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目的:构建赖型钩端螺旋体外膜蛋白LipL32基因真核表达载体并在COS-7细胞中表达,为钩端螺旋体DNA疫苗的研究和开发奠定基础。方法:从赖型钩端螺旋体017株全基因组中PCR扩增出目的基因,双酶切构建重组质粒pcDNA3.1-LipL32。脂质体转染法将重组质粒转染COS-7细胞,通过RT-PCR、Westernblot检测目的基因的表达。结果:成功构建了LipL32基因的真核表达载体,并在COS-7细胞中获得瞬时和稳定表达。结论:赖型钩端螺旋体外膜蛋白LipL32基因真核表达载体能在哺乳动物细胞内表达,为钩端螺旋体DNA疫苗的应用提供了实验依据。
OBJECTIVE: To construct an eukaryotic expression vector for Leptospira leptospiral outer membrane protein LipL32 gene and express it in COS-7 cells, which lays the foundation for the research and development of Leptospira DNA vaccine. Methods: The target gene was amplified by PCR from the whole genome of Leptospira interrogans strain 017. The recombinant plasmid pcDNA3.1-LipL32 was constructed by double enzyme digestion. The recombinant plasmids were transfected into COS-7 cells by liposome transfection method, and the expression of the target gene was detected by RT-PCR and Western blot. Results: The eukaryotic expression vector of LipL32 gene was successfully constructed and transiently and stably expressed in COS-7 cells. CONCLUSION: The eukaryotic expression vector of Leptospira interrogans lipoprotein LipL32 gene can be expressed in mammalian cells, providing an experimental basis for the application of Leptospira DNA vaccine.