the gene will usually be flanked by DNA
that does not flank the mammalian genomic DNA in the genome of the
source organism. In another example, a heterologous region is a
construct where the coding sequence itself is not found in nature
(e.g., a cDNA where the genomic coding sequence contains introns,
or synthetic sequences having codons different than the native
gene). Allelic variations or naturally-occurring mutational events
do not give rise to a heterologous region of DNA as defined herein.
The term "DNA construct", as defined above, is also used to refer
to a heterologous region, particularly one constructed for use in
transformation of a cell.
[0082] A cell has been "transformed" or "transfected" by exogenous
or heterologous DNA when such DNA has been introduced inside the
cell. The transforming DNA may or may not be integrated (covalently
linked) into the genome of the cell. In procaryotes, yeast, and
mammalian cells for example, the transforming DNA may be maintained
on an episomal element such as a plasmid. With respect to
eucaryotic cells, a stably transformed cell is one in which the
transforming DNA has become integrated into a chromosome so that it
is inherited by daughter cells through chromosome replication. This
stability is demonstrated by the ability of the eucaryotic cell to
establish cell lines or clones comprised of a population of
daughter cells containing the transforming DNA.
[0083] A "clone" is a population of cells derived from a single
cell or common ancestor by mitosis. A "cell line" is a clone of a
primary cell that is capable of stable growth in vitro for many
generations.
II. Description
[0084] Serum amyloid A (SAA) is an acute phase protein which is
produced in the liver and occurs at elevated levels in the serum of
mammals as part of the inflammatory response related to tissue
injury or infection. The inventors have discovered a unique isoform
of SAA that occurs at highly elevated levels in colostrum. Elevated
colostrum SAA in healthy cows returns to background levels found in
milk within four days after calving. Colostrum SAA by believed to
be produced locally (i.e., in mammary ductal epithelial cells), as
it in colostrum occurs independently of the blood concentration of
acute phase SAA (A-SAA) (in samples of colostrum, whey and serum
taken from five test cows, serum SAA was found to be in the range
of 15 .mu.g/ml, while in colostrum, SAA was elevated to levels in
the average range of 300 .mu.g/ml).
[0085] The unique isoform of SAA in colostrum may be fulfilling a
variety of functions relating to the general role of colostram in
the development of neonatal immunity. For instance, colostrum SAA
may act as vehicle for transport of lipids and immunoglobulins
across the endothelial membranes of gut and/or vasculature in the
newborn. It may be produced locally by the vascular endothelium
after injury, and serve as a vehicle for transport of
immunoglobulins into the intravascular space. In general, colostrum
SAA is likely to have anti-microbial activity (either directly or
indirectly) and to regulate the immune response in some manner.
Colostrum SAA also may be involved in tissue remodeling by inducing
enzymes involved in tissue repair and degradation, and by
regulating production of protective mucins in mucosal tissue.
[0086] An elevated level of SAA has been detected in the colostrum
of cows, horses, sheep and pigs. It has been purified from cow,
sheep and horse colostrum, using methods such as those described in
Example 3. Purified colostrum SAA from these sources was subjected
to N-terminal amino acid sequence analysis. These sequences are set
forth below, compared with the corresponding sequence of SAA3 from
rabbit synovial fibroblasts. TABLE-US-00001 Colostrum: Cow:
MWXTFLKEAGQGAKDMWRAY (SEQ ID NO:1) Sheep: WLLTFLKLEAG (SEQ ID NO:2)
Horse: RELKTFLKEAGQG (SEQ ID NO:4) Synovial fibroblasts: Rabbit
SAA3: REWLTFLKEAGQGAKDMWRAYSDMKEA (Part of SEQ ID NO:9)
[0087] The colostrum-derived SAAs share a unique amino acid
sequence in the amino-terminal (or N-terminal) end TFLK (TFLK
motif). The TFLK motif is not found in any of the serum-derived
SAAs from any mammal, but does share homology with SAA3 produced by
rabbit synovial fibroblasts. The human SAA3 pseudogene (not
expressed in serum or tissue) also comprises a deduced amino acid
sequence that contains the TFLK sequence motif.
[0088] Further analysis of tryptic digest fragments of horse
colostrum SAA revealed, however, that colostrum SAA is indeed a
unique SAA. Sequences of the tryptic fragments of horse colostrum
SAA are set forth below (SEQ ID NO: 4 is an alternate of SEQ ID
NO:3). TABLE-US-00002 REWFTFL (SEQ ID NO:3) EANYIGADKYFH (SEQ ID
NO:5) GNYDAAQRGPGGA (SEQ ID NO:6) VTDLFK (SEQ ID NO:7)
SGKDPNHFRPHGLPDKY (SEQ ID NO:8)
[0089] In FIG. 1, the five horse colostrum SAA tryptic fragment
sequences(SEQ ID NOS: 3-8) and the N-terminal sequences from cow
and sheep and horse colostrum SAA (SEQ ID NOS: 1-3) are shown in
alignment with the complete amino acid sequences of synovial
fibroblast SAA3 from rabbit (SEQ ID NO:9), horse serum SAA (SEQ ID
NO:10) and serum SAA1 from mink (SEQ ID NO:11). As can be seen from
the alignment, horse colostrum SAA shares regions of similarity
with each of rabbit synovial fibroblast SAA3, horse serum SAA and
mink serum SAA1, yet is distinct from each of these proteins.
[0090] Thus, the present invention provides a novel SAA isoform
isolated from colostrum. Although the horse, cow and sheep
colostrum SAAs are exemplified herein, the present invention
includes the colostrum SAA isoform from any mammalian species,
inasmuch as the present inventors have identified colostrum SAAs
from several mammalian species. Furthermore, as described in
greater detail below, nucleic acid molecules encoding colostrum
SAAs are also contemplated as being part of the present invention,
as are antibodies immunologically specific for this novel SAA
isoform.
[0091] According to the invention the cDNA of bovine colostrum
associated SAA has been determined making possible the production
of recombinant forms of the protein by methods known in the art and
disclosed herein. (SEQ ID NO:12) See FIG. 2, is the cDNA sequence
for the protein and the invention comprises this sequence as well
as conservatively modified variants. Further disclosed is the
active region of the protein which is highly conserved, as well as
the properties of the protein which make it useful for assays to
detect inflammation associated with mastitis or for pharmaceutical
preparations for treating gastrointestinal disorders by stimulating
mucin production.
[0092] Also according to the invention the genomic sequence of
bovine colostrum associated SAA has been determined including the
3' and 5' untranslated regions as well as several intron regions.
These noncoding genomic sequences are often required to obtain the
highest expression of the coding regions when using and expression
construct to obtain recombinant protein. Thus as disclosed herein
the invention comprises a nucleotide sequence encoding bovine
colostrum associated SAA that includes portions of noncoding
sequences as disclosed herein. Preferably these non coding regions
comprise those adjacent to exons and more preferably comprise from
about at least about 50 contiguous bases to maximize expression.
Even more preferred the entire no coding region or its
conservatively modified variant may be used. These noncoding
regions include bases 1-647, 738-2982, 3121-3510, 3674-4984 or
there conservatively modified variants of SEQ ID NO:31.
[0093] The following description sets forth the general procedures
involved in practicing the present invention. To the extent that
specific materials are mentioned, it is merely for purposes of
illustration and is not intended to limit the invention. Unless
otherwise specified, general cloning procedures, such as those set
forth in Sambrook et al., Molecular Cloning, Cold Spring Harbor
Laboratory (1989) (hereinafter "Sambrook et al.") or Ausubel et al.
(eds) Current Protocols in Molecular Biology, John Wiley & Sons
(1999) (hereinafter "Ausubel et al." are used.
A. Preparation of Colostrum SAA, Antibodies Specific for Colostrum
SAA and Nucleic Acid Molecules Encoding Colostrum SAA
1. Proteins and Antibodies
[0094] Colostrum SAA may be prepared in a variety of ways,
according to a variety of methods that have been developed for
purifying SAA from serum. One such method is set forth in Example
3. Variations in hydrophobic chromatography matrix systems and
eluants also may be employed, such as those described by Smith et
al. (Protein Expression & Purification 2: 158-161, 1991).
[0095] Alternatively, the availability of amino acid sequence
information, such as (SEQ ID NOS: 1-8), enables the isolation of
nucleic acid molecules encoding colostrum SAA. This may be
accomplished using anti-colostrum SAA antibodies to screen a cDNA
expression library from a selected species, according to methods
well known in the art. Alternatively, a series of degenerate
oligonucleotide probes encoding parts or all of (SEQ ID NOS: 1-8)
FIG. 1 may be used to screen cDNA or genomic libraries, as
described in greater detail below.
[0096] Once obtained, a cDNA or gene may be cloned into an
appropriate in vitro transcription vector, such a pSP64 or pSP65
for in vitro transcription, followed by cell-free translation in a
suitable cell-free translation system, such as wheat germ or rabbit
reticulocytes. In vitro transcription and translation systems are
commercially available, e.g., from Promega Biotech, Madison, Wis.
or BRL, Rockville, Md. The pCITE in vitro translation system
(Novagen) also may be utilized.
[0097] According to a preferred embodiment, larger quantities of
the proteins may be produced by expression in a suitable
procaryotic or eucaryotic system. For example, part or all of a
colostrum SAA-encoding DNA molecule may be inserted into a vector
adapted for expression in a bacterial cell (such as E. coli) or a
yeast cell (such as Saccharomyces cerevisiae), or a mammalian cell.
Such vectors comprise the regulatory elements necessary for
expression of the DNA in the host cell, positioned in such a manner
as to permit expression of the DNA in the host cell. Such
regulatory elements required for expression include promoter
sequences, transcription initiation sequences and, optionally,
enhancer sequences.
[0098] Colostrum SAA produced by gene expression in a recombinant
procaryotic or eukaryotic system may be purified according to
methods known in the art. In a preferred embodiment, a commercially
available expression/secretion system can be used, whereby the
recombinant protein is expressed and thereafter secreted from the
host cell, to be easily purified from the surrounding medium. If
expression/secretion vectors are not used, an alternative approach
involves purifying the recombinant protein by affinity separation,
such as by immunological interaction with antibodies that bind
specifically to the recombinant protein. Such methods are commonly
used by skilled practitioners.
[0099] The present invention also provides antibodies capable of
binding to colostrum SAA from one or more selected species.
Polyclonal or monoclonal antibodies directed toward part or all of
a selected colostrum SAA may be prepared according to standard
methods. Monoclonal antibodies may be prepared according to general
methods of Kohler and Milstein, following standard protocols. In a
preferred embodiment, antibodies are prepared, which react
immunospecifically with selected epitopes of colostrum SAA that
distinguish it from other SAAs.
2. Nucleic Acid Molecules
[0100] Once sequence information is obtained, nucleic acid
molecules encoding colostrum SAA may be prepared by two general
methods: (1) they may be synthesized from appropriate nucleotide
triphosphates, or (2) they may be isolated from biological sources.
Both methods utilize protocols well known in the art.
[0101] The availability of nucleotide sequence information enables
preparation of an isolated nucleic acid molecule of the invention
by oligonucleotide synthesis. Synthetic oligonucleotides may be
prepared by the phosphoramadite method employed in the Applied
Biosystems 38A DNA Synthesizer or similar devices. The resultant
construct may be purified according to methods known in the art,
such as high performance liquid chromatography (HPLC). Long,
double-stranded polynucleotides, such as a DNA molecule of the
present invention, must be synthesized in stages, due to the size
limitations inherent in current oligonucleotide synthetic methods.
Thus, for example, a long double-stranded molecule may be
synthesized as several smaller segments of appropriate
complementarity. Complementary segments thus produced may be
annealed such that each segment possesses appropriate cohesive
termini for attachment of an adjacent segment. Adjacent segments
may be ligated by annealing cohesive termini in the presence of DNA
ligase to construct an entire long double-stranded molecule. A
synthetic DNA molecule so constructed may then be cloned and
amplified in an appropriate vector.
[0102] Nucleic acid molecules encoding colostrum SAA also may be
isolated from mammalian species of interest using methods well
known in the art. Nucleic acid molecules from a selected species
may be isolated by screening cDNA or genomic libraries with
oligonucleotides designed to match a nucleic acid sequence specific
to a colostrum SAA-encoding gene. If the gene from a species is
desired, the genomic library is screened. Alternatively, if the
protein coding sequence is of particular interest, the cDNA library
is screened. In positions of degeneracy, where more than one
nucleic acid residue could be used to encode the appropriate amino
acid residue, all the appropriate nucleic acids residues may be
incorporated to create a mixed oligonucleotide population, or a
neutral base such as inosine may be used. The strategy of
oligonucleotide design is well known in the art (see also Sambrook
et al., Molecular Cloning, 1989, Cold Spring Harbor Press, Cold
Spring Harbor N.Y.).
[0103] Alternatively, PCR (polymerase chain reaction) primers may
be designed by the above method to encode a portion of colostrum
SAA protein, and these primers used to amplify nucleic acids from
isolated cDNA or genomic DNA. In a preferred embodiment, the
oligonucleotides used to isolate colostrum SAA-encoding nucleic
acids are designed to encode sequences unique to colostrum SAA, as
opposed to serum SAA.
[0104] In accordance with the present invention, nucleic acids
having the appropriate sequence homology with a colostrum
SAA-encoding nucleic acid molecule may be identified by using
hybridization and washing conditions of appropriate stringency. For
example, hybridizations may be performed, according to the method
of Sambrook et al. (1989, supra), using a hybridization solution
comprising: 5.times.SSC, 5.times. Denhardt's reagent, 1.0% SDS, 100
.mu.g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium
pyrophosphate and up to 50% formamide. Hybridization is carried out
at 37-42.degree. C. for at least six hours. Following
hybridization, filters are washed as follows: (1) 5 minutes at room
temperature in 2.times.SSC and 1% SDS; (2) 15 minutes at room
temperature in 2.times.SSC and 0.1% SDS; (3) 30 minutes-1 hour at
37.degree. C. in 1.times.SSC and 1% SDS; (4) 2 hours at
42-65.degree. in 1.times.SSC and 1% SDS, changing the solution
every 30 minutes.
