幽门螺杆菌Lpp20真核表达重组体的构建及其表达鉴定

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。

Construction of Eucaryotic Expression Vector for Lpp20 from Helicobacter pylori and Its Expression in Hela Cells

Objective To construct the recombinant plasmid of eucaryotic expression containing Lpp20 gene from Helicobacter pylori(H.pylori),and transfect it into Hela cells to express the Lpp20 protein.Methods Lpp20 gene was amplified from the genomic DNA of H.pylori by polymerase chain reaction(PCR),and the gene was inserted into eucaryotic expression vector pcDNA3.1(+).After identification by sequencing and restrictive enzyme digestion,the recombinant plasmid was transfected into Hela cells by using liposome.The expressed protein in Hela cells was verified by immunocytochemistry and Western-blot.Results The target gene Lpp20 segment about 528bp was obtained.The DNA sequence of Lpp20 gene constructed in pcDNA3.1(+) vector was consistent with the nucleotide sequence that had been published in GenBank.The specific protein Lpp20 expressed in Hela cells by pcDNA3.1(+) /Lpp20 was detected with Western blot and immunocytochemistry.Conclusions Lpp20 gene can be expressed in eukaryotic system which lays the foundation of studying its biological activities and the development of the H.pylori DNA vaccine.