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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Expression of Hepatitis B Virus S Gene in Pichia pastoris and Application of the Product for Detection of Anti-HBs Antibody

  • Hu, Bo (Department of Laboratory Medicine, The Third Affiliated Hospital of Sun Yat-sen University) ;
  • Liang, Minjian (Department of Laboratory Medicine, The Third Affiliated Hospital of Sun Yat-sen University) ;
  • Hong, Guoqiang (Department of Laboratory Medicine, The Third Affiliated Hospital of Sun Yat-sen University) ;
  • Li, Zhaoxia (Department of Laboratory Medicine, The Third Affiliated Hospital of Sun Yat-sen University) ;
  • Zhu, Zhenyu (Department of Biochemistry, Sun Yat-sen University of medicine) ;
  • Li, Lin (Department of Laboratory Medicine, The Third Affiliated Hospital of Sun Yat-sen University)
  • Published : 2005.11.30
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Abstract

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying ahexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5% of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100% with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.

Keywords

References

  1. Berzofsky, J. A. (1985) Intrinsic and extrinsic factors in protein antigenic structure. Science 229, 932-940 https://doi.org/10.1126/science.2410982
  2. Boue, O., Farnos, O., Gonzalez, A., Fernandez, R., Acosta, J. A., Valdes, R., Gonzalez, L. J., Guanche, Y., Izquierdo, G., Suarez, M., Dominguez, I., Machado, H., Rodriguez, M. and Lleonart, R. (2004) Production and biochemical characterization of the recombinant Boophilus microplus Bm95 antigen from Pichia pastoris. Exp. Appl. Acarol. 32, 119-128 https://doi.org/10.1023/B:APPA.0000018199.87122.e4
  3. Bretthauer, R. K. and Castellino, F. J.(1999) Glycosylation of Pichia pastoris-derived proteins. Biotechnol. Appl. Biochem. 30, 193-200
  4. Budkowska, A., Briantais, M. J., Dubreuil, P., Capel, F., Grangeot- Keros, L. and Pillot, J. (1985) Detection of antibodies directed against the pre-S gene product of hepatitis B virus: relationship between anti-pre-S response and recovery. Ann. Inst. Pasteur immunol. 136, 57-65 https://doi.org/10.1016/S0769-2625(85)80075-1
  5. Chen, Y. H., Chang, T. C. and Chang, G. G. (2004) Functional expression, purification, and characterization of the extra stable human placental alkaline phosphatase in the Pichia pastoris system. Protein Expr. Purif. 36, 90-99 https://doi.org/10.1016/j.pep.2004.03.006
  6. Heermann, K. H., Goldmann, U., Schwartz, W., Seyffarth, T., Baumgarten, H. and Gerlich, W. H. (1984) Large surface proteins of hepatitis B virus containing the pre-S sequence. J. Virol. 52, 396-402
  7. Kalidas, C., Joshi, L. and Batt, C. (2001) Characterization of glycosylated variants of beta-lactoglobulin expressed in Pichia pastoris. Protein Eng. 14, 201-207 https://doi.org/10.1093/protein/14.3.201
  8. Kim, M. W., Rhee, S. K., Kim, J. Y., Shimma, Y., Chiba, Y., Jigami, Y. and Kang, H. A. (2004) Characterization of Nlinked oligosaccharides assembled on secretory recombinant glucose oxidase and cell wall mannoproteins from the methylotrophic yeast Hansenula polymorpha. Glycobiology 14, 243-251 https://doi.org/10.1093/glycob/cwh030
  9. Lenassi, Z. A., Trobec, S., Gaberc-Porekar, V. and Menart, V. (2004) High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles. J. Biotechnol. 109, 115-122 https://doi.org/10.1016/j.jbiotec.2003.11.013
  10. Lin, J., Fido, R., Shewry, P., Archer, D. B. and Alcocer, M. J. (2004) The expression and processing of two recombinant 2S albumins from soybean (Glycine max) in the yeast Pichia pastoris. Biochim. Biophys. Acta. 1698, 203-212 https://doi.org/10.1016/j.bbapap.2003.12.001
