嗜肺军团菌重组蛋白momp-Linker-pip的重组质粒构建及鉴定

Construction and identification of recombinant plasmid containing momp-Linker-pip gene of Legionella pneumophila

目的利用自带Linker的引物,重组嗜肺军团菌pip和momp基因,构建其重组质粒pET-MLP,以获得其原核细胞表达产物并进行鉴定。方法首先制备足量的pET-pip、pET-momp以及空质粒pET-32a(+);通过PCR扩增pip、momp基因,将其定向克隆至pET-32a(+)并进行纯化筛选和鉴定;最后利用含有Linker的引物,将重组质粒pET-pip和pET-momp基因进行连接,完成pET-MLP的构建、筛选和鉴定。结果利用PCR、限制性核酸酶切电泳进行结果鉴定,于NCBI中将重组质粒的核酸序列与LP1型军团菌的相应序列对比,结果表明其同源性达到98%。结论实验成功构建、表达了重组质粒pET-MLP,为下一步以重组蛋白MLP作为诊断抗原进行嗜肺军团菌血清学检测奠定了基础。

Objective The recombinant plasmids pET-MLP of Legionella pneumophila pip and momp genes were constructed using primers with self-linkers to obtain their prokaryotic expression products and to characterize them.Methods The pET-pip,pET-momp and empty plasmid pET-32a(+)were first prepared in sufficient amounts;the pip and momp genes were amplified by PCR,cloned into pET-32a(+)and purified for screening and identification;finally,the recombinant plasmids pET-pip and pET-momp genes were ligated using primers containing Linker.The construction of pET-MLP were completed,screened and identified.Results The results were identified by PCR and restriction nuclease electrophoresis,and the nucleic acid sequences of the recombinant plasmid were compared with the corresponding sequences of Legionella pneumophila type LP1 in NCBI,and the results showed that the homology reached 98%.Conclusion The recombinant plasmid pET-MLP was successfully constructed and expressed,which laid the foundation for the next step of serological detection of Legionella pneumophila with recombinant protein MLP as the diagnostic antigen.