结核分支杆菌MTB41 DNA疫苗的构建及原核表达

Construction of the MTB41 DNA Vaccine of Mycobacterium tuberculosis and Its Cloning,Expression in Prokaryotic Expression Vector

为了构建结核分支杆菌MTB41DNA疫苗,同时将其克隆到原核表达载体中进行表达。用结核分支杆菌H37RV 株基因组DNA为模板,用PCR法对基因MTB41进行扩增,克隆到真核表达载体pJW4303 中,构建MTB41 重组真核质粒(DNA疫苗),进行了酶切鉴定和序列分析;克隆到原核表达载体pET22b 中,酶切和测序鉴定正确后转入E.coli BL21(DE3)PLysS 宿主菌株内中,诱导表达,进行SDS PAGE电泳分析。电泳发现转化了重组质粒的菌株有蛋白表达,所表达的蛋白质相对分子质量为41 ku,MTB41 DNA疫苗酶切鉴定和序列分析完全正确,确定含有正确的读码框,疫苗制备成功。DNA 疫苗的成功构建, 重组蛋白MTB41的成功表达,为结核病诊断、重组疫苗应用和免疫效应检测以及抗原、抗体的大规模制备打下了基础。

The purpose of this study is to construct the MTB41 DNA vaccine of Mycobacterium tuberculosis and clone it into prokaryotic expression vector to express. The gene encoding MTB41 was amplified from M.tuberculosis H37Rv chromosomal DNA by using PCR technique. PCR product was cloned seperately into the pJW4303 and pET22b, then transformed into E.coli DH5α strain, plasmid DNA was extracted and digested with enzymes.Plasmids containing the right insertion were sequenced to confirm their identity and retransformed the recombinant MTB41+pET22b into E.coli. BL21(DE3)PLysS strain. Bacterial lysates prepared from IPTG induced cultures were loaded directly onto SDS-PAGE. Upon IPTG induction, the recombinant MTB41+pET22b produced a protein with an apparent MW of 41 ku.In conclusion, we have successfully constructed MTB41 DNA vaccine and its protein has been expressed. It will establish the detecting of immunity effectiveness and preparation of the antigen and antibody of MTB41 protein on a large scale.