Modification And Secreted Expression Of Hepatitis B Virus PreS2/S Gene In Pichia Pastoris Expression System

At present, there are 3.5～4 hundred million chronic hepatitis B virus carriers in the world, among them ninety million people developed to liver diseases. About 1～2 million patients die of HBV infection and its following diseases every year. Our country is a highly endemic region of HBV infection, about 1.7 hundred million people were infected with HBV and 170 million people are suffering from chronic hepatitis. About 500 thousand people die of liver cancer or cirrhosiscaused by HBV every year.Inoculating HBV vaccine is an important mean for preventing hepatitis. The World Health Organization (WHO) recommended to inoculate infants in the country where hepatitis prevailed. At present, our country has integrated hepatitis B vaccination into immunization programs system.HBV DNA contains four open reading frames coding for S, P, C and X gene products respectively. Among them, the S gene product is currently deemed most antigenic. Therefore, the S gene is used for the production of HBV vaccine. Inadition to S protein, the hepatitis B viral envelope contains two less abundant surpace proteins with higher molecular weights: the middle protein (MP) and the large protein (LP). These larger polypeptides share the 226 amino acid (AA) residues of the S protein at the carboxy-terminus and posses additional redidues at the amino-terminus. The M protein contains a 55 AA extension of the S protein, named the preS2 region. The L protein contains an additional 108 or 109 residues (depending on the HBV subtype), called the preS1 region, amino-terminal to the preS2 region of the M protein. Several observations suggest that the addition of the preS regions to the S protein may result in a more immunogenic vaccine.However, there is a problem still unsolved, about 5～10% people vaccinated do not react or has low reaction. Recently, it was found that the preS gene product can be a candidate protein complement to such negative reaction responders. The preS2 polypeptide is of strong antigenicity and will help the body system to get immune to the HBV infection. Thus the preS2 gene will play an important role in the development of new HBV vaccines, which will be more effective.Pichia pastoris has been utilized widely as a heterologous gene expression system recently. There are several advantages such as high productivity, stable inherity, secretable product, and mature fermentation techniques. The ability of promoter, signal sequence and the selectivity of codon may be the factors which affect the level of expression. We rebulid preS2/S gene with preferred codons for P.pastori, and synthetize the new gene by the method of PCR extending, in order to improve expression of preS2/S protein in P. pastoris.This study is to construct a recombinant engineering yeast strain with molecular cloning technique.PCR primers were designed to amplify HBV wtpreS2/S and modpreS2/S gene from HBV DNA in vitro, following a routine sequencing assay to identify its nucleic acid sequence. Then these quence was analyzed and compared with the data from GeneBank, the result demonstrated that we have isolated the designated preS2/S clone. In order to obtain high level secreted expression of hepatitis B virus (HBV) preS2/S gene in Pichia Pastoris system, the HBV surface antigen (HBsAg) preS2/S gene was cloned into the secreted expression vector pPICZαB at first. Having chosen an expression vector-pPICZαB, we began to clone the preS2/S gene and modified preS2/S gene into its mutil-cloning site, and express the protein with a native N-terminus.After identification by digestion of restriction enzyme, the correct orientation of the recombinant expression vector was reconfirmed.The linear recombinant plasmid pPICZαB- preS2/S was then electroporoted into yeast Pichia Pastoris cells GS115. Multiinsertion transformants were screened with Zeocin resistance. After methanol induction, the expressed HBsAg M protein were analyzed by SDS-PAGE and ELISA. High level expression clones of wtpreS2/S and modpreS2/S were selected from about 208 positive colonies respectively. High level expression clones modpreS2/S 23 were selected from these positive colonies. Specific HBsAg protein expression could be detected with SDS-PAGE from 10×concent rated culture medium. The peak expression was on the 4th day after methanol induction. The molecular weight of the monomer was about 32kD.The stable-level recombinant was successfully expressed in P. pastoris.The recombinant plasmid pPICZαB-preS2/S could be induced to express the HBV MP by methanol in methanol-trophic yeast expression system.