  11. Milich, D. R, Thornton, G. B, Neurath, A. R., Kent, S. B., Michel, M. L, Tiollais, P. and Chisari, F. V. (1985) Enhanced immunogenicity of the pre-S region of hepatitis B surface antigen gene in yeast. Science 228, 1195-1199 https://doi.org/10.1126/science.2408336
  12. Murasugi, A., Kido, I., Kumai, H. and Asami, Y. (2003) Efficient production of recombinant human pleiotrophin in yeast, Pichia pastoris. Biosci. Biotechnol. Biochem. 67, 2288-2290 https://doi.org/10.1271/bbb.67.2288
  13. Murthy, T. V. (2004) Expression of GST-fused kinase domain of human Csk homologous kinase from Pichia pastoris facilitates easy purification. Biotechnol. Lett. 26, 443-449 https://doi.org/10.1023/B:BILE.0000018265.25289.6d
  14. Neurath, A. R., Kent, S. B., Parker, K., Prince, A. M., Strick, N., Brotman, B. and Sproul, P. (1986) Antibodies to a synthetic peptide from the pre-S 120-145 region of the hepatitis B virus envelope are virus-neutralizing. Vaccine 4, 35-37 https://doi.org/10.1016/S0264-410X(86)80001-9
  15. Neurath, A. R., Kent, S. B., Strick, N. and Parker, K. (1986) Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus. Cell 46, 35-37 https://doi.org/10.1016/0092-8674(86)90663-X
  16. Ouyang, J., Wang, J., Deng, R., Long, Q. and Wang, X. (2003) High-level expression, purification, and characterization of porcine somatotropin in Pichia pastoris. Protein Expr. Purif. 32, 28-34 https://doi.org/10.1016/S1046-5928(03)00216-X
  17. Peterson, D. L. (1981) Isolation and characterization of the majior protein and glycoprotein of hepatitis B surface antigen. J. Biol. Chem. 256, 6975-6983
  18. Raz, R., Dagan, R., Gallil, A., Brill, G., Kassis, I. and Koren, R. (1996) Safety and immunogenicity of novel mammalian cellderived recombinant hepatitis B vaccine containing PreS1 and PreS2 antigens in children. Vaccine 14, 207-211 https://doi.org/10.1016/0264-410X(95)00185-4
  19. Shih, J. W. and Gerin, J. L. (1977) Proteins of hepatitis B surface antigen: Amino acid composition of the Major polypeptids. Virology 21, 1219-1222
  20. Shouval, D., Ilan, Y., Adler, R., Deepen, R., Panet, A., Even- Chen, Z., Gorecki, M. and Gerlich, W. H. (1994) Improved immunogenicity in mice of mammalian cell-derived recombinant hepatitis B vaccine containing PreS1 and PreS2 antigens as composed with conventional yeast-derived vaccine. Vaccine 12, 1453-1459 https://doi.org/10.1016/0264-410X(94)90155-4
  21. Stibbe, W. and Gerlich, W. H. (1983) Structural relationships between minor and major proteins of hepatitis B surface antigen. J. Virol. 46, 626-628
  22. Stocker, J. W., Malavasi, F. and Trucco, M. (1981) Enzyme immunoassay for monoclonal antibodies; Immunological Methods. Ivan, L. and Benvenato, P. (eds.), pp. 301-303, Academic Press, New York, USA
  23. Valenzuela, P., Medina, A., Rutter, W. J., Ammerer, G. and Hall, B. D. (1982) Synthesis and assembly of hepatitis B virus surface antigen particles in the yeast. Nature 298, 347-350 https://doi.org/10.1038/298347a0
  24. Vanlandschoot, P., Van Houtte, F., Hoek, F., Nieuwland, R. and Leroux-Roels, G. (2003) Saccharomyces cerevisiae-derived HBsAg preparations differ in their attachment to monocytes, immune-suppressive potential, and T-cell immunogenicity. J. Med. Virol. 70, 513-519 https://doi.org/10.1002/jmv.10425
  25. Zhu, M., Cheng, J. and Hua, Z. C. (2003) Expression of kringle 5 domain of human plasminogen in Pichia pastoris. Prep. Biochem. Biotechnol. 33, 269-281 https://doi.org/10.1081/PB-120025370
  26. Zuckerman, J. N. and Zuckerman, A. J. (2003) Mutations of the surface protein of hepatitis B virus. Antiviral. Res. 60, 75-78 https://doi.org/10.1016/j.antiviral.2003.08.013

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