[0105] One common formula for calculating the stringency conditions
required to achieve hybridization between nucleic acid molecules of
a specified sequence homology (Sambrook et al., 1989, supra):
T.sub.m=81.5.degree. C.+16.6 Log[Na+]+0.41(% G+C)-0.63(%
formamide)-600/#bp in duplex
[0106] As an illustration of the above formula, using [N+]=[0.368]
and 50% formamide, with GC content of 42% and an average probe size
of 200 bases, the T.sub.m is 57.degree. C. The T.sub.m of a DNA
duplex decreases by 1-1.5.degree. C. with every 1% decrease in
homology. Thus, targets with greater than about 75% sequence
identity would be observed using a hybridization temperature of
42.degree. C. In a preferred embodiment, the hybridization is at
37.degree. C. and the final wash is at 42.degree. C., in a more
preferred embodiment the hybridization is at 42.degree. and the
final wash is at 50.degree., and in a most preferred embodiment the
hybridization is at 42.degree. C. and final wash is at 65.degree.
C., with the above hybridization and wash solutions.
[0107] The stringency of the hybridization and wash depend
primarily on the salt concentration and temperature of the
solutions. In general, to maximize the rate of annealing of the
probe with its target, the hybridization is usually carried out at
salt and temperature conditions that are 20-25.degree. C. below the
calculated T.sub.m of the of the hybrid. Wash conditions should be
as stringent as possible for the degree of identity of the probe
for the target. In general, wash conditions are selected to be
approximately 12-20.degree. C. below the T.sub.m of the hybrid. In
regards to the nucleic acids of the current invention, a moderate
stringency hybridization is defined as hybridization in
6.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100
.mu.g/ml denatured salmon sperm DNA at 42.degree. C., and wash in
2.times.SSC and 0.5% SDS at 55.degree. C. for 15 minutes. A high
stringency hybridization is defined as hybridization in
6.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100
.mu.g/ml denatured salmon sperm DNA at 42.degree. C., and wash in
1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes. A very
high stringency hybridization is defined as hybridization in
6.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100
.mu.g/ml denatured salmon sperm DNA at 42.degree. C., and wash in
0.1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes.
[0108] Nucleic acids of the present invention may be maintained as
DNA in any convenient cloning vector. In a preferred embodiment,
clones are maintained in plasmid cloning/expression vector, such as
pGEM-T (Promega Biotech, Madison, Wis.) or pBluescript (Stratagene,
La Jolla, Calif.), either of which is propagated in a suitable E.
coli host cell.
[0109] Colostrum SAA-encoding nucleic acid molecules of the
invention include cDNA, genomic DNA, RNA, and fragments thereof
which may be single- or double-stranded. Thus, this invention
provides oligonucleotides (sense or antisense strands of DNA or
RNA) having sequences capable of hybridizing with at least one
sequence, of a nucleic acid molecule of the present invention. Such
oligonucleotides are useful as probes for detecting colostrum
SAA-encoding genes or mRNA in test samples, e.g. by PCR
amplification.
B. Uses of Colostrum SAA Protein, Antibodies and Nucleic Acids
1. Proteins and Antibodies
[0110] Purified colostrum SAA, or fragments thereof, may be used to
produce polyclonal or monoclonal antibodies which may serve as
sensitive detection reagents for the presence and accumulation of
the proteins in cultured cells or tissues and in intact organisms.
Recombinant techniques enable expression of fusion proteins
containing part or all of a selected colostrum SAA. The full length
protein or fragments of the protein may be used to advantage to
generate an array of monoclonal or polyclonal antibodies specific
for various epitopes of the protein, thereby providing even greater
sensitivity for detection of the protein. In a preferred
embodiment, fragments of colostrum SAA that distinguish colostrum
SAA from serum SAAs are utilized for generating epitope-specific
antibodies.
[0111] Polyclonal or monoclonal antibodies immunologically specific
for colostrum SAA may be used in a variety of assays designed to
detect and quantitate the proteins. Such assays include, but are
not limited to, (1) immunoprecipitation followed by protein
quantification; (2) immunoblot analysis (e.g., dot blot, Western
blot) (3) radioimmune assays, (4) nephelometry, turbidometric or
immunochromatographic (lateral flow) assays, and (5) enzyme-coupled
assays, including ELISA and a variety of qualitative rapid tests
(e.g., dip-stick and similar tests).
[0112] Polyclonal or monoclonal antibodies that immunospecifically
interact with colostrum SAA can be utilized for identifying and
purifying such proteins. For example, antibodies may be utilized
for affinity separation of proteins with which they
immunospecifically interact. Antibodies may also be used to
immunoprecipitate proteins from a sample containing a mixture of
proteins and other biological molecules.
2. Nucleic Acids
[0113] Colostrum SAA-encoding nucleic acids may be used for a
variety of purposes in accordance with the present invention. The
DNA, RNA, or fragments thereof may be used as probes to detect the
presence of and/or expression of the genes. Methods in which
colostrum SAA-encoding nucleic acids may be utilized as probes for
such assays include, but are not limited to: (1) in situ
hybridization; (2) Southern hybridization (3) northern
hybridization; and (4) assorted amplification reactions such as
polymerase chain reactions (PCR) and reverse transcriptase-PCR
(RT-PCR).
[0114] The exemplified colostrum SAA-encoding nucleic acids of the
invention (e.g., cow, sheep, horse) may also be utilized as probes
to identify related genes from other species, including humans. As
is well known in the art and described above, hybridization
stringencies may be adjusted to allow hybridization of nucleic acid
probes with complementary sequences of varying degrees of
homology.
[0115] In addition to the aforementioned uses of colostrum
SAA-encoding nucleic acids, they are expected to be of utility in
the creation of transgenic cells, tissues and organisms.
[0116] The present invention provides novel purified and isolated
nucleic acid sequences encoding bovine colostrum associated SAA
protein. In presently preferred forms, the DNA sequences comprise
cDNA sequences encoding the novel SAA, or its conservatively
modified variants, which are present in colostrum, comprise the
active TFLK region and which possess the biological activity of the
proteins disclosed herein. In a more preferred embodiment the
nucleic acid sequence comprises at least about 93% identity to (SEQ
ID NO:12) or 92% identity of the encoded amino acid sequence.
Specifically, the sequence isolated is depicted in (SEQ ID NO:12).
Alternate DNA forms such as genomic DNA, and DNA prepared by
partial or total chemical synthesis from nucleotides as well as DNA
with deletions or mutations, is also within the contemplated scope
of the invention.
[0117] Further according to the invention genomic bovine colostrum
associated SAA sequences have been characterized and identified.
The genomic region including introns can be used to with the
colostrum SAA sequences and are often necessary to achieve the most
efficient expression of nucleotide sequences.
[0118] Association of DNA sequences provided by the invention with
homologous or heterologous species expression control DNA sequences
such as promoters, operators, regulators, and the like, allows in
vivo and in vitro transcription from mRNA which, in turn, is
susceptible to translation to provide the novel sodium channel
proteins of the invention, and related poly- and oligo-peptides in
large quantities. In a presently preferred DNA expression system of
the invention colostrum associated SAA encoding DNA is operatively
linked to a regulatory promoter DNA sequence allowing for in vitro
transcription and translation of the protein.
[0119] Incorporation of DNA sequences into prokaryotic and
eucaryotic host cells by standard transformation and transfection
processes, potentially involving suitable viral and circular DNA
plasmid vectors, is also within the contemplation of the invention
and is expected to provide useful proteins in quantities heretofore
unavailable from natural sources. Use of mammalian host cells is
expected to provide for such post-translational modifications (e.g.
truncation, glycosylation, and tyrosine, serine, or threonine
phosphorylation) as may be needed to confer optimal biological
activity on recombinant expression products of the invention as
more fully set forth hereinafter.
[0120] Most of the techniques which are used to transform cells,
construct vectors, extract messenger RNA, prepare cDNA libraries,
and the like are widely practiced in the art, and most
practitioners are familiar with the standard resource materials
which describe specific conditions and procedures. However, for
convenience, the following paragraphs may serve as a guideline.
Hosts and Control Sequences
[0121] Both prokaryotic and eucaryotic systems may be used to
express colostrum associated SAA encoding sequences; prokaryotic
hosts are, of course, the most convenient for cloning procedures.
Prokaryotes most frequently are represented by various strains of
E. coli; however, other microbial strains may also be used. Plasmid
vectors which contain replication sites, selectable markers and
control sequences derived from a species compatible with the host
are used; for example, E. coli is typically transformed using
derivatives of pBR322, a plasmid derived from an E. coli species by
Bolivar, et al, Gene (1977) 2:95. pBR322 contains genes for
ampicillin and tetracycline resistance, and thus provides multiple
selectable markers which can be either retained or destroyed in
constructing the desired vector. Commonly used prokaryotic control
sequences which are defined herein to include promoters for
transcription initiation, optionally with an operator, along with
ribosome binding site sequences, include such commonly used
promoters as the beta-lactase (penicillinase) and lactose (lac)
promoter systems (Chang, et al, Nature (1977) 198:1056) and the
tryptophan (trp) promoter system (Goeddel, et al, Nucleic Acids Res
(1980) 8:4057) and the lambda derived P.sub.L promoter and N-gene
ribosome binding site (Shimatake, et al, Nature (1981)
292:128).
[0122] In addition to bacteria, eucaryotic microbes, such as yeast,
may also be used as hosts. Laboratory strains of Saccharomyces
cerevisiae, Baker's yeast, are most used although a number of other
strains or species are commonly available. Vectors employing, for
example, the 2.mu. origin of replication of Broach, J. R., Meth Enz
(1983) 101:307, or other yeast compatible origins of replication
(see, for example, Stinchcomb, et al, Nature (1979) 282:39,
Tschumper, G., et al, Gene (1980) 10:157 and Clarke, L, et al, Meth
Enx (1983) 101:300) may be used. Control sequences for yeast
vectors include promoters for the synthesis of glycolytic enzymes
(Hess, et al, J Adv Enzyme Reg (1968) 7:149; Holland, et al,
Biochemistry (1978) 17:4900). Additional promoters known in the art
include the promoter for 3-phosphoglycerate kinase (Hitzeman, et al
J Biol Chem (1980) 255:2073). Other promoters, which have the
additional advantage of transcription controlled by growth
conditions and/or genetic background are the promoter regions for
alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,
degradative enzymes associated with nitrogen metabolism, the alpha
factor system and enzymes responsible for maltose and galactose
utilization. It is also believed terminator sequences are desirable
at the 3' end of the coding sequences. Such terminators are found
in the 3' untranslated region following the coding sequences in
yeast-derived genes.
[0123] Also according to the invention the promoter sequences
herein may be used with other heterolgous genes the production of
which is desired in a host cell or tissue. The colostrum SAA
promoter can be used as an inducible promoters to cause the
production of operably linked sequences in response to prolactin or
can be used to facilitate temporal and spatial expression of linked
gene sequences to the mammary tissue and excreted in milk of the
transgenic animal. Any gene sequence capable of being expressed in
a host cell may be operably linked to the promoter of the invention
and used in this manner. For example bovine serum albumin could be
operably linked to the colstrum SAA of the invention causing
transgenic BSA to be produced in the milk of said animal.
[0124] It is also, of course, possible to express genes encoding
polypeptides in eucaryotic host cell cultures derived from
multicellular organisms. See, for example, Axel, et al, U.S. Pat.
No. 4,399,216. These systems have the additional advantage of the
ability to splice out introns and thus can be used directly to
express genomic fragments. Useful host cell lines include VERO and
HeLa cells, and Chinese hamster ovary (CHO) cells. Expression
vectors for such cells ordinarily include promoters and control
sequences compatible with mammalian cells such as, for example, the
commonly used early and late promoters from Simian Virus 40 (SV 40)
(Fiers, et al, Nature (1978) 273:113), or other viral promoters
such as those derived from polyoma, Adenovirus 2, bovine papilloma
virus, or avian sarcoma viruses. The controllable promoter, hMT1I
(Karin, M., et al, Nature (1982) 299:797-802) may also be used.
General aspects of mammalian cell host system transformations have
been described by Axel (supra). It now appears, also that
"enhancer" regions are important in optimizing expression; these
are, generally, sequences found upstream or downstream of the
promoter region in non-coding DNA regions. Origins of replication
may be obtained, if needed, from viral sources. However,
integration into the chromosome is a common mechanism for DNA
replication in eucaryotes.
Transformations
[0125] Depending on the host cell used, transformation is done
using standard techniques appropriate to such cells. The calcium
treatment employing calcium chloride, as described by Cohen, S. N.,
Proc Natl Acad Sci (USA) 1972) 69:2110, or the rbCl2 method
described in Maniatis, et al, Molecular Cloning: A Laboratory
Manual (1982) Cold Spring Harbor Press, p. 254 and Hanahan, D., J
Mol Biol (1983) 166:557-580 maybe used for prokaryotes or other
cells which contain substantial cell wall barriers. For mammalian
cells without such cell walls, the calcium phosphate precipitation
method of Graham and van der Eb, Virology (1978) 52:546, optionally
as modified by Wigler, M., et al, Cell (1979) 16:777-785 maybe
used. Transformations into yeast maybe carried out according to the
method of Beggs, J. D. Nature (1978) 275:104-109 or of Hinnen, A.,
et al, Proc Natl Acad Sci (USA) (1978) 75:1929.
Vector Construction
[0126] Construction of suitable vectors containing the desired
coding and control sequences employs standard ligation and
restriction techniques which are well understood in the art.
Isolated plasmids, DNA sequences, or synthesized oligonucleotides
are cleaved, tailored, and relegated in the form desired.
[0127] The DNA sequences which form the vectors are available from
a number of sources. Backbone vectors and control systems are
generally found on available "host" vectors which are used for the
bulk of the sequences in construction. Typical sequences have been
set forth above. For the pertinent coding sequence, initial
construction may be, and usually is, a matter of retrieving the
appropriate sequences from cDNA or genomic DNA libraries. However,
once the sequence is disclosed it is possible to synthesize the
entire gene sequence in vitro starting from the individual
nucleoside derivatives. The entire sequence for genes or cDNA's of
sizable length, e.g., 500-1000 bp may be prepared by synthesizing
individual overlapping complementary oligonucleotides and filling
in single stranded nonoverlapping portions using DNA polymerase in
the presence of the deoxyribonucleotide triphosphates. This
approach has been used successfully in the construction of several
genes of known sequence. See, for example, Edge, M. D., Nature
(1981) 292:756; Nambair, K. P., et al, Science (1984) 223:1299;
Jay, Ernest, J Biol Chem (1984) 259:6311.
