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. 1990 Feb;58(2):366–372. doi: 10.1128/iai.58.2.366-372.1990

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

L C Iacono-Connors 1, C S Schmaljohn 1, J M Dalrymple 1
PMCID: PMC258464  PMID: 2105271

Abstract

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BEALL F. A., TAYLOR M. J., THORNE C. B. Rapid lethal effect in rats of a third component found upon fractionating the toxin of Bacillus anthracis. J Bacteriol. 1962 Jun;83:1274–1280. doi: 10.1128/jb.83.6.1274-1280.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brachman P. S., Gold H., Plotkin S. A., Fekety F. R., Werrin M., Ingraham N. R. Field Evaluation of a Human Anthrax Vaccine. Am J Public Health Nations Health. 1962 Apr;52(4):632–645. doi: 10.2105/ajph.52.4.632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chakrabarti S., Brechling K., Moss B. Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques. Mol Cell Biol. 1985 Dec;5(12):3403–3409. doi: 10.1128/mcb.5.12.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Davies J. C. A major epidemic of anthrax in Zimbabwe. Cent Afr J Med. 1982;28(12):291–298. [PubMed] [Google Scholar]
  5. Ezzell J. W., Ivins B. E., Leppla S. H. Immunoelectrophoretic analysis, toxicity, and kinetics of in vitro production of the protective antigen and lethal factor components of Bacillus anthracis toxin. Infect Immun. 1984 Sep;45(3):761–767. doi: 10.1128/iai.45.3.761-767.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fish D. C., Mahlandt B. G., Dobbs J. P., Lincoln R. E. Purification and properties of in vitro-produced anthrax toxin components. J Bacteriol. 1968 Mar;95(3):907–918. doi: 10.1128/jb.95.3.907-918.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Friedlander A. M. Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J Biol Chem. 1986 Jun 5;261(16):7123–7126. [PubMed] [Google Scholar]
  8. Green B. D., Battisti L., Koehler T. M., Thorne C. B., Ivins B. E. Demonstration of a capsule plasmid in Bacillus anthracis. Infect Immun. 1985 Aug;49(2):291–297. doi: 10.1128/iai.49.2.291-297.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hambleton P., Carman J. A., Melling J. Anthrax: the disease in relation to vaccines. Vaccine. 1984 Jun;2(2):125–132. doi: 10.1016/0264-410x(84)90003-3. [DOI] [PubMed] [Google Scholar]
  10. Ivins B. E., Ezzell J. W., Jr, Jemski J., Hedlund K. W., Ristroph J. D., Leppla S. H. Immunization studies with attenuated strains of Bacillus anthracis. Infect Immun. 1986 May;52(2):454–458. doi: 10.1128/iai.52.2.454-458.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ivins B. E., Welkos S. L. Cloning and expression of the Bacillus anthracis protective antigen gene in Bacillus subtilis. Infect Immun. 1986 Nov;54(2):537–542. doi: 10.1128/iai.54.2.537-542.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ivins B. E., Welkos S. L. Recent advances in the development of an improved, human anthrax vaccine. Eur J Epidemiol. 1988 Mar;4(1):12–19. doi: 10.1007/BF00152686. [DOI] [PubMed] [Google Scholar]
  13. KEPPIE J., SMITH H., HARRIS-SMITH P. W. The chemical basis of the virulence of Bacillus anthracis. III. The role of the terminal bacteraemia in death of guinea-pigs from anthrax. Br J Exp Pathol. 1955 Jun;36(3):315–322. [PMC free article] [PubMed] [Google Scholar]
  14. Leppla S. H. Production and purification of anthrax toxin. Methods Enzymol. 1988;165:103–116. doi: 10.1016/s0076-6879(88)65019-1. [DOI] [PubMed] [Google Scholar]
  15. Little S. F., Knudson G. B. Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig. Infect Immun. 1986 May;52(2):509–512. doi: 10.1128/iai.52.2.509-512.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Little S. F., Leppla S. H., Cora E. Production and characterization of monoclonal antibodies to the protective antigen component of Bacillus anthracis toxin. Infect Immun. 1988 Jul;56(7):1807–1813. doi: 10.1128/iai.56.7.1807-1813.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Machuga E. J., Calton G. J., Burnett J. W. Purification of Bacillus anthracis lethal factor by immunosorbent chromatography. Toxicon. 1986;24(2):187–195. doi: 10.1016/0041-0101(86)90121-2. [DOI] [PubMed] [Google Scholar]
  18. Mackett M., Smith G. L., Moss B. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7415–7419. doi: 10.1073/pnas.79.23.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matsuura Y., Possee R. D., Overton H. A., Bishop D. H. Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. J Gen Virol. 1987 May;68(Pt 5):1233–1250. doi: 10.1099/0022-1317-68-5-1233. [DOI] [PubMed] [Google Scholar]
