MXPA00003138A

MXPA00003138A - I(chlamydia) protein, gene sequence and uses thereof

Info

Publication number: MXPA00003138A
Application number: MXPA/A/2000/003138A
Authority: MX
Inventors: James W Jackson; John L Pace
Original assignee: Antex Biologics Inc
Priority date: 1997-10-02
Filing date: 2000-03-30
Publication date: 2001-06-26

Abstract

A high molecular weight ("HMW") protein of i(Chlamydia), the amino acid sequence thereof, and antibodies that specifically bind the HMW protein are disclosed as well as the nucleic acid sequence encoding the same. Also disclosed are prophylactic and therapeutic compositions, comprising the HMW protein, a fragment thereof, or an antibody that specifically binds the HMW protein or a portion thereof, or the nucleotide sequence encoding the HMW protein or a fragment thereof, including vaccines.

Description

CHLAMYDIA PROTEIN, SEQUENCE OF CENES AND USES THEREOF 1. FIELD OF THE INVENTION The present invention generally relates to a high molecular weight protein ("HMW") of Chlamydia, its amino acid sequence, and antibodies, including cytotoxic antibodies. , which bind specifically to the HMW protein. The invention further encompasses prophylactic and therapeutic compositions comprising the HMW protein, a fragment thereof, or an antibody that specifically binds to the HMW protein or a portion thereof or the nucleotide sequence encoding the HMW protein. or a fragment thereof, including vaccines. The invention further provides methods for preventing, treating and ameliorating disorders in mammals and birds related to Chlamydia infections and for inducing immune responses to Chlamydia. The invention further provides isolated sequences of nucleotides and degenerate sequences encoding the HMW protein, vectors having said sequences, and host cells containing said vectors. Diagnostic methods as well as sets of diagnostic elements are also included. 2. BACKGROUND OF THE INVENTION Chlamydias are prevalent human pathogens that cause disorders such as sexually transmitted diseases, respiratory diseases including pneumonia, neonatal conjunctivitis, as well as blindness. Chlamydia are intracellular bacteria that infect the epithelial lining of the lungs, tractogenital or conjunctiva. The most common species of Chlamydia include Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pecorum and Chlamydia pneumoniae. Recently, the newly designated Chlamydia species, C. pneumoniae (formerly C. trachomatis TWAR) has been implicated as a major cause of epidemic human neomitis and may perhaps play a role in atherosclerosis. Currently, 18 serovars of C. trachomatis are known to cause trachoma and a broad spectrum of sexually transmitted diseases: with serovars A, B and C being the most frequently associated with trachoma, while serovars DK are the most frequent cause of infections genitals. C. trachomatis is the leading cause of sexually transmitted disease in many industrialized countries, including the United States of America. While the exact incidence of C. trachomatis infection in the United States is unknown, current epidemiological studies indicate that more than 4 million chlamydial infections occur each year, compared to an estimated 2 million gonococcal infections. While all ethnic and socio-economic racial groups are affected, the highest prevalence of chlamydial infections occurs among young people, 12 to 20 years of age, who are sexually active. Most genito-urinary chlamydial infections are clinically asymptomatic. It is common for both men and women to have it for a long time. Up to 25% of men and 75% of women diagnosed with chlamydial infections have no obvious signs of infection. As a consequence, these asymptomatic individuals constitute a great reservoir that can sustain the transmission of people within the community. Far from being benign, a serious disease can develop from these infections, which include: urethritis, lymphogranuloma venereum (LGV), cervicitis, and epididymitis in men. Increased infections of the endocervix frequently cause endometritis, pelvic inflammatory disease (PID) and salpingitis that can cause tubal occlusion and cause infertility. C. trachomatis infection of neonates results from a perinatal exposure to the infected cervix of the mother. Nearly 70% of neonates born vaginally to mothers with chlamydial cericitis become infected during delivery. The mucous membranes of the eyes, oropharynx, tract-urogenital and rectum are the primary sites of infection. Chlamydial conjunctivitis has become the most common form of neonatal ophthalmia. Approximately 20% to 30% of exposed infants develop inclusion conjunctivitis within 14 days of delivery even after receiving prophylactic treatment with either silver nitrate or antibiotic ointment. C. trachomatis is also the leading cause of pneumonia in infants in the United States of America. Almost 10-20% of neonates born through an infected cervix will develop chlamydial pneumonia and will require some type of medical intervention. In developing countries, ocular infections of C. trachomatis provoke trachoma, a chronic follicular conjunctivitis with repeated formation of scars that causes the distortion of the eyelids and an eventual loss of sight. Trachoma is the leading global disease of blindness that can be prevented. The World Health Organization estimates that more than 500 million people in the world, including approximately 150 million children, currently suffer from active trachoma and more than 6 million people have been victims of blindness due to this disease. In developed countries, the costs associated with the treatment of chlamydial infections are enormous. In the United States of America, the annual cost of treating these diseases was estimated at 2.5-3 trillion in 1992 and projected to exceed 8 trillion by the year 2000. A potential solution to this health crisis would be an effective chlamydial vaccine. Several lines of evidence suggest that the development of an effective vaccine is feasible. Studies in both humans and primates have shown that short-term protective immunity against C. trachomatis can be produced by vaccinating with whole chlamydia. However, the protection is characterized by a short duration, specific for serovars, and due to mucosal antibody. In addition, in some people vaccinated, the disease was exacerbated when these people were naturally infected with a serovar different from that used for immunization. It was shown that this adverse reaction is due to a delayed-type hypersensitivity response. Thus, there is a need to develop a chlamydial vaccine based on a subunit capable of producing an effective immune response but without sensitization. Said subunit vaccine may require to be expressed at the mucosal level by neutralizing secretory IgA antibodies and / or cellular immune response to be effective. Efforts to develop subunit vaccines to date have focused almost exclusively towards larger outer membrane protein (MOMP). MOMP is an integral membrane protein approximately 401 kDa in size and comprising up to about 60% of infectious elementary body (EB) membrane protein (Cald ell, H.D., J.

Kromhout, and L. Schachter. 1981. Infect. Im un., 31: 1161- 1176). MOMP provides structural integrity to extracellular EB and is thought to function as a porin-like molecule when the organism is growing intracellularly and is metabolically active. With the exception of four variable domains exposed superficially (VDI-VDIV), MOMP is conserved to a large extent among all 18 serovars. MOMP is highly immunogenic and can cause a local neutralization of anti-Chlamydia antibodies. However, there are problems with this approach. Most of the mapping-specific neutralizing epitopes that have been mapped are located within the RV regions and therefore cause only serovar-specific antibody. Attempts to combine specific epitopes for serovars in various vaccine vectors (eg, poliovirus) to generate neutralizing cross-reactive neutralizing antibodies have had limited successes (Murdin, AD, Su H., Manning DS, Klein MH, Parnell MJ, and HD). Caldwell, 1993. Infect. Immun., 61: 4406-4414, Murdin, AD, H. Su, MH Klein, and HD Caldwell, 1995. Infect. Immun., 63: 1116-1121). Two other major outer membrane proteins in C. trachomatis, the proteins rich in 60 kDa and 12 kDa cysteines, as well as the lipopolysaccharide surface exposed, are highly immunogenic but, unlike MOMP do not induce a neutralizing antibody (Cerrone et al., 1991, Infect. Immun., 59: 79-90). Therefore, the need for a chlamydial vaccine based on novel subunits remains. 3. COMPENDIUM OF THE INVENTION It is an object of the present invention to provide a high molecular weight protein substantially purified and isolated from Chlamydia sp. ("HMW protein"), wherein the HMW protein has an apparent molecular weight of about 105-115 kDa, in accordance with that determined by SDS-PAGE, or a fragment or analogue thereof. Preferably, the HMW protein has substantially the amino acid sequence of any of SEQ ID Nos .: 2, 15 and 16. Preferred fragments of the HMW protein include SEQ ID Nos .: 3, 17, and 25-37. As used herein, the term "substantially the sequence" refers to the sequence being at least 80%, more preferably at least 90% and especially at least 95% identical with the reference sequence. Preferably, the HMW protein is an outer membrane protein. More preferably, the HMW protein from external Eembrane is located on the surface. Preferably, the HMW protein has a heparin binding domain. Preferably, the HMW protein has a porin-like domain. It is proposed that all Chlamydia species are included in this invention, however species such as Chlamydia trachomatis, Chlamydia psittaci, Chlamydia percorum and Chlamydia pneumoniae are preferred. The substantially purified HMW protein is at least 70% by pure weight, more preferably at least about 90% by pure weight, and can be in the form of an aqueous solution. Also included in this invention are recombinant forms of the HMW protein, where in transformed E. coli cells, the expressed recombinant form of the HMW protein has an apparent molecular weight of about 105-115 kDa, as determined by SDS-PAGE, an analogous fragment thereof. The term polypeptide derived from HMW encompasses fragments of the HMW protein; variants of the wild-type HMW protein or fragments thereof containing one or more deletions, insertions or substitutions of amino acids and chimeric proteins comprising a heterologous polypeptide fused to the C-terminus or the N-terminus or an internal segment of the whole or a part of the HMW protein. As used herein and in the claims, the term "HMW protein" refers to a recombinant purified native or purified high molecular weight protein of a Chlamydia species where the apparent molecular weight (in accordance with the determined SDS-PAGE) is of approximately 105-115 kDa. As used herein and in the claims, the term "rHMW protein" refers to recombinant HMW protein. Another object of the present invention is to provide a substantially pure, isolated nucleic acid molecule encoding a HMW protein or a fragment or analog thereof. Preferred is the nucleic acid sequence wherein the encoded HMW protein comprises the amino acid sequence of any of SEQ ID NO. 2, 15 and 16 or a fragment thereof, particularly SEQ ID Nos: 3, 17, 25-37. Also included is an isolated nucleic acid molecule comprising a DNA sequence of any of SEQ ID Nos: 1, 23-24 or a complementary sequence thereof; a fragment of the HMW DNA sequence having the nucleic acid sequence of any of SEQ ID Nos 4-14, 18-22 or the complementary sequence thereof; and a nucleic acid sequence that hybridizes under stringent conditions to any of the sequences described above. The nucleic acid that hybridizes under stringent conditions preferably has a sequence identity of about 70% with any of the sequences identified above, more preferably, about 90%. The production and use of derivatives and analogs of the HMW protein are within the scope of the present invention. In a specific embodiment, the derivative or analog is functionally active, i.e., capable of displaying one or more functional activities associated with a full-length wild-type HMW protein. As an example, said derivatives or analogs having the desired immunogenicity or desired antigenicity can be used, for example, in immunoassays, for immunization, etc. A specific embodiment refers to a fragment of HMW that may be linked by an anti-HMW antibody. Derivatives or analogues of HMW can be tested for the desired activity through methods known in the art. Particularly, HMW derivatives can be made by altering HMW sequences by substitutions, additions or removals that provide functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same amino acid sequence as the HMW gene can be employed in the practice of the present invention. These sequences include, but are not limited to, nucleotide sequences comprising all or a portion of genes altered by the replacement of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. In the same way, the HMW derivatives of the present invention include, but are not limited to, those containing as a primary amino acid sequence, all or a portion of the amino acid sequence of a HMW protein that includes altered sequences where Functionally equivalent amino acid residues are substituted by residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence may be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration. Substitutes of an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid. In a specific embodiment of the invention, proteins are provided that consist of or comprise an HMW protein fragment consisting of at least 6 (continuous) amino acids of the HMW protein. In other embodiments, the fragment consists of at least 7 to 50 amino acids of the HMW protein. In specific embodiments, such fragments are not greater than 35, 100 or 200 amino acids. Derivatives or analogues of HMW include, but are not limited to, molecules that comprise regions substantially homologous to HMW or fragments (e.g., in various embodiments, at least 60% or 70% or 80% or 90% or 95% identity in a amino acid sequence of identical size either when compared to a linear sequence where the alignment is carried out by a computer-homology program known in the art) or whose coding nucleic acid is capable of hybridizing to a HMW sequence coding, under strict conditions, moderately strict or not strict. By way of example and not limitation, computerized homology programs include the following: Basic Local Alignment Search Tool (BLAST) (ww.ncbi.nlm.nih.gov) (Altschul et al., 1990, J. of Molec. Biol ., 215: 403-410, "The BLAST Algorithm; Altschul et al., 1997, Nuc. Acids Res. 25: 3389-3402) a suitable heuristic search algorithm to search for similarity of sequences that gives significance using the statistical methods of Karlin and Altschul 1990, Proc. Nat'l Acad. Sci. USA, 87: 2264-68; 1993, Proc. Nat'l Acad. Sci. USA 90: 5873-77. Five specific BLAST programs perform the following tasks: 1) The BLASTP program compares an amino acid research sequence against a protein sequence database. 2) The BLASTN program compares a nucleotide research sequence against a database of nucleotide sequences. 3) The BLASTX program compares the conceptual translation products of six frames of a nucleotide research sequence (both strands) against a database of protein sequences. 4) The TBLASTN program compares a sequence of protein research against a database of translated nucleotide sequences in the six reading frames (both females). 5) The TBLASTX program compares the translations of six frames of a nucleotide search sequence against the translations of six frames of a nucleotide sequence database. Smith-Waterman (database: European Bioinformatics Institute wwwz.ebi.ac.uk/bic_sw/) (Smith-Waterma, 1981, J. of Molec. Biol. 147: 195-197) is a mathematically rigorous algorithm for alignments of sequences. FASTA (see Pearson et al., 1988, Proc. Nat'l Acad. Sci. USA, 85: 2444-2448) is a heuristic approach to the Smith-Waterman algorithm. For a general comment on the procedure and benefits of the BLAST, Smith-Waterman and FASTA algorithms, see Nicholas et al., 1998, "A Tutorial on Searching Sequence Databases and Sequence Scoring Methods" (www.psc.edu) and references cited here. The HMW derivatives and analogs of the present invention can be produced by various methods known in the art. The manipulations that result in its production can occur at the gene level or at the protein level. For example, the cloned HMW gene sequence can be modified by any of several strategies known in the art (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.) Cold Spring Harbor , New York). The sequence can be dissociated at appropriate sites with restriction endonuclease (s), followed by additional enzymatic modification, if desired, isolated, and ligated in vitro. In the production of the gene encoding an HMW derivative or analog, care must be taken to ensure that the modified gene remains within the same translation reading frame as HMW, uninterrupted by translational suspension signals, in the gene region. where the desired HMW activity is encoded. In addition, the nucleic acid sequence encoding HMW can be mutated iri vitro or in vivo to create and / or destroy translation, initiation, and / or termination sequences, or to create variations in coding regions and / or form new sites of restriction endonuclease or destroy pre-existing sites, to facilitate further in vitro modifications. Any mutagenesis technique known in the art can be employed, including, but not limited to, chemical mutagenesis, site-directed mutagenesis in vitro (Hutchinson, C, et al., 1978, J. Biol. Chem 253: 6551), use of ® linkers (Pharmacia), etc. Manipulations of the HMW sequence can also be performed at the protein level. Within the scope of the present invention are included fragments of HMW protein or other derivatives or analogs that are differentially modified during or after translation, for example, by glycosylation, lipidation, acetylation, phosphorylation, amidation, derivatization by protective groups. known blocking, proteolytic dissociation, binding to an antibody molecule or another cellular ligand, etc. Any of several chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc. In addition, analogs and derivatives of. HMW can be chemically synthesized. For example, a peptide that responds to a portion of an HMW protein that comprises the desired domain or that mediates the desired activity in vitro can be synthesized by the use of a peptide synthesizer. In addition, if desired, non-classical amino acids or chemical analogs of amino acids can be introduced as a substitution or addition in the HMW sequence. Non-classical amino acids include, but are not limited to, D-isomers of the common amino acids, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2- aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designed amino acids such as β -methylamino acids, Calfa-methyloacids, Nalpha-methylamino acids, and amino acid analogues in general. In addition, the amino acid can be D (dextrorotary) or L (levorotary). Another object of the present invention is to provide a recombinant expression vector adapted for the transformation of a host or for the administration of an HMW protein to a host, comprising the nucleic acid molecule of SEQ ID No: 1, 23 or 24 or any fragment of it. Preferably, the recombinant expression vector is adapted for transformation of a host and comprises an expression means operatively connected to the nucleic acid molecule for expression by the host of said HMW protein or fragment or analogue thereof. More preferably, it is the expression vector where the expression device includes a portion of nucleic acid encoding a leader sequence for the secretion of the host or an affinity domain connected to any N or C terminus of the protein or fragment or analogous to it. A further aspect of the invention includes a transformed host cell that contains an expression vector described above and the recombinant HMW protein or fragment or analogue thereof that can be produced by the transformed host cell. A further aspect of the invention is directed to an HMW protein that can be recognized by an antibody preparation that specifically binds to a peptide having the amino acid sequence of SEQ ID No. 2, 15-16 or a fragment or the like replaced conservatively of it. Antigenic and / or immunogenic compositions are another aspect of the invention where the compositions comprise at least one component selected from the following group: a) an HMW protein, where the molecular weight is about 105-115 kDa, in accordance with the determined by SDS-PAGE, or a fragment or analogue thereof; b) an isolated nucleic acid molecule encoding an HMW protein, or a fragment or analogue thereof; c) an isolated nucleic acid molecule having the sequence of SEQ ID Nos. 1, 22, 23 or 24, the complementary sequence thereof or a nucleic acid sequence that hybridizes under stringent conditions there or fragment thereof; d) an isolated recombinant HMW protein, or fragment or analogue thereof, that can be reproduced in a transformed host comprising an expression vector comprising a nucleic acid molecule according to the definitions in b) or c) and a means of expression operatively led to the nucleic acid molecule for expression by the host of said HMW protein or fragment or analogue thereof; e) a recombinant vector comprising a nucleic acid encoding an HMW protein or fragment or analogue thereof; f) a transformed cell comprising the vector of e) and optionally an adjuvant, and a pharmaceutically acceptable carrier or diluent, said composition produces an immune response when administered to a host. Preferred adjuvants include cholera holotoxin or subunits, thermally labile holotoxin from E. coli, subunits and mutant forms thereof, alum, QS21, and MPL. Particularly, alum, LTR192G, mLT and QS21 are preferred. Also included are methods for the production of an immune response in a mammal or a bird comprising administration to said mammal of an effective amount of the antigenic or immunogenic composition described above.

Another aspect of the invention focuses on antisera prepared against the antigenic or immunogenic composition of the invention, and antibodies present in antisera that specifically bind to HMW proteins or a fragment or analogue thereof. Preferably, the antibodies are linked to an HMW protein having the amino acid sequence of SEQ ID Nos: 2, 15-16 or fragment or a conservatively substituted analog thereof. Also included are monoclonal antibodies that specifically bind to an HMW protein, or a fragment or analogue thereof.

A further aspect of the invention includes pharmaceutical compositions and vaccines comprising an effective amount of at least one component selected from the following group: a) an HMW protein, where the isolated protein molecular weight is about 105-115 kDa, in accordance with that determined by SDS-PAGE, an analogous fragment thereof; b) an isolated nucleic acid molecule encoding an HMW protein, or a fragment or analogue thereof; c) an isolated nucleic acid molecule having the sequence of SEQ ID No .: 1, 22, 23 or 24, the complementary sequence thereof or a nucleic acid sequence that hybridizes under stringent conditions with it a fragment of the same; d) an isolated recombinant HMW protein, or analogous fragment thereof that can be reproduced in a transformed host comprising an expression vector comprising a nucleic acid molecule as defined in b) or c) and means of expression operatively coupled to the nucleic acid molecule for expression by the host of said HMW protein of a Chlamydia species or the analogous fragment thereof; e) a recombinant vector comprising a nucleic acid encoding an HMW protein or fragment or analogue thereof; f) a transformed cell comprising the vector of e), g) antibodies that specifically bind to the component of a), b), c), d) or e), and a pharmaceutically acceptable carrier or diluent thereof. The preferred compositions are effective vaccines at the mucosal level. The invention also includes a diagnostic reagent which may include one or more of the aforementioned aspects, such as for example the native HMW protein, the recombinant HMW protein, the nucleic acid molecule, the immunogenic composition, the antigenic composition, the antisera, the antibodies, the vector comprising the nucleic acid, and the transformed cell comprising the vector. Methods and sets of diagnostic elements for detecting Chlamydia or anti-Chlamydia antibodies in a test sample are also included, wherein the methods comprise the steps of: a) contacting said sample with an antigenic composition comprising HMW protein from Chlamydia or an analogous fragment thereof or immunogenic composition or antibodies to form Chlamydia antigen: in a complex of anti-Chlamydia antibodies, and in addition, b) detect the presence or measure the amount of said immunocomplexes formed during step a) as a indication of the presence of said Chlamydia or anti-Chlamydia antibodies in the test sample. The sets of diagnostic elements for detecting Chlamydia or Chlamydia antibodies comprise antibodies, or an antigenic or immunogenic composition comprising Chlamydia HMW protein or an analogous fragment thereof, a container device for contacting said antibody or composition with a test sample suspected of having Chlamydia or Chlamydia antibodies and a reagent medium for detecting or measuring the Chlamydia antigen: immunocomplexes of anti-Chlamydia antibodies formed between said antigenic or immunogenic composition or said antibodies and said sample of proof. A further aspect of the present invention offers methods for determining the presence of nucleic acids encoding the HMW protein or an analogous fragment thereof in a test sample, comprising the steps of: contacting the test bag with the nucleic acid molecule provided therein to produce duplexes comprising the nucleic acid molecule and any nucleic acid molecule encodes the HMW protein in the test sample and that can hybridize specifically therewith; and determine the production of duplex. The present invention also provides a set of diagnostic elements and reagents for determining the presence of nucleic acid encoding an HMW protein or fragment or analogue thereof in a sample, comprising: the nucleic acid molecule of 'compliance with the provided here; means for contacting the nucleic acid with the test sample to produce duplexes containing the nucleic acid molecule and any nucleic acid molecule encoding the HMW protein in the test sample - and which can hybridize specifically therewith; a means to determine the production of duplexes. Also included in this invention are methods for preventing, treating or ameliorating Chlamydia-related disorders in an animal, including mammals and birds that require such treatment, which comprises the distortion of an effective amount of the pharmaceutical composition or vaccine of the invention. Preferred disorders include a bacterial infection by Chlamydia, trachoma, conjunctivitis, urethritis, lymphogranuloma venereum (LGV), cervicitis, epididymitis, or endometritis, inflammatory pelvic disease (PID), salpingitis, tubal occlusion, infertility, cervical cancer, and atherosclerosis Preferred pharmaceutical compositions or vaccines include those formulated for administration in vivo to a host to provide protection against disease or treatment thereof, caused by a species of Chlamydia. Also preferred are compositions formulated as a microparticle, capsule, liposome preparation or emulsion. 4. / ABBREVIATIONS anti-HMW = antibody or HMW polypeptide antiserum. ATCC = American Type Culture Collection Immuno-reactive = capable of eliciting a cellular or humoral immune response. KDa = kilodaltons OG = n-octyl β-D-glucopyranoside or octyl glucoside. OMP = outer membrane protein OMPs • = outer membrane proteins PBS = phosphate-regulated salt solution. PAGE = Polyacrylamide gel electrophoresis Polypeptide = a peptide having any length, preferably a peptide having 10 or more amino acids. SDS = sodium dodecyl sulfate SDS-PAGE = sodium dodecylsulfate polyacrylamide gel electrophoresis. Nucleotide or nucleic acid sequences defined herein are represented by one letter symbols for the bases in the following manner: A (adenine) C (cytosine) G (guanine) T (thymine) U (uracil) M (A or C) R (A or G) W (A or T / U) S (C or G) Y (C or T / U) K (G or T / U) V (A or C or G, not T / U) H ( A or C or T / U, no G) D (A or G or T / U, no C) B (C or G or T / U, no A) N (A or C or G or T / U) or (unknown) Sequences of peptides and polypeptides defined herein, are represented by one letter symbols for amino acid residues in the following manner: A (alanine) R (arginine) N (asparagine) D (aspartic acid) C (cysteine) Q (glutamine) E (glutamic acid) G (glycine) H (histidine) I (isoleucine) L (leucine) K (lysine) M (methionine) F (phenylalanine) P (proline) S (serine) T (threonine) W ( tryptophan) Y (tyrosine) V (valine) X (unknown) The present invention can be better understood by reference to the following detailed description of the invention, examples not limiting specific embodiments of the invention and annexed figures. 5. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Western blot analysis of elementary bodies of C. trachomatis L2 (Ebs). Ebs purified by gradient were solubilized in sample buffer of standard Laemmli SDS-PAGE containing 2-mercaptoethanol, boiled for about 3 minutes and loaded on a 4-12% Tris-glycine gradient gel containing SDS and subjected to 100V electrophoresis. Immediately after the electrophoresis, the proteins were electroabsorbed in PVDF membranes at a temperature of 4 ° C for about 2.5 hours to about 50V. The blocked membrane was probed using a 1/5000 dilution of anti-rHMWP 'antibody (K196) for 1.5 hours at room temperature. After washing, the membrane was treated with a 1 / 5,000 dilution of goat anti-rabbit IgG antibody conjugated with HRO for 1 hour at room temperature. The absorption was developed using a standard TMB substrate system.

Three immunoreactive bands were detected in Ebs and RBs. One dot indicates HMW protein of approximately 105-115 kDa. Figure 2: Consensus Nucleic Acid Sequence that encodes the open reading frame of the HMW protein from C. trachomatis LGV L2. Figure 3: Deduced Amino Sequence Acids of the HMW protein from the open reading frame in PCR from C. trachomatis LGV L2. Figure: SDS-PAGE of partially purified recombinant HMW protein from C. trachomatis LGVL2 expressed in E.coli. SDS-PAGE standards were used as pre-stained and as molecular weight markers. The positions of the molecular weight markers in the gel are indicated on the left side and on the right side of the figure by lines to the molecular weights (kDa) of some of the markers. See text example for details. Band A: Mark 12 Markers of Wide molecular weight range (Novex); myosin, 200 Kdal; B-galactosidase, 116.3 Kdal; phosphorylase B, 97.4 Kdal; bovine serum albumin, -66.3 Kdal. Band B: Recombinant HMWP of C. trachomatis L2. Band C: SeeBlue Pre-stained Molecular Weight markers (Novex); myosin, 250 Kdal; bovine serum albumin, 98 Kdal; glutamic dehydrogenase, 64 Kdal.

