钩端螺旋体Lipl21基因真核质粒构建及在HeLa细胞的表达

目的构建钩端螺旋体外膜脂蛋白Lipl21基因片段的真核表达重组质粒,利用脂质体体外转染HeLa细胞,探讨其在体外真核细胞中的表达情况,为寻找新的预防钩端螺旋体病的候选疫苗分子提供实验依据. 方法应用PCR技术从钩端螺旋体黄疸出血群赖型56601株基因组模板中扩增Lipl21基因,纯化回收后克隆入pUCM-T载体,再亚克隆入真核表达载体pcDNA3.1（＋）,运用脂质体2000将重组体pcDNA3.1（＋）/LipL21转染入HeLa细胞,免疫组化法观察目的基因的表达. 结果双酶切及测序鉴定证明成功构建LipL21真核表达重组体pcDNA3.1（＋）/LipL21,DNA测序显示重组质粒含有561bp的目的基因片段,读码框架正确,无碱基错配及移码突变.重组质粒pcDNA3.1（＋）/LipL21在体外HeLa细胞中能有效表达目的蛋白LipL21. 结论成功构建了钩端螺旋体Lipl21基因真核表达质粒pcDNA3.1（＋）/Lipl21,且能够在体外真核细胞中表达,为进一步筛选新的预防钩端螺旋体病的候选疫苗分子奠定了一定的实验基础.

Construction of eukaryotic recombinant plasmid caontaining the Lip121 gene of leptospira and its expression in HeLa cells

Objective To construct the eukaryoctic expression recombinant plasmid containing the outer membrane Lipoprotein Lipl21 of the Leptospira interrogans serovar Lai, and transfect it into HeLa cells to express target protein Lipl21,to provide a new candidate antigen for preventing leptospirosis. Methods Lipl21 Gene was amplified from the genomic DNA of Leptospira interrogans serovar Lai ,strain Lai 56601 by polymerase chain reaction (PCR),and the gene was inserted into cloning vector pUCm-T. The inserted Lipl21 gene was subcloned into appropriate site of pcDNA3.1(+) vector.The positive clones were acquired after being identifid with restrictive enzymes and sequence analysis. After being sequenced with DNA auto-sequence analysis instrument, The amplified DNA sequence of Lipl21 was searched for alignment with NCBI Blast program, and the recombinant plasmid was transfected into HeLa cells using Liposome. Results The target gene Lipl21 segment about 561bp was obtained successfully. cells Immunocytochemistry analysis showed that the recombinant plasmid can be expressed in HeLa cells. Conclusion The recombinant plasmid of pcDNA3.1(+)/Lipl21 was constructed successfully,and can be expressed in HeLa cells,which provided the experimental basis for developing novel nucleic acid vaccine preventing leptospirosis.