【摘要】 目的:构建赖型钩端螺旋体LipL41基因的真核重组表达质粒、并对其表达进行检测。方法:以赖型钩端螺旋体017株基因组DNA为模板PCR扩增目的基因,克隆至原核表达质粒pGEX-4T-1上。测序分析后酶切,并连接至真核表达质粒pcDNA3上,构建真核重组表达质粒LipL41-pCDNA3。利用脂质体介导转染COS7细胞,提取细胞总RNA,RT-PCR检测其表达。结果:PCR扩增出1 011 bp大小的目的片段,序列分析显示它与Leptospira kirschneri的LipL41基因成熟肽序列同源性高达98%。酶切鉴定证实真核重组表达质粒LipL41-pCD-NA3构建成功,RT-PCR检测显示,LipL41-pCDNA3转染组在大约1kb处出现特异的扩增带,而空质粒组无此条带。结论:LipL41的真核重组表达质粒构建成功,并可在哺乳动物细胞中表达。更多还原

【Abstract】 AIM:To construct a eukaryotic expression vector expressing outer membrane lipoprotein LipL41 of Leptospira lai and express it in mammalian cell.METHODS: LipL41 gene was amplified by PCR from genome of Leptospira lai 017 strain,and was subcloned into vector pGEX-4T-1.After sequencing,LipL41 gene digested by restriction endonuclease and cloned into vector pcDNA3.After confirming the correctness of the eukaryotic recombinant vector by restrication enzyme digestion,it was transfected into COS7 cells by liposome.Its expression was analyzed by RT-PCR.RESULTS: A fragment of 1 011 bp was amplified,and sequence analysis showed it had a 98% homology with Leptospira kirschneri.The analysis of restriction enzyme indicated that the eukaryotic recombinant vector was correctly constructed.A specific amplified fragment was showed in the cells transfected with recombinant plasmid by RT-PCR,but the cell transfected with blank plasmid did not show this band.CONCLUSIONS: The LipL41 gene of Leptospira lai was successfully inserted into eukaryotic expression plasmid and the recombinant plasmid expressed the LipL41 mRNA.更多还原