Figure 5: maps of plasmids pAH306, pAH310, pAH312, pAH316 and. the open PCR reading frame. Figure 6: Predicted amino acid sequences of HMW protein for C. trachomatis L2, B and F. The sequence of C. trachomatis L2 is given in the top row and starts with the first residue of the mature protein, the N-sequences. eukaryotic glycosylation potentials E. They are underlined. A hydrophobic helical region flanked by proline-rich segments of adequate length to encompass the double layer of lipids is underlined and enclosed in parentheses. Differences of amino acids identified in serovars B and F are indicated below the protein sequence of MGP L2. Figure 7: Indirect fluorescent antibody staining of C. trachomatis Nll inclusion bodies (serovar F) using anti-rHMWP 'antibody. Panel A: post-immunization sera of rabbit K196. The inclusion bodies of Chlamydia show yellow staining. Panel B: K196 rabbit pre-immunization sera. 6. DETAILED DESCRIPTION OF THE INVENTION The term "antigens" and its related term "antigenic" as used herein and in the claims refer to a substance that specifically binds to an antibody or T cell receptor. Preferably said antigens are immunogenic The term "immunogenic" according to what is used herein and in the claims refers to the ability to induce an immune response, for example, an antibody and / or cellular immune response in an animal, preferably a mammal or bird. The term "host" as used herein and in the claims refers either in vivo in an animal or in vitro in cultures of mammalian cells. An effective amount of the antigenic, immunogenic, pharmaceutical composition, including, but not limited to, vaccine, should be administered, wherein "effective amount" refers to the amount that is sufficient to produce a prophylactic, therapeutic or desired improvement response in a patient, including but not limited to, an immune response. The amount required will vary, depending on the immunogenicity of the protein, fragment, nucleic acid or HMW derivative used, and the species and weight of the patient to whom it is administered, but can be determined using standard techniques. The composition elicits an immune response in a patient that produces antibodies, including HMW antiprotein antibodies and antibodies that are opsonizing or bactericidal. In preferred non-limiting embodiments of the invention, an effective amount of a composition of the invention results in an elevation of the antibody titer to at least three immunogenic, antigenic, pharmaceutical and vaccines can be administered to mucosal surfaces by, for example, the nasal, oral (intragastric), ocular, branquiolar, intravaginal or intrarectal routes. Alternatively, other modes of administration including suppositories and oral formulations may be desirable. In the case of suppositories, the binders and carriers can include, for example, polyalkylene glycols or triglycerides. Oral formulations may include incipients normally employed such as, for example, pharmaceutical grades of saccharin, cellulose and magnesium carbonate. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations or powders and contain from about 0.001 to 95% of the HMW protein. Immunogenic, antigenic, pharmaceutical and vaccine compositions are administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective, protective or immunogenic. In addition, the immunogenic, antigenic, pharmaceutical and vaccine compositions can be used in combination with or combined with one or more target molecules for administration to specific cells of the immune system, such as mucosal surface. Some examples include, but are not limited to, vitamin B12, bacterial toxins or fragments of >; the same, monoclonal antibodies and other specific focus lipids, proteins, nucleic acids or carbohydrates. The amount administered depends on the patient to be treated, including, for example, the ability of the patient's immune system to synthesize antibodies, and, if necessary, to produce the cell-mediated immune response. Accurate amounts of active ingredient that are required for administration depend on the judgment of the physician. However, suitable dosage ranges are readily determinable by a person skilled in the art and can be in the order of 0.1 to 1000 μg of the HMW protein, fragment or analogue thereof. Suitable regimens for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dose may also depend on the route (s) of administration and will vary according to the size of the host. The concentration of the HMW protein in an antigenic, immunogenic or pharmaceutical composition according to the present invention is generally from about 0.001 to 95%. A vaccine containing antigenic material from only one pathogen is a monovalent vaccine. Vaccines containing antigenic material of various pathogens are combined vaccines and also belong to the present invention.

Such combination vaccines contain, for example, a material of several pathogens and of several strains of the same pathogen, or of combinations of several pathogens. Antigenic, immunogenic, or pharmaceutical preparations, including vaccines, may comprise as an immunostimulatory material a nucleotide vector comprising at least a portion of the gene encoding the HMW protein, or the at least a portion of the gene may be used directly for immunization. To effectively induce humoral immune responses (HIR) and cell-mediated immunity (CMI), the immunogens are typically emulsified in adjuvants. Immunogevity can be significantly improved if the immunogen is coadministered with an adjuvant. Adjuvants can act by retaining the immunogen locally near the site of administration to produce a depot effect that facilitates a slow and sustained release of the antigen to the cells of the immune system. Adjuvants can also attract cells of the immune system to an immunogen reservoir and stimulate said cells to elicit immune responses. Many adjuvants are toxic, inducing granulomas, acute and chronic inflammation (Freud's complete adjuvant, FCA), cytolysis (saponins and Pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP). Even when FCA is a. Excellent adjuvant and widely used in research, can not be used for its use neither in human vaccines nor in veterinary vaccines due to its toxicity. Desirable characteristics of ideal adjuvants include: 1) absence of toxicity; 2) ability to stimulate the long-term immune response; 3) manufacturing simplicity and long-term storage stability; 4) ability to provoke either CMI or HIR or both in antigens administered by several routes, if required; 5) synergy with other adjuvants; 6) ability of selective interaction with populations of cells that present antigens (APC); (7) ability to specifically elicit appropriate immune responses specific for TH1 or Th2 cells; And (8) ability to selectively increase appropriate levels of antibody isotypes (e.g., IgA) against antigens. Immunostimulation agents or adjuvants have been used for many years to improve the immune responses of hosts for example to vaccines. Intrinsic adjuvants, such as lipopolysaccharides, are usually the components of killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators typically not covalently linked with antigens and are formulated to improve host immune responses. Thus, adjuvants have been identified that improve the immune response to parenterally administered antigens. Aluminum hydroxide and aluminum phosphate (commonly known as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum to increase antibody responses to diphtheria and tetanus toxoids is well known and an HbsAg vaccine has received alum as an adjuvant. Other extrinsic adjuvants may include saponins that form complexes with membrane protein antigens (Immunological stimulation complexes), pluronic polymers with mineral oil, mycobacteria killed in mineral oil, complete Freund's adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes. The international patent application, PCT / US95 / 09005 which is incorporated herein by reference describes mutant forms of heat labile toxin of ecolienterotoxigenic ("mLT"). U.S. Patent 5,057,540, which is incorporated herein by reference, discloses to the adjuvant, Qs21, a non-toxic fraction purified by HPLC of a saponin from the bark of the South American tree Quiliaja saponaria molina 3D-MPL is described in the patent. British 2, 220,211, and is incorporated herein by reference. US Patent No. 4,855,283 issued to Lockhoff et al on August 8, 1989, which is incorporated herein by reference, presents analogs of glycolipids that include N-glycosylamides, N-glycosylureas and N-glycosylcarbamamates, each of which is substituted in the sugar residue by an amino acid, as immunomodulators or adjuvants. Lockhoff reported that N-glycophospholipids and glycoloterolipids are capable of eliciting strong immune responses in both herpes simplex virus vaccine and pseudo rabies vaccines. Some glycolipids have been synthesized from long chain alkylamides and fatty acids directly linked to the sugars through the anomeric carbon atom, to mimic the functions of naturally occurring lipid residues. U.S. Patent No. 4,258,029 to Moloney, incorporated herein by reference, teaches that octadecyltyrosine hydrochloride (OTH) functioned as an adjuvant when complexes were formed with tetanus toxoid and poliovirus vaccine type I, II and III inactivated with alina. The lipidation of synthetic peptides has also been used to increase its immunogenicity. Accordingly, according to this invention, the immunogenic, antigenic, pharmaceutical compositions, including vaccines, comprising the HMW protein, or a fragment or derivative thereof or a nucleic acid encoding HMW or a fragment thereof or vector that it expresses itself may further comprise an adjuvant such as, for example, not limited to, alum, mLT, QS21 and all those listed above. Preferably, the adjuvant is selected from alum, LT, 3D-mPL, or Bacillus Calmette-Guerine (BCG) and mutated or modified forms of the foregoing, particularly MLT and LTR192G. The compositions of the present invention may also further comprise a pharmaceutically-arranged carrier, including, but not limited to, saline, bicarbonate, dextrose or other aqueous solution. Other suitable pharmaceutical carriers are described in Remigton's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field, which is incorporated herein by reference in its entirety. Immunogenic, antigenic and pharmaceutical compositions, including vaccines, can be administered in a suitable non-toxic pharmaceutical carrier, can be found in microcapsules, and / or can be found in a prolonged-release implant.

Immunogenic, antigenic and pharmaceutical compositions, including vaccines, can be administered at various intervals in order to maintain antibody levels and / or can be used in combination with other bacteriocidal or bacteriostatic methods. As used herein and in the claims, the term "antibodies" of the invention may be obtained by any conventional method known to one skilled in the art, such as, but not limited to, the methods described in Antibodies A. Laboratory Manual (E. Harlow, D. Lane, Cold Spring Harbor Laboratory Press, 1989) which is incorporated herein by reference in its entirety. The term "antibodies" is intended to include all forms, such as, but not limited to, polyclonal, monoclonal, purified IgG, IgM, IgA and fragments thereof, including, but not limited to fragments such as Fv, chain single Fv (scFV), F (ab ') 2, Fab fragments (Harlow and Leon, 1988, Antibody, Cold Spring Harbor); single chain antibodies (U.S. Patent No. 4,946,778) chimeric or humanized antibodies (Morrison et al., 1984, Proc. Nat'l Acad. Sci. USA 81: 6851); Neuberger et al., 1984, Nature 81: 6851) and regions of complementary determination (CDR), (see Verhoeyen and Windust, in Molecular Immunology 2nd edition, by BD Hames and DM Glover, IRL Press, Oxford University Press, 1996, in pages 283-325), etc. In general, an animal (a wide range of vertebrate species can be used, the most common being mice, rats, guinea pigs, bovines, pigs, hamsters, sheep, birds and rabbits) is immunized with the HMW protein or a sequence of nucleic acids or immunogenic fragment or derivative thereof of the present invention in the absence or presence of an adjuvant or any agent that increases the effectiveness of the immunogen and reinforced at regular intervals. The serum of the animal is tested for the presence of the desired antibody by any convenient method. The serum or blood of any animal can be used as the source of polyclonal antibodies. For monoclonal antibodies, the animals are treated in accordance with what is described above. When an acceptable antibody titre is detected, the animal is euthanized and the spleen removed aseptically for fusion. Spleen cells are mixed with a line of specifically selected immortal myeloma cells, and the mixture is then exposed to an agent, typically polyethylene glycol or the like, which promotes fusion of the cells. Under these circumstances, the fusion is carried out in a random selection and the resulting product is a mixture of cells fused together with non-fused cells of each type. The lines of myeloma cells used for fusion are specifically chosen in such a way that, by using the selection medium, such as HAT: hypoxanthine, aminopterin, and thymidine, the only cells that persist in the culture from of the fusion mixture are the cells that are hybrid between cells derived from the immunized donor and the myeloma cells. After fusion, the cells are diluted and cultured in the selective medium. The culture medium is sieved to determine the presence of. antibody having the desired specificity towards the chosen antigen. These cultures containing the antibody of choice are cloned by limiting dilution until it can be determined that the cell culture is a single cell in origin. Antigens, immunogens and immunoassays The HMW protein or nucleic acid encoding it, and fragments thereof are useful as an antigen or immunogen for the generation of anti-HMW protein antibodies or as an antigen in immunoassays including immunosorbent assays linked to enzyme (ELISA), radioimmunoassays (RIA) and other antibody binding assays not linked with enzyme or methods known in the art for the detection of antibacterial antibodies, anti-Chlamydia and anti-HMW protein. In ELISA assays, the HMW protein is immobilized on a selected surface, eg, a surface capable of binding proteins such as the wells of a polystyrene microtiter plate. After washing to remove incompletely absorbed HMW protein, a non-specific protein solution known to be antigenically neutral as to the test sample may be bound to the selected surface. This allows the blocking of non-specific absorption sites on the immobilization surface and consequently reduces the background caused by non-specific bonds of antisera on the surface. The immobilization surface is then placed in contact with a sample, such as for example clinical or biological materials, to be tested in a manner that causes the formation of an immunological complex (antigen / antibody). This may include diluting the sample with diluents such as bovine gamma globulin (BGG) solutions and / or phosphate buffered saline (PBS) / Tween. The sample is then allowed to incubate for 2 to 4 hours at temperatures within a range of about 20 ° C to 37 ° C. After incubation, the surface in contact with the sample is washed to remove the material that did not form immunocomplexes. The washing process may include washing with a solution such as PBS / Tween or a borate regulator. After the formation of the specific immunocomplexes between the test sample and linked HMW Xa protein, and after subsequent washing, the occurrence, and up to the amount of complex formation can be determined by subjecting the immunocomplex to a second antibody having specificity. for the first antibody. If the test sample is of human origin, the second antibody is an antibody having specificity for human immunoglobulins and in general IgG. To provide a means of detection, the second antibody can have an associated activity such as for example an enzymatic activity which generates, for example, a color development upon incubation with an appropriate chromogenic substrate. The detection is then achieved by detecting the generation of color. Quantification can be achieved by measuring the degree of color generation using, for example, a visible spectrophotometer and comparing it with an appropriate standard. Any other means of detection known to those skilled in the art can be employed. Another embodiment includes sets of diagnostic elements comprising all of the essential reagents required to carry out a desired immunoassay in accordance with the present invention. The set of diagnostic elements can be presented in a commercial package as a combination of one or several containers containing the necessary reagents. Said set of elements may comprise HMW protein or nucleic acid encoding it or fragment thereof, a monoclonal or polyclonal antibody of the present invention in combination with various components of conventional element sets. Components of conventional element sets will be readily apparent to those skilled in the art and are presented in several publications, including Antibodies A Laboratory Manual (E. Harlow, D. Lane, Cold Spring Harbor Laboratory Press, 1989) which is incorporated herein by reference In its whole. Components of conventional element assemblies may include elements such as, for example, microtiter plates, regulators to maintain the pH of the test mixture (such as, but not limited to, Tris, HEPES, etc.), second conjugated antibodies as for example anti-mouse IgG conjugated with peroxidase (or any other anti-IgG for the animal from which the first antibody was derived), and the like, and other standard reagents. NUCLEIC ACIDS AND THEIR USES The nucleotide sequences of the present invention, including DNA, and RNA and comprising a sequence encoding the HMW protein or a fragment or analogue thereof, can be synthesized using methods known in the art, such as, for example, by the use of conventional chemical approaches, or amplification by polymerase chain reaction (PCR) using convenient pairs of oligonucleotide primers and ligase chain reaction using a battery of contiguous oligonucleotides. The sequences can also allow the identification and cloning of the HMW protein gene from any Chlamydia species, for example to screen chlamydial genomic libraries or expression libraries. The nucleotide sequences encoding the HMW protein of the present invention are useful for their ability to selectively form duplex molecules with complementary segments of other protein genes. Depending on the applications, various hybridization conditions can be used to achieve several sequence identities. In specific aspects, nucleic acids comprising a sequence complementary to at least 10, 15, 25, 50, 100, 200 or 250 nucleotides of the HMW protein gene are provided (Figure 2). In specific embodiments, nucleic acids that hybridize with an HMW protein nucleic acid (e.g., having SEQ ID NO: 1, 23 or 24 sequences) under mild conditions, under mild, moderate or highly stringent conditions. For a high degree of selectivity, relatively strict conditions are used to form the duplexes, as for example, exemplary title and not in a limiting manner, conditions of low salt content and / or high temperature, such as those provided by 0.02 M NaCl. at 0.15 M at temperatures between approximately 50 ° C and 70 ° C. For some applications, less stringent hybridization conditions are required such as by way of example and not limitation, for example a salt of 0.15 M to 0.9 M, at temperatures lying within a range of about 20 ° C to 55 ° C. Hybridization conditions can also be stricter by adding increasing amounts of formamide, in order to destabilize the hybrid duplex. Thus, particular conditions of hybridization can be easily manipulated and generally through a method of choice according to the desired results. By way of example and not limitation, in general, convenient hybridization temperatures in the presence of 50% formamide are: 42 ° C for a probe having a homology of 95 to 100% with the target fragment, 37 ° C for a homology of 90 to 95% and 32 ° C for a homology of 70 to 90%. Mild, moderate and highly stringent conditions are well known to those skilled in the art and will vary predictably according to the base composition and the length of the particular nucleic acid sequence and according to the specific organism from which the derivative is derived. nucleic acid sequence. For guidelines regarding these conditions, see, for example, .Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Press, N.Y. pages 9.47-9.57; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. which is incorporated herein by reference. In the preparation of genomic libraries, DNA fragments are generated, some of which encode parts or all of Chlamydia HMW proteins. DNA can be dissociated at specific sites using several restriction enzymes. Alternatively, DNase can be used in the presence of manganese to fragment the DNA or the DNA can be physically cut, as for example by sonication. The DNA fragments can be size-separated by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis, column chromatography and sucrose gradient centrifugation. The DNA fragments can then be inserted into suitable vectors, including but not limited to, plasmids, cosmids, lambda or T4 bacteriophages, bacmids, and yeast artificial chromosome (YAC). (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, Glover, DM (ed), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, UK Vol. I, II.). The genomic library can be screened by hybridization of nucleic acid to determine the presence of labeled probes (Benton and Davis, 1977, Science 196: 180, Grunstein and Hogness, 1975, Proc, Nat. Acad.Sci.U.S.A. 72: 3961). Genomic libraries can be screened with labeled degenerate oligonucleotide probes corresponding to the amino acid sequence of any HMW protein peptide using optimal approaches well known in the art. In particular embodiments, the screening probe is a degenerate oligonucleotide corresponding to the sequence of SEQ ID NO: 4. In another embodiment, the screening probe may be a degenerate oligonucleotide corresponding to the sequence of SEQ ID NO: 5. In a further embodiment, any of the oligonucleotides SEQ ID NOs: 6-9, 12-14 and 18-21 are used as a probe. In additional embodiments, any of the sequences SEQ ID NOs: 1, 10-11, 22-24 and any fragment thereof, or complements of the sequence or fragments can be used as a probe. Preferred probes have 15 nucleotides or more. Clones in libraries with insert DNA encoding HMW protein in fragments thereof are hybridized with one or more degenerate oligonucleotide probes. Hybridization of oligonucleotide probes of this type in genomic libraries is carried out using methods known in the art. For example, hybridization with the two oligonucleotide probes mentioned above should be carried out in 2X SSC, 1.0% SDS at 50 ° C and washed using the same conditions. In another aspect, nucleotide sequence clones are encoded for part or all of the HMW protein or HMW derived polypeptides can also be obtained by screening Chlamydia expression libraries. For example, Chlamydia DNA or Chlamydia cDNA generated from RNA is isolated and random fragments are ligated preparations in an expression vector (eg, a bacteriophage, plasmid, phagemid or cosmid) such that the sequence inserted in the vector can be expressed by the host cell where it is then introduced into a vector. Various screening assays can be employed to select the expressed HMW protein or the HMW derived polypeptides. In one embodiment, the various anti-HMW antibodies of the invention can be used to identify the desired clones using methods known in the art. See, as for example, Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Appendix IV. Clones or plates from a library are contacted with the antibodies to identify the clones that bind. In a modality, colony or plates containing DNA encoding HMW protein or HMW-derived polypeptide could be detected using DYNA Beads according to Olsvick et al. , 29th ICAAC, Houston, Tex. 1989, which is incorporated herein by reference. Anti-HMW antibodies are crosslinked on tosylated M280 DYNA Beads, and these beads containing antibodies are then used for adsorption on colonies or plates expressing HMW protein or HMW derived polypeptide. Colonies or plates expressing HMW protein or HMW derived polypeptide are identified as the colonies or plates where the beads are packaged. Alternatively, anti-HMW antibodies can be immobilized unspecifically on a suitable support, such as for example Celite® resin or silica. This material can then be used for absorption in bacterial colonies expressing HMW protein or polypeptide derived from HMW according to that described in the previous paragraph. In another aspect, a polymerase chain reaction amplification can be used to produce a substantially pure DNA that encodes a part or all of the HMW protein from Chlamydia genomic DNA. Oligonucleotide primers, degenerate or otherwise, corresponding to known HMW protein sequences can be used as primers. In particular embodiments, an oligonucleotide, degenerate or otherwise, that encodes the peptide having an amino acid sequence of SEQ ID NO. 2, 3 or 15-17 or any part of. it can be used as the initiator 5 '. As examples of fragment, a 5 'primer can be made from any of the nucleotide sequences of SEQ ID NO. 4-7, 10, 12, 22-24 or any part thereof. Nucleotide, degenerate or other sequences that are complete reverse of SEQ ID NO. 11, 13 or 14 the 3 'initiator can be used. A polymerase chain reaction can be carried out, for example, by using a Perkin-Elmer Cetus thermal cycle device and Taq polymerase (Gene A p ™). One can choose to synthesize several different degenerate initiators, for use in polymerase chain reactions. It is also possible to vary the level of stringency of the hybridization conditions employed in the initiation of polymerase chain reactions to allow greater or lesser degrees of similarity between nucleotide sequences between the degenerate primers and the corresponding sequences in Chlamydia DNA. After a successful amplification of a segment of the HMW protein sequence coding, this segment can be cloned olecularly and sequenced, and used as a probe to isolate a complete genomic clone. This, in turn, will allow the determination of the complete nucleotide sequences of the gene, the analysis of its expression, and the production of its protein product for functional analysis with what is described below. ~~ In a clinical diagnostic mode, the nucleic acid sequences of the Hmw protein genes of the present invention can be employed with an appropriate indicator means, such as a label, for determination of hybridization. A wide variety of suitable indicator devices are known in the art, including radioactive, enzymatic or other linked indicators, such as for example labeled with avidin / biotin and digoxigenin, which can provide a detectable signal. In some diagnostic modalities, an enzyme label such as for example urease, alkaline phosphatase or peroxidase instead of a radioactive label can be used. In the case of enzymatic labels, calorimetric indicator substrates are known which can be used to provide a means visible to the human eye or spectrophotometrically to identify a specific hybridization with sample containing HMW protein gene sequences. The nucleic acid sequences of the HMW protein genes of the present invention are useful as hybridization probes in solution hybridizations and in modalities employing solid phase methods. In modalities that include solid phase procedures, the test DNA (or RNA) from the samples, such as chemical samples, including exudates, body fluids. { for example, serum, amniotic fluid, effusion of the middle ear, sputum, semen, urine, tears, mucus, bronchoalveolar lavage fluid) or tissues, is absorbed or otherwise fixed on a selected matrix or surface. The bound single-stranded nucleic acid is then subjected to specific hybridization with selected probes comprising the nucleic acid sequences of the genes encoding the HMW protein or fragments or analogs thereof of the present invention under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required according to, for example, the G + C contents, white nucleic acid type, nucleic acid source, hybridization probe size, etc. After washing the hybridization surface in such a way that the non-specifically bound probe molecules are removed, a specific hybridization is detected, or even quantified through the label. It is preferred to select portions of nucleic acid sequences that are conserved among Chlamydia species. The selected probe will have at least 15 base pairs and can be within a range of 30 to 90 base pairs.

