引入CpG基序的嗜肺军团菌lvgA基因真核表达质粒的构建及其在NIH3T3细胞中的表达

Construction of the eukaryotic expression plasmid containing lvgA gene flanked with CpG motifs of Legionella pneumophila and its expression in NIH3T3 cells

目的构建了引入CpG基序的lvgA基因的真核表达质粒pclvgA/CpG,并在NIH3T3细胞中表达。方法采用PCR方法将特定CpG基序作为核酸佐剂构建于嗜肺军团菌lvgA基因侧翼,定向克隆入真核表达载体pcDNA3.1/myc-his(+),构建重组质粒pclvgA/CpG,体外阳离子脂质体转染法转染NIH3T3细胞。结果通过测序证实真核表达质粒pclvgA/CpG中lvgA/CpG克隆片段长为657bp,推测lvgA基因编码蛋白大小约为27.7Ku,lvgA基因及两侧翼原设计CpG基序完整扩出,测序序列与设计序列完全符合。用免疫荧光检测重组质粒在NIH3T3细胞中瞬时表达。结论本实验成功构建了嗜肺军团菌pclvgA/CpG真核表达质粒,并在NIH3T3细胞检测到其瞬时表达信号。

Objective To construct the eukaryotic expression plasmid containing lvgA gene flanked with CpG motifs of Legionella pneumophila for its expression in NIH3T3 cells. Methods lvgA gene flanked with CpG motifs of Legionella pneumophila was amplified by PCR. The PCR products was inserted into the eukaryotic expression plasmid pcDNA3. 1/myc-his （＋） to construct the recombinant plasmid pclvgA/CpG, which was subsequently transfected into NIH3T3 cells via lipofection. Immunofluorescence analysis was carried out to detect the transient expression of the plasmid in the cells. Results Sequence analysis showed that the recombinant plasmid pclvgA/CpG contained the lvgA/CpG fragment with a length of 657 bp, encoding a protein of 27.7 Ku. Immunofluorescence analysis identified the transient expression of the recombinant plasmid pclvgA/CpG in NIH3T3 cells. Conclusion The lvgA gene flanked with CpG motifs of Legionella pneumophila has been constructed successfully, and the transient expression of the recombinant plasmid pclvgA/CpG can be detected in NIH3T3 cells.