伤寒沙门菌bcfD基因的克隆表达

Cloning of bcfD gene of Salmonella enterica serovar typhi and its expression

目的构建伤寒沙门菌BcfD基因的原核表达质粒,表达BcfD蛋白。方法PCR法从伤寒沙门菌Ty2菌株基因组中扩增bcfD目的基因,经克隆和亚克隆构建原核表达载体pET-30a-bcf,将该重组质粒转化E.coliBL21(DE3)并进行原核表达,用SDS-PAGE和免疫印迹分析表达蛋白。结果扩增到与bcfD基因预期大小相符的片段,测序证明与bcfD基因序列完全一致,SDS-PAGE显示该蛋白主要以包涵体的形式表达,相对分子质量为42×103,免疫印迹分析表明,表达产物与经用大肠杆菌吸附处理后的伤寒患者血清能进行免疫反应。结论成功构建了bcfD基因原核表达载体并在大肠杆菌中表达了目的蛋白。

Objective To construct the prokaryotic expression plasmid of bcfD gene of Salmonella enterica （S. enterica） serovar typhi and to express BcfD protein. Methods The bcfD gene of S. enterica serovar typhi strain ty2 was amplified by PCR and subcloned into procaryotic expression vector pET-30a. The recombinant plasmid pET-30a-bcf was transformed into E. coli BL21 （DE3） and its procaryotic expression was induced by IPTG. The expressed protein was analyzed by SDS-PAGE and Western bloting. Results The DNA segment was amplified to the expected size of bcfD gene which was confirmed by bcfD gene sequencing. SDS-PAGE showed that the BcfD protein was expressed as inclusion body with a molecular weight of about 42 kDa. Western blot analysis showed immune reaction of the expressed product with the typhoid sera treated with absorbent E. coli BL21 （DE3）. Conclusion The prokaryotic expression vector of bcfD gene we constructed can express the target protein in E. coli BL21 （DE3）.