EXPRESSION OF THE HMW PROTEIN GENE Plasmid vectors containing control and replicon sequences that are derived from species compatible with the host cell can be used for the expression of the genes encoding the HMW protein or fragment thereof in expression systems . Expression vectors contain all the necessary elements for the transcription and translation of the inserted protein coding sequence. The vector usually carries a replication site, as well as tagging sequences that are capable of providing a phenotype selection in transformed cells. For example, E. coli can be transformed using pBR322 which contains genes for ampicillin and tetracycline resistance cells. Other commercially available vectors are useful, including, but not limited to, pZERO, pTrc99A, pUC19, pUCld, pKK223-3, pEXl, pCAL, pET, pSPUTK, pTrxFus, pFastBac, pThioHis, pTrcHis, pTrcHis2, and pLEx. The plasmids or phage must also contain, or be modified to contain promoters that can be used by the host cell for expression of their own proteins. In addition, phage vectors containing control sequence and replicons that are compatible with the host can be used as a transformation vector relative to these hosts. For example, the phage in lambda GEM®-11 can be used to create recombinant phage vectors that can be used to transform host cells, such as E. coli LE392. Promoters commonly employed in recombinant DNA construction include the β-lactamase (penicillinase) promoter systems and lactose and other microbial promoters such as for example the T7 promoter system in accordance with that described in US Patent No. 4,952,496. Details relating to nucleotide sequences of promoters are known, allowing a person skilled in the art to functionally link them to genes. The particular promoter employed will generally depend on the desired results. In accordance with this invention, it is preferred to prepare the HMW protein by recombinant methods, particularly when the naturally occurring HMW protein as isolated from a culture of a Chlamydia species can include small amounts of toxic materials and other contaminants. This problem can be avoided by the use of a HMW protein recombinantly produced in heterologous systems that can be isolated from the host in such a way as to minimize contaminants in an isolated material. Particularly desirable hosts for expression in this regard include Gram-positive bacteria that do not have LPS and therefore do not have endotoxins. Such hosts include Bacillus species and may be particularly useful for the production of non-pyrogenic rHMW protein, fragments and analogues thereof. Various host-vector systems can be employed to express the protein coding sequence. These systems include, but are not limited to, mammalian cell systems infected with viruses (eg, vaccinia virus, adenovirus, etc.); insect cell systems infected with viruses (eg, baculovirus); microorganisms such as for example yeast vectors containing yeast, or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Hosts which are suitable for the expression of the HMW protein genes, fragments, analogs or variants thereof, may include E. coli, Bacillus species, Haemophilus, fungi, yeast, such as, for example, Saccharomyces pichia, Bordetella, or the baculovirus expression system can be used. Preferably, the host cell is a bacterium, and especially the bacterium is E. coli, B. subtilis or Salmonella. The expression elements of vectors vary in their strengths and specificities. According to the guest-vector system used, any of several suitable elements of transcription and translation may be employed. In a preferred embodiment, a chimeric protein comprising HMW protein or polypeptide sequence derived from HMW and a pre and / or pro sequence of the host cell is expressed. In other preferred embodiments, a chimeric protein comprising HMW protein or a sequence of HMW-derived polypeptides fused therewith, eg, an affinity purification peptide, is expressed. In additional embodiments, a chimeric protein is expressed comprising an HMW protein sequence or polypeptide sequence derived from HMW and a useful immunogenic peptide or protein. In preferred embodiments, the expressed HMW derived protein contains a sequence that forms either an outer surface epitope or the receptor binding domain of the native HMW protein. Any method of the art for inserting DNA fragments into a vector can be employed to construct expression vectors containing a chimeric gene consisting of appropriate transcription / translation control signals and the protein-coding sequences. These methods can include recombinant DNA in vitro and synthetic and recombinant techniques in vivo (genetic recombination). The expression of a nucleic acid sequence encoding HMW protein or polypeptide derived from HMW can be regulated by a second nucleic acid sequence such that the inserted sequence is expressed in a host transformed with the recombinant DNA molecule. For example, the expression of the inserted sequence can be controlled by any promoter / enhancer element known in the art. Promoters that can be employed to control the expression of inserted sequences include, but are not limited to, the SV40 initial promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter contained in the long terminal repeat. of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Nati. Acad. Sci. USA 78: 1441- 1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296: 39-42) for expression in animal cells; the ß-lactamase promoters (Villa-Kamaroff et al., 1978, Proc. Nati, Acad. Sci. USA 75: 3727-3731), tac (DeBoer et al., 1983, Proc. Nati. Acad. Sci. USA 80: 21-25), PL, or trc for "expression in bacterial cells (see also" Useful proteins from recombinant bacteria "in Scientific American, 1980, 242: 74-94), the promoter region of nopalinsintetase or the promoter of 35S RNA from cauliflower mosaic virus (Gardner et al., 1981, Nucí Acids, Res. 9: 2871), and the photosynthetic enzyme promoter, ribulosbiphosphatcarboxylase (Herrera-Estrella et al., 1984, Nature 310 : 115-120) for expression in implant cells, promoter elements from yeast or other fungi such as for example the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the promoter, pgk (phosphoglycerol kinase), the alkaline phosphatase promoter. Expression vectors containing HMW protein or HMW-derived polypeptide coding sequences can be identified to through three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences such as for example radioactivity with anti-HMW antibody. In the first approach, the presence of a foreign gene inserted into the expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to the protein ofEmbedded HMW or HMW derived polypeptide coding sequence. In the second approach, the recombinant / host vector system can be identified and selected based on the presence or absence of certain "marker" gene functions (eg, thymidine kinase activity, antibiotic resistance, transformation phenotype, body formation, etc.). occlusion in baculovirus, etc.) caused by the insertion of foreign genes in the vector. For example, if the HMW protein or HMW-derived polypeptide coding sequence is inserted into the 1-vector marker gene sequence, recombinants containing the insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression readers can be identified by assaying the foreign gene product expressed by the recombinant. Such assays may be based, for example, on the physical or functional properties of HMW protein or HWM-derived polypeptide in in vitro assay systems, for example, binding with HMW ligand or receptor or binding with anti-HMW antibody of the invention, either the ability of the host cell to haemagglutinate or the ability of the cell extract to interfere with hemagglutination by Chlamydia. Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art can be employed to propagate it. Once acceptable host and acceptable growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As explained above, the expression vectors that can be employed include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect virus such as baculovirus; yeast vectors; bacteriophage vectors (for example, lambda), and plasmid and plasmid DNA vectors, to name a few. In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or else recombinant expression readers can be identified by testing the foreign gene product expressed by the recombinant. Such assays may be based, for example, on the physical or functional properties of HMW protein or HWM-derived polypeptide in in vitro assay systems as an example, binding with HMW ligand or receptor or binding with anti-HMW antibody of the invention, either the ability of the host cell to haemagglutinate or the ability of the cell extract to interfere with hemagglutination by Chlamydia. Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art can be employed to propagate it. Once acceptable host and acceptable growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As explained above, the expression vectors that can be employed include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect virus such as baculovirus; yeast vectors; bacteriophage vectors (for example, lambda), and plasmid and plasmid DNA vectors, to name a few. In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the genetic product in the specific manner desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, the expression of the genetically engineered HMW protein or HMW derived from HMW can be controlled. In addition, different host cells have characteristic and specific mechanisms for post-translational translation and processing and protein modification. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the expressed foreign protein. Proteins, polypeptides, peptides, antibodies and nucleic acids of the invention are useful as reagents for clinical or medical diagnosis of infections with Chlamydia and for scientific research on the pathogenicity, virulence, and infectivity of Chlamydia, as well as defense mechanisms of the Guest. For example, the DNA and RNA of the invention can be used as probes to identify the presence of Chlamydia in biological samples by hybridization or amplification by polymerase chain reaction. DNA and RN can also be used to identify other bacteria that can encode a polypeptide related to the Chlamydia HMW protein. The proteins of the invention can be used to prepare polyclonal and monoclonal antibodies which can be used to further purify compositions containing the proteins of the invention by affinity chromatography. The proteins can also be used in standard immunoassays to screen for the presence of antibodies to Chlamydia in a sample. 7. BIOLOGICAL DEPOSITS Certain plasmids containing portions of the gene have the open reading frame of the gene and which encode the Chlamydia HMW protein described and referred to herein have been deposited with the American Type Culture Collection (ATCC) located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, in accordance with the Budapest Treaty and in accordance with 37 CFR 1.808 and prior to the filing of this application. The identifications of the respective portions of the genes present in these plasmids appear below. Samples of the deposited materials will be available to the public upon granting a patent based on the publication of the North American patent. The invention described and claimed herein should not be considered as limited by the scope of the deposited plasmids since the deposited mode is for the purpose of illustrating the invention only. Any equivalent or similar plasmid that encodes similar proteins or equivalents or fragments or analogs according to that described in this application are within the scope of the invention. Plasmid ATCC Accession No. Deposit Date PAH342 ATCC 985538 September 8, 1997 8. EXAMPLES The above disclosure generally describes the present invention. A more specific description is provided below in the following examples. The examples are described solely for the purpose of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances suggest. Even though specific terms were employed herein, such terms must be understood in a descriptive sense and not to limit the invention. Methods of molecular genetics, protein biochemistry and immunology used but not explicitly described in the dissemination and in the examples are widely reported in the scientific literature and are within the reach of experts in the field. 8.1 EXAMPLE 1: Isolation and purification of mature Chlamydia proteins McCoy cells were cultured either in standard tissue culture flasks of 225 cm2 or in Welsh revolving bottles (Cytodex microcarrier, Pharmacia) at a temperature of 37 ° C in C02 at 5% using a DMEM medium supplemented with fetal bovine serum free of 10% Chlamydia antibody, glucose and non-essential amino acids. Used bodies of C. trachomatis L2 (ATCC VR-902B) from infected McCoy cells. Basically, McCoy cells infected with C. trachomatis L2 (LGV) were sonicated and cell debris was removed by centrifugation. The supernatant containing Chlamydia elementary bodies (EBs) was then centrifuged and the pellet containing EBs was resuspended in a Hank balanced salt solution (HBSS). An RNase / DNase solution was added and incubated at a temperature of 37 ° C for one hour with occasional mixing. The solution containing EV was layered in a discontinuous density gradient (40%, 44% and 54%) of Angiovist 370 (mixture of diatrizoate melgumine and diatrizoate sodium, Berlex Laboratories, Wayne, NJ) and subjected to ultracentrifugation for separation of EBs in the gradient. After centrifugation, the EBS were harvested from the gradient between the interface of the Angiovist 370 layers of 44% and 54%. The EBs were washed in a phosphate-buffered saline solution and resuspended in HBSS. The purified EBs were extracted sequentially with 0.1% OGP (high ionic strength) in HBSS to remove peripheral surface protein and kept on ice. The same EB preparation was then extracted with 1.0% OGP, 10mM DTT, 1mM PMSF, 10mM EDTA, and in a 50mM Tris buffer pH 7.4. The extracts were dialyzed (3500 MWCO) to remove detergent and other reagents and polyophilization concentrates. The extracts containing protein were diluted in HBSS and passed on commercially available heparin-sepharose columns (HiTrap Col., Pharmacia). After the samples were applied to the heparin column, the non-adhered proteins were removed by washing with excess HBSS. The bound proteins were eluted in batches with PBS containing 2 M sodium chloride. The eluents were extensively dialyzed to remove salt and then lyophilized. The heparin binding proteins were fractionated according to size by SDS-PAGE and visualized by tension with silver or analyzed by the Western blot method. Proteins (s) of approximately 105-115 kDa present in moderate amounts were detected as shown in Figure 1. The isoelectric point of the native HMW protein was determined as 5.95. To obtain an N-terminal amino acid sequence, sufficient quantities of the HMW protein were subjected to electro absorption (>; 5 μg) in PVDF membrane (Applied Biosystems), and a Coomassie blue stain was applied. The immobilized HMW protein was released from the membrane and treated in situ with low levels of endopeptidase Lys-C, endopeptidase Arg-C and / or endopeptidase Glu-C to fragment the native protein. The resulting peptide fragments were purified by HPLC and their N-terminal amino acid sequences were terminated using an ABI 430 protein sequencer and standard protein sequencing methodologies. The N-terminal amino acid sequence is: EIMVPQGIYDGETLTVSFXY and is known as SEQ ID No. 3. When a PDB + SwissProt + PIR + GenPept database (> 145 K unique sequences) was searched with the N-terminal sequence of HMW proteins (20 residues) using rigorous correspondence parameters, no precise homology was found. Thus, the HMW protein is a novel chlamydial protein. Since this protein was isolated under conditions that would only release peripheral membrane proteins (for example Omp2), these data indicate that the HMW protein is a protein associated with the surface. 8.2 EXAMPLE 2: PREPARATION OF ANTIBODIES FOR WHOLE CHLAMYDIA Ebs To help characterize the HMW protein, hyperimmune rabbit antisera were prepared against total Ebs from C. trachomatis L2. Animal CDa received a total of three immunizations of approximately 250 μg of Ebs of chlamydia per injection (starting with a complete Freund's adjuvant and followed with an incomplete Freund's adjuvant) at intervals of approximately 21 days. In each immunization, approximately half of the material was administered intramuscularly (i.m.) and half was injected intranodally. Fourteen days after the third vaccine, a fourth boost of approximately 100 μg EBs i.m. and the animals were bled 7-10 days later. A 1: 100,000 titer was obtained in accordance with that determined by ELISA. 8.3 EXAMPLE 3: DETERMINATION OF POST-TRANSLATIONAL MODIFICATIONS Recently, several proteins associated with C. trachomatis membranes have been modified post-translationally. The EB proteins rich in 18 kDa and 32 kDa cysteine, which are lectin binding proteins, carry specific carbohydrate moieties (Swanson, A.F. and C.C Kuo, 1990. Infect. Immun., 58: 502-507). The incorporation of radiolabeled palmitic acid has been used to demonstrate that the type C protein, Mip-like trachomatis of approximately 27 kDa is lipidated (Lundemose, AG, DA Rouch, CW Penn, and JH Pearce, 1993. J. Bacteriol. : 3669-3671). Swanson et al. They discovered that MOMP of serovar L2 contains N-acetylglucosamine and / or N-acetylgalactosamine and these carbohydrate moieties mediate the MOMP binding on Hela cell membranes. To determine if the HMW protein is glycosylated, EBs were cultured in McCoy cells in the presence of tritiated galactose or glucosamine, subjected to affinity chromatography with deparin and the deparin binding proteins were analyzed by SDS-PAGE and autoradiographed. Briefly, McCoy cells are cultured in T225 bottles under standard conditions (DMEM + 10% FCS, 35 ml per bottle, 10% C02) at a confluence of approximately 90% and are inoculated with sufficient EBs to achieve an infection capacity of 90 % -100% After an infection period of 3 hours at a temperature of 37 ° C, cycloheximide (1 μg / ml) is added to inhibit the synthesis of host cell protein and the cultures are re-incubated for a further 4-6 hours. Approximately 0.5 Ci of tritiated galactose (D- [4-5-3H (N)] galactose, NEN) either glucose (D- [1, 6 H (N) glucosamine, NEN) is then added to each flask and the cultures allowed to incubate for 30- 40 additional hours Cells are harvested by scraping and the EBs are purified by gradient centrifugation. The HMW protein is isolated from 1.0% OGP surface extracts by affinity chromatography, eluted with NaCl and analyzed by SDS-PAGE using molecular weight markers labeled with 14 C (BRL) and then committed to autoradiography. Dried gels are exposed for 1-4 weeks to a Kodak X-AR film at a temperature of -70 ° C. To determine the post-synthesis lipid modification, serovar L2 of C. trachomatis is grown in monolayers of McCoy cells in accordance with standard procedures. Approximately 24 hours after infection, a conventional culture medium is removed (DMEM + 10% FCS) and said medium is replaced with a medium free of serum containing cycloheximide (1 μg / ml) and palmitic acid [U-14C] (0.5 mCi / vial T225, NEN), and incubated for an additional 16-24 hours to allow protein lipidation to occur. Surface EB extracts are prepared, heparin binding proteins are isolated and analyzed by autoradiography in accordance with what is described above. The functionality of glycosylated or lipidated portions is evaluated by the treatment of whole Ebs or surface extracts of OGP with appropriate glycosidases. After removal of carbohydrate, the extracts are subjected to affinity chromatography and SDS-PAGE to determine if the HMW protein retains the ability to bind with heparin sulfate. 8.4 EXAMPLE 4: CLONING OF THE N-END SEGMENT OF THE HMW PROTEIN GENE Degenerate oligonucleotides were designed based on the N-terminal sequence of the HMW protein and said oligonucleotides were synthesized. These oligonucleotides were then used to generate polymerase chain reaction products specific for genes that were used as hybridization probes to screen a C. Trachomatis L2? ZAPII DNA library to isolate the gene for the HMW protein. Briefly, appropriate segments of low degeneracy peptides were identified from the internal amino acid sequence and N-terminal amino acid sequence data through a computerized analysis (MacVector, IBI) and used to guide the design of chain reaction primer sets. of oligonucleotide polymerase specific for low degeneracy sequence. Employing the N-terminal primary sequence as a guide, four degenerate oligonucleotide probes complementary to the first six residues of the HMW EIMVPQ peptide (residues 1-6 of SEQ ID No. 3) were designed and used, and they comprise all possible combinations of nucleotides (equal total degeneration = 192 individual sequences) and used as forward amplification primers. SEQ ID No. 4 5 '-GAA-ATH-ATG-GNT-CCN-CAA-3' SEQ ID NO. 5'-GAA-ATH-ATG-GTN-CCN-CAG-3 'SEQ ID No. 6 5'-GAG-ATH-ATG-GTN-CCN-CAA-3' SEQ ID No.7"5'-GAG -ATH-ATG-GTN-CCN-CAG-3 'Two additional oligonucleotide probes representing the reverse complementary DNA sequence of the five internal residual peptides YDGET (residues 9-13 of SEQ ID No. 3), and comprising all the Possible nucleotide compositions (total degeneration = .128 individual sequences) have been designed and used as reverse application primers SEQ ID No. 8 5'-NGT-YTC-NCC-RTC-ATA-3 'SEQ ID No. 9 5'-NGT-YTC-NCC-RTC-GTA-3 'Oligonucleotides were synthesized on an ABI model 380B DNA synthesizer using a 0.2 μmol scale column (trityl, self-dissociation) and standard chemistry of phosphoramidite. Oligonucleotides were manually purified in C-18 syringe columns (OP, ABI columns). The purity and the yield were finished spectrophotometrically (relations 230/260/280). Standard amplification reactions by polymerase chain reaction (2 mM Mg2, 200 umol dNTPs, 0.75 AmpliTaq units, final volume 50 μl) were programmed using approximately 0.2 μg of L2 C DNA. Trachomatis (approximately 3X107 copies of the HMW protein gene in the case of a single copy) and approximately 100 pmol of each primer forward (N-end oligo) and reverse (internal oligo). Higher than normal initiator concentrations (~ 20 pmol / 50 μl) were used for amplification, at least initially, in order to compensate for initiator degeneracy. The amplification of white sequences was achieved using a thermal standard profile of three steps, 30 cycles, that is, 95 ° C, 30 seconds; 60 ° C, 45 seconds, 72 ° C, 1 minute. The amplification was carried out in sealed 50 μl glass capillary tubes using a term cycle device from Idaho Technologies. To verify that the polymerase chain reaction production generated during these amplification reactions were specific for the HMW protein coding sequence and were not "primer dimer" or other DNA amplification artifacts, amplimers were purified using columns Silica gel centrifuges (QIAGEN) were cloned into the pZERO polymerase chain reaction cloning vector (StrataGene) and subjected to direct DNA sequence analysis. The DNA sequence for the PCR products was determined using conventional dideoxy end sequencing chemistry and a modified T7 DNA polymerase (Sequeanse, USB). Briefly, each hardened double-stranded plasmid was denatured through a short course of NaOH. After neutralization, each denatured tempering was employed to program 4 separate sequencing reactions. Each reaction contained the universal forward sequencing primer M13 (21-mer) but a different ddNTP / dNTP terminating mixture (ie, A, G, C or T). The termination products were labeled by the inclusion of [alpha-35S] dATP in the reaction (approximately 50uCi / reaction, more than 3000Ci / mmol, Amersham). Individual extension products were denatured (formamide, approximately 95 ° C) and subjected to a high-resolution denaturing polyacrylamide gel electrophoresis (6% acrylamide, 8M urea, TAE regulator, approximately 500V, approximately 90 minutes). Sequencing gels were then transferred to filter paper (Whatmann 3MM), dried under vacuum, and then subjected to autoradiography at a temperature of -70 ° C for 24-72 hours. Base stairs were read manually from each gel and a consensus sequence was determined. Specific amplimers for HMW protein suitable for library screening and / or Southern blot analysis were produced by polymerase chain reaction and radiolabeled uniformly during the amplification process by the addition of [alpha-32P] dNTPs (approximately 50μCi each dNTP, Amersham , more than 5000 Ci / mmol) to the reaction mixture. Labeling reactions were carried out as before except that the reactions were performed in 0.5 ml microcentrifugation tubes using a Warm Cycle device. The unincorporated label and the amplification primers of the reaction mixture were removed using chromatography columns by centrifugation size exclusion (BioSpin 6 columns, BioRad). A highly redundant C. trachomatis serovar L2 DNA library (more than 50,000 primary clones) has been constructed by cloning fragmented fragments greater than or equal to 10 Kbp in size produced from a partial digestion of ECQRI of genomic DNA in the lambdaZAPII cloning vector (Stratagene). Polymerase chain reaction products specific for radiolabelled HMW protein were used to screen this library to determine the recombinant clones that carry all or part of the HMW protein coding sequence. Standard recombinant DNA procedures and methodologies were used for these experiments. All the phages that were hybridized with these probes were purified until homogeneity through sequential rounds of placement in dishes and sieves of hybridization. Once the reactive phages have been purified, phagemids containing inserts (pBluescript SK derivatives) of the phage of origin were rescued by excision by co-infection of the host cells with an appropriate auxiliary phage, for example, R408 or VCSM13 (Stratagene). Individual pharamids were further purified by plating on LB agar containing ampicillin (100 μg / ml) and individual colonies were selected.

To confirm that purified phagemid derivatives carried the HMW protein sequence, the plasmid DNA was prepared and used to program amplification reactions containing the sets of polymerase chain reaction primers specific for HMW protein. The presence of specific inserts for HMW protein was confirmed by the production of the polymerase chain reaction product of appropriate size. Plasmid pAH306 is a derivative containing HMW protein that was isolated by these methodologies. PHYSICAL CARTOGRAPHY OF pAH306 The pAH306 inserts were physically mapped and the HMW protein gene localization was determined using the appropriate six base restriction endonuclease (e.g., EcoRI, Hindi, BamHI, PstI, Smal, Kpnl, etc.), and sequences encoding HMW protein localized by Southern hybridization using specific polymerase chain reaction products for extreme Radiolabelled N as a probe. The orientation and magnitude of the HMW protein-specific sequences were determined by polymerase chain reaction analysis using sets of primers consisting of forward primers specific for HMW protein as well as complementary reverse primers of either the T3 promoter sequences. or T7 located in the cloning vector. It was determined that plasmid pAH306 contained a unique EcoRI fragment of approximately 6.6 Kbp of chlamydial origin. A directional polymerase chain reaction analysis of pAH306 demonstrated that this derivative encodes approximately 1.5 Kbp of the N-terminal region of the HMW protein gene. The DNA sequence for the HMW protein gene encoded in pAH306 was obtained for both strands through a conventional "sequence shift" coupled with asymmetric polymerase chain reaction cycle sequencing methodologies (ABI Prism Dye-Terminator Cycle Sequencing, Perkin-Elmer). Sequencing reactions were programmed with undigested plasmid DNA (approximately 0.5 μg / rxn) as annealing and appropriate sequencing primers specific for HMW protein (approximately 3.5 pmol / rxn). In addition to the annealing and the sequencing primer, each sequencing reaction (approximately 20 μl) contained the four different termination nucleotides of dNTPs (ie, A, G, C, and T) and the four corresponding ddNTPs (i.e. ddA, ddG, ddC and ddT); with each terminator conjugated with one of four different fluorescent dyes. Single-strand sequencing elongation products were terminated at random positions along the annealing by incorporating the ddNTP terminators labeled with dye. Finishing products labeled with fluorescent dye were purified using microcentrifugation size exclusion chromatography columns (Pri? Ceton Genetics), dried in vacuo, suspended in a Tempered Resuspension Regulator (Perkin-Elmer), denatured at 95 ° C during approximately 5 minutes, and resolved by high-resolution capillary electrophoresis in an automated ABI 310 DNA sequencer (Perkin-Elmer). DNA sequence data produced from individual reactions were collected and the relative peak fluorescence intensities were analyzed automatically on a PowerMax computer using a programmatic ABI sequence analysis (Perkin-Elmer). Individually autologous DNA sequences were manually edited for accuracy before being combined into a "series" of consensus sequences using a programmatic AutoAssembler (Perkin-Elmer). Both strands of the HMW protein gene segment encoded by pAH306 were sequenced and these data were compiled to create a composite sequence for the protein gene segment of HMW. The segment coding sequence of the HMW protein is listed as SEQ ID No .: 10 and is represented by nucleotides 382 to 1979 in Figure 2. A map of pAH306 is shown in Figure 5.

Database analysis (eg, primary amino acid homologies, hydrophobic profiles, N- / 0-glycosylation sites, functional / conformational domain analysis) and predicted amino acid sequences for the HMW protein are carried carried out using the programmatic GeneRunner and Intelligentics, and it was determined that the HMW protein was novel. 8.5 '. EXAMPLE 5: EXTREME C SEGMENT DONATION OF THE HMW PROTEIN GENE Chromosome displacement was employed to isolate the C-terminal portion of the HMW protein gene. A fragment of BamHI-EcoRI of approximately 0.6 Kbp distant from the N-terminus sequence of the mature HMW protein and close to the T3 promoter sequence of the vector was chosen as a probe for the initial chromosome shift. Briefly, pAH306 was digested to completion with BamHI and EcoRI and the digestion products were fractionated according to size by agarose gel electrophoresis (0.8% agarose in TAE buffer). The desired band of approximately 0.6 Kbp BamHI / EcoRI (b / E) was removed from the gel and purified using silica gel chromatography chromatography columns and commercially available reagents (QIAGEN): The purified 0.6 Kbp B / E fragment was radiolabelled with [α-dATP] (> 3000Ci / mmol, Amersham) through randomly initiated labeling methodologies using commercially available reagents (Boehringer Mannheim) and was used to test Southern blots of C. trachomatis L2 genomic DNA that had been digested to HindIII culmination. The 0.6 Kbp B / E probes from pAH306 was hybridized on a genomic fragment of HindIII of approximately 1.4 Kbp. Based on the restriction map experimentally derived from the HMW protein gene segment encoded in pAH306, this fragment encodes approximately 0.2 Kbp of the C-terminal HMW protein sequence. The radiolabelled 0.6 Kbp B / E fragment was subsequently used to test a moderately redundant C. trachomatis library (approximately 5,000 primary clones) to identify clones containing the approximately 1.4 Kbp HindIII fragment. Briefly, C. trachomatis L2 genomic DNA was digested to completion using an approximately 10 fold excess of the HindIII restriction endonuclease (approximately 10 units per 1 μg of genomic DNA, 37 ° C, 18-24 hours). The products of the dispersion were fractionated according to size by agarose gel electrophoresis (0.8% agarose, TAE) and DNA fragments ranging from approximately 1.0 Kbp to 2.0 Kbp were removed from the gel. Removed agarose bands containing the desired sizes of DNA fragments were dissolved in a solubilization / binding solution (QX1, QIAGEN) and purified using commercially available silica gel centrifuge columns (QUIAGEN). Purified 1.0-2.0 Kbp HindIII genomic fragments were then cloned into the SK-pBlueScript plasmid which had previously been digested to HindIII completion and then treated with calf intestinal iosphatase to avoid re-ligation of the vector. The vector / insert ligations were carried out in a final reaction volume of approximately 50 μl (50mMTris-HC1, pH 7.00, 10 mM NaCl, 1 mM ATP, 0.5 mM DTT) at a temperature of 25 ° C for approximately 16- 24 hours using T4 DNA ligase (approximately 10 units / reaction) and a molar ratio between vector and insert of approximately 1:10. After ligation, aliquots (approximately 50 ng of ligated DNA) were used to electroporate a competent E. coli host, such as E. coli TOP10. The electroporated cells were then placed in a dish on LB agar containing approximately 100 μg / ml of ampicillin to select clones containing plasmid. Approximately 1,000 ApR transformants containing plasmid were transferred directly from LB Ap agar plates into nylon membranes (HyBond N +, Amersham) by capillary action. After the transfer, dishes were re-incubated at a temperature of 37 ° C to regenerate viable colonies for further handling. The colonies transferred to membranes were used and the DNA was released by treating the colonies with a denaturing SDS / NaOH solution.

A solution of regulated NaCl 6 Tris was used to neutralize and stabilize the lysis material. The released DNA was immobilized on the membranes by UV irradiation. Standard recombinant DNA procedures and standard methodologies were used to test the colonies with the radiolabelled 0.6 Kbp B / E fragment and to identify the recombinant derivatives carrying the desired HindIII fragment of approximately 1.4 Kbp. Plasmid pAH310 was a derivative isolated by these methods and the coding segment of the HMW protein is represented by nucleotides 99-2401 in Figure 2. A restriction analysis using HindIII and EcoRI, individually and in combination, together with DNA sequence analysis of purified plasmid DNA confirms that pH310 encodes the expected HindIII fragment of approximately 1.4 Kbp. These analyzes also demonstrate that the approximately 1.4 Kbp insert consists of the same approximately 1.2 kbp HindIII-EcoRI fragment that is present in pAH306 and an approximately 0.2 Kbp EcoRI-HindIII fragment encoding the HMW protein-specific DNA. of terminal C. The EcoRI-HindIII fragment of approximately 0.2 Kbp (E / H) was chosen as the probe for the second displacement of chromosomes. Briefly, pAH310 was digested to completion with EcoRI and HindIII and the digestion products were fractionated in size by agarose gel electrophoresis (0.8% agarose in TAE regulator). The desired band of approximately 0.2 Kbp (E / H) was removed from the gel, purified, radiolabeled with [aP 32 p and used as a probe to identify clones in the original genomic library of C. trachomatis L2? ZAPII encoding the C-terminal segment of the HMW protein gene. Plasmid pAH316 is a derivative isolated by these methods. A restriction analysis of pAH316 showed that this derivative contains an insert of C. trachomatis L2 of approximately 4.5 Kbp consisting of two EcoR fragments of approximately 2.5 Kbp and approximately 2.0 Kbp in size. Southern hybridization analysis using the E / H fragment of approximately 0.2 Kbp as a probe located this sequence on the EcoRI fragment of approximately 2.5 Kbp of pAH316. Directional PCR analyzes employing pAH3156 plasmid DNA purified as annealed and sets of amplification primers specific for the E / H fragment of approximately 0.2 Kbp and vector sequences T3 and T7 demonstrated that pAH316 encodes the C-terminal segment of the HMW protein. The coding segment of the HMW protein is represented by nucleotides 1974 to 3420 in Figure 2, and appears in the list as SEQ ID No .: 11. 8.6 Example 6: PRODUCTION OF RECOMBINANT PROTEIN HMW TRUNCATED Half-end N of the HMW protein was cloned by polymerase chain reaction as a fragment of approximately 1.5 Kbp in a commercially available EcoRI expression plasmid pTrccHisB (Invitrogen). The forward primer employed in these reactions was designated 140FXHO (57-mer), listed as SEQ ID No. 18, and contains sequences complementary to the first 10 N-terminal residues of the mature HMW protein. In addition to the HMW protein coding sequences this forward primer also carries a unique Xhol restriction site located optimally upstream of the first residue of the mature HMW protein (Glu / E) for proper fusion on the purification domain by affinity (His) 6 encoded in the vector plasmid, and a fastener G / C of 6 5 'end bases for effective amplification and an internal spacer of 12 bases for recognition and effective endonuclease digestion.