[0128] Synthetic oligonucleotides are prepared by either the
phosphotriester method as described by Edge, et al, Nature (supra)
and Duckworth, et al, Nucleic Acids Res (1981) 9:1691 or the
phosphoramidite method as described by Beaucage, S. L., and
Caruthers, M. H., Tet Letts (1981) 22:1859 and Matteucci, M. D.,
and Caruthers, M. H., J Am Chem Soc (1981) 103:3185 and can be
prepared using commercially available automated oligonucleotide
synthesizers. Kinasing of single strands prior to annealing or for
labeling is achieved using an excess, e.g., approximately 10 units
of polynucleotide kinase to 1 nmole substrate in the presence of 50
mM Tris, pH 7.6, 10 mM MgCl.sub.2, 5 mM dithiothreitol, 1-2 mM ATP,
1.7y pmoles .gamma.32P-ATP (2.9 mCi/mmole), 0.1 mM spermidine, 0.1
mM EDTA.
[0129] Once the components of the desired vectors are thus
available, they can be excised and ligated using standard
restriction and ligation procedures.
[0130] Site specific DNA cleavage is performed by treating with the
suitable restriction enzyme (or enzymes) under conditions which are
generally understood in the art, and the particulars of which are
specified by the manufacturer of these commercially available
restriction enzymes. See, e.g., New England Biolabs, Product
Catalog. In general, about 1 .mu.g of plasmid or DNA sequence is
cleaved by one unit of enzyme in about 20 .mu.l of buffer solution;
in the examples herein, typically, an excess of restriction enzyme
is used to insure complete digestion of the DNA substrate.
Incubation times of about one hour to two hours at about 37.degree.
C. are workable, although variations can be tolerated. After each
incubation, protein is removed by extraction with
phenol/chloroform, and may be followed by ether extraction, and the
nucleic acid recovered from aqueous fractions by precipitation with
ethanol. If desired, size separation of the cleaved fragments may
be performed by polyacrylamide gel or agarose gel electrophoresis
using standard techniques. A general description of size
separations is found in Methods in Enzymology (1980)
65:499-560.
[0131] Restriction cleaved fragments may be blunt ended by treating
with the large fragment of E. coli DNA polymerase I (Klenow) in the
presence of the four deoxynucleotide triphosphates (dNTPs) using
incubation times of about 15 to 25 min at 20.degree. to 25.degree.
C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl2, 6 mM DTT and
0.1-1.0 mM dNTPs. The Klenow fragment fills in at 5'
single-stranded overhangs but chews back protruding 3' single
strands, even though the four dNTPs are present. If desired,
selective repair can be performed by supplying only one of the, or
selected, dNTPs within the limitations dictated by the nature of
the overhang. After treatment with Klenow, the mixture is extracted
with phenol/chloroform and ethanol precipitated. Treatment under
appropriate conditions with S1 nuclease or BAL-31 results in
hydrolysis of any single-stranded portion.
[0132] Ligations are performed in 15-50 .mu.l volumes under the
following standard conditions and temperatures: for example, 20 mM
Tris-Cl pH 7.5, 10 mM MgCl.sub.2, 10 mM DTT, 33 .mu.g/ml BSA, 10
mM-50 mM NaCl, and either 40 .mu.M ATP, 0.01-0.02 (Weiss) units T4
DNA ligase at 0 C. (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6
(Weiss) units T4 DNA ligase at 14.degree. C. (for "blunt end"
ligation). Intermolecular "sticky end" ligations are usually
performed at 33-100 .mu.g/ml total DNA concentrations (5-100 nM
total end concentration). Intermolecular blunt end ligations are
performed at 1 .mu.M total ends concentration.
[0133] In vector construction employing "vector fragments", the
vector fragment is commonly treated with bacterial alkaline
phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in
order to remove the 5' phosphate and prevent self-ligation of the
vector. Digestions are conducted at pH 8 in approximately 10 mM
Tris-HCl, 1 mM EDTA using about 1 unit of BAP or CIP per .mu.g of
vector at 60.degree. for about one hour. In order to recover the
nucleic acid fragments, the preparation is extracted with
phenol/chloroform and ethanol precipitated. Alternatively,
re-ligation can be prevented in vectors which have been double
digested by additional restriction enzyme digestion and/or
separation of the unwanted fragments.
[0134] For portions of vectors derived from cDNA or genomic DNA
which require sequence modifications, site specific primer directed
mutagenesis may be used (Zoller, M. J., and Smith, M. Nucleic Acids
Res (1982) 10:6487-6500 and Adelman, J. P., et al, DNA (1983)
2:183-193). This is conducted using a primer synthetic
oligonucleotide complementary to a single stranded phage DNA to be
mutagenized except for limited mismatching, representing the
desired mutation. Briefly, the synthetic oligonucleotide is used as
a primer to direct synthesis of a strand complementary to the
phage, and the resulting partially or fully double-stranded DNA is
transformed into a phage-supporting host bacterium. Cultures of the
transformed bacteria are plated in top agar, permitting plaque
formation from single cells which harbor the phage.
[0135] Theoretically, 50% of the new plaques will contain the phage
having, as a single strand, the mutated form; 50% will have the
original sequence. The resulting plaques are washed after
hybridization with kinased synthetic primer at a wash temperature
which permits binding of an exact match, but at which the
mismatches with the original strand are sufficient to prevent
binding. Plaques which hybridize with the probe are then picked,
cultured, and the DNA recovered.
Verification of Construction
[0136] Correct ligations for plasmid construction can be confirmed
by first transforming E. coli strain MC1061 obtained from Dr. M.
Casadaban (Casadaban, M., et al, J Mol Biol (1980) 138:179-207) or
other suitable host with the ligation mixture. Successful
transformants are selected by ampicillin, tetracycline or other
antibiotic resistance by using other markers depending on the mode
of plasmid construction, as is understood in the art. Plasmids from
the transformants are then prepared according to the method of
Clewell, D. B., et al, Proc Natl Acad Sci (USA) (1969) 62:1159,
optionally following chloramphenicol amplification (Clewell, D. B.,
J Bacteriol (1972) 110:667). Several mini DNA preps are commonly
used, e.g., Holmes, D. S., et al, Anal Biochem Acids Res (1979)
7:1513-1523. The isolated DNA is analyzed by restriction and/or
sequenced by the dideoxy nucleotide method of Sanger, F., et al,
Proc Natl Acad Sci (USA) (1977) 74:5463 as further described by
Messing, et al, Nucleic Acids Res (1981) 9:309, o4 by the method of
Maxam, et al, Methods in Enzymology (1980) 65:499.
Hosts Exemplified
[0137] Host strains used in cloning and prokaryotic expression
herein are as follows:
[0138] For cloning and sequencing, and for expression of
construction under control of most bacterial promoters, E. coli
strains such as MC1061, DH1, RR1, C600hfl, K803, HB101, JA221, and
JM101 can be used.
3. Assays Based on the Discovery of SAA in Colostrum
[0139] The discovery of a specific, constitutively expressed form
of SAA in colostrum enables a new way of detecting the presence of
colostrum in a sample containing a mixture of biological fluids
(e.g., colostrum and milk). For instance, since SAA is elevated in
colostrum and not in milk from normal mammary tissue, the
measurement colostrum SAA in a milk sample can be used to
differentiate colostrum from milk. Accordingly, in instances where
it is undesirable to have milk that contains colostrum (some
countries have laws to this effect), an immunological or
hybridization assay, as described above, may be used to detect
colostrum-tainted milk. Accordingly, in instances where it is
undesirable to have milk that contains colostrum, an immunological
or hybrization assay, as described above, may be used to detect
colostrum tainted milk.
[0140] Colostrum SAA also may be used for a variety of other
purposes. These include, but are not limited to its use as (1) a
carrier for delivery of molecules across the gut or vasculature,
(2) a nutritional supplement for development of the gut mucosa in
newborns, and (3) as a regulator of immune responses (via injection
or oral administration).
4. Pharmaceutical Preparations
[0141] According to the invention Applicant has discovered that the
colostrum associated SAA of the invention and more particularly its
active site, (the TFLK motif) stimulate mucin production in the
intestine. This is significant as mucins have been shown to have a
key role in the prevention and treatment of intestinal infections
and many probiotics act through inducing mucin production. See Mack
et al, "Probiotics inhibit enteropathogenic Escherechia coli
adherence in vitro by inducing intestinal mucin gene expression",
1999, Am J Physiol, 4 Part 1 G941-950, the disclosure of which is
incorporated herein by reference. Thus the invention also includes
pharmaceutical preparations for animals involving colostrum
associated SAA. Those skilled in the medical arts will readily
appreciate that the doses and schedules of pharmaceutical
composition will vary depending on the age, health, sex, size and
weight of the animal rather than administration, etc. These
parameters can be determined for each system by well-established
procedures and analysis e.g., in phase I, II and III clinical
trials.
[0142] For administration, the colostrum associated SAA can be
combined with a pharmaceutically acceptable carrier such as a
suitable liquid vehicle or excipient and an optional auxiliary
additive or additives. The liquid vehicles and excipients are
conventional and are commercially available. Illustrative thereof
are distilled water, physiological saline, aqueous solutions of
dextrose and the like.
[0143] In general, in addition to the active compounds, the
pharmaceutical compositions of this invention may contain suitable
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. Oral dosage forms encompass tablets, dragees, and
capsules. Preparations which can be administered rectally include
suppositories. Other dosage forms include suitable solutions for
administration parenterally or orally, and compositions which can
be administered buccally or sublingually.
[0144] The pharmaceutical preparations of the present invention are
manufactured in a manner which is itself well known in the art. For
example the pharmaceutical preparations may be made by means of
conventional mixing, granulating, dragee-making, dissolving,
lyophilizing processes. The processes to be used will depend
ultimately on the physical properties of the active ingredient
used.
[0145] Suitable excipients are, in particular, fillers such as
sugars for example, lactose or sucrose mannitol or sorbitol,
cellulose preparations and/or calcium phosphates, for example,
tricalcium phosphate or calcium hydrogen phosphate, as well as
binders such as starch, paste, using, for example, maize starch,
wheat starch, rice starch, potato starch, gelatin, gum tragacanth,
methyl cellulose, hydroxypropyhnethylcellulose, sodium
carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,
disintegrating agents may be added, such as the above-mentioned
starches as well as carboxymethyl starch, cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof, such as
sodium alginate. Auxiliaries are flow-regulating agents and
lubricants, for example, such as silica, talc, stearic acid or
salts thereof, such as magnesium stearate or calcium stearate
and/or polyethylene glycol. Dragee cores may be provided with
suitable coatings which, if desired, may be resistant to gastric
juices.
[0146] For this purpose concentrated sugar solutions may be used,
which may optionally contain gum arabic, talc,
polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide,
lacquer solutions and suitable organic solvents or solvent
mixtures. In order to produce coatings resistant to gastric juices,
solutions of suitable cellulose preparations such as
acetylcellulose phthalate or hydroxypropylmethylcellulose
phthalate, dyestuffs and pigments may be added to the tablet of
dragee coatings, for example, for identification or in order to
characterize different combination of compound doses.
[0147] Other pharmaceutical preparations which can be used orally
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer such as glycerol or
sorbitol. The push-fit capsules can contain the active compounds in
the form of granules which may be mixed with fillers such as
lactose, binders such as starches, and/or lubricants such as talc
or magnesium stearate and, optionally, stabilizers. In soft
capsules, the active compounds are preferably dissolved or
suspended in suitable liquids, such as fatty oils, liquid paraffin,
or liquid polyethylene glycols. In addition stabilizers may be
added. Possible pharmaceutical preparations which can be used
rectally include, for example, suppositories, which consist of a
combination of the active compounds with the suppository base.
Suitable suppository bases are, for example, natural or synthetic
triglycerides, paraffinhydrocarbons, polyethylene glycols, or
higher alkanols. In addition, it is also possible to use gelatin
rectal capsules which consist of a combination of the active
compounds with a base. Possible base material include for example
liquid triglycerides, polyethylene glycols, or paraffin
hydrocarbons.
[0148] Suitable formulations for parenteral administration include
aqueous solutions of active compounds in water-soluble or
water-dispersible form. In addition, suspensions of the active
compounds as appropriate oily injection suspensions may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils for example, sesame oil, or synthetic fatty acid esters,
for example, ethyl oleate or triglycerides. Aqueous injection
suspensions may contain substances which increase the viscosity of
the suspension, include for example, sodium carboxymethyl
cellulose, sorbitol and/or dextran, optionally the suspension may
also contain stabilizers.
[0149] In addition to administration with conventional carriers,
active ingredients may be administered by a variety of specialized
delivery drug techniques which are known to those of skill in the
art. The following examples are given for illustrative purposes
only and are in no way intended to limit the invention.
[0150] As used herein the term "an effective amount" shall mean an
amount of colostrum associated SAA sufficient to increase mucin
production so that adherence of pathogens to mucosal cells is
decreased as determined by the methods and protocols disclosed
herein.
[0151] According to the invention, the novel colostrum associated
SAA and more particularly its TFLK motif active site has been shown
to stimulate mucin production, more specifically MUC3. Mucin
production has been shown to inhibit the adherence of E coli, and
probiotic agents which do the same, have been shown to work through
stimulation of mucins. The colostrum associated SAA and/or peptide
can be used as a probiotic.
[0152] The significance of mucins in intestinal infections lies in
their ability to PREVENT the events necessary for infectious
organisms to cause disease.
[0153] Mucins are produced by intestinal epithelial cells and
secreted onto their surface. Thus, mucins are strategically located
between the epithelial cells of the gut and offending agents
ingested into the intestinal tract (i.e. infectious agents, noxious
substances).
[0154] Mucins also inhibit the adherence of bacteria to the
epithelial cells of the intestinal tract. Binding of bacteria to
the lining cells of the gut is the first step in invasion, toxin
delivery and development of diarrheal disease. If binding of the
enteric pathogens is inhibited then disease does not develop.
