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. 1989 Feb;57(2):487–494. doi: 10.1128/iai.57.2.487-494.1989

Cloning and characterization of a Chlamydia trachomatis L3 DNA fragment that codes for an antigenic region of the major outer membrane protein and specifically hybridizes to the C- and C-related-complex serovars.

E J Carlson 1, E M Peterson 1, L M de la Maza 1
PMCID: PMC313122  PMID: 2492261

Abstract

Chlamydia trachomatis L3 DNA was cloned and expressed in lambda gt11. A recombinant plaque that expressed an antigen that reacted with rabbit polyclonal antichlamydial L3 serum and with two monoclonal antibodies specific for serovars L3 and I was selected from this Chlamydia genomic library. The beta-galactosidase Chlamydia fusion protein was purified by immunoaffinity chromatography and injected into mice to produce monoclonal antibodies. These monoclonal antibodies reacted by Western (immuno-) blot with both the fusion protein and the major outer membrane protein from purified L3 elementary bodies. The chlamydial DNA fragment was shown by DNA sequence analysis to be 168 base pairs in length and to correspond to the constant regions 1 and 2 and the variable segment 1 of the major outer membrane protein gene. The recombinant chlamydial DNA fragment hybridized under stringent conditions by Southern and dot blot analysis exclusively with the DNA from the C- and C-related-complex C. trachomatis serovars.

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Selected References

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  1. Allan I., Cunningham T. M., Lovett M. A. Molecular cloning of the major outer membrane protein of Chlamydia trachomatis. Infect Immun. 1984 Sep;45(3):637–641. doi: 10.1128/iai.45.3.637-641.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Caldwell H. D., Kromhout J., Schachter J. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis. Infect Immun. 1981 Mar;31(3):1161–1176. doi: 10.1128/iai.31.3.1161-1176.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Caldwell H. D., Schachter J. Antigenic analysis of the major outer membrane protein of Chlamydia spp. Infect Immun. 1982 Mar;35(3):1024–1031. doi: 10.1128/iai.35.3.1024-1031.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  5. Hackstadt T., Todd W. J., Caldwell H. D. Disulfide-mediated interactions of the chlamydial major outer membrane protein: role in the differentiation of chlamydiae? J Bacteriol. 1985 Jan;161(1):25–31. doi: 10.1128/jb.161.1.25-31.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  7. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  8. Locker H., Frenkel N. BamI, KpnI, and SalI restriction enzyme maps of the DNAs of herpes simplex virus strains Justin and F: occurrence of heterogeneities in defined regions of the viral DNA. J Virol. 1979 Nov;32(2):429–441. doi: 10.1128/jvi.32.2.429-441.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mole S. E., Lane D. P. Use of simian virus 40 large T-beta-galactosidase fusion proteins in an immunochemical analysis of simian virus 40 large T antigen. J Virol. 1985 Jun;54(3):703–710. doi: 10.1128/jvi.54.3.703-710.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Newhall W. J., 5th Biosynthesis and disulfide cross-linking of outer membrane components during the growth cycle of Chlamydia trachomatis. Infect Immun. 1987 Jan;55(1):162–168. doi: 10.1128/iai.55.1.162-168.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Novotný J., Handschumacher M., Haber E., Bruccoleri R. E., Carlson W. B., Fanning D. W., Smith J. A., Rose G. D. Antigenic determinants in proteins coincide with surface regions accessible to large probes (antibody domains). Proc Natl Acad Sci U S A. 1986 Jan;83(2):226–230. doi: 10.1073/pnas.83.2.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Peterson E. M., Zhong G. M., Carlson E., de la Maza L. M. Protective role of magnesium in the neutralization by antibodies of Chlamydia trachomatis infectivity. Infect Immun. 1988 Apr;56(4):885–891. doi: 10.1128/iai.56.4.885-891.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Peterson E. M., de la Maza L. M. Characterization of Chlamydia DNA by restriction endonuclease cleavage. Infect Immun. 1983 Aug;41(2):604–608. doi: 10.1128/iai.41.2.604-608.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Peterson E. M., de la Maza L. M. Restriction endonuclease analysis of DNA from Chlamydia trachomatis biovars. J Clin Microbiol. 1988 Apr;26(4):625–629. doi: 10.1128/jcm.26.4.625-629.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Richmond S. J., Caul E. O. Fluorescent antibody studies in chlamydial infections. J Clin Microbiol. 1975 Apr;1(4):345–352. doi: 10.1128/jcm.1.4.345-352.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ripa K. T., Mårdh P. A. Cultivation of Chlamydia trachomatis in cycloheximide-treated mccoy cells. J Clin Microbiol. 1977 Oct;6(4):328–331. doi: 10.1128/jcm.6.4.328-331.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  19. Stephens R. S., Kuo C. C., Newport G., Agabian N. Molecular cloning and expression of Chlamydia trachomatis major outer membrane protein antigens in Escherichia coli. Infect Immun. 1985 Mar;47(3):713–718. doi: 10.1128/iai.47.3.713-718.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Stephens R. S., Mullenbach G., Sanchez-Pescador R., Agabian N. Sequence analysis of the major outer membrane protein gene from Chlamydia trachomatis serovar L2. J Bacteriol. 1986 Dec;168(3):1277–1282. doi: 10.1128/jb.168.3.1277-1282.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stephens R. S., Sanchez-Pescador R., Wagar E. A., Inouye C., Urdea M. S. Diversity of Chlamydia trachomatis major outer membrane protein genes. J Bacteriol. 1987 Sep;169(9):3879–3885. doi: 10.1128/jb.169.9.3879-3885.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stephens R. S., Wagar E. A., Schoolnik G. K. High-resolution mapping of serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis. J Exp Med. 1988 Mar 1;167(3):817–831. doi: 10.1084/jem.167.3.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tanaka T., Slamon D. J., Cline M. J. Efficient generation of antibodies to oncoproteins by using synthetic peptide antigens. Proc Natl Acad Sci U S A. 1985 May;82(10):3400–3404. doi: 10.1073/pnas.82.10.3400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tsang V. C., Peralta J. M., Simons A. R. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol. 1983;92:377–391. doi: 10.1016/0076-6879(83)92032-3. [DOI] [PubMed] [Google Scholar]
  26. Wang S. P., Grayston J. T. Immunologic relationship between genital TRIC, lymphogranuloma venereum, and related organisms in a new microtiter indirect immunofluorescence test. Am J Ophthalmol. 1970 Sep;70(3):367–374. doi: 10.1016/0002-9394(70)90096-6. [DOI] [PubMed] [Google Scholar]
  27. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. de la Maza L. M., Peterson E. M. Genital infections. Med Clin North Am. 1983 Sep;67(5):1059–1073. doi: 10.1016/s0025-7125(16)31166-x. [DOI] [PubMed] [Google Scholar]

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