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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 prokaryotic expression plasmid of BcfD gene of Salmonella typhi and express BcfD protein. Methods The bcfD gene was amplified by PCR from the genome of Typhi Ty2 strain. The prokaryotic expression vector pET-30a-bcf was constructed by cloning and subcloning. The recombinant plasmid was transformed into E.coli BL21 (DE3) -PAGE and Western blot analysis of expressed proteins. The result was consistent with the expected size of bcfD gene. The sequence of bcfD gene was confirmed by sequencing. The sequence of bcfD gene was identical with that of bcfD gene. SDS-PAGE showed that the protein was mainly expressed in inclusion bodies with the molecular weight of 42 × 103. The product can be immunized with the serum of typhoid patients after being adsorbed by Escherichia coli. Conclusion The prokaryotic expression vector of bcfD gene was successfully constructed and expressed in E. coli.