SEQ ID No. 18 5 '- AAG-GGC-CCA-ATT-ACG-CAG-AGC-TCG-AGA-GAA- ATT-ATG-GTT-CCT-CAA-GGA-ATT-TAC-GAT - 3' SEQ ID No. 19 5 '- CGC-TCT-AGA-ACT-AGT-GGA-TC - 3' The commercially available reverse sequencing primer SK (20mer, StrataGene), SEQ ID No. 19, which is complementary to the phagemid sequences downstream of the EcoRI site in pAH306, was used as a reverse amplification initiator in these reactions. To obtain acceptable yields of the HMW protein ORF product (approximately 1.5 Kbp), a polymerase chain reaction amplification was carried out using a mixture of thermostable DNA polymerase consisting of T. thermophilus DNA polymerase (Advantage Polymerase) , as the primary amplification polymerase and a minor amount of a high fidelity thermostable DNA second polymerase to promote an additional 5 '- 3' screening activity (CloneTech). An anti-Tth DNA polymerase antibody was added to the reaction mixture to provide automatic "hot start" conditions that promote the production of large amplimers greater than 2 KBP. Plasmid DNA aPh.306 purified using a commercially available alkaline / SDS system (QIAGEN) and silica gel centrifuge columns (QIAGEN) was used to program these amplification reactions (approximately 2.0 ng / reaction).

The amplimer of approximately 1.5 Kbp was purified from the unincorporated primers using columns - silica gel centrifuge and digested to completion using an excess of XhoI and EcoRI (approximately 10 units per one μG of DNA). The truncated HMW protein ORF of purified and digested N-terminus was then cloned into the commercially available expression plasmid pTrcHisB which had previously been digested with both XhoI and EcoRI (5: 1, insert: vector ratio). Aliquots of the ligation reaction were employed to electrotransform a suitable E. coli host (e.g. TOP10). Mini-prep of DNA from randomly selected ampicillin-resistant transformants were prepared, digested to completion with Hhol, EcoRI or a combination of both and examined to determine the presence and orientation of the ORF insert of truncated HMW protein. approximately 1.5 Kbp by agarose gel electrophoresis. The mini-prep of DNA from clones determined as carrying the Xhol / EcoRI insert of approximately 1.5 Kbp were prepared and used to program DNA sequencing reactions by asymmetric chain reaction of polymerases in order to confirm the fidelity of the formed union between the HMW protein fragment and the affinity purification domain (His) 6 of the expression vector. Plasmid pJJ36-J was a recombinant derivative isolated by these methods and is represented by nucleotides 446 to 1977 in Figure 2. The sequence of - amino acids deduced from the truncated fragment of the protein of HMW is represented by amino acids 29 to 532 in Figure 3 and appears in the list as SEQ ID No. 17. 8.7 EXAMPLE 7: DETERMINATION OF PRESENCE OF OTHER SPECIES Analysis of polymerase chain reaction were undertaken to establish the presence of the gene HMW in several strains of C. trachomatis clinically recognized and also other chlamydial species, for example C. pneumoniae. Strains of chlamydia trachomatis as frozen reserves from ATCC (Rockville, MD) were used to infect subconfluent monolayers (approximately 80%) of McCoy cells in accordance with standard procedures. The infected monolayers were either centrifuged in a Sorvall RT6000B centrifuge (approximately 1,300 revolutions per minute, 25 ° C, 30 minutes) and / or treated with dextran sulfate (approximately 50 μg / ml) at the time of infection to increase fixation Initial biovars of low infectious capacity (non-LGV) to host cells and therefore increase the final yield of EB. Approximately 48 hours later, infected monolayers were picked up by scraping and the host cells disrupted by their indication to release the elementary bodies (EBs). Total DNA was extracted from purified EBs (approximately 107-108) of each strain using . the K / Nonidet P40 proteinase method described by Denamur, et al., J. Gen. Microbiol. 137: 2525-2530 (1991L, which is incorporated herein by reference, and was further purified by phenol / chloroform extraction and salt precipitation.

Biotechnologies Inc. Chlamydia pneumoniae purified genomic DNA (AR-139). To determine the presence of the HMW protein gene in these strains, amplification reactions were programmed using total chlamydia DNA as annealed and the sets of oligonucleotide primers specific for HMW protein segment that appear in the following list. SEQ ID No. 20 5 '- ATG-GTT-CCT-CAA-GGA-ATT-TAC-G-3' SEQ ID No. 21 5 '- GGT-CCA-TCA-GCG-GGA-G-3' Briefly, standard PCR amplification reactions (2 mM Mg2 +, 100 micro mol dNTPs, 0.75 units AmpliTaq polymerase, 50 micro 1 volume) were programmed using approximately 15 micro 1 of crude extracts of C. trachomatis DNA (approximately 10 micro 1 of commercially available C. pneumoniae DNA) and about 20 pmol of each specific amplification primer for direct and reverse HMW protein of SEQ ID No. 20 and 21. Amplification of small target sequences (<; 2 Kbp) was achieved using a thermal profile of 32 cycles of three steps, that is, 95 ° C, 30 seconds; 60 ° C, 30 seconds, 72 ° C, 1 minute. Amplification of the longest white sequences for ORF cloning and sequencing was carried out using crude DNA extracts in identical form except that a combination of DNA polymerase enzyme Taba / Vent MAb was used (Advantage PCR, Clontech) at a temperature of 72 ° C. An extension time was used which corresponded to the size of the desired PCR product plus two minutes (i.e., desired PCR product = 6Kbp, extension time = 8 minutes). Both conventional and long-distance polymerase chain reactions were performed using 0.2 ml thin-walled polypropylene microcentrifuge tubes in an ABI 2400 thermal cycle device (PerkinElmer). After the thermal cycles, aliquots (approximately 20 micro 1) of the reactions and polymerase chain reaction products identified by 'standard agarose gel electrophoresis (0.8% agarose in TAE regulator) as well as bromide staining were analyzed. ethidium The results showed that the HMW protein is highly conserved in clinically relevant serovars; the HMW gene was present in all the strains of chlamydia tested, including serovars B, Ba, D, E, F, G, H, I, J, K, Li, L2 and MoPn and in C. pneumoniae. 8.8 EXAMPLE 8: DETERMINATION OF VARIATION OF SEQUENCES to establish the degree of variation of amino acid sequence and DNA between different strains of chlamydia, the gene for the HMW protein was cloned by PCR from a serovar of C. trachomatis (which represents the trachoma group of organisms) as from a serovar F of C. trachomatis (representing STD biovars of low infectious capacity) and were compared with the L2 sequence of C. trachomatis of HMW protein consensus. Briefly, LD-PCR was used to generate HMW protein-specific DNA fragments of approximately 6 Kbp and containing the complete coding sequence for the mature HMW protein. Amplification conditions for these LD-PCR exercises were in accordance with that described in Example 6. The reverse amplification primer used in these reactions (p316Kpn-RC, 56mer), listed as SEQ ID No. 13, is complementary to a sequence placed approximately 3Kbp downstream of the predicted HMW protein termination strand. As an aid for the cloning of the desired 6 Kbp amplimer, a unique restriction endonuclease site Kpnl 5 'to the chlamydial sequence was created in the p316Kpn-RC primer. The direct amplification primer used for these reactions (p306Kpn-F, 56mer) listed as SEQ ID No. 12, contains the complementary sequence of the first 10 amino acid residues (30 nucleotides) that specify the mature HMW protein as well as one that specifies a Kpnl site. P306Kpn-F was designed in such a way that the coding sequence of the end of the mature HMW protein could be bound in frame to a hexa-His agility domain encoded downstream of the highly efficient trc promoter in the E. expression vector. coli pTrcHisB (ClonTech) when the first of approximately 6 kbp was inserted into the Kpnl site of this vector. SEQ ID No. 12 5 '-AAG-GGC-CCA-ATT-ACG-CAG-AGG-GTA-CCG-AAA- TTA-TGG-TTC-CTC-AAG-GAA-TTT-ACG-AT-3' SEQ ID No. 13 5 '-AAG-GGC-CCA-ATT-ACG-CAG-AGG-GTA-CCC-TAA-GAA-GAA-GGC-ATG-CCG-TGC-TAG-CGG-AG-3' Protein products of HMW of approximately 6 Kbp were purified using silica gel column centrifuges (QIAGEN) and the fragments were subjected to two cycles of 8-10 hours of Kpnl digestion using a 10-fold excess of Kpnl (approximately 10 units per one micro g of purified fragment, 37 ° C). After the second digestion, the activity of the residual restriction enzyme was removed using QIAGEN centrifuge columns and the HMW protein fragments of approximately 6 Kbp Kpnl cloned in the pTrcHisB plasmid which had been previously digested until culmination with Kpnl and treated with phosphatase calf intestine to prevent a new vector ligation. HE. performed vector / insert ligations in a final reaction volume of approximately 50 micro 1 (50 nM Tris-HCl, pH 7.00, 10 mM NaCl, 1 mM ATP, 0.5 mM DTT) at a temperature of 25 ° C for approximately 2 hours using T4 DNA ligase (approximately 10 units / reaction) and a molar vector: insert ratio of approximately 1: 5. After ligation, aliquots (approximately 50 ng of ligated DNA) were used to electroporate a competent E. coli host, eg, E. coli TOP10. Transformants containing plasmid, were selected by placing in plates of electrotransformed cells on LB agar containing 100 micro g / ml of ampicillin. The ampicillin-resistant transformants (ApR) that appeared after an incubation period of about 14-24 hours at a temperature of 37 ° C were randomly collected and seeded in the same selective medium for purification. A single purified ApR colony from each initial transformant was used to inoculate approximately 5 ml of LB broth and grown overnight at a temperature of 37 ° C in a mild aeration incubator shaker (approximately 200 revolutions per minute). The cells from the broth cultures were harvested by centrifugation and used to prepare small amounts of plasmid DNA. Commercially available reagents (QIAGEN Plasmid Mini Kits) were employed for these plasmid extractions. Plasmid derivatives bearing inserts were presumptively identified by electrophoresis of undigested plasmid DNA on agarose gels (0.8% agarose in TAE regulator) and derivatives of larger size than the vector plasmid were identified. Derivatives containing inserts were confirmed and the orientation of the HMW protein inserts relative to the vector sequences were determined using appropriate restriction endonucleases (Kpnl, EcoRI, Hindi, BamHI, etc.), either separately or together in various combinations. The DNA sequence of the protein genes of HMW B and F of C. trachomatis were obtained for both females using "sequence displacement", the PCR cycle sequencing methodology of asymmetric dye terminator (ABI Prims Dye-Terminator Cycle Sequencing, Perkin-Elmer) described in example 4. Reactions were programmed with plasmid miniprep DNA and primers specific for the individual HMW protein sequence that were used in the sequencing of the HMW protein gene from the type strain. L2. DNA sequence data were collected using the ABI 310 Sequenator and analyzed automatically in a PowerMAC computer and an appropriate computer programmatic in accordance with that described in example 4. Individually autoanalyzed DNA sequences were manually edited for accuracy prior to their combination in a "series" of consensus sequences using a problematic AutoAssembler (Perkin-Elmer). Both strands of the HMW protein gene of serovars B and F of C. trachomatis were sequenced and these data were compiled to create composite consensus sequences for the protein genes of HMW and F of C. trachomatis. These sequences appear as SEQ ID Nos. 14 and 15. Figure 6 shows sequence comparisons of strains F and B of L2. 8.9 EXAMPLE 9: PRODUCTION OF RECOMBINANT PROTEIN To produce sufficient quantities of recombinant HMW protein for animal protection studies and immunogenicity, the HMW gene has been cloned by PCR in baculovirus and E. coli expression systems. Large amounts of rHMW protein are produced in an E.coli-based system as a chimeric fusion protein containing an N-terminal affinity purification domain (His) e. The complete open reading frame (ORF) of HMW protein was cloned by PCR from the genome of C.

Trachomatis L2 as a single Kpnl fragment and fused in the proper orientation and in the correct reading frame over the affinity purification domain (His) 6 encoded in the high expression plasmid vector pTrcHisB (CloneTech) in accordance with that described in Example 5. The affinity purification domain (His) 6 is part of a high expression locus consisting of highly efficient tac promoter (inducible by IPTG) and localized Shine and Delgarno consensus ribosome binding site (RBS). immediately upstream of the affinity purification domain of (His) 6. The HMW protein genes of C. trachomatis LGV L2, C. trachomatis B, and C. trachomatis F were cloned by polymerase chain reaction as fragments of 3.0Kbp. The direct primer (56-mer) used in these reactions was designated p306Hpn-F and contains sequences complementary to the first ten N-terminal amino acid residues of the mature HMW protein, listed as SEQ ID No. 12. In addition to the sequences of HMW protein coding, this direct initiator also carries a unique Kpnl restriction site located optimally upstream of the first residue of the mature HMW protein (Glu) for proper fusion with the affinity purification domain (His) β encoded in the vector plasmid, and the G / C fastener of 6 bases at the 5 'end for effective amplification and a 12 base internal spacer for effective endonuclease recognition / digestion. The reverse PCR primer, designated p316Kpn-3RC, contains a reverse complement sequence for a sequence of C. trachomatis located ~ 0.2kbp downstream of the HMW protein termination strand listed as SEQ ID No. 14. As in the case of p306Kpn.F, the reverse primer also contains a 5 'Kpnl restriction site for the C trachomatis sequence, a 6-base G / C fastener and an internal 12-base spacer. To obtain acceptable yields of the HMW protein ORF product (approximately 3,500 bp), polymerase chain reaction amplification was carried out using a mixture of thermostable DNA polymerases which consisted of T. thermophilus DNA polymerase as the amplification polymerase. and a minor amount of a second high-fidelity thermostable DNA polymerase to provide an additional 5 '-3' review activity (Advantage Polymerse, CloneTech). An anti-T ADFN polymerase antibody was added to the reaction mixture to provide automatic "hot start" conditions that promote the production of large amplimers (> 2 Kbp). The genomic DNA of several strains of C. trachomatis was isolated from EBs in accordance with that described with the example above and used to program these reactions. After amplification, the desired reaction products were purified from excess initiators using silica gel centrifuge columns commercially. • available and commercially available reagents (QUIAGEN) and were digested until their completion with a Kpnl excess (~ 10 units per 1 μg DNA). The HMW protein ORF purified and digested by Kpnl was then cloned into a pTrcHisB expression plasmid predigested with KpnI (5: 1, insert: vector ratio). Aliquots of the ligation reaction were then employed to electrotransform a suitable E. coli host (e.g., TOP10). Mini-prep DNA from ampicillin-resistant transformants taken at random were prepared, digested to completion with Kpnl, HindIII, or a combination of both and examined to determine the presence and orientation of the HMW protein ORF insert of ~ 3.2 Kbp using electrophoresis in alggarose gel and staining with ethyl bromide. Mini-prep DNA was used to program DNA sequencing reactions by symmetric polymerase chain reaction in accordance with that described in example (s) above to confirm the fidelity of the binding formed between the HMW protein fragment and the protein domain. purification by affinity (His) 6 of the vector. Plasmid pAH342 was a derivative isolated by these procedures, which contains ORF of HMW protein gene from C. Trichomatis L2 and is represented by nucleotides 446 to 3421 in Figure 2.

Recombinants were cultured in 2X-YT broth containing 100 μg / ml Ap to logarithmic phase medium (~ .5 OD60o) and induced with IPTG (final ImM) for an additional 4-5 hours to activate transcription from the TRC promoter of vectors. Cells were harvested by centrifugation and used from crude cells prepared by lysis using a French pressure cell. Alternatively, the expression of rHMW protein can be obtained by the use of a baculovirus expression system. Here, the HMW protein ORFs of C. trachomatis L2 and C. trachomatis F were cloned by polymerase chain reaction as PCR products of ~ 3 Kbp in a baculovirus transfer vector (e.g. pFtBacHTb) that had previously been digested to completion with Kpnl and treated with CIP to minimize the new vector ligation in essentially the same manner as described for pTrcHisB. The HMW protein expression cartridge generated in this cloning exercise (i.e., the baculovirus polyhedron promoter, affinity purification domain (His) 6 N-terminus, ORF protein gene of HMW) was then transferred to a baculovirus genome by site-specific transposition using a commercially available bacmid system (Bac-to-bac, Gibco). Briefly, the HMW protein baculovirus expression plasmid was used to transform competent E. coli DHLObac cells (Gibco) containing a bacmid (a baculovirus-plasmid hybrid replicon) of gentamicin resistance using standard transformation and methodologies. selection. Transformants in which the HMW protein expression cassette had been successfully transposed from the expression plasmid to the appropriate receptor site within the lacZ gene located in the bacmid replicon were identified using a blue-white IPTG / X selection. -gal standard. White GmR transformants were taken at random and seeded again for purification. Bacmid DNA was prepared from broth cultures by the method of Ish-Horowitz, N.A.R. 9: 2989-2993 (1981) incorporated herein by reference, and is used to transfect 9 Spodoptera frugiperda cells. After plaque purification, the recombinant HMW protein baculovirus is used to infect Spodoptera suspension cultures on a large scale. A yeast expression system is employed to generate a glycosylated form of HMW protein. 8.10 EXAMPLE 10: PURIFICATION OF RECOMBINANT PROTEIN A recombinant HMW protein was purified to homogeneity using standard preparations of immobilized metal affinity chromatography (IMAC). Previously, an E. coli strain having an expression plasmid containing an HMW protein gene was cultured in Luria broth in a 5 liter fermenter. (New Brunswick) a - a temperature of 37 ° C with moderate aeration to a logarithmic phase medium (~ 0.5 O.D.60o) and induced with IPTG (final IitiM) for 4-5 hours. A cell paste was collected, washed in PBS and stored at -20 ° C. Aliquots of frozen cell paste (~ 9-10 grams wet weight) were suspended in ~ 120 ml of D-PBS by mechanical agitation and used via passage through a French pressure cell (2X, 14,000 psi, 4). ° C). Soluble protein was then removed from the rHMW protein inclusion bodies by high speed centrifugation (~ 10,000Xg, 4 ° C, 30 minutes). The insoluble pellet containing rHMW protein was suspended in ~ 20ml of D-PBS ice temperature by homogenization and centrifuged as above. The washed rHMW protein inclusion bodies were then denatured by suspension in a sodium phosphate buffer (0.1 M, pH 7.0) containing 6 M gunidine hydrochloride and loaded onto a Ni2 + affinity column (1.5 cm X 25 cm, volume of bed ~ 15 ml) prepared from Fast-Flow Chelating Sepharose (Pharmacia) and loaded with Ni 2+ ions Zn by standard procedures. The unbound material was removed by washing the column with ~ 5-10 column volumes of a sodium phosphate buffer (0.1 M, pH 7.0) containing 8M urea. Recombinant HMW protein bound to the affinity resin by virtue of the affinity purification domain (His) 6 N-terminus was eluted using a sodium phosphate buffer of pH 4.0 / 8M urea (~ 20 ml). The eluted material was neutralized by the addition of 1.0 M Tris-HCl (~ 2.5 ml, pH 7.5) and dialyzed against the TE regulator containing SDS (0.5%) to remove the urea. The dialyzed material was concentrated using a Centricon-30 centrifugation concentrator (Amicon, 30,000 MWCO) and mixed with a 1/5 volume of 5X SDS gel sample buffer containing lmM 2-mercaptoethanol (Lam eli) and boiled at a temperature of 100 ° C for 5 minutes. Samples were loaded on Tris / glycine preparation acrylamide gels (4% stacking gel, 12% resolution gel, 30: 0.8 acrylamide: bis solution, 3mm thick). A pre-dyed molecular weight standard was made (SeeBlue, Novex) in parallel with the rHMW protein samples to identify size fractions in the gel. The area of the gel-containing proteins having molecular masses of ~ 50-70 Kdal was removed and the proteins were electro-eluted using an Elu-Trap device and membranes (S & S) in accordance with the manufacturer's specifications. The electro eluted protein was dialyzed to remove SDS. The protein concentration of the sample was determined using a Micro-BCA system (Pierce) and BSA as a concentration standard. The purity of the rHMW protein was determined using conventional SDS-PAGE and commercially available silver staining reagents (Silver Stain Plus, Novex) as shown in Figure 4.

The apparent molecular weight of isolated mature rHMW is approximately 105-115 kDa in accordance with that determined by SDS-PAGE. 8.11 EXAMPLE 11: PREPARATION OF PROTEIN ANTIBODIES HMW polyvalent antibodies directed against the HMW protein were generated by vaccination of rabbits with the purified HMW protein or fragments thereof. Each animal received a total of three immunizations of approximately 250μg of HMW protein or fragment thereof by injection, (starting with Freund's complete adjuvant and followed with incomplete Freund's adjuvant) at intervals of approximately 21 days. In each immunization, approximately half of the material was administered intramuscularly (i.m.) and half was injected intranodally. Fourteen days after the vaccination, a fourth booster of approximately lOOμg of HMW i.m. protein was applied. and the animals were bled 7-10 days later. The anti-HMW protein titers were measured by ELISA using a purified HMW protein (1.0 μg / pure) or EBs of C. trachomatis L2 (whole and crude protein extract) as capture ligand, specific IgG ELISA titer. Immunogen of 1/320, 000 were observed using truncated protein from purified rHMW and 1/2500 using either EBs or RBs. Serial dilutions of antiserum were performed in PBS and were tested by ELISA in duplicate. Anti-rabbit antibody conjugated with goat HRP diluted 1/1000 was used as the second reporter antibody in these assays. Titers are expressed as the highest dilution that shows a positive ELISA reaction, ie, an O.D.450 > 2SD above the average negative control value (pre-bled rabbit sera). Hyperimmune antisera were then used to test Western blots of crude EB or RB extracts as well as preparations of 1.0% OGP EB extracts to determine whether other serovars of C. trachomatis and chlamydia species express the HMW protein. Serovars of C. Trachomatis B, Ba, D, F, G, I, J, K, Li, L2, L3, and Chlamydia pneumoniae were tested and found to have a protein of an apparent molecular weight of 105-115 kDa that reacts with antiserum sera generated against HMW protein. 8.12 EXAMPLE 12: SURFACE LOCALIZATION The surface location of the HMW protein in different strains of Chlamydia and derivatives was examined by indirect fluorescence antibody (IFA). IFA was carried out employing the procedures generally known in the art using hyperimmune anti-HMW protein as the primary antibody. Hk cells infected with whole Ebs of one of the serovars of C. trachomatis L2, B and F, C. Pneomoniae or C. Psittaci are achieved through the following method. McCoy cells or Hak cells were grown to confluence in MEM media in 12mm layers in 24-well tissue culture plates and then inoculated by centrifugation with ~ 5X104 inclusion forming units (IFU) of the C. trachomatis strain NI1 (serovar F). After ~ 24 hours of incubation, the culture medium was removed and the infected cells were fixed in methanol for 10 minutes. The fixed monolayer was then washed with PBS (IX) to remove the fixative and overlay with >300 μl of truncated anti-60Kdal HMWP rabbit antibody that had been diluted ~ 1-100 in PBS. After a one-hour incubation with the primary antibody, the cells were previously washed with PBS and then incubated for approximately 30 minutes with a 1/200 dilution in an FITC-conjugated mouse anti-rabbit IgG antibody. The second antibody was diluted using a PBS solution containing 0.0091% Evans Blue as a counter stain to visualize the monolayer. The cells were washed twice in PBS to remove the secondary antibody, the caps were removed from the culture plates and mounted on microscope slides using a fluorescent mounting medium. Samples of identical cells stained with pre-mixed rabbit antibody or second antibody conjugated with FITC were only processed in parallel and served as controls for antibody specificity (negative). The counterstained samples were examined at a magnification of 1000 times with a Zeiss Axioskop photomicroscope equipped with plan-neoflur lenses. The results using C. trachomatis NI1 (serovar F) are shown in Figure 7. The results show that the increased fluorescence of the samples stained with HMW protein antibody compared to the controls confirmed the surface location of the HMW protein. In addition, the fluorescence of the samples stained with HMW protein antibodies shows the binding with the HMW protein located superficially from serovars L2, B and MoPn and C. Pneumoniae. 9 EXAMPLE: INVITRO NEUTRALIZATION MODEL The in vitro neutralization model has been used to show that the protective antiserum inhibited chlamydial infection (neutralization) of several tissue culture cell lines. Animal models are also essential to test the efficacy of vaccines with small animals (not priming) and also those of priming necessary for preclinical evaluation. The guinea pig of India has been used to study experimental genital and eye infections by the Indian guinea pig inclusion conjunctivitis agent (GPIC), C. psittaci. The mouse offers a consistent and reproducible model of genital tract infections using human genital tract isolates. The mouse model is a generally accepted preclinical assay and was used to evaluate MOM as a subunit vaccine. Another model is known as the trachoma infection primate model where it was shown that the induction of a secretory IgA was an essential component of the protection. The ability to vaccinate new antigen candidates from their unit is determined using the aforementioned generally accepted in vitro neutralization and animal model systems. As a preliminary exercise for animal protection studies, a hyperimmune anti-HMW antibody was evaluated for its ability to block the infectivity of several serovars of C. trachomatis (for example L2, B, F) in vitro. Even though McCoy cells were used to propagate Chlamydia, these cells could also allow antibody-mediated acceptance through Fe receptors. Therefore, to evaluate the inhibition of anti-HMW antibody to infection capacity, Hak cells that do not Fe receptors were used in these analyzes. The cells were plated in 24-well plates to a subconfluent monolayer (confluence of approximately 90% = 1X105 cells / ml) at a temperature of 37 ° C in 5% C02. Antibody-anti-HMW was diluted to approximately lOOμg / ml (total protein) in a sucrose-phosphate-glutamate regulator (SPG) and after serial dilution in SPG regulator. Frozen aliquots of pretitled Chlamydia was diluted in SPG buffer to approximately 2X104 IFU / ml. The Ebs were premixed with the anti-HMW antibody diluted to approximately 10-20 IFU / μl and incubated for 30 minutes at a temperature of 37 ° C on a shaking platform. The prepared Hak cells were washed in HBSS and then incubated with the Chlamydia anti-HMW / EB mixture in triplicate for each antibody using 500 IFU / ml. The plates were incubated for two hours at 37 ° C, and then the inoculum was removed and the dishes were washed three times with HBSS. A tissue culture medium containing 1 μg / ml of cyclohexamide was added and the dishes were incubated at a temperature of 37 ° C for approximately 24-36 hours in 5% C02 to allow development of the inclusion bodies. After incubation, the medium was removed and monolayers of cells were washed three times in PBS. The plates were then fixed in methanol for 20 minutes and washed again in PBS.

The cells were stained to visualize inclusions by incubation with anti-Chlamydia LPS antibody (diluted Approximately 1: 500, Virostat), the cells were washed three times in PBS, followed by incubation with goat secondary antibody conjugated with FITC for 30 minutes at a temperature of 37 ° C. The lids were washed, dried in the air, and mounted in glycerol in glass lids.

