The Expression,Purification And Application Of HBsAg

The Hepatitis B (HB) which caused by Hepatitis B virus (HBV) is the worldwide infectious disease. Hepatitis B surface antigen (HBsAg), which located in the HBV surface, can stimulate host-cells produce protective antibody-hepatitis B surface antibody (HBsAb). HBsAg is the major component of the vaccine. To judge whether the immune is successful or not, HBsAg is also the major component of the ELISA diagnostic agent. Large scale produce HBsAg is very important in diagnosis and protecting host-cells from HBV injure. The expression, primary purification and application of HBsAg are to be studied.The HBV S gene was amplified by PCR from the patient and cloned into the Pichia pastoris secreted expression vector pPICZa and cellular expression vector pPICZ by PCR, respectively. Recombinant plasmid pPICZaS and pPICZS were constructed respectively. After linearized by restriction enzyme SacI, the recombinants were transformed into GS115 competent cells by electroporation, respectively. Recombinants with insert were screened by different concentration of Zeocin. After induced with methanol, then analyzed by Western-blot, ELISA, the results indicated that HBsAg was expressed intracellular, but not secreted into medium although fused with a signal. The clone GS115-pPICZS that has high expression level was selected by increasing the concentration of Zeocin. After larger scale cultivated and further analyzed, the results indicated that the best harvest time point is induced after 96 hours, the expression level is 0.31g/L (40g pallet, wet weight). The comparative purified HBsAg was got through NI-NTA affinity chromatographer, which has finer immunity and can react with HBsAb. The clone is the good fungus for industrialized production. At the same time, in order to improve the expression level of HBsAg, the HBV Sgene was cloned into prokaryotic expression vector pRSETB. The recombinant plasmid pRSETBS was constructed. The recombinant was transformed into E.coil BL21(DE3)pLysS competent cells. After induced by different concentration of IPTG, the obvious HBsAg expression still can not be detected by Western-blot. Though host cell BL21(DE3)pLysS which suitable for toxic protein expression, HBsAg can not get high expression level because of its hydrophobic structure.