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目的 构建含小鼠白细胞介素 12双亚基及新霉素磷酸转移酶基因多顺反子逆转录病毒载体 ,并观察其在小鼠肝癌细胞中的表达。方法 利用脑心肌炎病毒 (EMCV)及脊髓灰质炎 (Polio)病毒内核糖体进入位点 (IRES) ,连接mIL 12p40及p35cDNAs和筛选基因新霉素磷酸转移酶 (NeoR) ,克隆至逆转录病毒载体pGCEN中 ,使三个基因同时受逆转录病毒载体 5′端LTR启动子控制 ,转录至同一mRNA转录本上 ,通过不同机制翻译成蛋白质 ,从而构建成多顺反子逆转录病毒载体 ,即pGCEN/mIL 12。在LipofectAMINE介导下将pGCEN/mIL 12转染包装细胞PA317,G418筛选 ,直至出现阳性克隆 ,挑取抗性克隆 ,扩大培养 ,收集上清 ,用小鼠成纤维细胞NIH3T3测定病毒滴度。然后用重组转录病毒感染小鼠肝癌细胞MM45T .Li,G418筛选 ,直至出现抗性克隆 ,扩大培养 ,对阳性克隆进行鉴定。结果 由PA317包装细胞产生的重组逆转录病毒的滴度为 5× 10 5CFU/ml,将其感染小鼠肝癌细胞MM45T .Li,后经PCR及Southernblot证明 ,外源基因已整合至小鼠肝癌细胞基因组中 ,RT PCR及Northernblot分析外源基因在mRNA水平上的表达 ,并证实mIL 12p40及p35cDNA和NeoR基因转录在同一mRNA上。ELISA显示mIL 12的表达量 48h为 10ng/ 10 6细胞。并且M45 /mIL 12培养上清能刺激人
Objective To construct a polycistronic retroviral vector containing murine interleukin - 12 double subunit and neomycin phosphotransferase gene and observe its expression in mouse liver cancer cells. Methods The encephalomyocarditis virus (EMCV) and polio virus internal ribosome entry site (IRES) were ligated with mIL 12p40 and p35 cDNA and the neomycin phosphotransferase (NeoR) gene was selected and cloned into retroviral vector pGCEN, so that the three genes simultaneously by the 5 ’end of the retroviral vector LTR promoter control, transcribed to the same mRNA transcripts, translated into proteins by different mechanisms to construct a polycistronic retroviral vector, that pGCEN / mIL 12. PGCEN / mIL12 was transfected into packaging cells PA317 and G418 with LipofectAMINE until the positive clones appeared. The resistant clones were picked and expanded. The supernatants were harvested and the virus titers were determined with mouse fibroblasts NIH3T3. Then, the mouse hepatoma cells MM45T .Li and G418 were infected with the recombinant virus until the resistant clones appeared and expanded to identify the positive clones. Results The recombinant retrovirus produced by PA317 packaging cells had a titer of 5 × 10 5 CFU / ml, which was then infected into MM45T .Li mouse hepatoma cells. After PCR and Southern blotting, it was proved that the foreign gene had been integrated into mouse hepatoma cells In the genome, RT PCR and Northern blot analysis of exogenous gene expression at the mRNA level, and confirmed that mIL 12p40 and p35cDNA and NeoR gene transcription in the same mRNA. ELISA showed that the expression level of mIL 12 was 10 ng / 10 6 cells for 48 h. And M45 / mIL 12 culture supernatant can stimulate people