问号状赖型钩端螺旋体LAg42膜蛋白基因的克隆及真核表达研究

目的:构建赖型钩端螺旋体lag42基因真核表达载体并转染哺乳动物细胞,为进一步研究奠定基础。方法:分别以问号状赖型钩体017株,56601株及双曲钩体PatocI株基因组为模板PCR扩增目的基因。构建lag42基因与质粒pcDNA3.1A 的重组真核表达质粒,克隆筛选并测序;通过脂质体介导将重组质粒转染入COS7细胞,用RT-PCR检测转染结果。结果:不同毒力赖型钩体均能扩增出约1100 bp的片段,而PatocI株则未能扩增出目的片段;PCR、双酶切及测序证实pcDNA3.1A -lag42构建成功;经RT-PCR检测证实重组质粒转染成功。结论:赖型钩体具有编码LAg42膜蛋白的基因,构建完成真核表达载体pcD-NA3.1A -lag42,并成功转染COS7细胞。

Cloning and eukaryotic expression of the LAg42 membrane protein gene of Leptospira interrogans serovar Lai

Objective:To construct a eukaryotic expression vector bearing lag42 gene from Leptospira(L.) Lai and transfect it intoCOS7 cells.Methods:The lag42 gene was amplified by PCR from genome of L.Lai 017 strain 56601strain and L.bilexa PatocI strain.The amplified DNA fragment was then cloned into vector pcDNA3.1A and DNA sequence was performed subsequently.Later,the recombinant plasmid pcDNA3.1A -lag42 was transfected intoCOS7 cells.RT-PCR was then used to check the transfection.Results:It was found that about 1100bp fragment was amplified in various virulent L.interrogans serovar Lai,but not in L.bilexa PatocI strain.There was a high homology of DNA sequence of the lag42 in various L.Lai.The recombinant plasmid was constructed triumphantly and successfully transfected into COS7 cells.Conclusion:These result suggested that lag42 gene is the conservative pathogenic mark in Leptospira,it may be associated with virulence and immunogenicity infection with pathogenic Leptospira.