含mIL-12基因多顺反子逆转录病毒载体构建及其在肝癌细胞中的表达  

Construction of polycistronic retroviral vector containing gene of mIL-12 and the expression in murine hepatoma cells

作　　者：唐展云[1] 赖冠华[1] 陈诗书[1] 

机构地区：[1]上海第二医科大学人类基因治疗研究中心

出　　处：《中华微生物学和免疫学杂志》2000年第2期93-97,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目!(39730440)

摘　　要：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 /mILObjective To construct a polycistronic retroviral vector containing both subunits cDNA of murine IL 12 and NeoR gene and investigate their expressions in murine hepatoma cells. Methods The polycistronic retroviral vector (pGCEN/mIL 12) was constructed in which both subunits cDNA of mIL 12 and NeoR gene were linked by internal ribosome entry site (IRES) from encephalomyocarditis (EMCV) and poliovirus. Two different IRES sequences were used to express both subunits of mIL 12 and NeoR gene from the same polycistronic mRNA . Retrovirus supernatant was generates by transfecting pGCEN/mIL 12 into amphotropic packaging PA317 cells with lipofectAMINE. Murine hepatoma cell line MM45T.Li infected with the retroviral supernatant was selected by G418. The positive clones were subsequently collected and used for measurement of in vitro expression or in vivo experiment. Results PCR and Southern blot showed the integration of the foreign genes in the G418 resistant clonies. Northern blot revealed a single mRNA transcript driven by 5′LTR promoter. The following measurement of mIL 12 including: proliferation of human peripheral blood mononuclear cells(PBMC) activated by PHA and stimulation of interferon γ induction in murine spleen cells(160U/ml). Conclusion The constructed polycistronic retroviral vector can express mIL 12 effectively in murine hepatoma, and can be potentially used in cancer gene therapy.

关 键 词：MIL-12基因 逆转录病毒载体 肝癌细胞 内核糖体 

分 类 号：R735.7[医药卫生—肿瘤]