[0155] Mucins have been shown to inhibit replication of
viruses.
[0156] Mucins are part of innate immunity and a basic defense
system of the gut. Thus, in comparison to the antibody/T-cell
driven acquired immune system, mucins provide advantages including:
immediate or rapidly inducible response to offending agents; broad
spectrum of action; locally effective; intact across animal
kingdom.
[0157] Increased production of mucins are possible by influences
outside of the intestinal cell.
[0158] Increased mucin secretion due to infectious agents is a
well-known clinical phenomenon. Mucin inhibits infectious
intestinal bacteria from attaching to intestinal cells and thus,
prevents infection. This is accomplished by mucins attaching to the
structures on the wall of the bacteria that would normally be used
to attach to the cells. Probiotic bacteria (non-infectious
bacteria) prevent attachment of infectious intestinal bacteria to
epithelial cells lining the intestinal tract. Secreted material
from probiotic bacteria cause intestinal cells to produce more
mucin and is the mechanism whereby probiotic agents prevent
infection.
[0159] Applicant has demonstrated that colostrum associated SAA is
present in the colostrum of mammalian species and is produced by
ductal epithelial cells of the mammary gland. Further, by amino
acid sequence analysis, that a portion of colostrum associated SAA
is preserved among many animal species. Applicant has synthesized a
10 amino acid peptide from bovine colostrum associated SAA, which
contains the TFLK motif, species-preserved region of the molecule.
This peptide increases the production of MUC3 in cells isolated
from the small intestine by activating the gene responsible for
mucin production. Applicant has shown that the intestinal mucin
genes are turned on very rapidly (within 30 minutes) by this
peptide. Further, the increase in mucin production by this peptide
is related to its concentration around the intestinal cells.
Experiments show low concentrations of colostrum associated SAA
peptide will not cause an increase in mucin production whereas too
much colostrum associated SAA peptide will decrease the gene driven
production of mucin. This phenomenon is very common in biological
systems and shows that it is a specific dose dependent effect. A
four amino acid region "TFLK" and the specific order of those four
amino acids within the colostrum associated SAA peptide are
responsible for stimulating mucin production. It has also been
shown that other peptides of the amyloid molecule unrelated to the
unique colostrum associated SAA region do not stimulate mucin
production.
[0160] This demonstrates pharmaceutical applications of this
peptide for numerous enteric pathologies. For example the
prevention of traveler's diarrhea. Many infectious organisms are
geographical in nature and travelers outside of their own areas
have usually not been previously exposed to these organisms, thus
have not developed immunity to them. Many people will take
antibiotics before traveling, but some antibiotics have deleterious
side effects and also organisms are becoming resistant to many
antibiotics.
[0161] Another other potential use would be to prevent dysentery
and other infectious diseases particularly for the military.
Vaccine development is proving to be problematic. For example,
failure of military recruits to take vaccination (anthrax vaccine)
and disease caused by vaccinations leading to the removal from the
market of the vaccine (rotavirus vaccine). Colostrum associated SAA
is to be a rapid, safe and effective means to reduce or prevent
intestinal-related infections.
[0162] Another example includes prevention or treatment of infant
diarrhea. Breast fed infants have far fewer infections than formula
fed infants. Since colostrum is a natural substance which is
beneficial to the infant and colostrum associated SAA is a
component of colostrum, it will be an invaluable natural addition
to formula. Such formulas are commonly commercially available such
as Infamel.TM., Similac.TM., Carnation Good Start.TM., and
Gerber.TM.. Probiotics have been shown to reduce severity and
shorten the recovery time for viral caused diarrhea. Thus, another
use for colostrum associated SAA would be for children with this
condition which would also have an economic impact by reducing
hospital stays and costs.
[0163] Yet another example includes the prevention or treatment of
necrotizing enterocolitis (NEC). This is a serious complication
that occurs in premature infants. With the various reproduction
techniques that are being used there has been an explosion in the
number of premature infants. Therapy for NEC has remained the same
for the last few decades. Since bacteria in the gut of the
premature infant have a major role in the development of NEC,
therapy for this condition consists of keeping the infant from
feeding, giving strong antibiotics and hoping that the bowel does
not perforate.
[0164] Another example includes the prevention of diarrhea in areas
of outbreaks. E. coli 0157:H7 outbreaks causing which can lead to
deaths from hemolytic-uremic syndrome. We have shown that mucin
production prevents E. coli from adhering to epithelial cells and
thus could prevent this infection.
[0165] Yet another example includes the treatment or prevention of
urinary tract infections. The bladder epithelial cells are very
similar to intestinal epithelial cells and are capable of producing
mucins. Therefore prevention of urinary infections, including
hospitalized patients with urinary catheters, would also be a use
for the pharmaceutical compositions of the invention.
[0166] Yet another example includes veterinary medicine, for the
prevention of infectious diarrhea in herd animals to allow for
removal of antibiotics from the feed.
[0167] Although this disclosure includes upregulation of intestinal
mucins, epithelial cells lining other mucosal surfaces, (e.g.
nasopharynx, bladder, etc.), also produce mucins. These mucins
function to prevent infections analogous to intestinal mucins, and
would also be effective targets for treatment according to the
invention.
[0168] The following examples are provided to describe the
invention in greater detail. They are intended to illustrate, not
to limit, the invention.
EXAMPLE 1
Comparative Analysis of SAA in Serum, Colostrum and Whey
[0169] The purpose of this study was to determine if SAA levels in
colostrum and whey corresponded to serum SAA levels in mastitis
symptomless and symptomatic cows.
[0170] Colostrum, whey and serum samples were obtained from a
challenge model study in which cattle were vaccinated against a
gram positive organism. Two sets of samples were utilized: one set
(4 cows) from vaccinated animals that displayed clinical symptoms
of mastitis, and the other set (5 cows) from vaccinated animals
showing no clinical symptoms. Sample designations are shown below:
TABLE-US-00003 Vaccinates - Non Clinical (NC) Vaccinates - Clinical
(C) NC Cow A C Cow A NC Cow B C Cow B NC Cow C C Cow C NC Cow D C
Cow D NC Cow E
Whey/colostrum samples were obtained from the quarter displaying
the clinical symptoms.
[0171] ELISA assays were conducted according to standard protocols,
e.g., as described by McDonald et al. (J. Immunol. Meth. 144:
149-155, 1991), using rat anti-SAA (human) monoclonal antibodies
that cross react with bovine SAA isoforms.
[0172] Results are shown in Table 1: TABLE-US-00004 TABLE 1
Comparison of Bovine Mastitis Sera, Whey and Colostrum SAA values
Nonclinical Samples: Clinical Samples: Whey/ Whey/ Sera Colost.
Sera Colost. Sample ug/ml ug/ml Sample ug/ml ug/ml NCA 3.9 2.3 CA
Day 0 1.3 0.8 Day 0 NCA 1.4 CA Day 14 1.9 Day 14 NCA 1.0 CA Day 28
1.1 Day 28 NCA 1.1 CA Day 42 0.8 Day 42 NCA 13.6 117.8 CA Calving
3.6 1108.0 calving NCA- 73.0 56.5 CA C + 14 12.3 0.8 C + 14 NCA-
11.8 3.4 CA C + 28 152.6 55.0 C + 28 NCB 2.6 2.3 CB Day 0 2.7 1.8
Day 0 NCB 1.4 CB Day 14 21.4 Day 14 NCB 1.2 CB Day 28 3.1 Day 28
NCB 0.8 CB Day 42 1.0 Day 42 NCB 4.6 346.2 CB Calving 17.2 291.0
calving NCB- 1.2 4.0 CB C + 14 26.7 1.7 C + 14 NCB- 0.8 7.3 CB C +
28 7.2 3.7 C + 28 NCC 3.1 1.8 CC Day 0 1.9 1.9 Day 0 NCC 1.8 CC Day
14 2.0 Day 14 NCC 1.4 CC Day 28 2.0 Day 28 NCC 0.9 CC Day 42 0.9
Day 42 NCC 24.5 15.8 CC Calving 18.0 5.8 Calving NCC 6.9 1.4 CC C +
14 167.9 30.0 C + 14 NCC 6.8 9.1 CC C + 28 1.3 8.4 C + 28 NCD 3.2
1.7 CD Day 0 2.7 3.4 Day 0 NCD 0.9 CD Day 14 1.6 Day 14 NCD 2.4 CD
Day 28 1.8 Day 28 NCD 0.8 CD Day 42 1.6 Day 42 NCD 13.8 484.4 CD
Calving 14.6 77.5 Calving NCD 1.3 1.6 CD C + 14 1629.0 999.5 C + 14
NCD 1.2 5.0 CD C + 28 33.1 2.5 C + 28 NCE 2.6 8.0 Day 0 NCE 1.6 Day
14 NCE 1.9 Day 28 NCE 1.0 Day 42 NCE 33.2 89.1 Calving NCE 7.8 0.6
C + 14 NCE 5.5 4.3 C + 28
[0173] As can be seen from the results set forth in Table 1, SAA
was present in high levels in the colostrum of cows at calving in
80% of the animals tested. SAA was not detected in whey samples of
most clinically healthy cows fourteen days later. SAA levels in
colostrum and whey were independent of the serum concentrations of
SAA. Serum levels of SAA at calving of control cows were normal (15
.mu.g/ml), whereas the average level in the colostrum at calving
was about 300 .mu.g/ml. In one cow, the colostrum SAA was as high
as 1100 .mu.g/ml. The mastitis challenged vaccinated cow CC
displayed highly elevated serum levels of SAA, but SAA levels in
the whey samples were almost normal. The vaccinated challenged cow
CD displayed high levels of SAA in serum and in whey.
EXAMPLE 2
Evaluation of SAA in Colostrum and Subsequent Serial Samplings of
Milk
[0174] The purpose of this study was to evaluate colostrum and
subsequent serial milk samplings to determine SAA content. Samples
were obtained from Holstein dairy cows at the University of
Nebraska--Lincoln Dairy Research Facility. Samples of colostrum
were taken at calving, and subsequent milk samples were taken twice
weekly for three weeks. Samples from all four udder quadrants were
pooled. Results are shown in Table 2. TABLE-US-00005 TABLE 2 SAA
Levels in Colostrum and Milk Samples Cow ID Sample Day SAA ug/ml 83
colostrum Calving 184.8 83 milk +4 0.2 83 milk +7 0.0 83 milk +11
0.0 83 milk +14 0.0 83 milk +18 0.0 83 milk +21 0.0 908 colostrum
Calving 135.6 908 milk +4 2.6 908 milk +7 9.1 908 milk +11 8.2 908
milk +14 2.0 908 milk +18 2.1 908 milk +21 3.6 961 colostrum
Calving 364.6 961 milk +4 0.3 961 milk +7 0.5 961 milk +11 0.0 961
milk +14 0.0 961 milk +18 0.0 961 milk +21 0.0
[0175] The results show that SAA is elevated in colostrum of normal
animals but in very low levels or not detected in normal milk
samples after colostrum has cleared.
EXAMPLE 3
Purification of SAA from Colostrum
[0176] The procedure set forth below can be used for purification
of SAA from serum, plasma, milk or colostrum from any animal
species. The procedure comprises two basic steps: the SAA is
purified to approximately 20% purity by hydrophobic chromatography,
then further purified to approximately 95% purity by SDS-PAGE and
electro-blotting.
[0177] Approximately 30 ml of octyl sepharose CL-4B (Pharmacia
#17-0790-01) was prepared by washing it with approximately 10
volumes (300 ml) water to remove any traces of ethanol. This may be
done by washing the gel in a sintered glass funnel (coarse, funnel
volume 600 ml) or by adding the water to the gel in a beaker, and
then allowing the gel to settle before pouring off the water and
rewashing the gel.
[0178] The final washes (2.times.40 ml) of the gel were with a
solution of 0.5M ammonium sulfate.
[0179] Prior to use, the colostrum was allowed to set at 4.degree.
C. to allow the lipid layer to separate from the aqueous layer,
since the lipid portion seemed to interfere with the purification
procedure. After the ammonium sulfate was poured off, 20 ml of the
4.degree. C. refrigerated colostrum with elevated levels of SAA
(preferably >1 .mu.g/ml) was added to the gel (in the beaker).
The suspension of colostrum and gel was swirled several times
during the one hour incubation at room temperature so to allow the
SAA to bind to the gel.
[0180] The gel was then poured into a 600 ml sintered glass funnel
(coarse) and the non-bound fraction was collected. This non-bound
fraction may be tested for SAA to determine the efficiency of
binding.
[0181] The gel was washed with 5-times 50 ml 50 mM Tris, 10 mM NaCl
buffer pH 7.6. The final wash should be clear.
[0182] The column was further washed with 2.times.50 ml of 30%
isopropanol in Tris/NaCl. These washes were most thorough when a
syringe with a 10 gauge needle was used to eject the
isopropanol/buffer solution onto the gel. The gel was thoroughly
mixed when this procedure was followed.
[0183] The SAA was eluted from the gel with a solution of 60%
isopropanol in TRIS/NaCl. Generally this was done in four elutions
of 10 ml each.
[0184] The eluates contained a variety of proteins, of which about
20% was SAA. In samples where the SAA was too dilute it was
concentrated by evaporating the isopropanol in a centrifugal
concentrator. (RC 1010, Jouan Inc.)
[0185] For further purification for sequencing or amino acid
analysis the proteins in the eluates were separated by SDS-PAGE and
transferred to PVDF membrane by electro-blotting.
[0186] The band which was identified as SAA by SAA specific
antibodies was then excised and used for sequencing.
EXAMPLE 4
Isolation of Colostrum Associated SAA cDNA
[0187] RNA Isolation: Total RNA for reverse
transcription-polymerase chain reaction (RT-PCR) was isolated from
mammary gland epithelial cells using TRIZOL (Gibco BRL) according
to manufacturers recommendations. The integrity of the RNA was
checked by fractionation on a 1% (wt/vol) agarose gel and
subsequent ethidium bromide staining.