The inclusions were counted in five fields through the midline of the cap in a Zeiss fluorescence photomys- teroscope. The results are reported as the percentage reduction of the cells that contain inclusion in relation to a control of heterogeneous body (pre-bled rabbit sera). 10. EXAMPLE: VACCINE EFFICACY (mouse model of Salpingitis and fertility) 10.1 METHODS 10.1.1. IMMUNIZATION AND CHALLENGE The Tuffrey murine infertility model was used to evaluate the efficacy of rHMWP in protecting animals against Salpingitis induced by Chlamydia trachomatis and infertility. Three groups of 17 female HeOuJ C3H mice (~ 6 weeks of age, Jackson Labs) were used for this evaluation. The test group of 17 animals was immunized at week 0, 2 and 3 by intranasal administration of ~20 μl of a vaccine formulation containing approximately 10-12 μl of gel purified rHMWP and ~ 5 μg mLT (SmithKline Beecham) as an adjuvant. Prior to immunization the rats were subjected to sedation using an anesthetic mixture consisting of 16% Ketaject and 16% Xylaject in 68% PBS without pyrogen (100μl i.p. / animal). The sedated animals were placed on their backs and, using a standard laboratory pipette, the vaccine formulation was administered; ~ 10 μl of the vaccine solution by nose with a waiting period of 5-10 minutes between applications. Two groups of 17 female mice (per test group) were immunized similarly but with a preparation containing only 5 μg of mLT (ie, adjuvant only, not antigen). One of these groups was subsequently challenged with C. trachomatis (infected fictitious immunity) and served as the negative fertility control while the other group was not challenged (fictitious immunization, dummy infection) and served as the positive fertility control. At 4 weeks, all animals received an i.p. single progesterone (2.5 μg in PBS free of pyrogen, DepoProvera, Upjohn) to stabilize the uterine epithelium. At week 5, animals immunized with rHMWP and animals in the negative control group were injected by bilateral intrauterine inoculation with ~5X105 inclusion formation units (IFU) of C. trachomatis NI1 (serovar F) _ in lOOμl of a sucrose phosphate glutamate regulator (SPG). To mimic the manipulations of the reproductive tract experienced by the other two groups, the animals in the positive control group were inoculated bilaterally with lOOμl of an extract of McCoy cells that did not contain C. trachomatis. In week 7, 5-7 animals from each group were sacrificed by means of asphyxia with C02 and the entire genital tract (both the upper and lower reproductive tract) was removed for histopathology analysis. In week 9, the remaining females of each group were placed in cages with male C3H mice of 8-10 weeks of age during a procreation period of two months to evaluate fertility (one male for every two females per cage with weekly rotation of the males within each group, animals from different experimental groups were not mixed). Palpation and periodic weighing were used to determine when the animals in each pair were pregnant. The parameters used to estimate the fertility of the group were: F, the number of mice that had baits at least once during the reproduction period divided by the total number of mice in this study group; M, the number of mice born (live or dead) divided by the number of baits produced in this group during the breeding period; and N, the total number of mice born (stillbirths or live) divided by the total number of mice in this group. -10.1.2 HISTOPATHOLOGY The genital tracts were treated for 24 hours or more in a Bouin's fixative, dehydrated progressively in 50%, 70% and 100% methanol, tied in toluene, and placed in paraffin or placed directly in a compound of OCT (Tissue-TEK, Miles) and subsequently frozen in liquid nitrogen. Tissue sections (approximately 6 μm) were stained with hematoxylin and eosin (after deparaffinization of the samples fixed with Bouin's fixative). Inflammatory changes in the oviducts and ovaries were scored as follows: 0, non-apparent inflammatory reaction; 1, some mononuclear cells infiltrate the periovarian space or the submucosa of the oviduct; 2, same as 1, but to a greater extent; 3, same as in 2, but with a thick oviductal submucosa and presence of inflammatory cells in the lumen of the oviduct; 4, like 3, but to a greater extent. Inflammation in the neck of the womb / vagina was rated based on the level of the intrahepitelial infiltrate observed. 10.1.3. DETERMINATION OF SPECIFIC HUMORAL RESPONSES FOR rHMWP Blood samples were collected periodically during the immunization and challenge phases by retro-orbital bleeding and serum prepared by centrifugation. Vaginal secretions were collected by repeated injection of 50 μl of sterile PBS into the vagina with a standard laboratory pipette device and immediately removing the solution. They took 2 to 3 injection / withdrawal cycles. The quantification of antibody response (Ab) by ELISA was carried out as described in section 8.11. Plates Microwell ELISA (Maxisorb, - NUNC) to determine Ab levels were coated overnight at a temperature of 4 ° C with approximately 0.5-1.0 μg of gel-purified rHMWP per well in carbonate buffer / 10 mM bicarbonate (pH 9.6), washed with PBS containing 0.1% Tween-20 (wash buffer) and blocked for approximately 1 hour at a temperature of 30 ° C with a PBS solution containing 3% BSA. For the determination of serum IgG levels specific for antigens, test sera were serially dissolved in a wash buffer containing 0.5% BSA and aliquots (100 μl) incubated in wells coated with antigen for approximately 2 hours at a time. temperature of 37 ° C. the data were then washed and incubated for approximately 41 hours at a temperature of 37 ° C with a second antibody (Sigma) of goat anti-mouse IgG conjugated with horseradish peroxidase (HRP). The goat anti-mouse IgA secondary antibody conjugated with HRP was used to detect the presence of IgA specific for rHMWP in vaginal secretions. After incubation with the appropriate secondary Ab, the plates were washed and incubated for approximately 20-30 minutes at room temperature with TMB substrate (sigma). The reactions were stopped by the addition of 2M H2SO4 and the absorbance was determined at 450 nm in a Molecular Devices SpectroMax microplate reader. Titers were determined as the reciprocal number of the sample dilution corresponding to an optical density of 1.0 at 450 nm. 10.1.4. DETERMINATION OF SPECIFIC CELLULAR RESPONSES FOR rHMWP Groups of 6 female C3H HeOuj mice (Jackson Labs) were submitted to sedation and immunized at weeks 0, 2, and 3 by means of intranasal administration of a rHMWP + mLT vaccine in accordance with that described in section 10.1.3. At weeks 4 and 5 immediately before treatment with progesterone and intrauterine challenge, respectively, three animals from each group were sacrificed by asphyxia with C02 and the spleens were aseptically removed and single suspensions of cells were prepared using conventional methodologies. Spleen cells from immunized animals were analyzed separately. For both the positive control group (fictitious immunization and fictitious infection) and for the negative control group (fictitious immunization, real infection) the passage cells were combined and tested for restimulation. To measure the proliferation of spleen cells, spleens were ground (5 to 10 turns) in 5 ml RPMI 1640 Glutamax I supplemented with 10% fetal calf serum, 25 mM HEPES, 50 U / ml penicillin, 50 μg / ml streptomycin, 1 mm sodium pyruvate, non-essential amino acids and 50 μM 2-mercaptoethanol (Gibco-BRL). Live cells were counted by staining with Trypan Blue and diluted in the same medium to reach a density of 1.0 - 2.0X106 cells / ml (Falcon 2063 polypropylene tubes). Triplicate cultures were established in 96 well round bottom culture dishes (Nunclon, Nunc) employing approximately 5X105 responder cells per well in 200 μl of medium. The cells were stimulated with 1.0 μg / ml rHMWP (antigen-specific proliferation) or with 4 μg / ml concavalin A (Boerhinger Mannhein) as positive stimulation control; Non-restimulated cell cultures were used as a negative control of cell activation. After 72 to 96 hours of incubation at a temperature of 37 ° C in 5% C02, the cells were labeled used for approximately 18 hours with 1.0 μCi 3 H-thymidine (Amersham) per well. The pulsed cells were harvested on fiberglass sheets using a Tomtec cell harvester (Mk III) and counted for beta emission in a Wallac 1450 Trilux 3-channel liquid scintillation counter. The stimulation index (SI) for a sample (individual or combined) was defined as the mean of the uptake by T cells stimulated with ConA or 3H-thymidine antigen for triplicate wells divided by the mean of unstimulated uptake for triplicate wells. The Stimulation indices for both antigen-specific proliferation (specific for rHMWP) and specific proliferation for ConA were determined. 10.2. RESULTS 10.2.1 EFFECTS ON MOUSE FERTILITY AFTER A HETEROTYPIC CHALLENGE Evidence that mucosal immunization with rHMWP combined with mLT can provide protection against infertility caused by a human clinical isolate of C. trachomatis (strain NIl, serovar F) appears in Table 1. The animals immunized with rHMWP had a significantly higher fertility rate (70%, that is, number of fertile females in group / total number of animals in the group) than the animals in the negative control group (30% , fictitious immunization and real infection). Similarly, the immunized group containing rHMWP produced more offspring and showed greater group fecundity than what was observed in the negative control group (51 vs. 24 offspring and 5.1 ± 4.7 vs. 2.4 ± 4.6 in terms of fertility scores, respectively ). As a group, animals immunized with the rHMWP vaccine had a fertility rate, a total number of offspring, and a fertility score comparable to what was observed in the positive control group that was fictitiously infected (fertility rate). : 80%, total number of offspring: 56, fertility rating: 4.9 ± 2.7). The protection against C. trachomatis-induced infertility obtained in this experiment also demonstrates the usefulness of rHMWP to provide protection through biovars and through serovars against C. trachomatis-induced disease. The recombinant HMWP antigen used in this experiment was cloned from a strain belonging to the group of C. trachomatis lymphogranuloma venereum (LGV) (strain L2) that causes systemic infections as well as more localized mucosal infections of the eye and genital tract. C. trachomatis challenges the organism used in these experiments, strain NI1 is a serovar F organism that belongs to the biovar of trachoma that causes numerous infections of the urogenital tract. Table 1. Fertility evaluations observed after approximately 2 reproduction cycles. Group Number Percentage Number Fecundity of of group of animals animals young (average + by fertile standard deviation) Immunized 10 70 p = 51 5.10 ± 4.68 with rHMWP 0.0892 p = 1.105" Immunization 10 80 p = 56 4.90 ± 2.70 Fiction 0.035 p = 0.078 Fictitious infection (positive control) Immunization 10 30 24 2.40 ± 4.61 fictitious real infection (negative control) 1 Average number of offspring per group. 2 Fisher's exact test, one-sided, 95% confidence interval, values are given as a relation to the negative control. 3 Student's t test, unpaired, Gaussian distribution, 95% confidence interval. The p values are given in relation to the negative control. 10.2.2. EFFECTS ON IMMUNOLOGICAL CELLULAR RESPONSE The specific activation by rHMWP of the cellular immune system was demonstrated using a conventional spleen cell proliferation assay. When spleen cells were tested during week 4 (immediately before treatment with progesterone) (table 2) and week 5 (approximately 7 days after treatment with hormones but before the intrauterine challenge) (table 3), all samples collected from Animals immunized with rHMWP developed a strong proliferative immune response specific for antigen. Stimulation Indices (Sis) specific for antigens obtained before treatment with progesterone from animals immunized with rHMWP were equal to or greater than the stimulation indices obtained through mitogenic stimulation ConA (mean values for stimulation with antigen and ConA obtained from 3 animals immunized with rHMWP: 26.2 vs 18.4, respectively). Spleen cells obtained from both fictitiously immunized animals and animals that were not exposed to either rHMWp or mLT antigens did not respond to a new stimulation in vitro with the rHMWP material, thus establishing the specificity of the proliferative response observed in immunized animals. A treatment with progesterone did not affect the specific proliferative response to antigens observed in animals immunized with rHMWP. Specific stimulation indices for antigens obtained with spleen cells obtained after hormonal treatment were higher than those observed through mitogenic stimulation (mean values for stimulation by antigens and ConA obtained from 3 animals immunized with rHMWP: 92.4 vs 37.8, respectively ). Other samples collected from animals immunized in a fictitious manner or not exposed neither to rHMWP antigen nor to mLT did not demonstrate any antigen-specific proliferative response. Table 2 Proliferation of specific cells for rHMWP before hormonal treatment Group Proliferation cell stimulation index (cpm) (cpm treated / cpm not Not treated / ConA / treated) RHMWP ConA / rHMWPImmunized animal 1557/20739/65741 13.3 / 42.2 with rHMWP # 1 Animal immunized 1508/26975/28361 17.9 / 18.8 with rHMWP # 2 Animal immunized 1238/29991/23453 24.0 / 18.9 with rHMWP # 3 Immunized Animals 1687/30546/1292 18 / < 1.0 fictitiously (combined) Animals not exposed nor 335/23886/838 71 / 2.5 antigen of rHMWP or mLT (combined) Table 3 Specific cell proliferation for rHMWP after hormonal treatment Group Cell proliferation stimulation index (cpm) (cpm treated / cpm not Not treated / ConA / treated) RHMWP ConA / rHMWP Immunized animal 767/15934/97458 20.8 / 127.0 with rHMWP # 1 Animal immunized 546/17212/28172 31.5 / 51.6 with rHMWP # 2 Animal immunized 297/18139/29300 61.1 / 98.6 with rHMWP # 3 Immunized Animals 273/18094/150 66.3 / < 1.0 in a fictitious manner (combined) Animals not exposed nor 345/16740/1341 48.5 / 3.9 to antigen of rHMWP or to mLT (combined) 10.2.3. EFFECTS ON IMMUNE HUMORAL RESPONSE To demonstrate that immunization with full-length rHMWP produces a humoral immune response, IgG titers were measured by ELISA on sera collected during week 5 immediately before challenge (ie, approximately 2 weeks after challenge). the third immunization). As shown in Table 4, immunization of C3H mice with three doses of approximately 10-12 μg of rHMWP produced detectable levels of anti-rHMWP IgG in all animals. Vaginal secretions of these animals were also collected at the same time and tested for the presence of anti-rHMWP mucosal IgG. Specific vaginal IgA was detected for antigen in three animals (table 4). Table 4 Specific humoral response for rHMWP Animal Titration according to Immunization Presence IgG ELISA Vaginal IgA with rHMWP serum anti-rHMWP anti-rHMWP 4.4 5,000 4.5 6,000 4.6 12,000 4.7 130 4.8 100 4.9 54,000 4.10 670 4.11 100 4.12 570 4.13 100,000 4.14 4,500 4.15 400 4.16 1,600 4.17 2,500 4.18 700 4.19 70,000 4.20 500 4.21 2,000 4.22 18,000 4.23 3,000 Mean ± Standard deviation 18.5 + 29 11. EXAMPLE: CONSTRUCTION OF PJJ701 A plasmid containing the HMWP gene of C. trachomatis L2 total was constructed by selective removal of the site EcoRI upstream to the N-terminus of HMWP in pAH306 (described in section 8.4). This was achieved by digestion of pAH306 to completion with Xhol and then by re-ligating the plasmid to create pAH306-Xho? -1. The EcoRi fragment of approximately 2.5 Kbp of pAH316 (described in section 8.5) containing the C-terminus of The remaining HMWP was isolated from agarose preparation gels that had been loaded with a complete EcoRI digestion of pAH316. A section of agarose gel containing the appropriate fragment of approximately 2.5 Kbp was then excised, dissolved with Nal regulator and purified from residual agarose by chromatography on a hydroxyapatite centrifuge column (QIAGEN). The EcoRI fragment of purified C-terminus HMWP was then ligated into the unique EcoRi site of pAh306-Xho? -l located at the 3 'end of the N-terminal coding sequence of HMWP, using T4 DNA ligase and employing standard protocols in molecular biology E. Coli ToplO cells were transformed with an aliquot of pAH306-Xo? -l (vector treated with phosphatase and digested with EcoRI) and the ligation reaction of EcoRI C-terminal fragment of 2.5 Kbp and selected recombinants in 2X / YT agar containing 100 μg / ml ampicillin. Transformants resistant to ampicillin were taken at random. Plasmid DNA was isolated from individual Ap R transformants using a Qiagen Mini-Prep plasmid DNA isolation system and screened for the presence of plasmid larger than pAH306-Hho? -l by conventional gel electrophoresis of agarose and staining with ethidium bromide. Derivatives of pAH306-Hho? -l carrying the 2.5 Kbp HMWP fragment in the appropriate orientation that could allow the expression of full-length HMWP were identified by restriction analysis using EcoRI and / or Xhol. Plasmid pAH374 was an isolated derivative of this experiment. A site-directed mutagenesis procedure based on polymerase chain reaction (Quick-Change Site-Directed Mutagenesis System, Stratagene) was used to effect a desired DNA change, namely the removal of the Ndel site within the HMWP coding sequence of pAH374. Initiators of utagénicos polymerase chain reaction, 41 bases in length and complementary to the sequencing that contained the Ndel site and designated 140-Nde-FX and 14-NdeRCX were designed to eliminate the Ndel recognition site but without changing the sequence of corresponding protein coding. The sequences of the two mutagenic polymerase chain reaction primers used to remove the Ndel site in pAH374 are shown below. 140-Nde-FX (SEQ ID NO: 38) 5 '-GGG TTT GGG AAT CAG CAC ATG AAA ACC TCA TAT AA TTT GC - 3'. . . . . 14-Nde-RCX (SEQ ID NO: 39) 5 '-GCA AAT GTA TAT GAG GTT TTC ATG TGC TGA TTC CCA AAC CC-3' After mutagenesis of Pfu DNA polymerase (Stratagene) and Dpnl digestion, to dissociate any plasmid parental pAH374 not altered, mutated plasmid DNA was transformed into E. coli XLl-Blue. Transformants containing plasmid were selected on 2X-YT agar containing 100 μg / ml ampicillin. Antibiotic-resistant transformants were randomized and screened for the presence of plasmids of the same size as pAH374. The identity of the plasmids isolated from the transformants was determined by digestion with restriction enzymes using E. cori. The absence of Ndel site in these plasmids was determined by digestion using Ndel. To verify the loss of the Ndel site of HMWP and to ensure that no unwanted changes occurred in the DNA sequence in this region during the mutagenesis procedure, mutagenized plasmids were further subjected to DNA sequence analysis employing a sequencing primer specific for localized sequences. upstream of the Ndel site. Plasmid pAH374-Nde? -l was a plasmid isolated in this experiment. A DNA fragment encoding HMWP of C. trachomatis L2 without the internal Ndel site, plasmid pAH37 -Nde? -l, was amplified by polymerase chain reaction from programmed reactions with plasmid pAH374-Nde? -l (approximately 50ng ) and the initiators 306-Nde-Metl and 312H6Xbal. The Met6 306N primer was designed to contain a central N site to direct cloning in pMG81. The Ndel site at 306NdeMetl spliced the initial ATG codon for HMWP signal sequence and was flanked by a 20 base G / C fastener on the 5 'side and complementary sequences to the first 15 residues of the HMWP signal sequence on the side 3' . The 312H6Xbal primer was designed to contain sequences complementary to the C-terminus of HMWP followed by a motif (CAT) 6 that specifies an affinity purification domain of hexahistidine. This primer also contained two UAA termination codons, an Xbal recognition sequence, and a 20-base G / C fastener at the 3 'end of the primer. The sequences of the polymerase chain reaction initiators 306NdeMetl and 312H6Xbal appear below. The amplification conditions by polymerase chain reaction described in section 8.4 were used to generate the HMWP Ndel-Xbal gene cassette. After amplification, the product of the polymerase chain reaction was purified using hydroxyapatite centrifuge columns (QiaGen) and digested overnight at a temperature of 37 ° C with an approximately 10-fold excess of Ndel and Xbal to generate the " "outlets" required at the ends of the fragment. The digested fragment was again purified using centrifugation columns and approximately 250 ng ligated to approximately 50 ng of plasmid DNA p completely digested previously with Ndel and Xbal and subsequently treated with CIP to avoid a new vector ligation. An aliquot of the ligation reaction was used to transform E. coli strain AR58 which had previously been made competent by the method of Lederberg and Cohen. Transformants were selected on 2X-YT agar containing 40 μg / ml kanamycin sulfate. Due to the temperature-induced promoter in p, the transformed cells were grown at 30 ° C, the kanamycin-resistant transformants were taken randomly and screened for the presence of plasmids ~ 3.0 Kbp larger in size than p. The presence of p derivatives containing inserts was confirmed by analysis with restriction enzymes using Ndel, Xbal, EcoRI and Ncol. Plasmid pJJ701 was a plasmid isolated from this exercise. 12. EXAMPLE 16: PRODUCTION OF COMPLETE LENGTH rHMWP FROM AR58 (PJJ701) One milliliter of a frozen stock of E. coli strain AR58 containing the plasmid pJJ701 was used to inoculate approximately 100 ml of 2X-YT broth containing 40 μg / ml kanamycin and cultured overnight at a temperature of 30 ° C to prepare a fermentor seed culture. Approximately 20 ml of the seed culture overnight was then used to inoculate a New Brunswick Bioflow 3000 fermentor loaded with approximately 2.01 of 2X-YT broth containing 40 μg / ml kanamycin. The AR58 culture (pJJ701) was cultivated at a temperature of 30 ° C with vigorous aeration until obtaining an O.D.62s value of 0.5-0.6. The expression of rHMWP was induced by increasing the temperature of the fermenter culture at about 30 ° C to 42 ° C. Incubation at elevated temperature proceeded for about 4-5 hours. At the end of the induction period, the culture of E. Coli, with some cells showing classical recombinant protein inclusion bodies, was harvested by continuous flow centrifugation using a Heraeus Contifuge 28RS centrifuge. After centrifugation, a cell mass was scraped from the centrifuge container and stored at a temperature of -70 ° C until processing. Approximately 15 gm of the frozen cell paste of R58 (pJJ71) was resuspended by vortexing and trituration in about 40 ml of a 10 mM sodium phosphate buffer at ice temperature, pH7.3. Once suspended, lysozyme (chicken egg albumin, Sigma) and Dnasa I (bovine pancreas, Sigma) were added at final concentrations of 1.0 mg / ml and 0.01 mg / ml, respectively, and the mixture was incubated on ice during 30 - 45 minutes. The cells were disrupted by two sequential passages through a SLM Aminco French pressure cell (~ 14 Kpsi, pre-cooling diameter: 1") pre-cooled (~ 4 ° C.) The used cell was then centrifuged for 5 minutes ~ 500Xg ( 4 ° C) in a Sorvall SS34 rotor to remove unbroken cells Insoluble material containing rHMWP (in pellets) was isolated by centrifugation for 45 minutes at ~ 20,000Xg (4 ° C) in a Sorval SS34 rotor. from the centrifugation was discarded and the insoluble fraction was stored at -20 ° C in the form of pellets.To selectively extract the contaminating proteins and remove the endotoxin, the insoluble pellet containing rHMWP was thawed on ice and washed twice with 10 ml of a PBS regulator containing 2.0% Triton X-100. The washing was carried out at room temperature and the suspension of the pellet containing gelatinous rHMWP was achieved by vortex and homogenization. generation in a conventional glass fabric mill. The insoluble material containing rHMWP was recovered after # washing by centrifugation at ~10, OOOXg for 20 minutes (room temperature) in a rotor to Sorvall SS34. The insoluble material was then washed (again by vortexing and homogenization) 2 times with 10 ml of a 4.0 M urea solution containing 2.0 M NaCl. The washed rHMWP material was recovered by centrifugation in accordance with the above. The insoluble rHMWP fraction was further washed twice with 10 ml of a PBS solution containing 1.0% of Zwittergent 3-14 (Sigma). The rHMWP pellet recovered after centrifugation of the final wash solution was then solubilized for two hours at room temperature in a standard Laemelli SDS-PAGE regulator containing 4M urea. solubilized rHMWP was fractionated by size in a single protein band of approximately 100 Kdal by electrophoresis through a Tris / glycine / SDS preparation gel in ~ 14 cm X ~ 20 cm X ~ 3 mm 10% polyacrylamide (36 : 1, acrylamide: bis-acrylamide). A 4% polyacrylamide stacking gel formed using a 5-well preparation comb, with approximately 500 μl / well, was polymerized at the top of the resolving gel. Electrophoresis was performed in a BioRad Protean unit for ~ 12 hours at ~ 22 ° C (~ 80 - 85 volts, constant voltage) using a conventional Tris / glycine / SDS regulator (BioRad). Pre-stained molecular weight standards (SeeBlue, Novex) were loaded in a parallel band and were used to measure the degree and separation efficiency of the protein species. After electrophoresis, the gel sandwich was disarmed and a vertical section of the rHMWP sample band adjacent to the molecular weight markers was removed and labeled with Coomassie Blue R250 to visualize the rHMWP band. The stained section was then repositioned in the remaining non-stained preparation gel and the acrylamide strip containing rHMWP was identified and removed. RHMWP was eluted from the gel section using an EluTrap electroelution device from Schleicher and Schuell. The electroelusion was carried out in accordance with the recommendations of the manufacturers except that SDS regulator was used at a force of 1/4 (Novex) as the elution regulator. The elusion was carried out at ~ 40 mA for ~ 12-14 hours, at room temperature. At the end of the elution period, the polarity of the cell was reversed for approximately 2-3 minutes to remove the eventual protein absorbed on the BT1 membrane. The solution containing rHMWP was removed from the collection chamber and stored in a conical polypropylene tube at a temperature of 4 ° C. The excess SDS detergent was removed using * an SDS precipitation system (SDS-OUT Precipitation kit, Pierce Chemical). Removal of excess detergent from the gel eluted protein solution was achieved in accordance with the manufacturer's protocol. RHMWP without detergent was diluted approximately 15-fold with a sterile endotoxin-free 10 μM sodium phosphate buffer (pH 7.4) and concentrated to approximately 1.0 mg / ml by ultrafiltration in an Amicon stirred concentration cell using a YM30 ultrafiltration membrane. Residual endotoxin was removed from the concentrated rHMWP solution by a polymyxtin B Affi.Prep Polymyxin Matrix (BioRad) treatment. The Affi-Prep treatment was carried out overnight at a temperature of 4 ° C in a batch mode in accordance with the recommendations of the manufacturers. The concentrated protein concentration of rHMWP treated with polymyxin B was determined using the Micro BCA method (Pierce Chem.) And BSA as standard. Purified rHMWP (~ 0.9-1.2 mg / ml protein concentration) was evaluated for the purity, identity and residual load of endotoxin by SDS-PAGE, Western blot, and a calorimetric endotoxin assay (BioWhittaker), respectively. The gel-purified rHMWP material had a purity greater than 95% as a single band of the expected molecular size (~ 110 Kdal) through gel analysis and reacted vigorously with K196 antibody specific for rHMWP in Western blots. The residual endotoxin was calculated to be 0.05 EU / μg or less.

