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Modification and Secreted Expression of Hepatitis B Virus PreS2/S Gene in Pichia Pastoris Expression System

Author: WangYuDi
Tutor: KongWei
School: Jilin University
Course: Biochemistry and Molecular Biology
Keywords: Wild-type preS2 / S ( wtpreS2 / s ) Modified preS2 / S ( modpreS2 / s ) Pichia pastoris GS115 Secretory expression
CLC: R392
Type: Master's thesis
Year: 2007
Downloads: 50
Quote: 0
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Abstract


Intends to get the hepatitis B virus surface antigen preS2 / S stability in in Pichia Pastoris yeast secretory expression . The Pichia pastoris first preference codon adr subtype preS2 / S gene modification . Then after the preS2 / S gene with the modification of the preS2 / S gene recombinant yeast secretory type expression vectors (pPICZαB) on , to form a recombinant plasmid , electrically transferred GS115 yeast cells , respectively, to obtain 208 recombinant sub , after PCR screening , shake flask fermentation screening and enzyme-linked immunosorbent assay (ELISA) testing, access to the integration of the positive clones stably expressing strains . Wild-type preS2 / S recombinant S antigen ELISA test results positive rate was 80.6% , modified preS2 / S recombinant S- antigen ELISA test results positive rate was 96.8% . Identification of positive clone strain modpreS2 / S after 23 fermentation induced expression , fermentation supernatant by SDS-PAGE electrophoresis , a specific protein bands , expression product of a monomer molecular weight of about 32,000 Da , and is higher on the fourth day expression levels . Proteins (middle protein, MP), the purified fermentation supernatant obtained the expected size of the hepatitis B virus surface antigen , S antigen ELISA test was strongly positive , the preS2 antigen weak positive . These results suggest that the preS2 / S gene stable secretion expression in Pichia expression system .

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