[0188] First Strand cDNA Synthesis: First strand cDNA synthesis was
performed using SuperScript II RNase H-Reverse Transcriptase (Gibco
BRL) essentially as described by the manufacturer. Briefly, 5 .mu.g
of total RNA was mixed together with RNase-free sterile water and
20 .mu.M of the cDNA1-T14 primer
(5'-GTTGTCGACTGTAGTGGAGT.sub.14-3') (SEQ ID NO:14) to obtain a
final volume of 12 .mu.L. The reaction mixture was incubated for 10
minutes at 75.degree. C. and then incubated at room temperature for
10 minutes. The mixture was then placed on ice while 4 .mu.L of
5.times. First Strand Buffer (Gibco BRL), 2 .mu.L of 0.1 M DTT, and
1 .mu.L of 10 mM dNTP mix (10 mM dATP, 10 mM dGTP, 10 mM dCTP, and
10 mM dTTP at neutral pH) was added. The contents of the reaction
were gently mixed and incubated at 42.degree. C. for 2 minutes.
SuperScript II RNase H-Reverse Transcriptase (200 Units) was added
to the reaction. Following gently mixing, the reaction was
incubated at 42.degree. C. for 1 hour. The reverse transcriptase
was inactivated by heating the mixture to 70.degree. C. for 15
minutes. To remove RNA complementary to the cDNA, 2 Units of
Escherichia coli RNase H (Gibco BRL) was added and the mixture was
incubated at 37.degree. C. for 20 minutes. The reaction mixture was
stored at -20.degree. C. until needed for second strand cDNA
synthesis.
[0189] Second Strand cDNA Synthesis and Polymerase Chain Reaction:
Second strand cDNA synthesis and amplification of the double
stranded cDNA was performed using either Platinum Taq DNA
Polymerase High Fidelity (Gibco BRL) or AmpliTaq DNA Polymerase (PE
Applied Biosystems), each according the manufacturer's
recommendations for the respective DNA polymerase. The PCR
reactions (50 .mu.L) with Platinum Taq DNA Polymerse High Fidelity
contained 5 .mu.g of the cDNA previously described, 20 .mu.M of the
forward primer, 20 .mu.M of the reverse primer, 5 .mu.L 10.times.
High Fidelity PCR Buffer (Gibco BRL), 1 .mu.L 10 mM dNTP mix, 2
.mu.L 50 mM magnesium sulfate, 1 Unit Platinum Taq DNA Polymerase
High Fidelity (Gibco BRL), and sterile water to obtain a final
volume of 50 .mu.L. The thermal cycling parameters with Platinum
Taq DNA Polymerase High Fidelity (Gibco BRL) were 40 cycles for 30
seconds at 94.degree. C., 15-30 seconds at 45-56.degree. C., and
1-4 minute at 50.degree. C.
[0190] The PCR reactions (50 .mu.L) with AmpliTaq DNA Polymerase
(PE Applied Biosystems) contained 5 .mu.g of the cDNA previously
described, 20 .mu.M of the forward primer, 20 .mu.M of the reverse
primer, 5 .mu.L 10.times. GeneAmp Buffer containing 15 mM magnesium
chloride (PE Applied Biosystems), 1 .mu.L 10 mM dNTP mix, 1.3 Units
AmpliTaq DNA Polymerase (PE Applied Biosystems), and sterile water
to obtain a final volume of 50 .mu.L. The thermal cycling
parameters with AmpliTaq DNA Polymerase (PE Applied Biosystems)
were initiated with a hot start followed by 40 cycles for 1 minute
at 95.degree. C., 30 seconds at 50.degree. C., and 1 minute at
72.degree. C., and then 1 cycle for 15 minutes at 72.degree. C.
[0191] The initial oligonucleotide primers suitable for PCR
amplification of colostrum associated SAA cDNA were designed by
back-translating the amino acid sequence obtained from colostrum
associated SAA amino-terminus and tryptic digested fragments (see
FIG. 2). The forward degenerate primer F1C
(5'-ACNTTYCTNAARGARGCNGGNCA-3') (SEQ ID NO:15) and reverse
degenerate primer R3B (5'-GAAGTGRTTGGGGTCTTTGCCACT-3') (SEQ ID
NO:16), which correspond to amino-terminal residues TFLKEAGQ (SEQ
ID NO:17) and carboxy-terminal residues SGKDPNHF (SEQ ID NO:18) in
the mature colostrum associated SAA protein, respectively, were
used in PCR for the initial amplification of the 300 bp middle cDNA
sequence for colostrun associated SAA. The 5' cDNA sequence for
colostrum associated SAA was obtained by PCR and subsequent DNA
sequencing using the forward primer M5RT2
(5'-AGCACAGGCAGCTCAGCTTCACCAGGA-3') (SEQ ID NO:19) and the reverse
primer M5GW2 (5'-GAAGTATTTGTCTGCACCCCTGTAGTTGGCTTCTT-3') (SEQ ID
NO:20). The M5RT2 primer was based on SAA cDNA sequences deposited
in GenBank and the M5GW2 primer was based on the 300 bp colostrum
associated SAA cDNA sequence previously described (see FIG. 2). The
3' cDNA sequence for colostrum associated SAA was obtained by PCR
and subsequent DNA sequencing using the forward primer M3GW2
(5'-CTGTTTAAGGGTATGACCAGGGACCAGGTACG-3') (SEQ ID NO:21) and the
reverse primer CDNA1 (5'-GTTGTCGACTGTAGTGGAG-3') (SEQ ID NO:22).
The M3GW2 primer was also based on the previously described 300 bp
colostrum associated SAA cDNA sequence (see FIG. 2) and the CDNA1
primer was identical to the first 19 nucleotides of the primer
CDNA1-T14 used in first strand cDNA synthesis (see above for
CDNA1-T14 sequence).
[0192] Cloning of colostrum associated SAA cDNA: The resulting 300
bp RT-PCR product obtained with AmpliTaq DNA Polymerase (PE Applied
Biosystems) using the degenerate oligonucleotides F1C and R3B was
agarose gel purified using Qiagen's QIAquick Gel Extraction Kit and
cloned into Invitrogen's pCRII-TOPO vector, according to the
manufacturer's recommendations. The TOPO cloning reaction was
transformed into E. coli TOP10 and plated on Luria-Bertani
containing 50 .mu.g/mL kanamycin and X-Gal, as recommended by
Invitrogen. Putative positive colonies were screened using the M13
Forward (-20) and M13 Reverse primer in PCR. Re-amplified inserts
were fractionated in a 2% (wt/vol) agarose gel and visualized by
ethidium bromide staining along with an appropriate DNA size
marker.
[0193] DNA Sequencing and Computer Analysis of colostrum associated
SAA cDNA: The cloned 300 bp colostrum associated SAA cDNA sequence
was re-amplified with the M13 Forward (-20) and M13 Reverse primers
in high-fidelity PCRs. The 5' and 3' region of the colostrum
associated SAA cDNA sequence was reamplified in high-fidelity
RT-PCRs using the M5RT2/M5GW2 and M3GW2/CDNA1 primer pairs,
respectively. The resulting amplicons were purified using Qiagen's
Qiaquick PCR Purification System and sequenced in both directions
by the DNA sequencing facility at Iowa State University (Ames,
Iowa) in an automated ABI 377 DNA sequencer. The SP6 and T7-2
primers were used in the sequencing of the cloned 300 bp colostrum
associated SAA cDNA. The primers M5RT2 and M5GW2 were used for
sequencing of the 5' region of colostrum associated SAA cDNA and
the primers M3GW2 and CDNA1 were used for sequencing of the 3'
region of colostrum associated SAA cDNA. The DNA sequence was
analyzed using the Wisconsin Genetics Computer Group (GCG) Package
Version 10.1 (Madison, Wis.) SeqEd, PileUp, and BLASTX programs.
The amino-terminal signal peptide cleavage site was identified by
using the SignalP (version 1.1) program (Nielsen et al., 1997).
[0194] RT-PCR Detection of colostrum associated SAA and not Acute
Phase SAA mRNA Expression by Bovine Mammary Gland Epithelial Cells:
As previously described in detail, a 300 bp RT-PCR product was
obtained from bovine mammary gland epithelial cells using the
primer CDNA1-T14 for first strand synthesis from the mRNA present
and then subsequent usage of the primers F1C and R3B for second
strand synthesis and amplification. The figures show the nucleotide
sequence obtained for this 300 bp RT-PCR product. This nucleotide
sequence correlated with the peptide sequencing data obtained from
the colostrum-associated and bovine mammary gland-associated SAA
isoform (see FIG. 2).
[0195] The forward degenerate primer F2
(5'-GACATGTGGMGAGCCTACTCYGACATG-3') (SEQ ID NO:23) and reverse
degenerate primer R3B (previously described) were used in RT-PCR
for amplification of A-SAA cDNA. The forward primer F2 corresponds
to amino-terminal residues DMWRAYSDM (SEQ ID NO:24) in the acute
phase SAA (A-SAA) protein (SWISS-PROT accession number P35541) and
the reverse primer R3B corresponds to the carboxy-terminal residues
SGKDPNHF (SEQ ID NO:25) in both the A-SAA protein and the colostrum
associated SAA protein. Subsequent cloning and nucleotide
sequencing of the resulting 267 bp cDNA sequences correlated with
colostrum associated SAA cDNA, strongly suggesting that colostrum
associated SAA and not A-SAA transcripts were present.
[0196] The restriction endonuclease XhoI site was found to be
present in the cDNA sequence of bovine A-SAA (data not shown), but
was not found in the cDNA sequence of bovine colostrum associated
SAA. XhoI restriction endonuclease digestion of the 300 bp and 267
bp CDNA sequences previously described did not cleave either of
these two RT-PCR products. This result additionally suggested that
only colostrum associated SAA and not A-SAA mRNA was transcribed by
bovine mammary gland epithelial cells.
[0197] To further verify that colostrum associated SAA and not
A-SAA mRNA was expressed by the bovine mammary gland epithelial
cells, the forward colostrum associated SAA-specific primer M3GW2
(previously described and shown in FIG. 2) and reverse CDNA1 primer
(previously described) were again used in RT-PCR. In addition, the
forward A-SAA-specific primer S3GW1
(5'-TAAGGGTACGACCAGTGGCCAGGGTCA-3') (SEQ ID NO:26), corresponding
to residues FKGTTSGQGQ (SEQ ID NO:27) in mature A-SAA) and the
reverse CDNA1 primer (previously described) were used in RT-PCR.
However, no RT-PCR product was observed using the forward
A-SAA-specific primer and reverse CDNA1 primer, further confirming
no expression or the low abundance of A-SAA mRNA expression by
bovine mammary gland epithelial cells.
EXAMPLE 5
Colostrum-SAA Production by Bovine MAC-T Mammary Epithelial
Cells
[0198] Bovine MAC-T mammary gland epithelial cells were obtained
from ATCC (CRL-10274) and cultured according to recommended
conditions (Turner, J D and Huynh H. Immortalized bovine mammary
epithelial cell line. U.S. Pat. No. 5,227,301 dated Jul. 13, 1993).
MAC-T cells were cultured on Dulbecco's Modified Eagles Media
(DMEM) supplemented with 10% Fetal Calf Serum (FCS), 5 .mu.g/ml
insulin, 1 .mu.g/ml hydrocortisone and fungizone. Cells were
incubated at 37.degree. C. with 5% CO.sub.2. For colostrum-SAA
production the cells were seeded onto type I Collagen coated
plates. After 14 hours of incubation, the cells were washed twice
with Dulbecco's phosphate buffered saline (DPBS) and incubated in
media (DMEM, 5 .mu.g/ml insulin, 1 .mu.g/ml hydrocortisone and 2.5%
FCS) supplemented with prolactin from sheep pituitary gland (5
.mu.g/ml) for the stimulation of colostrum-SAA production.
Approximately one half of the media was replaced daily with fresh
prolactin supplemented media. Standard ELISA for the quantitation
of SAA (FIG. 3) was used to assay aliquots of the growth media
collected on the different days for the presence of
colostrum-SAA.
[0199] Cells were kept in culture for 41 days in media supplemented
with prolactin. Levels of colostrum-SAA production reached a
maximum of almost 3000 ng/ml.
[0200] Purification of colostrum associated SAA from cell culture
fluid. Colostrum-SAA was purified from cell culture fluid by
affinity chromatography. Briefly, an affinity column was prepared
by coupling a monoclonal antibody with specificity to SAA to
cyanogen bromide activated sepharose 4B. Treating the column with
50 mM Tris, 0.1 NaCl, 0.2 M glycine pH 8 buffer blocked residual
active groups on the gel. The column was then washed with 50 mM
Tris, 0.1 M NaCl pH 7.2 buffer to remove any excess uncoupled
protein. Approximately 50-ml culture fluid was passed over the
column. The column was washed with a Tris-NaCl buffer to remove any
nonbound proteins that were trapped in the column. The proteins
were then eluted from the column with 0.1 M glycine-HCl pH 2.8. The
fractions were neutralized immediately. All fractions were assayed
by ELISA to determine which fraction contained the maximum amount
of colostrum associated SAA and also by western blot to assess the
total protein content of the fractions. (FIG. 4).
[0201] Determining the amino acid sequence of the purified protein.
The fraction containing the greatest amount of the colostrum-SAA
was subjected to 12% SDS-PAGE and electroblotted onto a PVDF
membrane by a Mini Trans-Blot system (BioRad Laboratories). A
section of one lane of the membrane was cut off and was stained
with the monoclonal antibody to SAA to verify the presence of the
colostrum-SAA. The remainder of the membrane was stained for five
minutes in a solution of methanol:water (40:60) containing 0.5%
(wt/vol) Bromphenol Blue, and destained in a solution of
methanol:water (50:50). The colostrum SAA proteins (identified by
the monoclonal antibody stain) were excised from the membrane. The
protein was deblocked using pfu pyroglutamate aminopeptidase
(TaKaRa Biochemicals) followed by N-terminal sequencing using Edman
degradation. Sequencing was performed on a Procise 491 made by
PE-Biosystems through the University of Nebraska Medical Center's
Protein Core Facility. The N-terminal sequence for colostrum-SAA
was present (FIG. 5).
