赖型钩端螺旋体LipL32-HlyX融合基因真核表达载体的构建及在COS7细胞中融合表达的研究

Construction of Eukaryotic Recombinant Vector and Expression in COS7 Cell of LipL32-HlyX Fusion Gene from Leptospira Serovar Lai

从赖型钩端螺旋体017株全基因组中通过聚合酶链反应(Polymerase chain reaction,PCR)分别扩增出脂蛋白32(LipL32)和溶血素-X(HlyX)基因,应用重叠延伸PCR技术(Gene splicing by overlap extension PCR,SOEPCR)获得LipL32-HlyX融合基因。以pcDNA3.1为载体,LipL32-HlyX为目的基因,双酶切构建重组质粒。将重组质粒转化大肠杆菌DH5α,经双酶切、PCR及测序鉴定证实重组质粒构建成功。脂质体转染法将构建成功的重组质粒转染COS7细胞,RT-PCR扩增出约2000bp的目的基因,Western blotting分析发现在75KD处出现特异性的阳性条带。结果证实赖型钩端螺旋体LipL32-HlyX融合基因真核表达载体能在哺乳动物细胞中表达,为钩体DNA疫苗的研究和开发奠定了基础。

This study was conducted to construct eukaryotic recombinant vector of LipL32- HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR （gene splicing by overlap extension PCR）. The fusion gene was then cloned into pcDNA3.1 by restrction nuclease digestion. Having been transformed into Ecoli DHSα, the recombiant plasmid was identified by restrction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then trans- feeted into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2 000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HIyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.