Prokaryotic Expression Of Mycobacterium Antigen 85B And The Preparation Of Polyclonal Antibody

1. ObjectiveIntravesical instillation of BCG is one of the most effective treatment and recurrence prevention approach of bladder cancer. However, as the BCG dose are large and Incertitude of curative effect, so this therapy is associated with some complications such as disseminated mycobacteriosis disease,which suggesting that this treatment must be improved. antigen 85B (Ag85B) is one gene of the mycobacterium secreted protein, which is primary antigen of mycobacterium tuberculosis. It provide a new way for the theament of bladder cancer. Our department is developing a new method for treating and preventing the recurrence of transitional bladder cancer with this antigen.The objective of our study is to prepared polyclonal anti-GST/Ag85B antibody, With the antibody, Ag85B expression can be monitored throughout in vitro and in vivo gene transfer studies, the polyclonal antibody can be used in further biological therapy with Ag85B gene.2. Methods and resultsIn the present study, first, To construct the recombinant prokaryotig plasmid DNA expression vector encoding mycobacterium tuberculosis antigen 85B. Fusion protein GST-Ag85B was expressed after IPTG induction and isolated and purified from the total proteins of E . coli BL21 transformed by the recombinant plasmid pGEX-Ag85B; Then, The polyclonal antibody was got by immunizing rabbits with this purified fusion protein, and the quality of the antibody was identified. The main results and conclusions are as follows:⑴ The gene encoding secreted form of Ag85B was amplified from the vector pUC18- Ag85B by restriction endonuclease digestion with EcoRⅠplus Sal, and was correctly inserted into sites cut with the same restriction endonucleases of eukaryotic expression vector pGEX-4T-1, which was confirmed by partial nucleotide sequencing and restriction endonuclease digestion. ⑵ By induction of IPTG, the E. coli BL21 harbouring the the constructed expressed recombinant proteins,which was inclusion bodies. The inclusion bodies were washed denatured and renatured. Sepharose High Performance ion exchange were used for the Purification. The purity of the fusion protein was detected with SDS-PAGE,which was confirmed to have a purity of more than 95%.⑶ Antiserum was obtained by programmed immunization of rabbits with the purified protein and purified through ammonium sulfate precipitation and ion exchange. The specificity of polyclonal anti-GST/Ag85B antibody was determined by ELISA. ⑷ Preliminary western blot result indicated that the polyclonal anti-GST/Ag85B antibody could recognize Ag85B/IL-II fusion protein which was prepared by our department,and showed high specificity.3. ConclusionGST/Ag85B fusion protein successfully expressed in E. coli and purified polyclonal prepared polyclonal antibody antibody with high specificity is prepared and applied successfully in preliminary investigation. the prepared polyclonal antibody provide the necessary material for further study.