[0202] Isoelectric Focusing (IEF) of SAA from serum, colostrum and
cell culture fluid. The PROTEIN IEF Cell (BioRad) was used for the
isoelectric focusing of the various SAA preparations. Ready Strip
IPG Strips (BioRad Laboratories) with a pH range of 3-10 were used
for the IEF. The second dimension (2-D) of the protein analysis was
done by subjecting the IPG strips to 12% SDS-PAGE gel and
electroblotting onto a PVDF membrane. The strips and blots were
done in duplicate so that one of the blots could be stained with a
protein stain, Coomassie Brilliant Blue, (CBB) and the other
stained with the anti-SAA monoclonal antibody for the
identification of the SAA protein isoforms. The samples also
contained internal IEF standards (BioRad Laboratories) so that
isoelectric point (pI) of each SAA isoforms could be determined. By
comparing the antibody stained spots to the spots of the standards
stained with CBB the apparent pI of the SAA isoforms could be
determined. All of these procedures were done according to the
protocol recommended by the manufacturer.
[0203] The proteins subjected to the IEF and 2-D analysis were
either affinity purified as described for the cell culture fluid
or, in the case of the serum only semi-purified by hydrophobic
chromatography. SAA is a highly hydrophobic molecule and will bind
readily to Octyl Sepharose beads and then under the appropriate
conditions can be eluted off the matrix. Briefly, serum with an
elevated SAA level was tumbled for one hour with an equal volume of
Octyl Sepharose CL-4B gel to allow for hydrophobic binding of the
proteins from the serum to the gel. The gel was washed with
Tris-HCl buffer to remove any proteins that were just trapped
within the matrix. The proteins bound to the gel were eluted by 60%
isopropanol in Tris-NaCl buffer. These eluates contain a variety of
proteins of which about 20% is SAA. An aliquot of this preparation
or the affinity purified Colostrum-SAA from the cell culture fluid
or the colostrum was loaded onto the IPG strips and then standard
procedures were followed for the IEF and the 2-D gel.
[0204] After analyzing the stained gels it was determined that the
colostrum-SAA from both colostrum and MAC-T cell culture fluid had
a pI of greater than 8 and was estimated to have an apparent pI of
9.4-9.6. The SAA from the serum contained of three isoforms with
apparent pI values of approximately 7.0, 5.8 and 5.5. There was no
isoform in the serum that matched the pI of the colostrum-SAA (FIG.
6).
EXAMPLE 6
Functional Roles of Colostrum-SAA
[0205] A remarkable feature of human physiology is that the mucosal
epithelial cells that line the intestinal tract are in contact with
a vast number of microbes and yet the incidence of infection and
inflammatory complications is low. This suggests that local host
protective mechanisms include highly effective, broad-spectrum,
non-inflammatory antimicrobial defenses. Whereas the acquired
immune system develops an effective response, it does so over a
period of days or weeks and in infants the acquired immune system
is immature and not fully functional. In contrast, the innate
immune system of the intestinal tract is continual or immediately
inducible to many potential pathogens introduced into the
intestinal tract and brought into close proximity to the mucosal
epithelial cells and functional at birth. Intestinal innate
immunity includes first-line host-defense elements that range from
simple inorganic molecules such as nitric oxide to natural killer
cells. There are also a number of molecules produced by the
epithelial cells that comprise an effector arm of the innate immune
system. These include the relatively small antimicrobial peptides
and more complex glycoprotein molecules such as mucins.
[0206] Mucins are secreted and cell-surface high-molecular-weight
glycoproteins synthesized by epithelial cells of a number of organ
systems including the intestinal tract. The strategic
interpositioning between the intestinal lumen and the underlying
mucosal epithelial cells of the intestinal tract has suggested that
mucins have a number of important biological functions. In the
intestinal tract, mucins protect against viral infections by
inhibition of viral replication and enhancing viral clearance from
the intestinal tract. Bacterial pathogens are prevented from
adherence to intestinal epithelial cells. Adherence of
enteropathogens is the crucial first step required for subsequent
invasion, colonization or toxin delivery. Inhibition of adherence
of enteric pathogens to intestinal epithelial cells by mucins could
be by means of steric hindrance. Applicant's previous work and that
of others has also shown that specific mucin-bacterial interactions
could also be an important mechanism whereby mucins effect benefit
for the host. However, regardless of the mechanism, prevention of
mucosal infections is an important function of mucins.
[0207] Different mucin genes have been identified and to date,
twelve human mucin genes have been identified. However, MUC3 mucin
is the predominant intestinal mucin. It has previously been shown
that the MUC3 mucins are effective in preventing the adherence of
enteropathogenic Escherichia coli (EPEC) to intestinal epithelial
cells. Applicant also showed that agents such as non-harmful
bacteria that normally colonize the intestinal tract (i.e.
probiotics) inhibit EPEC epithelial cell adherence and do so by the
upregulation of intestinal mucin genes. It is also well known that
breast-feeding infants are far less susceptible to infectious
diarrhea than formula fed infants. There are a number of theories
why this is so, but it is hypothesized that milk-associated amyloid
(colostrum SAA) may be an important inducer of MUC3 gene
expression. Applicant has evaluated MUC3 mRNA expression using an
in vitro human cell culture assay system. In this system,
intestinal cells incubated with the N-terminal peptide sequence of
colostrum SAA have shown increased MUC3 mRNA expression as compared
to control cells grown without the addition of colostrum SAA to the
cell culture medium. To further explore this finding, Applicant has
evaluated the functional specificity of the N-terminal peptide
sequence of colostrum SAA by evaluation of MUC3 expression in the
same in vitro assay using colostruni SAA N-terminal peptide
sequences that have been randomly scrambled and a colostrum SAA
peptide sequence downstream of the N-terminal sequence. If the
expression of MUC3 is increased then intestinal cells grown in the
presence of colostrum SAA should have a greater capacity to inhibit
adherence of bacterial pathogens. This will be studied using EPEC
in an in vitro assay system pre-incubated with colostrum SAA in the
cell culture medium. Enteropathogenic E. coli are non-invasive,
non-toxin producing pathogens that have been recognized as a
significant cause of diarrhea in third world countries and in day
care settings in developed countries. Future studies will evaluate
the benefits of colostrum SAA in in vivo studies as well as
characterized animal equivalent to human EPEC. Colostrum associated
SAA provides a means to naturally upregulate the innate protective
mechanisms of the human intestine and would provide a novel form of
therapy to a common problem that occurs all too often in the third
world leads to death of infants and in the developed world
countries and leads to significant morbidity and cost. Furthermore,
prevention of intestinal infections for traveler's or those having
to live in conditions with altered sanitary practices would also
reduce morbidity. Thus, this therapy offers an effective, natural,
non-drug/chemical therapy.
[0208] In order to address possible functional roles of
colostrum-SAA, the Applicant synthesized, on a standard amino acid
synthesizer, a 10 amino acid region of the molecule from bovine
that represented the N-terminal portion of the mature protein
containing the conserved TFLK. The peptide consisted of the
following amino acids: MWGTFLKEAG (SEQ ID NO:30) (Named
"N-terminal"). Since it was anticipated that the TFLK amino acids
would be the critical elements of the peptide, we also constructed
a peptide in which those four amino acids were scrambled in their
order MWGLTKFEAG (SEQ ID NO:28). (Named "Limited Scramble"). For
controls we synthesized two peptides, one in which the amino acids
in the entire N-Terminal peptide were arranged in random order
GKFAWEGMTL (SEQ ID NO:29) (Named "Total Scramble") and a 10 amino
acid peptide in which the first 7 residues were from the C-terminal
region of bovine SAA DAAQRGPQQA (SEQ ID NO:33) (Named
"C-terminal").
[0209] These four peptides were used in a cell culture assay
designed to evaluate them for their properties of inducing mucin
(MUC) mRNA production, either MUC3 or MUC2 according to the methods
described by Mack et al. (Biochem. Biophys. Res. Commun. 199:
1012-1018, 1994 and Am J Physiol. Vol. 4, part 1, pg. G841-950,
1999).
[0210] N-Terminal Peptide Titration MUC3. Intestinal epithelial
cells, Mack et al. 1994, 1999, were exposed to the N-terminal 10
amino acid bovine colostrum-SAA peptide (SEQ ID NO:30) at various
concentrations for 30 minutes incubation at 37.degree. C. The cells
were incubated an additional hour following replacement of the test
medium with fresh medium without peptide and then the total
cellular mRNA isolated and analyzed for MUC3 specific mRNA. FIG. 7
shows that the N-terminal 10 amino acid, bovine Colostrum-SAA
"N-terminal" peptide containing the TFLK motif stimulated the
production of MUC3 mRNA up to 1-1/2 times that of base line control
levels (significance of P<0.0002). The optimum concentration was
50 .mu.g/ml medium (see FIG. 7). TABLE-US-00006 ANOVA Table for
MUC3/28sRNA ration DF Sum of Squares Mean Square F-Value P-Value
Lambda Power colostrum SAA 5 87931.344 17586.269 6.670 .0002 33.349
.996 Concentration Residual 37 97557.446 2636.688
[0211] TABLE-US-00007 Means Table for MUC3/28sRNA ration Effect:
Colostrum SAA Concentration Count Mean Std. Dev. Std. Err. 0 8
100.000 0.000 0.000 1 7 105.429 28.254 10.679 10 7 105.857 69.163
26.141 25 5 145.000 24.576 10.991 50 8 220.500 71.889 25.417 100 8
103.125 60.326 21.329
[0212] MUC3 Stimulation. MUC3 stimulating activity of the
N-terminal 10 amino acid bovine colostrum-SAA peptide was compared
to the activity of the "Limited Scramble", the "Total Scramble" and
"C-Terminal peptides". Optimum time and temperature of incubation
and concentration of peptides was for 30 minutes at 37.degree. C.
at 50 .mu.g/ml respectively. Data in FIG. 8 shows that the original
N-Terminal peptide was the only peptide that stimulated MUC3 mRNA
statistically significantly over the control values (p<0.008).
Important note is that the lack of stimulation by the "Limited
Scramble" peptide, in which only the novel TFLK sequence was
rearranged strongly implies that this motif may be the key element
in conferring biological activity and perhaps a rationale for it
being conserved amongst species (see FIG. 8). TABLE-US-00008 ANOVA
Table for MUC2 mRNA/28s RNA ratio DF Sum of Squares Mean Square
F-Value P-Value Lambda Power Colostrum SAA 4 6974.667 1743.667
5.081 .0039 20.322 .932 Peptides Residual 25 8580.000 343.200
[0213] TABLE-US-00009 Mean Table for MUC2 mRNA/28s RNA ratio
Effect: Colostrum SAA Peptides Count Mean Std. Dev. Std. Err.
N-terminal 6 101.167 21.876 8.931 Limited Scramble 6 87.167 24.078
9.830 Total Scramble 6 65.833 20.605 8.412 C-terminal 6 67.500
15.268 6.233 Control 6 100.000 0.000 0.000
[0214] MUC2 Stimulation. To address whether the N-Terminal 10 amino
acid bovine colostrum-SAA peptide would stimulate mRNA synthesis
for MUC2 production, intestinal epithelial cells were cultured
under conditions favoring MUC2 expression rather than MUC3. All
peptides were used at concentrations and conditions that were
previously optimized for MUC3 stimulation. As shown in FIG. 9, none
of the peptides stimulated the production of MUC2 mRNA. When
comparing the N-Terminal 10 amino acid peptide that stimulated MUC3
to control baseline levels, the values were not significantly
different. To show that the lack of MUC2 stimulation was not due to
culture conditions, cells were exposed to the N-Terminal 10 amino
acid bovine colostram-SAA peptide at 2.times. and 5.times. optimum
levels for MUC3 (100 and 500 .mu.g/ml respectively). Additionally,
conditions were changed to 2.times. the optimum MUC3 incubation
time (1 hr). None of these changes resulted in MUC2 mRNA increase
over control values. The evidence strongly indicates specificity of
function in stimulating the production of a mucin produced mainly
in the small intestine (MUC3) over one produced primarily in the
large intestine (MUC2) (see FIG. 9). TABLE-US-00010 ANOVA Table for
MUC3 mRNA/28s RNA ratio DF Sum of Squares Mean Square F-Value
P-Value Lambda Power Colostrum SAA 4 25215.200 6303.800 4.387 .0080
17.550 .886 Peptides Residual 25 35919.500 1436.780
[0215] TABLE-US-00011 Mean Table for MUC3 mRNA/28s RNA ratio
Effect: Colostrum SAA Peptides Count Mean Std. Dev. Std. Err.
N-terminal 6 174.833 22.266 9.090 Limited Scramble 6 95.000 38.063
15.539 Total Scramble 6 109.333 56.874 23.219 C-terminal 6 111.333
44.774 18.279 Control 6 100.000 0.000 0.000
[0216] The present invention is not limited to the embodiments
described above, but is capable of modification within the scope of
the appended claims.
EXAMPLE 7
Colostrum-SAA Production by Bovine MAC-T Mammary Epithelial Cells
after Stimulation of by Lipopolysaccharide (LPS)
[0217] Bovine MAC-T mammary gland epithelial cells were obtained
from ATCC (CRL-10274) and cultured according to recommended
conditions (Immortalized bovine mammary epithelial cell line. U.S.
Pat. No. 5,227,301 dated Jul. 13, 1993). MAC-T cells were cultured
on Dulbecco's Modified Eagles Media (DMEM) supplemented with 10%
Fetal Calf Serum (FCB), 5 .mu.g/ml insulin, 1 .mu.g/ml
hydrocortisone and fungizone. Cells were incubated at 37.degree. C.
with 5% CO.sup.2. Typically, for colostram-SAA production the cells
were seeded at 2.5.times.10.sup.5 cells/cm.sup.2 onto type I
Collagen coated plates. After 14 hours of incubation, the cells
were washed twice with Dulbecco's phosphate buffered saline (DPBS)
and incubated with media (DMEM, 5 .mu.g/ml insulin, 1 .mu.g/ml
hydrocortisone and 2.5% FCS) supplemented with lipopolysaccharide
from E. coli. The concentration of LPS added to the media for the
stimulation of colostrum-SAA production ranged from 0 to 5
.mu.g/ml. Essentially all of the media was replaced daily with
fresh LPS supplemented media. Standard ELISA for the quantitation
of SAA (FIG. 11) was used to assay aliquots of growth media
collected daily for the presence of the colostrum-SAA.
[0218] For the production of colostrum-SAA, the cells could also be
cultured on regular tissue culture plates (non-collagen coated).
