赖型钩端螺旋体外膜蛋白真核表达载体构建及表达的初步研究  

Construction of eukaryotic expression vector and preliminary expression of outer envelope protein from Leptospiria serovar Lai

作　　者：晏菊芳[1] 鲍朗[1] 胡昌华[1] 李学敏[1] 张会东[1] 

机构地区：[1]华西医科大学感染免疫研究室,成都610041

出　　处：《中国人兽共患病杂志》2002年第4期23-27,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金 (批准号 :3 0 0 70 670 );四川省学术带头人基金资助

摘　　要：目的　测定所克隆的强毒力赖型钩端螺旋体OmpL1基因的序列 ,构建能够在哺乳动物细胞中表达赖型钩体OmpL1外膜蛋白的重组质粒。 方法　首先对自行构建的含有赖型钩端螺旋体OmpL1基因的重粗质粒 pAC -OmpL1进行插入片段的序列分析 ,然后将该质粒略作修改 ,使更适于在真核细胞中表达目的蛋白 ,同时将OmpL1基因亚克隆入另一个质粒pcDNA3中 ,采用限制性内切酶分析所得质粒的正确性。最后以脂质体转染法将两个重组质粒转入COS7细胞 ,以RT -PCR分析导入质粒的表达。结果　pAC -OmpL1中插入的赖型钩体OmpL1基因全长 96 0bp ,具有完整的阅读框架 ,与流感伤寒型钩体OmpL1基因同源性达 88%。限制性内切酶分析显示 ,质粒pAC -OmpL1的修改已达目的 ,OmpL1基因也按正向插入到pcDNA3中。RT -PCR结果显示 ,重组质粒转染组在约 1kb处出现特异的扩增带 ,而空白质粒载体组无此条带。 结论　赖型钩端螺旋体OmpL1基因与流感伤寒型的高度同源 ,该基因已成功地插入到两个真核表达质粒载体中 。Aim To determine the sequence of the cloned OmpL1 gene from Leptospria serovar Lai strain 017 and construct its mammalian cell expression plasmid Mehtods Firstly,the OmpL1 gene inserted in the recombinant plasmid pAC OmpL1 was sequenced Then the plasmid was modified for its better expression in eukaryotic cell and the OmpL1 gene was subcloned into another vector pcDNA3 After confirming the correctness of the modified plasmid and the new contructed recombinet plasmid by restrication enzyme analysis,they were transfected into COS7 cell instantly with liposome The expression of the two plasmids was analysed by RT PCR Results The OmpL1 gene of L serovar Lai inserted in the pAC OmpL1 is 960bp in length and has complete open reading frame The neucletide sequence share 88% similarity with L serovar alstoni The analysis of restrication enzyme indicated that the plasmid pAC OmpL1 has been modified as expected and the OmpL1 gene was also correctly inserted in the plasmid pcDNA3 The result of RT PCR showed that the cell transfected with recombinant plasmid got a specific amplified fragment with 1kb,but the cell transfected with blank plasmid vector had not this band Conclusion The OmpL1 gene of L serovar Lai has high homology with L serovar alstoni The gene has been successfully inserted into two eukaryotic expression pasmids respectively and these two recombinant plasmids can express the OmpL1 mRNA

关 键 词：钩端旋体 质粒 真核细胞 基因序列 基因表达 钩体病 

分 类 号：R377.5[医药卫生—病原生物学] R514.4[医药卫生—基础医学]