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目的构建含幽门螺杆菌(Hp)Lpp20基因的真核表达重组体,并鉴定其在Hela细胞中能否有效表达,为进一步开展Lpp20核酸疫苗与该蛋白的生物学功能的研究打下基础。方法抽提Hp标准菌株26695基因组DNA,应用聚合酶链式反应(PCR)技术从基因组DNA扩增Lpp20基因,经过一系列酶切、连接反应将其克隆入真核表达载体pcD-NA3.1(+),转入大肠杆菌,筛选阳性克隆,通过PCR和酶切反应鉴定;用脂质体法将构建好的重组载体pcDNA3.1(+)-Lpp20转染Hela细胞,通过免疫细胞化学法及Western印迹法检测其在Hela细胞中表达的Lpp20蛋白。结果成功扩增出长约528 bp的Lpp20基因,测序结果表明扩增出的Lpp20基因与Hp Lpp20序列一致,PCR和酶切鉴定结果证实Lpp20基因正确克隆入真核表达载体pcDNA3.1(+),并经免疫细胞化学法和Western印迹法检测到重组体可在Hela细胞中有效表达。结论成功构建了Lpp20基因的Hp真核表达重组体,并检测到其在Hela细胞中可表达出免疫反应性良好的蛋白,为进一步探索其免疫作用及生物学功能奠定了基础。
Objective To construct a recombinant eukaryotic expression vector containing Lp20 gene of Helicobacter pylori (Hp) and identify its expression in Hela cells, which lays the foundation for the further study of Lpp20 nucleic acid vaccine and its biological function. Methods The genomic DNA of Hp strain 26695 was extracted and the Lpp20 gene was amplified from genomic DNA by polymerase chain reaction (PCR). After a series of digestion and ligation reactions, the Lpp20 gene was cloned into the eukaryotic expression vector pcD-NA3.1 +) Into Escherichia coli. The positive clones were screened and identified by PCR and restriction enzyme digestion. The constructed recombinant plasmid pcDNA3.1 (+) - Lpp20 was transfected into Hela cells by lipofection method, Western blotting was used to detect the expression of Lpp20 protein in Hela cells. Results The Lpp20 gene of about 528 bp in length was successfully amplified. The sequencing results showed that the amplified Lpp20 gene was consistent with the Hp Lpp20 sequence. PCR and restriction enzyme analysis confirmed that the Lpp20 gene was cloned into the eukaryotic expression vector pcDNA3.1 (+). , And the recombinant was detected by immunocytochemistry and Western blotting in Hela cells. Conclusion The Hp eukaryotic expression recombinant of Lpp20 gene was successfully constructed and its immunoreactive protein could be expressed in Hela cells, which laid a foundation of further exploration of its immune function and biological function.