Cells were kept in culture for 40 days on these plates in media
supplemented with LPS at 10 .mu.g/ml with a daily replacement of
the media. Levels of colostrum-SAA production reached a maximum of
almost 30 .mu.g/ml.
[0219] Aliquots of the culture fluid from the MAC-T cells collected
at various times over the period of 27 days were analyzed for
protein content. The proteins from these samples were separated by
SDS-PAGE on a 10% gel and then silver stained. The protein profile
changed over the period of 27 days. Although the individual
proteins were not identified, changes in overall banding patterns
could be observed, particularly between the molecular weight range
of 15-25 kDa. Here, several proteins could be detected by day 3 but
then were no longer detectable by day 18. The production of
colostrum-SAA, as determined by ELISA since on the gel SAA was
obscured by the heavy band at 15 kDa, increased in concentration
from 0.001 .mu.g/ml on day 1 to approximately 25 .mu.g/ml by day
27.
Purification of Colostrum-Associated SAA from Cell Culture
Fluid.
[0220] Colostrum-SAA was purified from cell culture fluid by
affinity chromatography. Briefly, an affinity column was prepared
by coupling monoclonal antibody with specificity to SAA to cyanogen
bromide activated sepharose 4B. Treating the column with 50 mM
Tris, 0.1 M NaCl, 0.2 M glycine pH 8 buffer blocked residual active
groups on the gel. The column was then washed with 50 mM Tris, 0.1
M NaCl pH 7.2 buffer to remove any excess uncoupled protein.
Approximately 50 ml culture fluid was passed over the column. The
column was then washed with the Tris-NaCl buffer to remove any
non-bound proteins that were trapped in the column. A 0.1 M
glycine-HCl pH 2.8 buffer was used to elute the proteins from the
column. The fractions were neutralized immediately. All fractions
were assayed by ELISA to determine which fractions contained the
maximum amount of colostrum associated SAA.
Isoelectric Focusing (IEF) of SAA from Colostrum and Cell Culture
Fluid.
[0221] The PROTEIN IEF Cell (BioRad) was used for the isoelectric
focusing of the SAA preparations. Since both colostrum and media
from MAC-T cells stimulated by prolactin contain the same isoform,
only the SAA purified form colostrum was used for the comparison.
The samples of SAA had been prepared by affinity purification.
Ready strip IPG Strips (BioRad Laboratories) with a pH range of
3-10 and 7-10 were used for the IEF. The second dimension (2-D) of
the protein analysis was done by subjecting the IPG strips to 12%
SDS-PAGE gel and electroblotting onto a PVDF membrane. The blots
were stained with anti-SAA antibody for the identification of the
SAA protein isoforms. By comparing the spots stained with the
antibody on the blot of the 2D gel from the SAA purified from
colostrum and the SAA from the culture fluid of MAC-T cells
stimulated with LPS, the similarities of the isoforms could be
determined. Each sample was first analyzed on a pH 3-10 IPG strip
for the first dimension of separation and then a subsequent sample
analyzed on a pH 7-10 IPG strip.
[0222] After analyzing the stained gels it was determined that the
SAA purified from the culture fluid of MAC-T cells stimulated with
LPS contains only one isoform of SAA with the pI of 9.4-9.6. This
is identical to the pI of the SAA purified from colostrum and not
to the isoforms associated with the acute phase response. (FIG. 4).
LPS is a compound that normally elicits an inflammatory response.
SAA that is produced by the MAC-T cells as a result of LPS
stimulation is the same as the colostrum SAA produced as a result
of hormonal (prolactin) stimulation of the MAC-T cells and also the
same isoform that is present in colostrum.
EXAMPLE 8
Bovine Colostrum Serum Amyloid A 3 Genomic Sequence
[0223] Cloning Of Sequence Identification No. 13: The cDNA and
amino acid sequence for bovine M-SAA3 (MAA) was obtained as
previously described herein and has been deposited in the GenBank
database under Accession No. AF335552. The nucleotide sequence of
the introns, promoter, and 3' flanking regions for the bovine
M-SAA3 gene were determined by PCR amplification and genomic
walking procedures (Universal Genome Walker Kit; Clontech). The
primary and nested secondary M-SAA3 gene-specific primers used in
the genomic walking procedures were designed according to the
manufacturer's recommendations and were initially complimentary to
either the 5' or 3' region of the bovine M-SAA3 cDNA previously
described. Primary PCRs were carried out in 25 .mu.L volumes
containing 200 .mu.M of adapter primer (AP1), 200 .mu.M
deoxynucleoside triphosphates, 1.1 mM magnesium acetate, 15 mM
potassium acetate, 40 mM Tris-HCl (pH 9.3), 1 U of rTth DNA
polymerase XL (Perkin-Elmer), and approximately 25 ng of
adapter-ligated bovine genomic DNA digested with either StuI, ScaI,
HincIII, or SspI. The thermal cycling parameters used were 7 cycles
for 15 seconds at 94.degree. C. and 3 minutes at 72.degree. C., 37
cycles for 15 seconds at 94.degree. C. and 3 minutes at 67.degree.
C., and then 1 cycle for minutes at 67.degree. C. Secondary PCRs
were carried out in 50 .mu.L volumes containing 1 .mu.L of a 1:50
dilution of the appropriate primary PCR mixture, adapter primer
AP2, and either a forward or reverse nested M-SAA3 gene-specific
primer. The other reaction components and thermal cycling
parameters were the same as those used for primary PCR.
[0224] Nucleotide Sequencing And Computer Analysis Of Genomic
Sequence: The resulting 2.4 kb StuI, 1.7 kb ScaI, 1.8 kb HincII,
and 1.5 kb SspI secondary PCR products obtained from genomic
walking, in addition to PCR products obtained using primer-walking
methodology, were sequenced by the DNA Sequencing Facility at
either Iowa State University or at the University of Nebraska
Medical Center using the AP2 primer and/or M-SAA3 gene-specific
primers. The DNA sequence from several independent high-fidelity
PCR products was analyzed using the Wisconsin Genetics Computer
Group (GCG) Package (Version 10.1, Madison, Wis.). Assembly of the
overlapping amplicons provided the following nucleotide sequence
for the promoter, exons, introns, and 3' flanking region of the
bovine M-SAA3 gene (SEQ ID NO: 31) See FIG. 10. The TATA box is
double underlined in the promoter region for bovine
mammary-associated serum amyloid A 3 (M-SAA3, MAA). The additional
three upstream nucleotides (single underlined) from the TATA box
are also conserved in most "milk protein" TATA boxes. Putative
consensus STAT5 DNA-binding sites at -1741 to -1749 and at -2184 to
-2193 are italicized in bold type and denoted above the sequence.
Other possible transcription factor DNA-binding sites are
underlined and the corresponding transcription factor for this
consensus sequence is identified above the sequence (MatInspector
V2.2/TRANSFAC 4.0, Quandt et al., 1995). The transcriptional start
site is underlined and indicated above the nucleotide with +1. The
beginning and ending of the three introns are denoted with an arrow
above these regions. The start and stop codons are underlined and
indicated above the nucleotides. The encoded amino acids are
denoted under the double-stranded DNA sequence. The presumed signal
peptide cleavage site to remove the leader sequence predicted by
the SignalP program (Version 1.1) (Nielsen et al., 1997) with 100%
certainty is denoted by an inverted triangle. The polyadenylation
signal (-6307 to -6312) is underlined and the probable site for
cleavage and polyadenylation is indicated with a double arrow.
References
[0225] Nielsen H., Engelbrecht J., Brunak S., von Heijne G. 1997.
Identification of prokaryotic and eukaryotic signal peptides and
prediction of their cleavage sites. Protein Engineering 10:1-6.
[0226] Quandt K., Frech K., Karas H., Wingender E., Werner T. 1995.
MatInd and MatInspector--New fast and versatile tools for detection
of consensus matches in nucleotide sequence data. Nucleic Acids
Research 23:4878-4884.
Sequence CWU 1
1
34 1 20 PRT Bos taurus MISC_FEATURE 3 X can be any amino acid 1 Met
Trp Xaa Thr Phe Leu Lys Glu Ala Gly Gln Gly Ala Lys Asp Met 1 5 10
15 Trp Arg Ala Tyr 20 2 10 PRT Ovis aries 2 Trp Leu Leu Thr Phe Leu
Lys Glu Ala Gly 1 5 10 3 7 PRT Equus caballus 3 Arg Glu Trp Phe Thr
Phe Leu 1 5 4 13 PRT Equus caballus 4 Arg Glu Leu Lys Thr Phe Leu
Lys Glu Ala Gly Gln Gly 1 5 10 5 12 PRT Equus caballus 5 Glu Ala
Asn Tyr Ile Gly Ala Asp Lys Tyr Phe His 1 5 10 6 13 PRT Equus
caballus 6 Gly Asn Tyr Asp Ala Ala Gln Arg Gly Pro Gly Gly Ala 1 5
10 7 6 PRT Equus caballus 7 Val Thr Asp Leu Phe Lys 1 5 8 17 PRT
Equus caballus 8 Ser Gly Lys Asp Pro Asn His Phe Arg Pro His Gly
Leu Pro Asp Lys 1 5 10 15 Tyr 9 27 PRT Oryctolagus cuniculus 9 Arg
Glu Trp Leu Thr Phe Leu Lys Glu Ala Gly Gln Gly Ala Lys Asp 1 5 10
15 Met Trp Arg Ala Tyr Ser Asp Met Lys Glu Ala 20 25 10 110 PRT
Equus caballus 10 Leu Leu Ser Phe Leu Gly Glu Ala Ala Arg Gly Thr
Trp Asp Met Ile 1 5 10 15 Arg Ala Tyr Asn Asp Met Arg Glu Ala Asn
Tyr Ile Gly Ala Asp Lys 20 25 30 Tyr Phe His Ala Arg Gly Asn Tyr
Asp Ala Ala Lys Arg Gly Pro Gly 35 40 45 Gly Ala Trp Ala Ala Lys
Val Ile Ser Asp Ala Arg Glu Asn Phe Gln 50 55 60 Arg Phe Thr Asp
Arg Phe Ser Phe Gly Gly Ser Gly Arg Gly Ala Glu 65 70 75 80 Asp Ser
Arg Ala Asp Gln Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys 85 90 95
Asp Pro Asn His Phe Arg Pro His Gly Leu Pro Asp Lys Tyr 100 105 110
11 129 PRT Mustela vison 11 Met Lys Leu Phe Thr Gly Leu Ile Phe Cys
Ser Leu Val Leu Gly Val 1 5 10 15 Ser Ser Gln Trp Tyr Ser Phe Ile
Gly Glu Ala Val Gln Gly Ala Trp 20 25 30 Asp Met Tyr Arg Ala Tyr
Ser Asp Met Arg Glu Ala Asn Tyr Lys Asn 35 40 45 Ser Asp Lys Tyr
Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Gln Arg 50 55 60 Gly Pro
Gly Gly Ala Trp Ala Ala Lys Val Ile Ser Asp Ala Arg Glu 65 70 75 80
Arg Ser Gln Arg Val Thr Asp Leu Phe Lys Tyr Gly Asp Ser Gly His 85
90 95 Gly Val Glu Asp Ser Lys Ala Asp Gln Ala Ala Asn Glu Trp Gly
Arg 100 105 110 Ser Gly Lys Asp Pro Asn His Phe Arg Pro Ser Gly Leu
Pro Asp Lys 115 120 125 Tyr 12 396 DNA Bos taurus CDS (1)..(396) 12
atg aac ctt tcc acg ggc atc att ttc tgc ttc ctg atc ctg ggc gtc 48
Met Asn Leu Ser Thr Gly Ile Ile Phe Cys Phe Leu Ile Leu Gly Val 1 5
10 15 agc agc cag aga tgg ggg aca ttc ctc aag gaa gct ggt caa ggg
gct 96 Ser Ser Gln Arg Trp Gly Thr Phe Leu Lys Glu Ala Gly Gln Gly
Ala 20 25 30 aaa gac atg tgg aga gct tac caa gac atg aaa gaa gcc
aac tac agg 144 Lys Asp Met Trp Arg Ala Tyr Gln Asp Met Lys Glu Ala
Asn Tyr Arg 35 40 45 ggt gca gac aaa tac ttc cac gcc cgt gga aac
tat gac gct gcc cga 192 Gly Ala Asp Lys Tyr Phe His Ala Arg Gly Asn
