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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-epitope gene of hepatitis C virus (HCV). Methods The human ubiquitin gene (Ub) and the HCV-PAH-A2 restricted multi-epitope gene (Mep) were synthesized and cloned into the prokaryotic expression plasmid pRSET-A to construct the prokaryotic expression plasmid pRSET-Ub-Mep. The double-stranded plasmid pRSET-Ub-Mep was digested with BamHⅠand HindⅢ, and the multi-epitope gene Ub-Mep was obtained and subcloned into eukaryotic expression vector pcD-NA3.1 (-) to construct recombinant eukaryotic expression vector pcDNA3.1 -) - Ub-Mep was transfected into C1R-AAD cells. After pressure screening with G418, C1R-AAD cell clones stably expressing Ub-Mep fusion protein were obtained by limiting dilution. The stably transfected cells were detected by RT-PCR Ub-Mep gene mRNA transcription; Indirect immunofluorescence and Western blot were used to detect the expression of Ub-Mep protein in stable transfected cells. Results The constructed eukaryotic expression plasmid pcDNA3.1 (-) - Ub-Mep was confirmed by restriction enzyme digestion. The expression of Ub-MepmRNA and protein was detected in stably transfected C1R-AAD cells. Conclusion C1R-AAD cell clones stably expressing HCVHLA-A2 restricted multi-epitope antigens have been established and target cells have been established to further study the cellular immune response induced by HLA-A2 restricted multi-epitope genes.