钩端螺旋体lipL21基因真核表达质粒的构建及其在HeLa细胞中的表达

Construction of the eukaryotic expression plasmid with the lipL21 gene of Leptospirae and its expression in HeLa cells

目的构建钩端螺旋体外膜脂蛋白LipL21基因片段的真核表达重组质粒,利用脂质体体外转染HeLa细胞,探讨其在体外真核细胞中的表达情况,为寻找新的预防钩端螺旋体病的候选疫苗分子提供实验依据。方法应用PCR技术从钩端螺旋体黄疸出血群赖型56601株基因组模板中扩增lipL21基因,纯化回收后克隆入pUCM-T载体,再亚克隆入真核表达载体pcD-NA3.1(+),运用脂质体2000将重组体pcDNA3.1(+)-lipL21转染入HeLa细胞,细胞免疫组化法观察目的基因的表达。结果双酶切及测序鉴定证明成功构建了lipL21真核表达重组体pcDNA3.1(+)-lipL21,DNA测序显示重组质粒含有561bp的目的基因片段,读码框架正确,无碱基错配及移码突变。重组质粒pcDNA3.1(+)-lipL21体外在HeLa细胞中能有效表达目的蛋白LipL21。结论成功构建了钩端螺旋体lipL21基因真核表达质粒pcDNA3.1(+)-lipL21,且能够在体外真核细胞中表达;为进一步筛选新的预防钩端螺旋体病的候选疫苗分子奠定了一定的实验基础。

In order to provide a new candidate antigen for the preparation of vaccine against leprospirosis, the eukaryotic expression plasmid with outer membrane lipopvotein gene lipL21 of Leptospira interrogans serovar. Lai was constructed and the recombinant plasmid was transfected into HeLa cells to express the target protein LipL21. The lipL21 gene was amplified from the genomie DNA of L. interrogans serovar. Lai ,strain Lai 56601 by PCR; the amplified gene was inserted into cloned vector pUCm-T, and the inserted gene was then subcloned to appropriate site of eukaryotic expression vector pcDNA3.1 （＋）. Aherwards, the recombinant plasmid peDNA3, 1（＋）-lipL21 was tranfected to HeLa cells by using the liposome 2000. As identified with double restriction enadonuelese digestion and sequence analysis, the eukaryotic expression recombinant pcDNA3.1 （＋）-lipL21 was successfully constructed. The DNA sequencing revealed that this recombinant plasmid contained the target gene fragment of 561 bp in size and the target protein LipL21 could be dfieiently expressed in HeLa ceils as demonstrated by the immunocytoehemical analysis. The results obtained in the present study can provide an experimental basis of the development of novel vaccine against leptospirosis.