Claims

CLAIMS An HMW protein of Chlamydia species isolated where the apparent molecular weight is approximately 105-115 kDa, in accordance with that determined by SDS-PAGE, or a fragment or analogue thereof. The protein according to claim 1 which is substantially purified. The protein according to claim 1 wherein the Chlamydia species is Chlamydia trachomatis, Chlamydia pecorum, Chlamydia psittaci or Chlamydia pneumoniae. The protein according to claim 1 having an amino acid sequence illustrated in SEQ ID No: 2, 15 or 16 or a fragment or analog thereof conservatively substituted therewith. The fragment according to claim 4 having an amino acid sequence illustrated in SEQ ID No: 3, | 7 or 25-37. The protein according to claim 1 recognizable by an antibody preparation that specifically binds to a peptide having an amino acid sequence of SEQ ID No: 2, 15 or 16 or a fragment or analog thereof conservatively substituted therefor. An isolated nucleic acid molecule encoding the HMW protein of claim 1 or a fragment or analog thereof. The nucleic acid molecule according to claim 7 wherein the Chlamydia species is Chlamydia trachomatis, Chlamydia pecorum, Chlamydia psittaci or Chlamydia pneumoniae. The nucleic acid molecule according to claim 7 wherein the encoded protein has the amino acid sequence of SEQ ID No: 2, 15 or 16 or a conservatively substituted fragment or analogue thereof. An isolated nucleic acid molecule having a sequence selected from the group consisting of: a) a DNA sequence of SEQ ID No: 1, 23 or 24, or a complementary sequence or fragment thereof; b) a DNA sequence encoding an HMW protein having the amino acid sequence of SEQ ID No: 2, 15 or 16 or fragment thereof; c) a DNA sequence encoding a deduced amino acid sequence of SEQ ID No: 2, 15 or 16 or the complementary or degenerate sequence or a fragment thereof; and d) a nucleic acid sequence that hybridizes under stringent conditions to any of the sequences defined in a), b) or c). 11. A recombinant expression vector adapted for transformation of a host comprising the nucleic acid molecule of claim 7 or 10. 12. A recombinant expression vector adapted for transformation of a host comprising the nucleic acid molecule in accordance with claim 7 or 10 and an expression means operatively coupled to the nucleic acid molecule for expression by the host of HMW protein or a fragment or analogue thereof. The expression vector according to claim 12, wherein the expression means includes a portion of nucleic acid encoding a leader sequence for secretion from the host of the HMW protein or fragment or analog thereof. 14. A transformed host cell containing an expression vector of claim 12. 15. A transformed host cell containing an expression vector of claim 13. 16. An isolated recombinant protein or fragment or analogue thereof which can be produced by the transformed host of claim 14. 17. An isolated recombinant protein or fragment or analogue thereof that can be produced by the transformed host of claim 15. 18. A recombinant vector for administration of an HMW protein or fragment or analogue thereof to a host comprising the nucleic acid molecule of claim 7 or 10. An immunogenic composition, comprising at least one component selected from the group consisting of: a) an isolated HMW protein, wherein the apparent molecular weight is approximately 105-115 kDa, in accordance with that determined by SDS-PAGE, or a fragment or the like replaced conservatively of it; b) an isolated nucleic acid molecule encoding a HMW protein of a), or a fragment or analogue thereof; c) an isolated nucleic acid molecule having the sequence of SEQ ID Nos. 1, 23 or 24, the complementary sequence or a nucleic acid sequence that hybridizes under stringent conditions there or fragment thereof; d) an isolated recombinant HMW protein, or fragment or analogue thereof, that can be reproduced in a transformed host comprising an expression vector comprising a nucleic acid molecule as defined in b) or c) and a means of expression operatively coupled to the nucleic acid molecule for expression by the host of said HMW protein or fragment or analogue thereof; e) a recombinant vector comprising a nucleic acid sequence of b) or c) encoding an HMW protein or fragment or analogue thereof; Y "f) a transformed cell comprising the vector of e) and optionally an adjuvant, and a pharmaceutically acceptable carrier or diluent, said composition producing an immune response when administered to a host." An antigenic composition, comprising at least one selected component Within the group consisting of: a) an isolated HMW protein, where the apparent molecular weight is approximately 105-115 kDa, in accordance with that determined by SDS-PAGE, or a fragment or analogue thereof; an isolated nucleic acid molecule encoding an HMW protein of a), or a fragment or analogue thereof, c) an isolated nucleic acid molecule having the sequence of SEQ ID Nos. 1, 22, 23 or 24, the complementary or degenerate sequence thereof or a nucleic acid sequence that hybridizes under stringent conditions there; d) an isolated recombinant HMW protein, or fragment or analogue thereof, that can of being reproduced in a transformed host comprising an expression vector comprising a nucleic acid molecule according to that defined in b) or c) and expression means operably coupled to the nucleic acid molecule for expression by the host of said HMW protein or the fragment or analogue thereof; e) a recombinant vector, comprising a nucleic acid sequence of b) or c) encoding an HMW protein or fragment or analogue thereof; and f) a transformed cell comprising the vector of e) and optionally an adjuvant, said composition produces an immune response when administered to a host. . A method for producing an immune response in an animal, comprising administering to said animal an effective amount of the antigenic composition of claim 20 or of the immunogenic composition of claim 19. The method according to claim 21 wherein the animal is mammal or bird. . Antisera prepared against the antigenic composition of claim 20 or the immunogenic composition of claim 19. . Antibodies present in the antisera of claim 23 that specifically bind to an HMW protein or a fragment or analogue thereof. . A diagnostic reagent selected from the group consisting of: the protein of claim 1, the nucleic acid molecule of claim 10, the immunogenic composition of claim 20, the antigenic composition of claim 19, the antisera of the claim 23, the vector of claim 12, the transformed cell of claim 14, or the antibodies of claim 24.. A method for detecting anti-Chlamydia antibodies in a test sample, comprising the steps of: a) contacting a sample with the HMW protein of claim 1, the antigenic composition of claim 20 or the immunogenic composition of the claim 19 to form, in the presence of said antibodies, immunocomplexes of Chlamydia antigen: anti-Chlamydia antibody, and in addition, b) detect- the presence of the amount of said immunocomplexes formed during step a) or measure the amount of said immunocomplexes formed during step a) as an indication of the presence of said anti-Chlamydia antibodies in the test sample. . A set of diagnostic elements for detecting antibodies to Chlamydia, said set of elements comprising the HMW protein of claim 1, the antigenic composition of claim 20 or the immunogenic composition of claim 19, a container for contacting said protein or composition with a test sample which is suspected of having said antibodies and reagents to detect or measure the Chlamydia antigen immunocomplexes: anti-Chlamydia antibody formed between said protein or composition and said antibodies. . A method to detect the presence of Chlamydia in a test sample, comprising the steps of: a) contacting a test sample with the antibodies of claim 24 for a time sufficient to allow said antibodies to bind to Chlamydia, if present, and to form immunocomplexes Chlamydia: anti-viral antibodies; Chlamydia, and in addition, b) either detect the presence of the amount of said immunocomplex formed during step a) or measure the amount of said immunocomplex formed during step a) as an indication of the presence of said Chlamydia in the sample of proof. . A set of diagnostic elements for detecting the presence of Chlamydia, said set of elements comprises the antibodies of claim 24, the container for contacting said antibodies with a test sample suspected of having said Chlamydia and reagents for detect or measure immunocomplexes Chlamydia: anti-Chlamydia antibody formed between said antibodies and said Chlamydia. A pharmaceutical composition comprising an effective amount of at least one component selected from the group consisting of: a) a HMW protein, where the apparent molecular weight is about 105-115 kDa, as determined by SDS-PAGE , or a fragment or analogue thereof; b) an isolated nucleic acid molecule encoding an HMW protein of a), or a fragment or analogue thereof; c) an isolated nucleic acid molecule having the sequence of SEQ ID Nos. 1, 23 or 24, the complementary or degenerate sequence therein or a nucleic acid sequence that hybridizes under stringent conditions there; d) an isolated recombinant HMW protein, or a fragment or analogue thereof, which can be produced in a transformed host comprising an expression vector comprising a nucleic acid molecule according to that defined in b) or c) and means of expression operatively coupled with the nucleic acid molecule for expression by the host of said HMW protein or fragment or analogue thereof; e) a recombinant vector, comprising a nucleic acid sequence of b) or c) encoding an HMW protein or fragment or analogue thereof; f) a transformed cell comprising the vector of e) and g) antibodies that specifically bind to the component of a), b), c), d), e) or f), and optionally a pharmaceutically acceptable carrier or diluent. A vaccine composition comprising an effective amount of at least one component selected from the group consisting of: a) an HMW protein, where the apparent molecular weight is about 105-115 kDa, as determined by SDS- PAGE, or a fragment or analogue thereof; b) an isolated nucleic acid molecule encoding an HMW protein of a), or a fragment or analogue thereof; c) an isolated nucleic acid molecule having the sequence of SEQ ID Nos. 1, 23 or 24, the complementary or degenerate sequence, or a nucleic acid sequence that hybridizes under stringent conditions there; d) an isolated recombinant HMW protein, or a fragment or analogue thereof, which can be produced in a transformed host comprising an expression vector comprising a nucleic acid molecule according to that defined in b) or c) and means of expression operatively coupled to the nucleic acid molecule for expression by the host of said HMW protein or fragment or analogue thereof; e) a recombinant vector, comprising a nucleic acid sequence of b) or c) encoding a HMW protein or fragment or analogue thereof; f) a transformed cell comprising the vector of e) and g) antibodies that specifically bind to the component of a), b), c), d), e) of), and optionally an adjuvant, and a pharmaceutically acceptable carrier or diluent acceptable, where the vaccine produces an immune response when administered to a host. 32. A method for preventing, treating or ameliorating a Chlamydia-related disorder in a host requiring such treatment, comprising administering to a host an effective amount of the pharmaceutical composition of claim 30 or of the vaccine composition of claim 31. The method according to claim 32, wherein the disorder is selected from the group consisting of a bacterial infection by Chlamydia, conjunctivitis, urethritis, lymphogranuloma venereum (LGV), cervicitis, epididimitis, endometritis, pelvic inflammatory disease (PID), salpingitis, tubal occlusion, infertility, cervical cancer, arteriosclerosis and atherosclerosis. 34. The method according to claim 33 wherein the host is a bird or mammal. 35. The composition according to any of claims 19, 20, 30 or 31 formulated for in vivo administration to a host to provide protection against disease caused by a species of Chlamydia. 36. The composition of any of claims 19, 20, 30 or 31 wherein the species is selected from the group consisting of Chlamydia trachomatis, Chlamydia pecorum, Chlamydia psittaci and Chlamydia pneumoniae. 37. The composition of any of claims 19, 20, 30 or 31 formulated as a microparticle, capsule or liposome preparation. 38. The protein of any of claims 1, 4, 6, 16 and 17 wherein the protein binds to heparin or heparin sulfate. 39. The protein of any of claims 1, 4, 6, 16 or 17, wherein the protein is an outer membrane protein. 40. A method for determining the presence of nucleic acid encoding an HMW protein or a fragment or analog thereof in a sample, comprising the steps of: a) contacting a sample with the nucleic acid molecule of the claim 7 or 10 or any fragment or complement thereof to produce duplexes comprising the nucleic acid molecule and any nucleic acid molecule that encodes the HMW protein in the sample and that can hybridize specifically therewith; and b) determine the production of duplexes. 41. A set of diagnostic elements for determining the presence of nucleic acid encoding an HMW protein or fragment or analogue thereof in a sample, comprising: a) the nucleic acid molecule of claim 7 or 10 or any fragment of the same or complement thereof; b) means for contacting the nucleic acid with the sample to produce duplexes comprising the nucleic acid molecule and any nucleic acid encoding the HMW protein in the sample and can hybridize specifically therewith; y) means to determine the production of duplex. SUMMARY OF THE INVENTION _ A high molecular weight protein ("HMW") of Chlamydia, its amino acid sequence, and antibodies that bind specifically to the HMW protein, as well as the nucleic acid sequence encoding it, are presented. Prophylactic and therapeutic compositions are also disclosed, comprising the HMW protein, a fragment thereof, either -u antibody that specifically binds to the HMW protein or part thereof, or the nucleotide sequence encoding the HMW protein or a fragment thereof, including vaccines. LIST OF SEQUENCES < 11C > ANTEXBIOLOGICS, INC. < 12C > PROTEIN OF CHLAMYDIA, SEQUENCE OF GENES AND USES OF THE THEMSELF < 13C > 7969-076 < 14C > < 141 > < 150 > 08 / 942,596 < 151 > 1997-10-02 < 160 > 41 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 4435 < 212 > AND < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: recombinant expression vector < 401 > 1 • gggcaaaact cttccccccg ggatttatat gggaaagggg aaactttggc ccgtattcaa 60 gcgcc = CGGG ttttggggcg gaatgaattt tttcgttccg gaaaaagtaa ttccccggga 120 scgtagggta tcggtttcat aggctcgcca aatgggatat aggtggaaag gtaaaaaaaa 180 ctgagccaag caaaggatag agaagtcttg taatcatcgc aggttaaagg ggggatgtta 2.0 ttttagcctg caaatagtgt aattattgga tcctgtaaag agaaaaggac gaatgcgctg 300 catttattga aagataagaa tattaaatta ttaatttttt atgaagcgga gtaattaatt 360 ttatctctca gcttttgtgt gatgcaaacg tctttccata agttctttct ttcaatgatt 420 ctagcttatt cttgctgctc tttaaatggg gggggatatg cagcagaaat catggttcct 480 caaggaattt acgatgggga gacgttaact gtatcatttc cctatactgt tataggagat 540 ccgagtggga ctactgtttt ttctgcagga gagttaacat taaaaaatct tgacaattct 600 attgcagctt tgcctttaag ttgttttggg aacttattag ggagttttac tgttttaggg 660 agaggacact cgttgacttt cgagaacata cggacttcta caaatggggc agctctaagt 720 aatagcgctg ctgatggact gtttactatt gagggtttta aagaattatc cttttccaat 780 tgcaattcat tacttgccgt actgcctgct gcaacgacta ataagggtag ccagactccg 840 .acgacaacat ctacaccgtc taatggtact atttattcta aaacagatct tttgttactc 900 aataatgaga agttctcatt ctatagtaat ttagtctctg gagatggggg agctatagat 960 gctaagagct taacggttca aggaattagc aagctttgtg tcttccaaga aaatactgct 1020 caagctgatg ggggagcttg tcaagtagtc accagtttct ctgctatggc taaccjaggct 1080 ttgtagcgaa cctattgcct tgttgcagga gtaagagggg gagggattgc tgctgttcag 1140 gatgggcagc agggagtgtc atcatctact tcaacagaag atccagtagt aagtttttcc 1200 agaaatactg cggtagagtt tgatgggaac gtagcccgag taggaggagg gatttactcc 1260 tacgggaacg ttgctttcct gaataatgga aaaaccttgt ttctcaacaa tgttgcttct 1320 ttgctgctaa cctgtttaca gcaaccaaca agtggacagg cttctaatac gagtaataat 1380 tacggagatg gaggagctat cttctgtaag aatggtgcgc aagcaggatc caataactct 1440 ggatcagttt cctttgatgg agagggagta gttttcttta gtagcaatgt agctgctggg 1500 aaagggggag ctatttatgc caaaaágctc tcggttgcta actgtggccc tgtacaattt 1560 tcgctaatga ttaaggaata tggtggagcg atttatttag gagaatctgg agagctcagt 1620 ttatctgctg attatggaga tattattttc gatgggaatc ttaaaagaac agccaaagag 1680 aatgctgccg atgtta ATGG cgtaactgtg tcctcacaag ccatttcgat gggatcggga 1740 gggaaaataa cgacattaag agctaaagca ttctctttaa gggcatcaga tgatcccatc 1800 acggaaataa gagatggcaa ccagccagcg cagtcttcca aacttctaaa aattaacgat 1860 ggtgaaggat acacagggga tattgttttt gctaatggaa gcagtacttt gtaccaaaat 1920 gttacgatag agcaaggaag gattgttctt cgtgaaaagg caaaattatc agtgaattct 1980 ctaagtcaga caggtgggag tctgtatatg gaagctggga gtacatggga ttttgtaact 2040 ccacaaccac cacaacagcc tcctgccgct aatcagttga tcacgctttc caatctgcat 2100 ttgtctcttt cttctttgtt agcaaacaat gcagttacga atcctcctac caatcctcca 2160 gcgcaagatt ctcatcctgc agtcattggt agcacaactg ctggttctgt tacaattagt 2220 gggcctatct tttttgagga tttggatgat acagcttatg ataggtatga ttggctaggt 2280 aaatcaatgt tctaatcaaa cctgaaatta cagttaggga ctaagccccc agctaatgcc 2340 ccatcagatt tgactctagg gaatgagatg cctaagtatg gctatcaagg aagctggaag 2400 cttgcgtggg atcctaatac agcaaataat ggtccttata ctctgaaagc tacatggact 2460 aaaactgggt ataatcctgg gcctgagcga gtagcttctt tggttccaaa tagtttatgg 2520 ggatccattt tagatatacg atctgcgcat tcagcaattc aagcaagtgt ggatgggcgc 2580 tcttattgtc gaggattatg ggtttctgga gtttcgaatt tcttctatca tgaccgcgat 2640 gctttaggtc agggatatcg gtatattagt gggggttatt ccttaggagc aaactcctac 2700 tttggatcat cgatgtttgg tctagcattt accgaagtat ttggtagatc taaagattat 2760 gtagtgtgtc gttccaatca tcatgcttgc ataggatccg tttatctatc tacccaacaa 2820 gctttatgtg gatcctattt gttcggagat gcgtttatcc gtgctagcta cgggtttggg 2880 aatcagcata tgaaaacctc atatacattt gcagaggaga gcgatgttcg ttgggataat 2940 aactgtctgg ctggasagat tggagcggga ttaccgattg tgattactcc atctaagctc 3000 tatttgaatg agttgcgtcc tttcgtgcaa gctgagtttt cttatgccga tcatgaatct 3060 aaggcgatca tttacagagg agctcgggca ttcaagagcg gacatctcct aaatctatca 3120 gttcctgttg gagtgaagtt tgatcgatgt tctagtacac atcctaataa atatagcttt 3180 atggcggctt atatctgtga tgcttatcgc accatctctg gtactgagac aacgctccta 3240 tcccatcaag agacatggac aacagatgcc tttcatttag caagacatgg agttgtggtt 3300 agaggatcta tgtatgcttc tctaacaagt aatatagaag Tatat ggcca tggaagatat 3360 gagtatcgag atgcttctcg aggctatggt ttgagtgcag gaagtagagt ccggttctaa 3420 aaatattggt tagatagtta agtgttagcg atgccttttt ctttgagatc tacatcattt 3480 tgttttttag cttgtttgtg ttcctattcg tatggattcg cgagctctcc tcaagtgtta 3540 acgcctaatg taaccactcc ttttaaggga gacgatgttt acttgaatgg agactgcgct 3600 tttgtcaatg tctatgcagg agctgaagaa ggttcgatta tctcagctaa tggcgacaat 3660 ccggacaaaa ttaacgatta ccatacatta tcatttacag attctcaagg gccagttctt 3720 caaaattatg ccttcatttc agcaggagag acacttactc tgagagattt ttcgagtctg 3780 atgttctcga aaaatgtttc aagggaatga ttgcggagaa tctccgggaa aaccgtgagt 3840 atttccggag caggcgaagt gattttctgg gataactccg tggggtattc tcctttatct 3900 actgtgccaa cctcatcatc aactccgcct gctccaacag ttagtgatgc tcggaaaggg 3960 tctatttttt ctgtagagac tagtttggag atctcaggcg tcaaaaaagg ggtcatgttc 4020 gataataatg ccgggaattt cggaacagtt tttcgaggta agaataataa taatgctggt 4080 ggtggaggca gtgggttccg ctacaccatc aagtacgact tttacagtta aaaactgtaa 4140 agggaaagtt tctttcacag ataacgtagc ctcttgcgga ggcggagtgg tttataaagg 4200 cattgtgctt ttcaaagaca atgaaggagg catattcttc cgagggaaca cagcatacga 4260 tgatttaagg attcttgctg ctactaatca ggatcagaat acggagacag gaggcggtgg 4320 aggagttatt tgctctccag