CAJ | 학술논문

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,定向克隆人真核表达载体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/CpC中lvgA/CpC克隆片段长为657 bp,推测lvgA基因编码蛋白大小约为27.7 Ku,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.
목적 구건료인입CpG기서적lvgA기인적진핵표체질립pclvgA/CpG,병재NIH3T3세포중표체.방법 채용PCR방법 장특정CpG기서작위핵산좌제구건우기폐군단균lvgA기인측익,정향극륭인진핵표체재체pcDNA3.1/myc-his(+),구건중조질립pclvgA/CpG,체외양리자지질체전염법전염NIH3T3세포.결과 통과측서증실진핵표체질립pclvgA/CpC중lvgA/CpC극륭편단장위657 bp,추측lvgA기인편마단백대소약위27.7 Ku,lvgA기인급량측익원설계CpG기서완정확출,측서서렬여설계서렬완전부합.용면역형광검측중조질립재NIH3T3세포중순시표체.결론 본실험성공구건료기폐군단균pclvgA/CpG진핵표체질립,병재NIH3T3세포검측도기순시표체신호.
Objective To construct the eukaryotic expression plasmid containing IvgA gene flanked with CpG motifs of Legionella pneumophila for its expression in NDOT3 cells. Methods IvgA 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 IvgA 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.