Abstract

This study aimed at exploring the method of secretory expression of hepatitis B virus L gene and Mycobacterium tuberculosis Ag85B fusion gene in Pichia pastorisGS115, and preliminary tests of L-Ag85B fusion protein immunoreactivity. The hepatitis B Virus genome extracted from HBsAg positive serum and genome of M. tuberculosis H37Rv was used as template to amplify L and Ag85B gene. L-Ag85Bfusion gene was cloned into vector pPICZαC and the expression plasmid pPICZαC-L-Ag85B was constructed. After linearization, the plasmid was transformed into P. pastoris GS115 by electroporation. SDS-PAGE and Western blot were used to analyze the expression of protein. The purified fusion protein L-Ag85B was respectively assayed with serum containing anti-HBsAg or anti-Ag85B by ELISA. The recombinant vector was confirmed by sequencing and enzyme digestion. The results of SDS-PAGE and Western blot showed that the recombinant protein was induced by methanol and stably expressed in P. pastoris, while it has specific reaction with the serum containing anti-HbsAg or anti-Ag85B. However, the successful construction of a recombinant yeast expression vector containing gene coding L protein and Ag85B gene, and expressed in P. pastoris, lays a foundation for further researches on immunogenicity and immune protection by the new type of Hepatitis B- Tuberculosis combined vaccine.

Key words: Hepatitis B virus, mycobacterium tuberculosis, L gene, Ag85B gene, Pichia pastoris, combined vaccine.