ABSTRACT
Hepatitis B virus (HBV) infection can cause the severe threat to the health of the people around the world. It depends upon the development of efficient diagnostic reagent and vaccine to prevent the prevalence of HB. In this study, we constructed the high expression recombinant Pichia pastoris and performed the screening tests in shake flasks to obtain the optimal values of several key fermentation parameters. Based on their effects on the growth and expression level of recombinant strains, FBS was the optimal industrial medium. The optimal values for the dissolve oxygen (DO), the final concentrations of methanol and the pH values were 50 mL, 1% (V/V) and 5.4-6.0, respectively. The optimal values of the parameters simulated in shake flasks were successfully scaled up to 10 L bioreactors to achieve high-throughput production: 310 OD600 in biomass and 27 mg/L in recombinant HBsAg. The expressed recombinant HBsAg in P. Pastoris was confirmed by SDS-PAGE and Western blotting. Electron microscopy examination showed that the purified protein could be self-assembled to 22 nm virus-like particles. The results provided a basis for industrial scale-up production of diagnostic reagent and vaccine of next generation against HB.
Subject(s)
Humans , Bioreactors , Fermentation , Hepatitis B Surface Antigens , Genetics , Pichia , Genetics , Metabolism , Recombinant Proteins , GeneticsABSTRACT
Objective To search for the gene encoding specific antigen of Ancylostoma caninum that induces host′s protective immunity. \ Methods\ A lambda ZAPⅡ cDNA library of A.canium was screened with sera from dogs \{immunized\} subcutaneously with hookworm larvae(L3). After sequencing, insert gene (AcAg) from positive clones was \{ligated\} into PUC18 and PET28C. Recombinant pET28C plasmid was induced to expressed protein in the E.coli BL21. The \{characteristic\} of recombinant protein is analyzed by SDS\|PAGE and Western bloting assay. \ Results \ Five positive \{clones\} were obtained, and proved to be the same. The insert gene (AcAg) in pET28C vector expressed a recombinant \{protein\} of about 43 kDa. Using Western bloting assay, this protein was recognized by the sera from dog immunized with \{Ancylostoma\} caninum third\|stage infected larvae and used for screening library. \ Conclusion \ The AcAg, which \{exhibits\} 35% homologous to Caenorhabditis elegans gene unc\|89, is a novel specific antigen of A.caninum. Its ability to elicit the \{protective\} immunity and the probability of the recombinant protein as a vaccine need to be further evaluated.