atgattctgt aaagtttgaa ggcaataaag gttctattgt 4380 ttttgattac aactttgcaa aaggcagagg cggaagcatc ctaacgaaag AATTC 4435 < 210 > 2 < 211 > 1012 < 212 > PRT < 213 > Chlamydia < 400 > 2 Met Gln Thr Ser Phe His Lys Phe Phe Leu Ser Met He Leu Ala Tyr 1 5 10 15 Being Cys Cys Ser Leu Asn Gly Gly Gly Tyr Ala Glu Wing He Met Val 20 25 30 Pro Glp Gly He Tyr Asp Gly Glu Thr Leu Thr Val Ser Phe Pro Tyr 35-40 45 Thr Val He Gly Asp Pro Ser Gly Thr Thr Val Phe Ser Wing Gly Glu 50 55 60 Leu Thr Leu Lys Asn Leu As Asn Ser He Ala Ala Leu Pro Leu Ser 65 70 75 80 Cys Phe Gly Asn Leu Leu Gly Ser Phe Thr Val Leu Gly Arg Gly His 85 90 95 Being Leu Thr Phe Glu Asn He Arg Thr Ser Thr Asn Gly Ala Ala Leu 100 105 110 Being Asn Being Wing Wing Asp Gly Leu Phe Thr He Glu Gly Phe Lys Glu 115 120 125 Leu Ser Phe Ser Asn Cys Asn Ser Leu Leu Ala Val Leu Pro Ala Ala 130 135 140 Thr Thr Asn Lys Gly Ser Gln Thr Pro Thr Thr Thr Ser Thr Pro Ser 145 150 155 160 Asn Gly Thr He Tyr Ser Lys Thr Asp Leu Leu Leu Leu Asn Asn Glu 165 170 175 Lys Phe Ser Phe Tyr Ser Asn Leu Val Ser Gly Asp Gly Gly Wing 180 185 190 Asp Wing Lys Ser Leu Thr Val Gln Gly He Ser Lys Leu Cys Val Phe 195 200 205 Gln GI Asn Thr Wing Gln Wing Asp Gly Gly Wing Cys Gln Val Val Thr 210 215 220 Ser Phe Ser Wing Met Wing Asn Glu Wing Pro He Wing Phe Val Wing Asn 225 230 235 240 Val Wing Gly Val Arg Gly Gly Gly Wing Wing Val Gln Asp Gly Gln 245 250 255 Gln Gly Val Being Ser Thr Ser Thr Glu Asp Pro Val Val Ser Phe 260 265 270 Ser Arg Asn Thr Wing Val Glu Phe Asp Gly Asn Val Wing Arg Val Gly 275 280 285 Gly Gly He Tyr Ser Tyr Gly Asn Val Wing Phe Leu Asn Asn Gly Lys 290 295 300 Thr Leu Phe Leu Asn Asn Val Ala Ser Pro Val Tyr He Ala Ala Lys 305 310 315 320 Gln Pro Thr Ser Gly Gln Wing Ser Asn Thr Ser Asn Asn Tyr Gly Asp 325 330 335 Gly Gly Wing He Phe Cys Lys Asn Gly Wing Gln Wing Gly Ser Asn Asn 340 345 350 Ser Gly Ser Val Ser Phe Asp Gly Glu Gly Val Val Phe Phe Ser Ser 355 360 365 Asn Val Wing Wing Gly Lys Gly Gly Wing He Tyr Wing Lys Lys Leu Ser 370 375 380 Val Wing Asn Cys Gly Pro Val Gln Phe Leu Arg Asn He Wing Asn Asp 385 390 395 400 Gly Gly Wing He Tyr Leu Gly Glu Be Gly Glu Leu Ser Leu Ser Wing 405 410 415 Asp Tyr Gly Asp He He Phe Asp Gly Asn Leu Lys Arg Thr Wing Lys - 420 425 430 Glu Asa Wing Wing Asp Val Asn Gly Val Thr Val Ser Ser Gln Wing He 435 440 ns Ser Met Gly Ser Gly Gly Lys He Thr Thr Leu Arg Ala Lys Ala Gly 450 455 460 His G3.n He Leu Phe Asn Asp Pro He Glu Met Wing Asn Gly Asn Asn 465 470 475 480 Gln Pro Wing Gln Ser Ser Lys Leu Leu Lys He Asn Asp Gly Glu Gly 485 490 495 Tyr Thr Gly Asp He Val Phe Wing Asn Gly Be Ser Thr Leu Tyr Gln 500 505 510 sn Val Thr He Glu Gln Gly Arg He Val Leu Arg Glu Lys Wing Lys 515 520 525 Leu Ser Val Asn Ser Leu Ser Gln Thr Gly Gly Ser Le- Tyr Met Glu 530 535 540 Wing Gly Ser Thr Trp Asp Phe Val Thr Pro Gln Pro Pro Gln Gln Pro 545 550 555 • 560 Pro Ala Ala Asn Gln Leu He Thr Leu Ser Asn Leu His Leu Ser Leu 565 570 575 Be Ser Leu Leu Ala Asn Asn Ala Val Thr Asn Pro Prc Thr Asn Pro 580 585 590 Pro Wing Gin Asp Ser His Pro Wing Val He Gly Ser Thr Thr Wing Gly 595 600 6C5 Ser Val Thr He Ser Gly Pro He Phe Phe Glu Asp Read Asp Asp Thr 610 615 620 Wing Tyr Asp Arg Tyr Asp Trp Leu Gly Ser Asn Gln Lys He Asn Val 625 630 635 640 Leu Lys Leu Gln Leu Gly Thr Lys Pro Pro Wing Asn Ala Pro Ser Asp 645 650 655 Leu Thr Leu Gly Asn Glu Met Pro Lys Tyr Gly Tyr Gln Gly Ser Trp 660 665 670 Lys Leu Wing Trp Asp Pro Asn Thr Wing Asn Asn Gly Pro Tyr Thr Leu 675 '680 685 Lys Wing Thr Trp Thr Lys Thr Gly Tyr Asn Pro Gly Pro Glu Arg Val 690 695 700 Wing Ser Leu Val Pro Asn Ser Leu Trp Gly Ser He Leu Asp He Arg 705 710 715 720 Ser Ala His Ser Ala He Gln Ala Ser Val Asp Gly Arg Ser Tyr Cys 725 730 735 Arg Gly Leu Trp Val Ser Gly Val Ser Asn Phe Phe Tyr His Asp Arg 740 745 750 Asp Wing Leu Gly Gln Gly Tyr Arg Tyr He Ser Gly Gly Tyr Ser Leu 755. 760 765 Gly Wing Asn Being Tyr Phe Gly Being Being Met Phe Gly Leu Wing Phe Thr 770 775 780 Glu Val Phe Gly Arg Ser Lys Asp Tyr Val Val Cys Arg Ser Asn His 785 790 795 800 HIs Ala Cys He Gly Ser Val Tyr Leu Ser Thr Gln Gln Ala Leu Cys 805 810 815 Gly Ser Tyr Leu Phe Gly Asp Wing Phe He Arg Wing Being * v- Gly Phe 820 825 63C Gly Asn Gln His Met Lys Thr Ser Tyr Thr Phe Wing Glu Glu 835 Ser ASD 840 845 Val Arg Trp Asp Asn Asn Cys Leu Wing Gly Glu He Gly Wing Gly Leu 850 855 860 Pro He Val He Thr Pno Ser Lys Leu Tyr Leu Asn Glu Leu 865 Arg Pro 870 875 880 Phe Val Gln Ala Glu Phe Ss? Tyr Ala Asp His Glu Ser Phe Thr Glu 885 890 895 Glu Gly Asp Gln Al to Arg Ala Phe Lys Ser Gly His Leu Leu Asn Leu 900 905 910 Ser Val Pro Val Gly Val Lys Phe Asp Arg Cys Ser Ser Thr His Pro 915 920 925 Asn Lys Tyr Ser Phe Ket Wing Wing Tyr He Cys Asp Al 930 to Tyr Arg Thr 935 940 He Ser Gly Thr Glu Thr Thr Leu Leu Ser His Gln Glu 1 945 'nr Trp Thr 950 955 960 T r Asp Ala Phe His Leu Ala Arg His Gly Val Val Val Arg Gly Ser 965 970 975 «- ti. A s «. Ihr - ", __ Glu yal ^ w and HÍJ ^ ^ 985 990 Tyr Glu Tyr Arg Asp Wing Ser Arg Gly Tyr Gly Leu Ser Wing Gly Ser 1000 1005 Arg Val Arg Phe 1010 < 210 > 3 < 211 > 20 < 212 > PRT < 213 > Chlamydia < 400 > 3 Glu He Met Val Pro Gln Gly He Tyr Asp Gly Glu Thr Leu Thr Val 5 10 15 Ser Phe Xaa Tyr 20 < 210 > 4 < 211 > 18 < 212 > DNA < 213 > Chlamydia < 400 > 4 gaaathatgg tnccncaa 18 < 210 > 5 < 211 > 18 < 212 > DNA < 213 > Chlamydia < 400 > 5 gaaathatgg tnccncag 18 < 210 > 6 < 211 > 18 < 212 > DNA < 213 > Chlamydia < 400 > 6 18 gagathatgg tnccncea < 210 > 7 < 211 > 18 < 212 > DNA < 213 > Chlamydia < 400 > 7 18 gagathatgg tnccnc-? G < 210 > 8 < 211 > 15 < 212 > DNA < 213 > Chlamydia < 400 > 8 ngtytcnccr tcata 15 < 210 > 9 < 211 > 15 < 212 > DNA 10 < 213 > Chlamydia < 400 > 9 ngtytcnccr tcgta 15 < 210 > 10 < 211 > 1511 < 212 > DNA < 213 > Chlamydia < 400 > 10 gaaatcatgg ttcctc.__.gg aatttacgat ggggagacgt taactgtatc atttccctat 60 actgttatag gagatc gag tgggactact gttttttctg caggagagtt aacattaaaa 120 aatcttgaca attctarrgc agctttgcct ttaagttgtt ttgggaactt attagggagt 180 tttactgttt tagggacagg acactcgttg actttcgaga acatacggac ttctacaaat 240 ggggcagctc taagta = ag cgctgctgat ggactgttta ctattgaggg ttttaaagaa 300 ttatcctttt ccaattr aa ttcattactt gccgtactgc ctgctgcaac gactaataag 360 ggtagccaga ctccgae ac aacatctaca gtactattta ccgtctaatg ttctaaaaca 420 gatcttttgt tactcaetaa tgagaagttc tcattctata gtaatttagt ctctggagat 480 tagatgraa gggggagcta gagcttaacg gttcaaggaa ttagcaagct ttgtgtcttc 540 caagaaaata ctgctcaagc tgatggggga gcttgtcaag tagtcaccag tjttctctgct 600 atggctaacg aggctcc at tgcctttgta gcgaatgttg caggagtaag agggggaggg 660 attgctgctg ttcag TTTS gcagcaggga gtgtcatcat ctacttcaac agaagatcca- 720 tttccaraaa gtagtaagtt tactgcggta gagtttgatg ggaacgtagc ccgagtagga 780 ggagggattt actcctargg gaacgttgct ttcctgaata atggaaaaac cttgtttctc 840 aacaatgttg ctt ctcrtgt ttacattgct gctaagcaac caacaagtgg acaggcttct 900 ataattacgg aatacgagta asatggagga gctatcttct gtaagaatgg tgcgcaagca 960 ggatccaata actctggatc agtttccttt gatggagagg gagtagtttt ctttagtagc 1020 aatgtagctg ctgggaaagg gggagctatt tatgccaaaa agctctcggt tgctaactgt 1080 ggccctgtac aatttttaag gaatatcgct aatgatggtg gagcgattta tttaggagaa 1140 tctggagagc tcagtttatc tgctgattat ggagatatta ttttcgatgg gaatcttaaa 1200 agaacagcca aagagaatgc tgccgatgtt aatggcgtaa ctgtgtcctc acaagccatt 1260 tcgatgggat cgggagggaa aataacgaca aagcagggca ttaagagcta tcagattctc 1320 tttaatgatc ccatcgagat ggcaaacgga aataaccagc cagcgcagtc ttccaaactt 1380 acgatggtga ctaaaaatta aggatacaca ggggatattg tttttgctaa tggaagcagt 1440 actttgtacc aaaatgttac gatagagcaa ggaaggattg ttcttcgtga aaaggcaaaa ttatcagtga 1500 to 1511 < 210 > 11 < 211 > 1444 11 < 212 > DNA < 213 > Chlamydia < 400 > 11 ttctetaagt eagacaggtg ggagtctgta tatggaagct gggagtacat gggattttgt 60 aactccacaa ccaccacaac agcctcctgc cgctaatcag ttgatcacgc tttccaatct 120 gcatttgtct ctttcttctt tgttagcaaa caatgcagtt acgaatcctc ctaccaatcc 180 tccagcgcaa gattctcatc ctgcagtcat tggtagcaca actgctggtt ctgttacaat 240 tagtgggcct atcttttttg aggatttgga tgatacagct tatgataggt atgattggct 300 caaaaaatca aggttctaat atgtcctgaa attacagtta gggactaagc ccccagctaa 360 tgccccatca gatttgactc tagggaatga gatgcctaag tatggctatc aaggaagctg 420 gaagcttgcg tgggatcc to atacagcaaa taatggtcct tatactctga aagctacatg 480 gactaaaact gggtataatc ctgggcctga gcgagtagct tctttggttc caaatagttt 540 attttacata atggggatcc gcattcagca tacgatctgc attcaagcaa gtgtggatgg 600 gcgctcttat tgtcgaggat tatgggtttc tggagtttcg aatttcttct atcatgaccg 660 cgatgcttta ggtcagggat atcggtatat tagtgggggt tattccttag gagcaaactc 720 tcatcgatgt ctactttgga atttaccgaa ttggtctagc gatctaaaga gtatttggta 780 tgtcgttcca ttatgtagtg ttgcatagga atcatcatgc tatctaccca tccgtttatc 840 acaagcttta tgtggatcct atttgttcgg agatgcgttt atccgtgcta gctacgggtt 900 tgggaatcag catatgaaaa cctcatatac atttgcagag gagagcgatg TGCTs tggga 960 taataactgt ctggctggag agattggagc gggattaccg attgtgatta ctccatctaa 1020 gctctatttg aatgagttgc gtcctttcgt gcaagctgag ttttctta tg ccgatcatga 1080 atcttttaca gaggaaggcg atcaagctcg ggcattcaag agcggacatc tcctaaatct 1140 atcagttcct gttggaytga agtttgatcg atgttctagt acacatccta ataaatatag 1200 ctttatggcg gcttatat t gtgatgctta tcgcaccatc tctggtactg agacaacgct 1260 cctatcccat caagagacat ggacaacaga tgcctttcat ttagcaagac atggagttgt 1320 ggttagagga tctatgtatg cttctctaac aagtaatata gaagtatatg gccatggaag 1380 atatgagtat cgagatgctt ctcgaggcta tggtttgagt gcaggaagta gagtccggtt 1440 CTAA 1444 < 210 > 12 < 211 > 56 < 212 > DNA < 213 > Chlamydia < 400 > 12 aagggcccaa ttacgcagag ggtaccgaaa ttatggttcc tcaaggaatt tacgat 56 < 210 > 13 < 211 > 56 12 < 212 > DNA < 213 > Chlamydia < 400 > 13 aagggcccaa ttacgcagag ggtaccctaa gaagaaggca tgccgtgcta gcggag 56 < 210 > 14 < 211 > 57 < 212 > DNA < 213 > Chlamydia < 400 > 14 aagggcccaa ttacgcagag ggtaccggag agctcgcgaa tccatacgea taggaac 57 < 210 > 15 < 211 > 1013 < 212 > PRT < 213 > Chlamydia < 400 > 15 Met Gln Thr Ser Phe His Lys Phe Phe Leu Ser Met He Leu Ala Tyr 1 5 10 15 Ser Cys Cys Ser Leu Asn Gly Gly Gly Tyr Wing Wing Gla He Met Val 20 25 30 Pro Gln Gly He Tyr Asp Gly Glu Thr Leu Thr Val Ser Fhe Pro Tyr 35 40 45 Thr Val He Gly Asp Pro Ser Gly Thr Thr Val Phe Ser Wing Gly Glu 50 55 60 Leu Thr Leu Lys Asn Leu Asp Asn Ser He Ala Wing Leu Pro Leu Ser 65 70 75 80 Cys Phe Gly Asn Leu Leu Gly Ser Phe Thr Val Leu Gly Arg Gly His - 85 90 95 13 Ser Leu Thr Phe Glu Asn He Arg Thr 'Ser Thr Asn Gly Ala Ala Leu 100 105 110 Ser Asp Ser Wing Asn Ser Gly Leu Phe Thr He Glu Gly Phe Lys Glu 115 120 125 Leu Ser Phe Ser Asn Cys Asn Pro Leu Leu Ala Val Leu Pro Ala Ala 130 135 140 Thr Thr Asn Asn Gly Ser Gln Thr Pro Ser Thr Thr Ser Thr Pro Ser 145 150 155 160 Asn Gly Thr He Tyr Ser Lys Thr Asp Leu Leu Leu Leu Asn Asn Glu 165 170 175 Lys Phe Ser Phe Tyr Ser Asn Ser Val Ser Gly Asp Gly Gly Wing He 180 185 190 Asp Wing Lys Ser Leu Thr Val Gln Gly He Ser Lys Leu Cys Val Phe 195 200 205 Gln Glu Asn Thr Wing Gln Wing Asp Gly Gly Wing Cys Cln Val Val Thr 210 215 220 Ser Phe Ser Ala Ket Wing Asn Glu Wing Pro He Wing Phe Val Wing Asn 225 230 235 240 Val Ala Gly Val Arg Gly Gly Gly He Ala Ala Gl Gl.n Asp Gly Gln 245 250 255 Gln Gly Val Being Ser Thr Being Thr Glu Asp Pro Val Val Being Phe 260 265 270 Being Arg Asn Thr Wing Val Glu Phe Asp Gly Asn Val Wing Arg Val Gly 275 280 285 Gly Gly He Tyr Ser Tyr Gly Asn Val Wing Phe Leu Asa Asn Gly Lys 290 295 300 Thr Leu Phe Leu Asn Asn Val Ala Ser Pro Val Tyr He Ala Ala Glu 305 310 315 320 Gln Pro Thr Asn Gly Gln Wing Ser Asn Thr Ser Asp Asn Tyr Gly Asp 325 330 335 14 Gly Gly Wing He Phe Cys Lys Asn Gly Wing Gln Wing Wing Gly Ser Asn 340 '345 350 Asn Ser Gly Ser Val Ser Phe Asp Gly Glu Gly Val Val Phe Phe Ser 355 360 365 Being Asn Val Ala Ala Gly Lys Gly Gly Ala He Tyr Ala Lys Lys Leu 370 375 380 Ser Val Ala Asn Cys Gly Pro Val Gln Leu Leu Gly Asn He Ala Asn 385 390 395 400 Asp Gly Gly Wing He Tyr Leu Gly Glu Be Gly Glu Leu Ser Leu Ser 405 410 415 Wing Asp Tyr Gly Asp Met He Phe Asp Gly Asn Leu Lys Arg Thr Wing 420 425 430 Lys Glu Asn Ala Ala Asp Val Asn Gly Val Thr Val Ser Ser Gln Ala 435 440 445 I Be Ket Gly Be Gly Gly Lys He Thr Thr Leu Arg Ala Lys Wing 450 455 460 Gly Eis Gin He Leu Phe Asn Asp Pro He Glu Met Wing Asn Gly Asn 465 470 475 480 Asn Gln Pro Wing Gln Ser Ser Glu Pro Leu Lys He Asn Asp Gly Glu 485 490 495 Gly Tyr Thr Gly A = p He Val Phe Wing Asn Gly Asn Ser Thr Leu Tyr 500 505 510 Gln Asr. Val Thr He Glu Gln Gly Arg He Val Leu Arg- Glu Lys Wing 515 520 525 Lys Leu Ser Val Asn Ser Leu Ser Gln Thr Gly Gly Ser Leu Tyr Met 530 535 540 Glu Ala Gly Ser Thr Leu Asp Phe Val Thr Pro Gln Pro Pro Gln Gln 545 550 555 560 Pro Pro Ala Ala Asn Gln Ser He Thr Leu Ser Asn Leu His Leu Ser 565 570 575 15 Leu Be Ser Leu Lea Wing Asn Asn Wing Val Thr Asn Pro Pro Thr Asn '580 585 590 Pro Pro Wing Gln Asp Ser His Pro Wing Val He Gly Ser Thr Thr Wing 595 600 605 Gly Ser Val Thr He Ser Gly Pro He Phe Phe Glu Asp Leu Asp Asp 610 615 620 Thr Wing Tyr Asp Arg Tyr Asp Trp Leu Gly Ser Asn Gln Lys He Asp 625 630 635 640 Val Leu Lys Leu Gln Leu Gly Thr Gln Pro Pro Wing Asn Ala Pro Ser 645 650 655 Asp Leu Thr Leu Gly Asn Glu Met Pro Lys Tyr Gly Tyr Gln Gly Ser 660 665 670 Trp Lys Leu Wing Trp Asp Pro Asn Thr Wing Asn Asn Gly Pro Tyr Thr 675 680 685 Leu Lys Wing Thr Trp Thr Lys Thr Gly Tyr Asn Pro Gly Pro Glu Arg 690 695 700 Val Wing Ser Leu Val Pro Asn Ser Leu Trp Gly Ser He Leu Asp He 705 710 • 715 720 Arg Ser Ala His Ser Ala He Gln Ala Ser Val Asp Gly Arg Ser Tyr 725 730 735 Cys Arg Gly Leu Trp Val Ser Giy Val Ser Asn Phe Phe Tyr His Asp 740 745 750 Arg Asp Ala Leu Gly Gln Gly Tyr Arg Tyr He Ser Gly Gly Tyr Ser 755 760 765 Leu Gly Wing Asn Being Tyr Phe Gly Being Being Met Phe Gly Leu Wing Phe 770 775 780 Thr Glu Val Phe Gly Arg Ser Lys Asp Tyr Val Val Cys Arg Ser Asn 785 790 795 800 His His Wing Cys He Gly Ser Val Tyr Leu Ser Thr Lys Gln Wing Leu 805 810 815 16 Cys Gly Ser Tyr Val Phe Gly Asp Wing Phe He Arg Wing Ser Tyr Gly 820 825 830 Phe Gly Asn Gln His Met Lys Thr Ser Tyr Thr Phe Wing Glu Glu Ser 835 840 845 Asp Val Cys Trp Asp Asn Asn Cys Leu Val Gly Glu He Gly Val Gly 850 855 860 Leu, Pro He Val He Thr Pro Ser Lys Leu Tyr Leu Asn Glu Leu Arg 865 870 875 880 Pro Phe Val Gln Wing Glu Phe Ser Tyr Wing Asp His Glu Ser Phe Thr 885 890 895 Glu Gla Gly Asp Gln Wing Arg Wing Phe Arg Ser Gly His Leu Ket Asn 900 905 910 Leu Ser Val Pro Val Gly Val Lys Phe Asp Arg Cys Ser Ser Thr His 915 920 925 Pro Asn Lys Tyr Ser Phe Met Gly Ala Tyr He Cys Asp Ala Tyr Arg 930 935 940 Thr He Ser Gly Thr Gln Thr Thr Leu Leu Ser His Gln Glu Thr Trp 945 950 955 960 Thr Thr Asp Wing Phe Eis Leu Wing Arg His Gly Val He Val Arg Gly 965 970 975 Ser Met Tyr Ala Ser Leu Thr Ser Asn He Glu Val Tyr Gly His Gly 980 985 990 17 Arg Tyr Glu Tyr Arg Asp Thr Ser Arg Gly Tyr Gly Leu Ser Wing Gly 995 1000 1005 Ser Lys Val Arg Phe 1010 < 210 > 16 < 211 > 1013 < 212 > PRT < 213 > Chlamydia < 400 > 16 Met Gln Thr Ser Phe Kis Lys Phe Phe Leu Ser Met He Leu Wing Tyr 1 5 10 15 Ser Cys Ser Leu Thr Gly Gly Gly Tyr Wing Ala Glu He Met Val 20 25 30 Pro Gln Gly He Tyr Asp Gly Glu Thr Leu Thr Val Ser Phe Pro Tyr 35 40 45 Thr Val He Gly Asp Pro Ser Gly Thr Thr Val Phe Be Wing Gly Glu 50 55 60 Leu Thr Leu Lys Aso Leu Asp Asn Ser Wing Ala Wing Leu Pro Leu Ser 65 70 75 80 Cys Phe Gly Asn Leu Leu Gly Ser Phe Thr Val Leu Gly Arg Gly His 85 90 95 Being Leu Thr Phe Glu Asn He Arg Thr Ser Thr Asn Gly Ala Ala Leu 100 105 110 Being Asp Being Wing Asn Being Gly Leu Phe Thr He Glu Gly Phe Lys Glu 115 120 125 Leu Being Phe Being Asn Cys Asn Being Leu Leu Wing Val Leu Pro Wing Wing 130 135 140 18 Thr Thr Asn Asn Gly Ser Gln Thr Pro Thr Thr Thr Ser Thr Pro Ser 145 150 155 160 Asn Gly Tpr He Tyr Ser Lys Thr Asp Leu Leu Leu Leu Asn Asn Glu 165 170 175 Lys Phe Ser Phe Tyr Ser Asn Leu Val Ser Gly Asp Gly Gly Thr He 180 185 190 Asp Ala Lys Ser Leu Thr Val Gln Giy He Ser Lys Leu Cys Val Phe 195 200 205 Gln Glu Asr. Thr Ala Gln Ala Asp Gly Gly Ala Cys Gis Val Val Thr 210 215 220 Being Phe Being Ala Ket Ala Asn Glu Ala Pro He Ala Phe He Ala Asn 225 230 235 240 Val Wing Gly Val Arg Gly Gly Gly Wing Wing Val G1-. Asp Gly Gln 245 250 255 Gln Gly Val Being Ser Thr Ser Thr Glu Asp Pro Val Val Ser Phe 260 265 270 Ser Arg Asn Thr Ala Val Glu Phe Asp Gly Asn Val Ala Arg Val Gly 275 280 285 Gly Gly He Tyr Ser Tyr Gly Asn Val Wing Phe Leu Asn Asn Gly Lys 290 295 300 Thr Leu Phe Leu Asn Asn Val Ala Ser Pro Val Tyr He Ala Ala Glu 305 310 315 320 Gln Pro Thr Asn Gly Gln Wing Being Asn Thr Being Asp Asn Tyr Gly Asp 325 330 335 Gly Gly Wing He Phe Cys Lys Asn Gly Wing Gln Wing Wing Gly Ser Asn 340 345 350 Handle Be Gly Ser Val Be Phe Asp Gly Glu Gly Val Val Phe Phe Ser * 355 360 365 Be Asn Val Ala Ala Gly Lys Gly Gly Ala He Tyr Ala Lys Lys Leu 370 375 380 19 Ser Val Ala Asn Cys Gly Pro Val Gln Phe Leu Gly Asn He Ala Asn 385 '390' 395 400 Asp Gly Gly Wing He Tyr Leu Gly Glu Be Gly Glu Leu Ser Leu Ser 405 410 415 Wing Asp Tyr Gly Asp He He Phe Asp Gly Asn Leu Lys Arg Thr Wing 420 425 430 Lys Glu Asn Ala Ala Asp Val Asn Gly Val Thr Val Ser Ser Gln Ala 435 440 445 He Be Met Gly Be Gly Gly Lys He Thr Thr Leu Arg Ala Lys Wing 450 455 460 Gly Eis Gln He Leu Phe Asn Asp Pro He Glu Met Wing Asn Gly Asn 465 470 475 480 Asn Gin Pro Wing Gln Ser Ser Glu Pro Leu Lys He Asn Asp Gly Glu 485 490 495 Gly Tyr Thr Gly Asp He Val Phe Wing Asn Gly Asn .Ser Thr Leu Tyr 500 505 510 Gln Asn Val Thr He Glu Gln Gly Arg He Val Leu Arg Glu Lys Wing 515 520 525 Lys Leu Ser Val Asn Ser Leu Ser Gln Thr Gly Gly Ser Leu Tyr Met 530 535 540 Glu Ala Gly Ser Thr Leu Asp Phe Val Thr Pro Gln Pro Pro Gln Gln 545 550 555 560 Pro Pro Ala Ala Asn Gln Leu He Thr Leu Ser Asn Leu His Leu Ser 565 570 575 Leu Be Ser Leu Leu Ala Asn Asn Ala Val Thr Asn Pro Pro Thr Asn 580 585 590 Pro Pro Wing Gln Asp Ser His Pro Wing Val He Gly Ser Thr Thr Wing 595 600 605 Gly Pro Val Thr He Ser Gly Pro Phe Phe Phe Glu Asp Leu Asp Asp 610 615 620 20 Thr Ala Tyr Asp Arg Tyr Asp Trp Leu Gly Ser Asn Gln Lys He Asp 625 630 635 640 Val Leu Lys Leu Gln Leu Gly Thr Gln Pro Ser Wing Asn Ala Pro Ser 645 650 655 Asp Leu Thr Leu Gly Asn Glu Met Pro Lys Tyr Gly Tyr Gln Gly Ser 660 665 670 Trp Lys Leu Wing Trp Asp Pro Asn Thr Wing Asn Asn Gly Pro Tyr Thr 675 680 685 Leu Lys Wing Thr Trp Thr Lys Thr Gly Tyr Asn Pro Gly Pro Glu Arg 690 695 700 Val Ala Ser Leu Val Pro Ace? Ser Leu Trp Gly Ser He Leu Asp He 705 710 715 720 Arg Ser Ala Kis Ser Ala He Glr. Ala Ser Val Asp Gly Arg Ser Tyr 725 730 735 Cys Arg Gly Leu Ti Val Ser Gly Val Ser Asn Phe Ser Tyr His Asp 740 745 750 Arg Asp Ala Leu Gly Gln Gly Tyr Arg Tyr He Ser Gly Gly Tyr Ser 755 760 765 Leu Gly Wing Asn Being Tyr Phe Gly Being Being Met Phe Gly Leu Wing Phe 770 775 780 Thr Glu Val Phe Gly Arg Ser Lys Asp Tyr Val Val Cys Arg Ser Asn 785 790 795 800 His His Wing Cys He Gly Ser Val Tyr Leu Ser Thr Lys Gln Wing Leu 805 810 815 Cys Gly Ser Tyr Leu Phe Gly Asp Wing Phe He Arg Wing Ser Tyr Gly 820 825 830 Phe G? And Asn Gln Eis Met Lys Thr Ser Tyr Thr Phe Wing Glu Glu Ser 835 840 845 - 21 Asp Val Arg Trp Asp Handle Handle Cys Leu Val Gly Glu He Gly Val Gly 850 855 860 Leu Pro He Val Thr Thr Pro Ser Lys Leu Tyr Leu Asn Glu Leu Arg 865 870 875 880 Pro Phe Val Gln Wing Glu Phe Ser Tyr Wing Asp His Glu Ser Phe Thr 885 890 895 Glu Glu Gly Asp Gln Wing Arg Wing Phe Arg Ser Gly His Leu Met Asn 900 905 910 Leu Ser Val Pro Val Gly Val Lys Phe Asp Arg Cys Ser Ser Thr His 915 920 925 Pro Asn Lys Tyr Ser Phe Met Gly Wing Tyr He Cys Asp Wing Tyr Arg 930 935 940 Thr He Ser Gly Thr Glr. Thr Thr Leu Leu Ser His Gln Glu Thr Trp 945 £ 50 555 960 Thr Thr Asp Wing Phe Kie Leu Wing Arg Kis Gly Val He Val Arg Gly 965 970 975 Ser Ket Tyr Ala Ser Leu Thr Ser Asn He Glu Val Tyr Gly His Gly 980 985 990 Arg Tyr Glu Tyr Arg As? Thr Ser Arg Gly Tyr Gly Leu Ser Wing Gly 995 1000 1005 Ser Lys Val Arg Phe 1010 < 210 > 17 < 211 > 505 < 212 > PRT < 213 > Chlamydia < 400 > 17 22 Glu He Ket Val Pro Gla Gly He Tyr Asp Gly Glu Thr Leu Thr Val 1 5? O 15 Be Phe Pro Tyr Thr Val He Gly Asp Pro Be Gly Thr Thr Val Phe 0. 