  20. Meynell G. G., Meynell E. The biosynthesis of poly d-glutamic acid, the capsular material of Bacillus anthracis. J Gen Microbiol. 1966 Apr;43(1):119–138. doi: 10.1099/00221287-43-1-119. [DOI] [PubMed] [Google Scholar]
  21. Mikesell P., Ivins B. E., Ristroph J. D., Dreier T. M. Evidence for plasmid-mediated toxin production in Bacillus anthracis. Infect Immun. 1983 Jan;39(1):371–376. doi: 10.1128/iai.39.1.371-376.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miyamoto C., Smith G. E., Farrell-Towt J., Chizzonite R., Summers M. D., Ju G. Production of human c-myc protein in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1985 Oct;5(10):2860–2865. doi: 10.1128/mcb.5.10.2860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moss B., Flexner C. Vaccinia virus expression vectors. Annu Rev Immunol. 1987;5:305–324. doi: 10.1146/annurev.iy.05.040187.001513. [DOI] [PubMed] [Google Scholar]
  24. PUZISS M., MANNING L. C., LYNCH J. W., BARCLAYE, ABELOW I., WRIGHT G. G. Large-scale production of protective antigen of Bacillus anthracis in anaerobic cultures. Appl Microbiol. 1963 Jul;11:330–334. doi: 10.1128/am.11.4.330-334.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Panicali D., Paoletti E. Construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4927–4931. doi: 10.1073/pnas.79.16.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. STANLEY J. L., SARGEANT K., SMITH H. Purification of factors I and II of the anthrax toxin produced in vivo. J Gen Microbiol. 1960 Feb;22:206–218. doi: 10.1099/00221287-22-1-206. [DOI] [PubMed] [Google Scholar]
  27. STANLEY J. L., SMITH H. Purification of factor I and recognition of a third factor of the anthrax toxin. J Gen Microbiol. 1961 Sep;26:49–63. doi: 10.1099/00221287-26-1-49. [DOI] [PubMed] [Google Scholar]
  28. Smith G. E., Ju G., Ericson B. L., Moschera J., Lahm H. W., Chizzonite R., Summers M. D. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8404–8408. doi: 10.1073/pnas.82.24.8404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Smith G. E., Summers M. D., Fraser M. J. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1983 Dec;3(12):2156–2165. doi: 10.1128/mcb.3.12.2156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smith H., Stoner H. B. Anthrax toxic complex. Fed Proc. 1967 Sep;26(5):1554–1557. [PubMed] [Google Scholar]
  31. THORNE C. B., GOMEZ C. G., HOUSEWRIGHT R. D. Synthesis of glutamic acid and glutamyl polypeptide by Bacillus anthracis. II. The effect of carbon dioxide on peptide production on solid media. J Bacteriol. 1952 Mar;63(3):363–368. doi: 10.1128/jb.63.3.363-368.1952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. THORNE C. B., MOLNAR D. M., STRANGE R. E. Production of toxin in vitro by Bacillus anthracis and its spearation into two components. J Bacteriol. 1960 Mar;79:450–455. doi: 10.1128/jb.79.3.450-455.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Turnbull P. C., Broster M. G., Carman J. A., Manchee R. J., Melling J. Development of antibodies to protective antigen and lethal factor components of anthrax toxin in humans and guinea pigs and their relevance to protective immunity. Infect Immun. 1986 May;52(2):356–363. doi: 10.1128/iai.52.2.356-363.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Uchida I., Sekizaki T., Hashimoto K., Terakado N. Association of the encapsulation of Bacillus anthracis with a 60 megadalton plasmid. J Gen Microbiol. 1985 Feb;131(2):363–367. doi: 10.1099/00221287-131-2-363. [DOI] [PubMed] [Google Scholar]
  35. Vodkin M. H., Leppla S. H. Cloning of the protective antigen gene of Bacillus anthracis. Cell. 1983 Sep;34(2):693–697. doi: 10.1016/0092-8674(83)90402-6. [DOI] [PubMed] [Google Scholar]
  36. Welkos S. L., Friedlander A. M. Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice. Microb Pathog. 1988 Aug;5(2):127–139. doi: 10.1016/0882-4010(88)90015-0. [DOI] [PubMed] [Google Scholar]
  37. Welkos S. L., Lowe J. R., Eden-McCutchan F., Vodkin M., Leppla S. H., Schmidt J. J. Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis. Gene. 1988 Sep 30;69(2):287–300. doi: 10.1016/0378-1119(88)90439-8. [DOI] [PubMed] [Google Scholar]
  38. Wood W. I., Gitschier J., Lasky L. A., Lawn R. M. Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1585–1588. doi: 10.1073/pnas.82.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. ZWARTOUW H. T., SMITH H. Polyglutamic acid from Bacillus anthracis grown in vivo; structure and aggressin activity. Biochem J. 1956 Jul;63(3):437–442. doi: 10.1042/bj0630437. [DOI] [PMC free article] [PubMed] [Google Scholar]

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