摘要
目的建立稳定表达丙型肝炎病毒(HCV)HLA-A2限制性多表位基因的C1R-AAD细胞克隆。方法合成人泛素基因(Ub)和HCVHLA-A2限制性多表位基因(Mep),分别克隆入原核表达质粒pRSET-A,构建多表位抗原基因的原核表达质粒pRSET-Ub-Mep。用BamHⅠ和HindⅢ双酶切质粒pRSET-Ub-Mep,得到复合多表位基因Ub-Mep,亚克隆入真核表达载体pcD-NA3.1(-),构建重组真核表达质粒pcDNA3.1(-)-Ub-Mep,转染至C1R-AAD细胞,G418压力筛选后,通过有限稀释法获得稳定表达Ub-Mep融合蛋白的C1R-AAD细胞克隆,采用RT-PCR法检测稳定转染细胞中Ub-Mep基因mRNA的转录;间接免疫荧光法和Westernblot法检测Ub-Mep蛋白在稳定转染细胞中的表达。结果重组真核表达质粒pcDNA3.1(-)-Ub-Mep经酶切鉴定证明构建正确;在稳定转染的C1R-AAD细胞中可检测到Ub-MepmRNA和蛋白水平的表达。结论已建立了稳定表达HCVHLA-A2限制性多表位抗原的C1R-AAD细胞克隆,为进一步研究HLA-A2限制性多表位基因诱导的细胞免疫应答建立了靶细胞。
Objective To establish a C1R-AAD cell clone stably expressing HLA-A2 restricted multi-epitopes gene of hepatitis C virus（HCV）.Methods Human ubiquitin（Ub）and HCV HLA-A2 restricted multi-epitopes（Mep）genes were synthesized and cloned into prokaryotic expression vector pRSET-A.The constructed recombinant plasmid pRSET-Ub-Mep was digested with BamHⅠ and Hind Ⅲ,and the obtained complex multi-epitopes gene Ub-Mep was subcloned into eukaryotic expression vector pcDNA 3.1（）-.The constructed recombinant plasmid pcDNA 3.1（-）-Ub-Mep was transfected to C1R-AAD cells,based on which the cell clone stably expressing Ub-Mep protein were obtained by G418 pressure screening and limiting dilution method.The transcription of Ub-Mep gene in stably transfected cells was determined by RT-PCR,and the expression of Ub-Mep protein by IFA and Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmidpcDNA3.1（-）-Ub-Mepwasconstructed correctly.The expressions of Ub-Mep at mRNA and protein levels were proved in the C1R-AAD cells stably transfected with the recombinant plasmid.Conclusion A C1R-AAD cell clone stably expressing HLA-A2 restricted multi-epitopes gene of HCV was established,which provided target cells for further study on cellular immune response induced by HLA-A2 restricted multi-epitopes gene.
出处
《中国生物制品学杂志》
CAS
CSCD
2010年第7期683-686,共4页
Chinese Journal of Biologicals
基金
国家自然科学基金项目(C0700760)
关键词
丙型肝炎病毒
表位
真核细胞
基因表达
转染
Hepatitis C virus（HCV）
Epitopes
Eukaryotic cells
Gene expression
Transfection