25 30 Be Wing Gly Glu Leu Thr Leu Lys Asn Leu Asp Asn Ser He Wing Wing 35 40 45 Leu Pro Leu Ser Cys Phe Gly Asn Leu Leu Gly Ser Phe Thr Val Leu 50 55 60 Gly Arg Gly His Ser Leu Thr Phe Glu Asn He Arg Thr Ser Thr Asn € 5 70 75 80 Gly Ala Ala Leu Ser Asn Ser Ala Ala Asp Gly Leu Phe Thr He Glu 85 90 95 Gly Phe Lys Glu Leu Ser Phe Ser Asn Cys Asn Ser Leu Leu Ala Val 100 105 110 Leu Pro Wing Wing Thr Thr Asn Lys Gly Ser Gln Thr Pro Thr Thr Thr 115 120 125 Ser Thr Pro Ser Asa Gly Thr He Tyr Ser Lys Thr Asp Leu Leu Leu 130 135 140 Leu Asn Asn Glu Lys Phe Ser Phe Tyr Ser Asn Leu Val Ser Gly Asp 145 150 155 160 Gly Gly Wing He Asp Wing Lys Ser Leu Thr Val Gln Gly He Ser Lys 165 170 175 Leu Cys Val Phe Gln Glu Asn Thr Wing Gln Wing Asp Gly Gly Wing Cys 180 '185 190 Gln Val Val Thr Ser Phe Ser Ala Ket Ala Asn Glu Ala Pro He Wing 195 200 205 Phe Val Wing Asn Val Wing Gly Val Arg Gly Gly Gly He Ala Wing Val 210 215 220 Gln Asp Gly Gln Gln Gly Val Being Ser Thr Being Thr Glu Asp Pro 225 230 235 240 23 Val Val Ser Phe Ser Arg Asn Thr Wing Val Glu Phe Asp Gly Asn Val 245 250 255 Wing Arg Val Gly Gly Gly He Tyr Ser Tyr Gly Asn Val Wing Phe Leu 260 265 270 Asn Asn Gly Lys Thr Leu Phe Leu Asn Asn Val Ala Ser Pro Val Tyr 275 280 285 He Wing Wing Lys Gln Pro Thr Ser Gly Gln Wing Being Asn Thr Ser Asn 290 295 300 Asn Tyr Gly Asp Gly Gly Wing He Phe Cys Lys Asn Gly Wing Gln Ala 305 310 315 320 0 Gly Ser Asn Asn Ser Gly Ser Val Ser Phe Asp Gly Glu Gly Val Val 325 330 335 Phe Phe Ser Ser Asn Val Ala Wing Gly Lys Gly Gly Wing He Tyr Wing 340 345 350 ? -ys Lys Leu Ser Val Wing Asn Cys Gly Pro Val Gln Phe Leu Arg Asn 355 360 365 5 He Wing Asn Asp Gly Gly Wing He Tyr Leu Gly Glu Ser Gly Glu Leu 370 375 380 Ser Leu Ser Wing Asp Tyr Gly Asp He He Phe Aso Gly Asn Leu Lys 385 390 395 400 Arg Thr Ala Lys Glu Asn Ala Ala Asp Val Asn Gly Val Thr Val Ser 405 410 415 Q "Ser Gla Wing He Met Met Gly Ser Gly Gly Lys He Thr Thr Leu Arg 420 425 430 Wing Lys Wing Gly His Gln He Leu Phe Asn Asp Pro He Glu Met Wing 435 440 445 Asn Gly Asn Asn Gln Pro Wing Gln Being Lys Leu Leu Lys He Asn 450 455 460 5 24 Asp Gly Giu Gly Tyr Thr Gly Asp He Val Phe Wing Asn Gly Ser Ser 465 < 70. 475 480 Thr Leu Tyr Gln Asn Val Thr He Glu Gln Gly Arg He Val Leu Arg 485 490 495 Glu Lys Wing Lys Leu Ser Val Asp Ser 500 505 < 210 > 18 < 211 > 57 < 212 > DNA < 213 > Chlamydia < 400 > 18 aagggcccaa ttacgcagag ctcgagagaa attatggttc ctcaaggaat ttacgat 57 < 210 > 19 < 211 > 20 < 212 > DNA < 213 > Chlamydia < 400 > 19 cgctctagaa ctagtgg = tc 20 < 210 > 20 < 211 > 22 < 212 > DNA < 213 > Chlamydia < 400 > 20 25 atggttcctc aaggaa tta cg 22 < 210 > 21 < 211 > 19 < 212 > DNA < 213 > Chlamydia < 400 > 21 ggtcccccat cagccggsg 19 < 210 > 22 < 211 > 1515 < 212 > DNA < 213 > Chlamydia < 400 > 22 gaaatcatgg ttcctcaagg aatttacgat ggggagacgt taactgtatc atttccctat 60 actgttatag gagatccrag tgggactact gttttttctg caggagagtt aacattaaaa 120 aatcttgaca attctattgc agctttgcct ttaagttgtt ttgggaactt attagggagt 180 tttactgttt taggga agg acactcgttg actttcgaga acatacggac ttctacaaat 240 ggggcagctc taagtaatag cgctgctgat ggactgttta ctattgaggg ttttaaagaa 300 ttatcctttt ccaattgcaa ttcattactt gccgtactgc ctgctgcaac gactaataag 360 ggtagccaga ctccgac ac aacatctaca ccgtctaatg gtactattta ttctaaaaca 420 gatcttttgt tactcaataa tgagaagttc tcattctata gtaatttagt ctctggagat 480 tagatgctaa gggggagcta gagcttaacg gttcaaggaa ttagcaagct ttgtgtcttc 540 caagaaaata ctgctcaagc tgatggggga gcttgtcaag tagtcaccag tttctctgct 600 atggctaacg aggctcctat tgcctttgta gcgaatgttg caggagtaag agggggaggg 660 attgctgctg ttcaggatgg gcagcaggga gtgtcatcat ctacttcaac agaagatcca 720 gtagtaagtt tttccageaa tactgcggta gagtttgatg ggaacgtagc ccgagtagga 780 ggagggattt actcctacgg gaacgttgct ttcctgaata atggaaaaac cttgtttctc 840 aacaatgttg cttctc c gt ttacattgct gctaagcaac caaeaagtgg acaggcttct 900 aatacgagta ataattacgg agatggagga gctatcttct gtaagaatgg tgcgcaagca 960 ggatccaata actctggstc agtttccttt gatggagagg gagtagtttt ctttagtagc 1020 aatgtagctg ctgggaaagg gggagctatt tatgccaaaa agctctcggt tgctaactgt 1080 ggccctgtac aatttttaag gaatatcgct aatgatggtg gagcgattta tttaggagaa 1140 tctggagagc tcagtttatc tgctgattat ggagatatta ttttcgatgg gaatcttaaa 1200 agaacagcca aagagaatgc tgccgatgtt aatggcgtaa ctgtgtcctc acaagccatt 1260 26 tcgatgggat cgggagggaa aataacgaca ttaagagcta aagcagggca tcagattctc 1320 tttaatgatc ccatcgagat ggcaaacgga aataaccagc cagcgcagtc ttccaaactt 1380 • ctaaaaatta acgatggtga aggatacaca ggggatattg tttttgctaa tggaagcagt 1440 actttgtacc aaaatgttac gatagagcaa ggaaggattg ttcttcgtga aaaggcaaaa 1500 ttatcagtga attct 1515 < 21Q > 23 < 211 > 3354 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: recombinant expression vector < 400 > 23 atgcaaacgt ctttccataa gttctttctt tcaatgattc tagcttattc ttgctgctct 60 ttaaatgggg gggcgtatgc acaaatcatg gttcctcaag gaatttacga tggggagacg 120 ttaactg at catttcccra tactg.tata ggagatccga gtgggactac tgttttttct 180 gcaggagagt taacgttaaa aaatcttgac aattctattg cagctttgcc tttaagttgt 240 tttgggaact tattagggag ttttactgtt ttagggagag gacactcgtt gactttcgag 300 acgga aaca cttctacaaa tggagctgca ctaagtgaca gcgctaatag cgggttattt 360 actattgagg gttttaaaca attatct tt tccaattgca acccattact tgccgtactg 420 cctgcrgcaa cgact Cataa tggcagccag actccgtcga caacatctac accgtctaat 480 ggtactattt to tctaaaac agatcttttg ttactcaata atgagaagtt ctcattctat 540 agtaattcag t tctgga? a tgggggagct atagatgcta agagcttaac ggttcaagga 600 atcagcasgc tttgtgtctt ccaagaaaat actgctcaag ctgatggggg agcttgtcaa 660 gtagtcacca gtttctctgc taeggctaac saggctccta ttgcctttgt agcgaatgtt 720 gcaggagtaa gagggggagg gattgctgct gttcaggatg ggcagcaggg agtgtca ca 780 tctacttcaa cagaagatcc agtagtaagt ttttccagaa atactgcggt agagtttgat 840 gggaacgtag cccgagtagg aggagggatt tactcctacg ggaacgttgc tttcctgaat 900 aatggaaaaa ccttgtttct caacaatgtt gcttctcctg tttacattgc tgctgagcaa 960 ccaacaaatg gacaggcttc taatacgagt gataattacg gagatggagg agctatcttc 1020 tgtaagaatg gtgcgcaagc agcaggatcc aataactctg gatcagtttc ctttgatgga 1080 gagggagtag ttttctttag tagcaatgta gctgctggga aagggggagc tatttatgcc 1140 cggttgctaa aaaaagctct ctgtggccct gtacaactct tagggaatat cgctaatgat 1200 ggtggagcga ttta tttagg agaatctgga gagctcagtt tatctgctga ttatggagat 1260 atgattttcg atgggaatct taaaagaaca atgctgccga gccaaagaga tgttaatggc 1320 gtaactgtgt cctcacaagc catttcgatg ggatcgggag ggaaaataac gacattaaga 1380 gctaaagcag ggcatcagat tctctttaat gatcccatcg agatggcaaa cggaaataac 1440 agtcttccga cagccagcgc acctctaaaa attaacgatg gtgaaggata cacaggggat 1500 27 attgtttttg ctaatggaaa cagtactttg taccaaaatg ttacgataga gcaaggaagg 1560 attgttcttc gtgaaaaggc aaaattatca gtgaattctc taagtcagac aggtgggagt 1620 ctgtatatgg aagctgggag tacattggat tttgtaactc cacaaccacc acaacagcct 1680 cctgccgcta atcagtcgat cacgctttcc aatctgcatt tgtctctttc ttctttgtta 1740 gcaaacaatg cagttacgaa tcctcctacc aatcctccag CGC * aagattc tcatcctgca 1800 gtcattggta gcacaactgc tggttctgtt acaattagtg ggcctatctt ttttgaggat 1860 cagcttatga ttggatgata taggtatgat tggctaggtt ctaatcaaaa aatcgatgtc 1920 ctgaaattac agttagggac tcagccccca gctaatgccc catcagattt gactctaggg 1980 aatgagatgc ctaagtatgg ctatcaagga agctggaagc tcctaataca ttgcgtggga 2040 gcaataatg gtcc ttatac tctgaaagct acatggacta aaactgggta taatcctggg 2100 cctgagcgag tagcttcttt ggttccaaat agtttatggg gatccatttt agatatacga 2160 cagcaattca tctgcgcatt agcaagtgtg gatgggcgct cttattgtcg aggattatgg 2220 gtttctggag tttcgaattt cttctatcat gaccgcgatg ctttaggtca gggatatcgg 2280 tatattagtg ggggttattc cttaggagca aactcctact ttggatcatc gatgtttggt 2340 ctagcattta ctgaagtatt tggtagatct aaagattatg tagtgtgtcg ttccaatcat 2400 catgcttgca taggatccgt ttatctatct accaaacagg ctttatgtgg atcttatgtg 2460 tttggagatg cgtttattcg tgctagctac atcagcatat gggtttggga gaaaacctca 2520 tatacatttg cagaggagag cgatgtttgt tgggataata actgtctggt tggagagatt 2580 ggagtgggat taccgattgt gattactcca tctaagctct atttgaatga gttgcgtcct aag 2640 ttcatg ctgagttttc ttatgccgat catgaatctt ttacaga ga aggcgatcaa 2700 gctcgggcat tcaggagtgg acatctcatg aatctatcag ttcctgttgg agtaaaa-.tt 2760 ga cgatgtt ctag ACACA ccctaataaa tatagcttta tgggggctta tatctgtgat 2820 ccatctctgg gcttatcgca gactcagaca acactcctat cccatcaaga gacatggaca 2880 acagatgcct ttcatttgg c aagacatgga gtcatagtta gagggtctat gtatgcttct 2940 ctaacaagca atatagaagt atatggccat ggaagatatg tacttctcga agtatcgaga 3000 ggttatggtt tgagtgcagg aag aaagtc cggttctaaa aatattggtt agatagttaa 3060 gtgttagcga tgcctttttc tttgagatct acatcatttt gttttttagc ttgtttgtgt 3120 tcctattcgt atggattcgc gagctctcct caagtgttaa cacctaatgt aaccactcct 3180 acgatgttta tttaaggggg cttgaatgga gactgcgctt ttgtcaatgt ctatgcaggg 3240 gcagagaacg gctcaattat ctcagctaat ggcgacaatt taacgattac cggacaaaac 3300 catacattat catttacaca ttctcaaggg ccagttcttc aaaattagcc ttca 3354 < 210 > 24 < 211 > 3324 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: recombinant expression vector < 400 > 24 28 atgcaaacgt ctttccataa gttctttctt tcaatgattc tagcttattc ttgctgctct 60 ttaagtgggg gggggtatgc agcagaaatc atgattcctc aaggaattta- cgatggggag 120 acgttaactg tatcatttcc ctatactgtt ataggagatc cgagtgggac tactgttttt 180 tctgcaggag agttaacgtt aaaaaatctt gacaattcta ttgcagcttt gcctttaagt 240 tgttttggga acttattagg gagttttact gttttaggga gaggacactc gttgactttc 300 oagaacatac ggacttctac aaatggagct gcactaagtg acagcgctaa tagcgggtta 360 tttactattg agggttttaa agaattatct ttttccaatt gcaactcatt acttgccgta 420 ctgcctgctg caacgactaa taatggtagc cagactccga cgacaacatc tacaccgtct 480 aatggtacta tttattctaa_ ttgttactca aacagatctt ataatgagaa gttctcattc 540 tatagtaatt tagtctctgg agatggggga actatagatg ctaagagctt aacggttcaa 600 ggaattagca agctttgtgt cttccaagaa aatactgctc aagctgatgg gggagcttgt 660 caagtagtca ccagtttctc tgctatggct aacgaggctc ctattgcctt tatagcgaat 720 gttgcaggag taagaggggg agggattgct gctgttcagg atgggcagca gggagtgtca '780 tcatctactt caacagaaga agtttttcca tccagtagta gaaatactgc ggtagagttt 840 gatgggaacg tagcccgagt aggaggaggg atttactcct acgggaacgt tgctttcctg 900 aataatggaa aaaccttgtt tctcaacaat gttgcttctc ctgtttacat tgctgctgag 960 caaccaacaa atggacaggc ttctaatacg agtgataatt acggagatgg aggagctatc 1020 atggtgcgca ttctgtaaga agcagcagga tccaataact ctggatcagt ttcctttgat 1080 ggagagggag tagttttctt tagtagcaat gtagctgctg ggaaaggggg agctatttat 1140 gccaaaaagc tctcggttgc taactgtggc cctgtacaat tcttagggaa tatcgctaat 1200 gatggtggag cgatttattt aggagaatct ggagagctca gtttatctgc tgattatgga 1260 tcgatgggaa gatattattt tcttaaaaga acagccaaag agaatgctgc cgatgttaat 1320 ggcgtaactg tgtcctcaca agccatttcg atgggatcgg gagggaaaat aacgacatta 1380 cagggcatca agagctaaag gattctcttt aatgatccca tcgagatggc aaacggaaat 1440 aaccagccag cgcagtcttc cgaacctcta aaaattaacg atggtgaagg atacacaggg 1500 gatatt ttt ttcctaargg aaacagtac tgtaccaaa atgttacgat ag ccaagga 1560 aggattgttc ttcgtgaaaa ggcaaaatta tcagtgaatt ctctaagtca gacaggtggg 1620 agtctgtata tggaagctgg cagtacattg gattttgtaa ctccacaacc accacaacag 1680 cctcctgccg ctaatcagt t gatcacgctt tccaatctgc atttgtctct ttcttctttg 1740 ttagcaaaca atgcagttac gaatcctcct accaatcctc cagcgcaaga ttctcatcct 1800 gcagtcattg gtagcacaac tgctggtcct gtcacaatta gtgggccttt cttttttgag 1860 gatttggatg atacagctta tsataggtat gattggctag gttctaatca aaaaatcgat 1920 gtcctgaaat tacagttagg gactcagccc tcagctaatg tttgactcta ccccatcaga 1980 tgcctaagta Sggaatgaga tggctatcaa ggaagctgga agcttgcgtg ggatcctaat 2040 atggtcctta acagcaaata tactctgaaa gctacatgga ctaaaactgg gtataatcct 2100 gggcctgagc gagtagc tc tttggttcca aatagtttat ggggatccat tttagatata 2160 cgatctgcgc attcagcaat tcaagcaagt gtggatgggc gctcttattg tcgaggatta 2220 H 9a9t tC-aa 'tetcctat catgaccgcg atgctttagg tcagggatat 2280 ata_.a cgg gtgggggt to ttccttagga gcaaactcct actttggatc atcgatgttt 2340 ggtctagcat ttaccgaagt atttggtaga tctaaagatt atgtagtgtg tcgttccaat 2400 catcatgCtt gCatagga: c cgttt.tct, tctaccaaac aagctttatg tggatcctat 2460 ttgttcggag atgcgtt at ccgtgctagc tacgggtttg ggaaccagca tatgaaaacc 2520 tcatacacat ttgcagacga cagcgatgtt cg ttgggata ataactgtct ggttggagag 2580 attggagtgg gattacccat tgtgactact ccatctaagc tctatttgaa tgagttgcgt 2640 29 cctttcgtgc aagctgagtt ttcttatgcc gatcatgaat cttttacaga ggaaggcgat 2700 caagctcggg cattcaggag tggtcatctc atgaatctat cagttcctgt tggagtaaaa 2760 tttgatcgat gttctagtac acaccctaat aaatatagct ttatgggggc ttatatctgt 2820 gatgcttatc gcaccatctc tgggactcag acaacactcc tatcccatca agagacatgg 2880 acaacagatg cctttcattt ggcaagacat ggagtcatag ttagagggtc tatgtatgct 2940 ctaacaa tc gcaatataga agtatatggc catggaagat atgagtatcg agatacttct 3000 cgaggttatg gtttgagtgc aggaagtaaa gtccggttct aaaaatattg gttagatagt 3060 taagtgttag cgatgccttt ttctttgaga tctacatcat tttgtttttt agcttgtttg 3120 tgttcctatt cgtatggatt cgcgagctct cctcaagtgt taacacctaa tgtaaccact 3180 ccttttaagg gggacgatgt ttacttgaat ggagactgcg tgtctatgca ctttagtcaa 3240 ggggcagaga acggctcaat tatctcagct aatggcgaca atttaacgat taccggacaa 3300 aaccatgcat tatcatttac agat 3324 < 210 > 25 < 211 > 65 < 212 > PRT < 213 > Chlamydia < 400 > 25 Pro Tyr Thr Val He Gly Asp Pro Ser Gly Thr Thr Val Phe Ser Ala 1 5 10 15 Gly Glu Leu Thr Leu Lys Asn Leu Asp Asn Be He Wing Wing Pro Leu 20 25 30 Ser Cys Phe Gly Asn Leu Leu Gly Ser Phe Thr Val Leu Gly Arg Gly 35 40 45 His Ser Leu Thr Phe Glu Asn He Arg Thr Ser Thr Asn Gly Ala Wing 50 55 60 Leu 65 < 210 > 26 < 211 > 24 30 < 212 > PRT < 213 > Chlamydia < 400 > 26 Ala Ala Asn Gln Leu He Thr Leu Ser Asa Leu His Leu Ser Leu Ser 1 5 10 15 Ser Leu Leu Ala Asn Asn Ala Val 20 < 210 > 27 < 211 > 8 < 212 > PRT < 213 > Chlamydia < 400 > 27 Gly Tyr Thr Gly Asp He Val Phe 1 5 < 210 > 28 < 211 > 7 < 212 > PRT < 213 > Chlamydia < 400 > 28 Tyr Gly Asp He He Phe Asp 1 * 5 < 210 > 29 < 211 > 63- < 212 > PRT 31 < 213 > Chlamydera cerviniventris _ < 400 > 29 Gly Tyr Ala Ala Glu He Met Val Pro Gln Gly He Tyr Asp Gly Glu 1 5 10 15 Thr Leu Thr Va l Ser Phe Pro Tyr Thr Val He Gly Asp Pro Ser Gly 20 25 30 Thr Thr Val Phe Ser Wing Gly Glu Leu Thr Leu Lys Asn Leu Asp Asn 35 40 45 Be He Ala Ala Leu Pro Leu Ser Cys Phe Gly Asn Leu Leu Gly 50 55 60 < 210 > 30 < 211 > 22 < 212 > PRT < 213 > Chlamydia < 400 > 30 Met Wing Asn Gly Asn Asn Gln Pro Wing Gln Ser Ser Lys Leu Leu Lys 1 5? O 15 He Asn Asp Gly Glu Gly 20 < 210 > 31 < twenty-one? > 14 < 212 > PRT < 213 > Chlamydia < 400 > 31 32 Wing Handle Gly Be Ser Thr Leu Tyr Gln Asn Val Thr He Glu 1 5 10 < 210 > 32 < 211 > 10 c < 212 > PRT < 213 > Chlamydia < 400 > 32 Lys Leu Ser Val Asn Ser Leu Ser Gln Thr 1 5 10 < 210 > 33 < 211 > 45 < 212 > PRT < 213 > Chlamydia < 400 > 33 Val He Gly Ser Thr Thr Wing Gly Ser Val Thr He Ser Gly Pro He 1 5 10 15 Phe Phe Glu Asp Leu As? Asp Thr Ala Tyr Asp Arg Tyr As? Trp Leu 20 25 30 Gly Ser As :. Glr. Lys He Asn Val Leu Lys Leu Gln Leu 35 40 45 < 210 > 34 < 211 > 64 < 212 > PRT < 213 > Chlamydia < 400 > 34 33 Val He Gly Ser T .-. R Thr Wing Gly Ser Val Thr He Ser Gly Pro He 1 5 10 15 Phe Phe Glu Asp Leu Asp Asp Thr Ala Tyr Asp Arg Tyr Asp Trp Leu 20 25 30 Gly Ser Asn Gln Lys He Asa Val Leu Lys Leu Gla Leu Gly Thr Lys 35 40 45 Pro Pro Wing Asn Wing Pro Being Asp Leu Thr Leu Gly Asr. Glu Met Pro 50 55 60 < 210 > 35 < 211 > 10 < 212 > PRT < 213 > Chlamydia < 400 > 35 Asp Pro Asn Thr Ala Asa Asn Gly Pro Tyr 10 < 210 > 36 < 211 > 458 < 212 > PRT < 213 > Chlamydia < 400 > 36 Gly Gly Wing Cys Gln Val Val Thr Ser Phe Ser Wing Met Wing Asn Glu 1 5 10 15 Wing Pro He Wing Phe Val Wing Asn Val Wing Gly Val Arg Gly Gly Gly 20 25 * 30 He Wing Wing Val Gla Asp Gly Gln Gln Gly Val Ser Ser Ser Thr Ser 35 40 45 34 Thr Glu Asp Pro Val Val Ser Phe Ser Arg Asn Thr Wing Val Glu Phe 50 55 60 Asp Gly Asa Val Wing Arg Val Gly Gly Gly He Tyr Ser Tyr Gly Asn 65 70 75 80 Val Ala Ph »Leu Asn Asn Gly Lys Thr Leu Phe Leu Asn Asn Val Wing 85 90 95 'Ser Pro Val Tyr He Wing Wing Lys Gln Pro Thr Ser Gly Gln Wing Ser 100 105 110 Asn Thr Ser Asn Asn Tyr Gly Asp Gly Gly Wing He Phe Cys Lys Asn 115 120 125 Gly Wing Gn Wing Gly Being Asn Asn Being Gly Ser Val Being Phe Asp Gly 130 135 140 Glu Gly Val Val Phe Phe Ser Ser Asn Val Ala Wing Gly Lys Gly Gly 145 150 155 160 Ala He Tyr Ala Lys Lys Leu Ser Val Ala Asn Cys Gly Pro Val Gln 165 170 175 Phe Leu Arg Asn He Wing Asn Asp Gly Gly Wing He Tyr Leu Gly Glu 180 185 190 Ser Gly Glu Leu Ser Leu Ser Wing Asp Tyr Gly Asp He He Phe A = p 1S5 200 205 Gly Asn Leu Lys Arg Thr Wing Lys Glu Asn Ala Ala Asp Val Asn Gly 210 215 220 Val Thr Val Ser Ser Gln Ala He Ser Met Gly Ser Gly Gly Lys He 225 '230 235 240 Thr Thr Leu Arg Wing Lys Wing Gly His Gln He Leu Phe Asn Asp Pro 245 250 255 He Glu Ket Wing Asn Gly Asn Asn Gln Pro Wing Gln Ser Ser Lys Leu 260 265 270 35 Leu Lys He Asa Asp Gly Glu Gly Tyr Thr Gly Asp He Val Phe Wing 275 280 285 Asn Gly Be Ser Thr Leu Tyr Gln Asn Val Thr He Glu Gln Gly Arg 290 295 300 He Val Leu Arg Glu Lys Ala Ly = Leu Ser Val Asn Ser Leu Ser Gln 305 310 315 320 Thr Gly Gly Ser Leu Tyr Met Glu Wing Gly Ser Thr Trp Asp Phe Val 325 '330 335 Thr Pro Glr. Pro Pro Gln Gln Pro Pro Ala Wing Asn Gln Leu He Thr 340 345 350 Leu Ser Asa Leu His Leu Ser Leu Ser Ser Leu Leu Ala Asa Asa Ala 355 360 365 Val Thr Asn Pro Pro Thr Asa Pro Pro Wing Gln Asp Ser His Pro Wing 370 375 380 Val He Gly Ser Thr Thr Wing Gly Ser Val Thr He Ser Gly Pro He 385 - 390 395 400 Phe Phe Gl Asp Leu Asp Asp Thr Wing Tyr Asp Arg Tyr Asp Trp Leu 405 410 415 Gly Ser Asa Gln Lys He Asn Val Leu Lys Leu Gln Leu Gly Thr Lys 420 425 430 Pro Pro Wing Asn Wing Pro As Asp Leu Thr Leu Gly Asn Glu Met Pro 435"440 445 Lys Tyr Gly Tyr Gln Gly Ser Trp Lys Leu 450 455 < 210 > 37 < 211 > 325 < 212 > PRT 36 < 213 > Chlamydia < 400 > 37 Leu Lys Ala Thr Tr? Thr Lys Thr Gly Tyr Asa Pro Gly Pro Glu Arg 1 - - - 5 - - 10 15 Val Ala Ser Leu Val Pro Asn Ser Leu Trp Gly Ser He Leu Asp He 20 25 30 Arg Ser Ala His Ser Ala He Gln Ala Ser Val Asp Gly Arg Ser Tyr 35 40 45 Cys Arg Gly Leu Tr? Val Ser Gly Val Ser Asn Phe Phe Tyr Eis Asp 50 = 5 60 Arg Asp Ala Leu Gly Gln Gly Tyr Arg Tyr He Ser Gly Gly Tyr Ser 65 70. 75 80 Leu Gly Wing Asn Being Tyr Phe Gly Being Being Met Phe Giy Leu Wing Phe 85 90 95 Thr Glu Val Phe Gly Arg Ser Lys Asp Tyr Val Val Cys Arg Ser Asn 100 105 110 His His Wing Cys He Gly Ser Val Tyr Leu Ser Thr Gln Gln Wing Leu 115 120 125 Cys Gly Ser Tyr Leu Phe Gly Asp Wing Phe He Arg Wing Ser Tyr Gly 130 135 140 Phe Gly Asn Gln His Met Lys Thr Ser Tyr Thr Phe Wing Glu Glu Ser 145 150 155 160 Asp Val Arg Trp Asp Asn Asn Cys Leu Wing Gly Glu He Gly Wing Gly 165 170 175 Leu Pro He Val He Thr Pro Ser Lys Leu Tyr Leu Asn Glu Leu Arg 180 185 190 Pro Phe Val Gln Wing Glu Phe Ser Tyr Wing Asp His Glu Ser Phe Thr * 195 200 205- Glu Glu Gly Asp Gln Ala Arg Ala Phe Lys Ser Gly His Leu Leu Asn 210 215 220 37 Leu Ser Val Pro Val Gly Val Lys Phe Asp Arg Cys Ser Ser Thr His 225 230 235 240 Pro Asn Lys Tyr Ser Phe Met Wing Wing Tyr He Cys Asp Wing Tyr Arg 245 250 255 Thr He Ser Giy Thr Glu Thr Thr Leu Leu Ser His Gln Glu Thr Trp 260 265 270 Thr Tnr Asp Ala Phe His Leu Ala Arg His Gly Val Val Val Arg Gly 275 280 285 Ser Met Tyr Ala Ser Leu Thr Ser Asn He Glu Val Tyr Gly His Gly 290 295 300 Arg Tyr Glu Tyr Arg Asp Wing Ser Arg Gly Tyr Gly Leu Ser Wing Gly 305 310 315 320 Be Arg Val Arg Phe 32S < 210 > 38 < 211 > 41 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: initiator < 400 > 38 ttt: a atcagcacat gaaaacctca tatacatttg c t 41 < 210 > 39 < 211 > 41 < 212 > DNA < 213 > Artificial Sequence < 220 > 38 < 223 > Description of Artificial Sequence: initiator < 400 > 39 gcaaatgtat atgaggtttt catgtgctga ttcccaaacc c 41 < 210 > 40 < 211 > 55 < 212 > DNA < 213 > Artificial Sequence < 2 ¿0 > < 223 > Description of Artificial Sequence: initiator < 400 > 40 aagggcccaa ttacgcagac atatggaaac gtctttccat aagttctttc tttca 55 < 210 > 41 < 211 > 80 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: initiator < 400 > 41 aagggcccaa ttacgcagag tctagattat taatgatgat gatgatgatg gaaccggact 60 ctacttcctg cactcaaacc '80