【摘要】 建立稳定表达HIV-1p24MEG复合多表位基因的p815细胞克隆。设计引物,以HIV-1标准株全长cDNA序列为模板,PCR扩增获得p24基因片段;合成改造后的多个表位基因MEG并且与p24片段相连接,克隆入pcDNA3.1(+)。在阳离子聚合物作用下重组真核质粒转染的p815(H-2d)细胞,以G418压力筛选,RT-PCR检测mRNA表达,间接免疫荧光检测蛋白表达。通过PCR获得了HIV-1p24片段,获得了与多表位基因连接后的p24MEG融合基因,成功构建了p24MEG基因的重组真核表达载体pcDNA3.1(+)/p24MEG。转染p815细胞后,RT-PCR检测到融合蛋白mRNA表达,间接免疫荧光显示p815细胞内有融合蛋白的表达。结论:建立了稳定表达HIV-1p24MEG融合蛋白的p815细胞克隆,为评价多表位抗原p24MEG在BALB/c小鼠体内诱导的细胞免疫应答奠定基础。更多还原

【Abstract】 The P815 cell clone stably expressing of HIV-1p24MEG fusion multi-epitope gene was constructed and screened.p24 gene was amplified by PCR,with the plasmid containing full-length of cDNA of HIV-1 standard strain as the template.The p24 gene and the gene coding cryptic epitopes by amino modified acid Substitution were inserted into pcDNA3.1(+),obtained the recombinant plasmid pcDNA3.1(+)/p24MEG.The P815(H-2d)cells were transfected with pcDNA3.1(+)/p24MEG by liposome.After screening by G418,positive cell clone was obtained,which were assessed by RT-PCR and IFA.p24 gene fragment and recombinant plasmid pcDNA3.1(+)/p24MEG coding multi-CTL epitope gene and p24 gene were obtained successfully.After transfection,were used to screen the mRNA and protein of p24MEG fusion protein could be found in RT-PCR and IFA respectively.Conclusion:construction the P815 cell clone stably expressing of HIV-1p24MEG fusion protein to evaluate the cellular immune responses in BALB/c mice induced by p24MEG protein.更多还原