Tyr Asp Ala Ala Arg 50 55 60 agg gga cct ggg ggt gcc tgg gct gct
aaa gtg atc agt aac gcc aga 240 Arg Gly Pro Gly Gly Ala Trp Ala Ala
Lys Val Ile Ser Asn Ala Arg 65 70 75 80 gag act att cag gga atc aca
gac cct ctg ttt aag ggt atg acc agg 288 Glu Thr Ile Gln Gly Ile Thr
Asp Pro Leu Phe Lys Gly Met Thr Arg 85 90 95 gac cag gta cgg gag
gat tcg aag gcc gac cag ttt gcc aac gaa tgg 336 Asp Gln Val Arg Glu
Asp Ser Lys Ala Asp Gln Phe Ala Asn Glu Trp 100 105 110 ggc cgg agt
ggc aaa gac ccc aac cac ttc aga cct gct ggc ctg cct 384 Gly Arg Ser
Gly Lys Asp Pro Asn His Phe Arg Pro Ala Gly Leu Pro 115 120 125 gac
aag tac tga 396 Asp Lys Tyr 130 13 131 PRT Bos taurus 13 Met Asn
Leu Ser Thr Gly Ile Ile Phe Cys Phe Leu Ile Leu Gly Val 1 5 10 15
Ser Ser Gln Arg Trp Gly Thr Phe Leu Lys Glu Ala Gly Gln Gly Ala 20
25 30 Lys Asp Met Trp Arg Ala Tyr Gln Asp Met Lys Glu Ala Asn Tyr
Arg 35 40 45 Gly Ala Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp
Ala Ala Arg 50 55 60 Arg Gly Pro Gly Gly Ala Trp Ala Ala Lys Val
Ile Ser Asn Ala Arg 65 70 75 80 Glu Thr Ile Gln Gly Ile Thr Asp Pro
Leu Phe Lys Gly Met Thr Arg 85 90 95 Asp Gln Val Arg Glu Asp Ser
Lys Ala Asp Gln Phe Ala Asn Glu Trp 100 105 110 Gly Arg Ser Gly Lys
Asp Pro Asn His Phe Arg Pro Ala Gly Leu Pro 115 120 125 Asp Lys Tyr
130 14 131 PRT Bos taurus 14 Met Asn Leu Ser Thr Gly Ile Ile Phe
Cys Phe Leu Ile Leu Gly Val 1 5 10 15 Ser Ser Gln Arg Trp Gly Thr
Phe Leu Lys Glu Ala Gly Gln Gly Ala 20 25 30 Lys Asp Met Trp Arg
Ala Tyr Gln Asp Met Lys Glu Ala Asn Tyr Arg 35 40 45 Gly Ala Asp
Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Arg 50 55 60 Arg
Gly Pro Gly Gly Ala Trp Ala Ala Lys Val Ile Ser Asn Ala Arg 65 70
75 80 Glu Thr Ile Gln Gly Ile Thr Asp Pro Leu Phe Lys Gly Met Thr
Arg 85 90 95 Asp Gln Val Arg Glu Asp Ser Lys Ala Asp Gln Phe Ala
Asn Glu Trp 100 105 110 Gly Arg Ser Gly Lys Asp Pro Asn His Phe Arg
Pro Ala Gly Leu Pro 115 120 125 Asp Lys Tyr 130 15 33 DNA
artificial sequence cDNA1-T14 primer 15 gttgtcgact gtagtggagt
tttttttttt ttt 33 16 23 DNA artificial sequence forward degenerate
primer F1C 16 acnttycyna argargcngg nca 23 17 24 DNA artificial
sequence reverse degenerate primer for colostrum associated SAA 17
gaagtgrttg gggtctttgc cact 24 18 8 PRT artificial sequence amino
terminal residues of colostrum SAA used to design primers 18 Thr
Phe Leu Lys Glu Ala Gly Gln 1 5 19 8 PRT artificial sequence
carboxy terminal residues of colostrum SAA used to design primers
19 Ser Gly Lys Asp Pro Asn His Phe 1 5 20 27 DNA artificial
sequence forward primer for colostrum associated SAA 20 agcacaggca
gctcagcttc accagga 27 21 35 DNA artificial sequence reverse primer
for colostrum associated SAA 21 gaagtatttg tctgcacccc tgtagttggc
ttctt 35 22 32 DNA artificial sequence forward primer for colostrum
associated SAA 22 ctgtttaagg gtatgaccag ggaccaggta cg 32 23 19 DNA
artificial sequence cDNA-1 primer 23 gttgtcgact gtagtggag 19 24 27
DNA artificial sequence forward degenerate primer used to amplify
SAA 24 gacatgtggm gagcctactc ygacatg 27 25 9 PRT artificial
sequence sequence used to design primer for SAA 25 Asp Met Trp Arg
Ala Tyr Ser Asp Met 1 5 26 8 PRT artificial sequence sequence used
to design SAA specific primer 26 Ser Gly Lys Asp Pro Asn His Phe 1
5 27 27 DNA artificial sequence forward SAA primer 27 taagggtacg
accagtggcc agggtca 27 28 10 PRT artificial sequence sequence from
SAA used to design primer 28 Phe Lys Gly Thr Thr Ser Gly Gln Gly
Gln 5 10 29 10 PRT artificial sequence limited scramble for
experiment 29 Met Trp Gly Leu Thr Lys Phe Glu Ala Gly 1 5 10 30 10
PRT artificial sequence randomized experimental control sequence 30
Gly Lys Phe Ala Trp Glu Gly Met Thr Leu 1 5 10 31 10 PRT artificial
sequence N-terminal region of bovine colostrum SAA 31 Met Trp Gly
Thr Phe Leu Lys Glu Ala Gly 1 5 10 32 4984 DNA Bos taurus
misc_feature 3191, 3222, 3229, 3323, 3402 n can be a, t, c, or g 32
gagtatataa agcaccggcc ccgtctccca ggcaggcagc acaggcagct cagcttcacc
60 aggagcctca gcaggagggc acggccacag gtgaggtgct agaactctcc
aacacttttc 120 ctcttcggag actctctctt cagcagcatt cttgcgctgc
agcccaactc tgcttccttc 180 ctgaatctac tgttctgacc attagaatcc
accagattga gcacttcagg gagtagggct 240 catcttgtct gcatcttctg
tgcaggcagc gatggggtga gcacgcaggc cacagacaca 300 tgtgcctcgt
tcacctcgtc tcgtatcaca gagaggcagc atgaacacac tcctcttgcc 360
tttgggaaac ttgcagtgca gctgggtctc agggctgata gaggatgact ggactggaaa
420 gtggtttatg ctaaaagcac gttgcaatcc ttcacacagg aaatcattgg
gattccaaga 480 tttcatatgg aaataagagc tggatcctct gtgttacaac
ctatcgtctg tctactgaga 540 taaaattcag aggggtttat gttcggaatg
taagagtgta tccacattac aactcagccc 600 caagacctgt cattcttgat
tgactccgct catctctctg ttgcaggatg aacctttcca 660 cgggcatcat
tttctgcttc ctgatcctgg gcgtcagcag ccagagatgg gggacattcc 720
tcaaggaagc tggtcaaggt aaggaccaaa ggatgggcca ggggaggctg tgtctgcttc
780 cccaggattg acctgagcag aggacacatc cccacagggc aaaggccaca
ggtgggcaga 840 aaagaagctt agttttcatg gtagcacttc ccgaagcttt
tctggccagc tttgcactct 900 tttaggggat ccccaagccc gaggtcacat
aaagtttggg ccccaacttt cagcaggagt 960 gaggaagaca tctggggggc
aaggtatctg ttgccaaaat accagtaagg ctctgctacc 1020 gcctcgtggg
caactagaga tggctcattt ccaagtctcc tgtagccatg aagtgggtgc 1080
aaccgctgaa tacttataaa taaaatactt gattttttag tagctgccca ggactgtcta
1140 agagctttat atgcaggaat cgactcgttt tccccctcag ggtttaatcc
ttgagtcctg 1200 caatgtaggg accatcaccc cttatcagag aacctgctgc
cccaagagat taagataggg 1260 tccaacatcc tccagcagag caggattgaa
cccagcatcc tgagaccttg ctgttgactt 1320 cggcccttct actgcctccc
agacaagagt acacgtggag ggtgaggggt ctgtgaacac 1380 gcatcctggt
ctttatctga gcagatggca gagagtgggg gttgctgcct ttggaaggaa 1440
acccgataga gctcccctcc ccacagtaaa tggcagcatg agtttccttg atgatggttc
1500 tgctgaggct gagacctggc gagaatccta tagcaagaga tatagacctc
actagccaga 1560 gcaaactggc cataatttat ttcccaaaac tatttggtgt
tattattttt ctgtgataat 1620 tgctgaataa ttgttttaag catttgttct
taattccatc taaattcaca caggcccaga 1680 taaaagtatc ttttcatctc
ttaggtcagt gttgttcaag gggcactcta ggatgacttg 1740 catgagaatt
aaccgtggtc tgggtgcttt gtggaatgca ggtgcctgga tccacacaca 1800
gtcccttccc tgaaccacag tccctggggc tgtctgcaaa tctgtccatt attgagcacc
1860 ccacttgatt ttgtgcacag taaacactga gaaccactac cttgttttgc
acccaaggga 1920 caaatatgtc gtgcatttgg aagcacttat taaacaactc
tagactccag ggaactattt 1980 aaatctgtaa ctcagggtgc atagctatag
taagaatatc atagccctca accaaactat 2040 ttttctgaac agtggaaata
gctaacacct aaaataaaga taagttatct catagagata 2100 ttacataaac
tattattata atccatgtta tattttcctc ttccctaatg agctaatcat 2160
ttaaaccttt gccattttat tctatttagg ttgggttttc tgtccatgcc tccctgatct
2220 ccatccaact ttatttattt ttttgcccta ctcttctaag gaccagagag
gtgatagtat 2280 agtgagcacc gacaatgttc cataaactca acctgtattt
cctcagttct tctgcataac 2340 caccctgagg gaggcattac tcctccattt
tactggagag gacactgaac tttagagctg 2400 gtgggtcagt tgcccttttt
ctgcatctga ttaccctgtt tcttcaaagc cctcttaggg 2460 agctcacctt
tatcacctgc tgatttaatt ctgacggttg cccatgtgca aacatgccct 2520
gagtattcag atgtactcag gcccgagtta gtccccaggg ctggatttct ccccttgacc
2580 agctgggagt atcctatatc cacagccttt ctcagtatcg tcattctcaa
gctctgatca 2640 gagcctctcc tgcgtctttc caggtggagg ttcattgtat
aagcaaacat cccttaaaga 2700 aagcattgac cgcttcttca cagacatcac
acacctccag aaacaaagtt ctaacagact 2760 tagaatgaaa tcaaacagaa
taaaccttgc atcaagtgtg atactcacaa cttcagatca 2820 gggaaggaag
tgagaagtaa agaagtattc atttcaagcc aataaaataa tctccaaggg 2880
cttggtcgaa ggctgaaacc taaaatcagt gggaggaaat gatttatttc tctttcacca
2940 aaacatgatc acattcatat catcattttc ttttcttccc aggggctaaa
gacatgtgga 3000 gagcttacca agacatgaaa gaagccaact acaggggtgc
agacaaatac ttccacgccc 3060 gtggaaacta tgacgctgcc cgaaggggac
ctgggggtgc ctgggctgct aaagtgatca 3120 ggtaccaggg tccctgggga
tgcagggatg ggtgagcaga gcttggctgc ctaggacaac 3180 ctggaagggc
naagccttgg agaactttcc tgtaggctgt gngccctcnt cctcttaccc 3240
accttcctgc tctgtgccca ctgtgaagtc tgaggggctg aagagcagag caacttggtg
3300 ggacaggcga ctctccaccc ttnctctatg ggtgctgttc acccagcaca
gggctgaggt 3360 gggctgagcc tgaggagcct cagggttgta gcccctcttt
cnttggctcc tctcagagtc 3420 attgatccct tggaaagagg agagatgggg
agggtggggc tgtggctcat agtcctggat 3480 taatcccctc cgtgccctct
tcctttccag taacgccaga gagactattc agggaatcac 3540 agaccctctg
tttaagggta tgaccaggga ccaggtacgg gaggattcga aggccgacca 3600
gtttgccaac gaatggggcc ggagtggcaa agaccccaac cacttcagac ctgctggcct
3660 gcctgacaag tactgagctg cctctctctc tgctcaggag atggggctgt
gagtccccaa 3720 ggacagggac actgacctag agagttctct gtcctcagaa
ggaagcagat ctaataaatg 3780 cttaagagat ggaatactga gactgtgtgt
cattcttggt ataaggacag cctgttagtt 3840 ccaggactga tggccggaca
ccgacgtgaa ggctgagcct gtgcctgtgt gtttggttct 3900 ggcacacaat
ctcagcatca ttcaggacag acgccctctg cagccttccc taatcagacc 3960
cgccccctcc ccagacccct ctggtgacac gggggccatt tccaggccct tcactgtcag
4020 gccttctcac tccctgccgt tgtgtcctgt ccccttctct gtccccaggt
ctagtcccct 4080 agcctgtcct ctgtgctctc tgtgtggggc atggacacag
gaggactgga tggtggaatc 4140 ctgctccaga aactgccacc tggatctcct
gttcatttct cagcagcacc tacaagtaca 4200 actatgagcc agtttctgtc
tgtgcatccg gaactgcctc cagtgctgtt cccttccctc 4260 tcttttcctt
gccttataca agttcccagg aacaaacatg tcaaggagtg gaggaataat 4320
ggcaacatga aaattcagag ccaggcgcct ttgtttgcct tggatatgat tcatgtcctc
4380 gagaggaagt cgttttcccc tcctggtcct ttctcaaccc agggaagcca
gcagcagtta 4440 ctttttattg aggaaaacag tgtctcttat ggaagggagt
tgggtctgtt agagcacagg 4500 aattatgagt gactctgtga gtcataacaa
tgctgaatat gtaaacgcat acatacacat 4560 aaataatgca catgaattat
agagattatg ataaaataaa aattgataaa tgtatcagaa 4620 ccacaagcag
aaattcataa tggaaaataa aagggtgtat catgaataaa gtcataatgg 4680
attcagtaat cttcatgttc catattccat ctgttgttgc tgttgttcag tcactcagtc
4740 atgttgactc ttagggaccc catggactgc agcatgccaa gtttccctgt
ccttcactat 4800 cacttggagt ttgctcaaac tcatgtccat tgagtctgtg
atgccattca accacctcat 4860 cctctgtggc ctccttgtcc tcctgccgtc
agtctttccc agcgtaaggg tcttttccag 4920 tgagtcagct gtttgcagca
gttggctaaa gaatggagct tcagcatcag tctttccaat 4980 caat 4984 33 131
PRT Bos taurus misc_feature N can be A, C, T, or G 33 Met Asn Leu
Ser Thr Gly Ile Ile Phe Cys Phe Leu Ile Leu Gly Val 1 5 10 15 Ser
Ser Gln Arg Trp Gly Thr Phe Leu Lys Glu Ala Gly Gln Gly Ala 20 25
30 Lys Asp Met Trp Arg Ala Tyr Gln Asp Met Lys Glu Ala Asn Tyr Arg
35 40 45 Gly Ala Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala
Ala Arg 50 55 60 Arg Gly Pro Gly Gly Ala Trp Ala Ala Lys Val Ile
Ser Asn Ala Arg 65 70 75 80 Glu Thr Ile Gln Gly Ile Thr Asp Pro Leu
Phe Lys Gly Met Thr Arg 85 90 95 Asp Gln Val Arg Glu Asp Ser Lys
Ala Asp Gln Phe Ala Asn Glu Trp 100 105 110 Gly Arg Ser Gly Lys Asp
Pro Asn His Phe Arg Pro Ala Gly Leu Pro 115 120 125 Asp Lys Tyr 130
34 10 PRT artificial sequence peptide of residues in C-terminal
region of bovine SAA used as instrumental control 34 Asp Ala Ala
Gln Arg Gly Pro Gln Gln Ala 1 5 10
* * * * *