CAJ | 학술논문

目的:构建卡氏肺孢子菌p55-v3及p55-v0抗原基因真核表达载体,mRNA水平鉴定其在COS-7细胞中的表达.方法:以卡氏肺孢子菌总RNA为模板,RT-PCR技术扩增p55-v3及p55-v0抗原基因,连接至pTA2载体,再克隆到pVAX1真核表达载体,构建重组质粒pVAX-p55-v3和pVAX-p55-v0.重组质粒在感受态DH5α大肠杆菌中大量扩增后,转染至COS-7细胞,RT-PCR鉴定其在mRNA水平的表达.结果:构建的重组质粒经酶切及测序鉴定,与文献报道的同源性分别达到99.8%和99.9%;其编码的氨基酸与文献报道的同源性为100%.RT-PCR显示p55-v3和p55-v0成功转染至COS-7细胞,并在其中进行基因表达.结论:本研究成功构建了pVAX-p55-v3和pVAX-p55-v0重组质粒,并在COS-7细胞中表达,为进一步阐明p55-v3的免疫保护功能以及核酸疫苗的研究提供了基础.
목적:구건잡씨폐포자균p55-v3급p55-v0항원기인진핵표체재체,mRNA수평감정기재COS-7세포중적표체.방법:이잡씨폐포자균총RNA위모판,RT-PCR기술확증p55-v3급p55-v0항원기인,련접지pTA2재체,재극륭도pVAX1진핵표체재체,구건중조질립pVAX-p55-v3화pVAX-p55-v0.중조질립재감수태DH5α대장간균중대량확증후,전염지COS-7세포,RT-PCR감정기재mRNA수평적표체.결과:구건적중조질립경매절급측서감정,여문헌보도적동원성분별체도99.8%화99.9%;기편마적안기산여문헌보도적동원성위100%.RT-PCR현시p55-v3화p55-v0성공전염지COS-7세포,병재기중진행기인표체.결론:본연구성공구건료pVAX-p55-v3화pVAX-p55-v0중조질립,병재COS-7세포중표체,위진일보천명p55-v3적면역보호공능이급핵산역묘적연구제공료기출.
Objective:To construct eukaryotic expression plasmids of pneumocystis carinii p55-v3 and p55-v0 antigenic genes and to identify their expression in COS-7 cells at mRNA level.Methods:Pneumocystis carinii total RNA was used as the template to amplify p55-v3 and p55-v0 antigenic gene by RT-PCR.The products were connected to pTA2 vector and then cloned in pVAX1 eukaryotic expression vector to construct recombinant plasmids as pVAX-p55-v3 and pVAX-p55-v0.After propagated in E.coli DH5α,the recombinant plasmids were transfected into COS-7 cells.After 24 h incubation,the RT-PCR was performed to identify the mRNA expression of p55-v3 and p55-v0 antigenic gene.Results:The recombinant plasmids were qualified by restrictive endonuclease digestion and sequencing.And when compared with that in GenBank,the homology of p55-v3 antigenic gene was 99.9% in nucleotides and 100% in amino acid.The homology between p55-v0 antigenic gene and the one reported previously in nucleotide and amino acid seguence were 99.8% and 100%.The results of RT-PCR confirmed that p55-v3 and p55-v0 antigenic genes were transfected into COS-7 cells successfully and the genes were expressed in the cells.Conclusion:In this study,the recombinant plasmids of pVAX-p55-v3 and pVAX-p55-v0 are conducted successfully and expressed in the COS-7 cells,which provide a basis for clarification of immunologic function of p55-v3 and study of DNA vaccine.