The Cloning, Expression, Purification And Identification Of Bacillus Anthracis Protective Antigen

Objective:Selecting the A16R strain of Bacillus Anthracis as the temple to clone the Pag gene expressed PA (protective antigen), and constructing the plasmid expression PA protein. This study will further promote the research of recombination-protein vaccine of Bacillus Anthracis.Methods:Pag gene was amplified by PCR. The primers were designed with the multi-clone sites in the expression plasmid Pet-32a and the Pag gene. The fragment was cloned or subcloned into the Pet-32a expression vector so called Pet-32a-PA and then was transfected into E.coli BL21(DE3). The protein was identified by ELISA and Western-blot.Results:DNA sequences analysis and double restriction enzyme digestion confirmed the expression plasmids successfully constructed. The protein was detected in E.coli BL21(DE3). The protein was expressd as the inclusion-body. After purifying and refolding the protein, we got the protein that could be detected and identified by ELISA and Western-blot.Conclusions:We successfully constructed Pet-32a-PA plasmids, which could produce PA as inclusion body. After purifying and refolding, we got the fuctional protein identified by ELISA and Western-blot which support the research of sub-unit vaccine of Bacillus Anthracis...