球形幽门螺杆菌vacA基因表达质粒构建及表达

Construction and expression of prokaryotic expression plasmid of vacA gene of coccoid helicobacter pylori

目的构建球形幽门螺杆菌vacA基因的重组表达质粒,初步观察其在E.coli中的表达。方法采用亚克隆技术,用BamHⅠ和SacⅠ从重组质粒pMD-18T-vacA上切下vacA基因,插入表达载体pET32a(+)质粒,转化大肠埃希菌BL21,在氨苄青霉素阳性的LB平板上筛选阳性重组子,并经双酶切及PCR扩增鉴定。重组质粒pET32a(+)-vacA转化大肠埃希菌,(IPTG)诱导表达后进行(SDS-PAGE)电泳和凝胶扫描定量分析。结果重组质粒酶切和PCR鉴定与预期结果相符,成功构建携带vacA基因的重组原核表达质粒pET32a(+)-vacA。核酸序列测定及同源性分析证实,表达质粒pET32a(+)-cagA中所含vacA基因与GenBank中的vacA序列同源性达到99.2%。vacA基因在大肠埃希菌中诱导表达后获得约156 kD蛋白,蛋白含量占全菌体蛋白含量15.5%。结论成功构建球形H.pylori vacA基因重组表达质粒,并获得高效表达,为研究该基因蛋白的生物学特性及DNA疫苗研制奠定了基础。

Objective To construct pmkaryotic expression plasmid of vacA gene of coccoid Helicobacter pylori. Methods VacA gene was digested with restriction endonuclesses from recombinant plasmid pMD-18T-vacA and was inserted into expression vector pET32a（ ＋ ） by subclone technique, then the recombinants were transformed into E. coli BL21 and identified by restriction endonucleases digestion and PCR. Then the geneticaly engineered bacteria of including pET32a（ ＋ ）vacA plasmicls were induced by IPTG, the expression product was analyzed by SDS-PAGE and densitometric scanning. Results The positive recombinant plasmids pET32a（ ＋ ）vacA were identified by restriction endonucleases digestion and PCR, in accordance with the expected results. Sequence analysis showed that the vacA gene homology of coccoid. H. pylori and reported in GenBand was 99.2 %. Plasmid pET23a（ ＋ ） vacA could express a specific 156KD a protein in E. coli BL21, the protein accouterd for 15.5 % of total protein of recombinant bacterial. Conclusion The prokaryotic expression plasmids, which contain vacA gene of coccoid H. pylori are successfully constructed. Plasmid pET32a（ ＋ ） vacA can express specific protein in E. coli BL21. This work will help studying the biologic character of VacA protein and DNA vaccine against H. pylori infection.