The Prokaryotic Expression Of MutA And Analyze It's Antibacterial Activity

AIM: To obtain mutA gene ( coding the mature peptide mutacin I) segments of Streptococcus Mutan (MS) CH43, express it efficiently in E.coli DH5 a and analyze the induced protein's antibacterial activities to S.mutans 10449 8148 and S.sobrinus 176. METHODS: (l)mutA gene segments were amplified by PCR with specific primers from genome of MS CH43 strain. The purified PCR products were ligated into the sequencing vector pMD18-T .the white clone were selected and sequenced . then the correct sequenced gene was subcloned into corresponding restriction MCS of pProEX-HTb to generate expression vectors pProEX-HTb-mutA . Through the recombinant DNA technology , the expression vectors containing the aimed gene was transformed into E.coli.DH5a, and induced by IPTG .the mutA protein was expressed. (2)In order to get high express yield, we optimize the induce condition, such as the concentration of IPTG the induce time and the induce jucture. Then purify the induced protein via Ni-NTA purification system and identified it by western-blot (mouse anti-his monoclonal antibody) . (3)the induced mutA protein is inclusion body , so we unfolding it and renature mutA protein, after that detect its...