CAJ | 학술논문

目的构建大鼠源肺孢子菌抗原p55 基因片段真核表达质粒,并研究该质粒在真核细胞COS-7中的有效表达.方法以含有p55全长基因的质粒为模板,通过PCR 扩增p55基因片段,克隆至pGEM-T 载体中,经酶切后再将p55基因重组至真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒pcDNA3.1(+)-582,经限制性内切酶酶切分析及测序鉴定正确后,应用脂质体转染技术转染入COS-7细胞,RT-PCR 检测其基因转录情况,SDS-PAGE 鉴定其表达相应的目的蛋白质情况.结果成功扩增了p55基因片段,酶切和测序证明正确构建了重组真核表达质粒pcDNA3.1(+)-582,RT-PCR 和SDS-PAGE 方法证实该片段能在COS-7细胞中有效表达目的蛋白质.结论成功构建了大鼠源肺孢子菌p55基因片段的真核表达质粒载体,并在COS-7 细胞中表达,为进一步进行核酸疫苗的研究奠定了基础.
목적 구건대서원폐포자균항원p55 기인편단진핵표체질립,병연구해질립재진핵세포COS-7중적유효표체.방법 이함유p55전장기인적질립위모판,통과PCR 확증p55기인편단,극륭지pGEM-T 재체중,경매절후재장p55기인중조지진핵표체재체pcDNA3.1(+)중,구건중조진핵표체질립pcDNA3.1(+)-582,경한제성내절매매절분석급측서감정정학후,응용지질체전염기술전염입COS-7세포,RT-PCR 검측기기인전록정황,SDS-PAGE 감정기표체상응적목적단백질정황.결과 성공확증료p55기인편단,매절화측서증명정학구건료중조진핵표체질립pcDNA3.1(+)-582,RT-PCR 화SDS-PAGE 방법증실해편단능재COS-7세포중유효표체목적단백질.결론 성공구건료대서원폐포자균p55기인편단적진핵표체질립재체,병재COS-7 세포중표체,위진일보진행핵산역묘적연구전정료기출.
To construct the eukaryotic expression plasmid containing the p55 gene fragment of Pneumocystis and to investigate the efficient expression in COS-7 cells, the gene fragment conaining the whole length of p55 gene was used as template to amplify this fragment with PCR and the amplified fragment was then cloned to vector pGEM-T. After enzyme digestion, p55 gene was cloned to the eukaryotic expression vector pcDNA3.1(+) to construct the plasmid pcDNA3.1(+)-582. This plasmid was then transfected to the eukaryotic expression cells COS-7 and PCR and SDS-PAGE assays were used to confirm the presence of target protein in these cells. In these ways, the eukaryotic expression vector for the p55 gene of Pneumocystis of rats was successfully constructed and expressed in COS-7 cells, thus providing the basis for further studies on the nucleic acid vaccine.