细菌内同源重组法制备mIL-12腺病毒载体及其体外高效表达  

Constrction of Recombinant Adenovirus Vector Containing mIL-12 Using the Method of Homogenous Recombination in Bacteria and Its Expressiion in vitro with High Efficient

作　　者：魏道严[1] 唐展云[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,上海200025

出　　处：《中国生物化学与分子生物学报》2000年第6期716-721,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家"8 6 3"高技术计划基金! (86 3-10 2 -0 5 );国家自然科学基金! (3970 440 )资助

摘　　要：利用细菌内同源重组法构建含 m IL- 1 2双顺反子重组腺病毒载体 ,其中由 polio IRES连接的 m IL- 1 2 p40和 p35双亚基目的基因片段用 Not +Xho 酶自真核表达载体 pc DNA3/m IL- 1 2中切出 ,亚克隆至经同样酶切的腺病毒穿梭质粒中 ,形成转移质粒 p Adtrack- CMV/m IL- 1 2 ,对之线性化后与腺病毒基因组质粒 p Adeasy- 1共转化 BJ51 83菌 ,抽提经鉴定含目的基因的重组体质粒DNA,转染 2 93细胞 ,包装成重组体腺病毒 Ad- m IL - 1 2 . Southern杂交证实 ,p40和 p35基因已被克隆至 Ad- m IL - 1 2基因组中 ;RT- PCR结果显示 ,Ad- m IL- 1 2感染的 MM45T· Li肿瘤细胞中有p40和 p35基因的表达 ,ELISA检测感染 48h细胞的上清中 m IL- 1 2的含量达 88ng每 1 0 6细胞 ,其体外能刺激小鼠脾脏淋巴细胞的增殖 ,提高 NK细胞活性及诱导 干扰素的产生 .结果表明 ,利用细菌内同源重组法制备的 Ad- m IL- 1 2重组腺病毒载体是一种方便、高效、成功率高的方法 ,所制备的重组体腺毒在体外能有效表达具有生物活性的 m IL- 1 2 ,为今后的体内应用奠定了基础 .In order to construct recombinant adenovirus vector containing mIl 12 bicistron,the two subunits of p40 and p35 of mIL 12 gene linked by poliovirus internal ribosome entry site(IRES)were liberated from the vector of pcDNA3/mIL 12 via Not Ⅰ+ Xho I digestion,and subcloned into shuttle vector of pAdtrack CMV,forming transfer vector of pAdtrack CMV/mIL 12.Linearized pAdtrack CMV/mIL 12 was cotransformed into BJ5183 cells with adenovirual genomic plasmid of pAdeasy 1.The DNA of identified recombinant plasmid was extracted,and 293 cells transfected to package adenovirus.Southern blot showed that p40 and p35 subunits had been subcloned into Ad mIL 12.RT PCR demonstrated that there were p40 and p35 subunits expression in MM45T·Li tumor cells infected with Ad mIL 12,resulted in the secretion of mIL 12 in culture supernatant which could stimulate splenocyte proliferation,augment NK cytotoxicity of splenocytes and induce murine IFN γ production.These results indicate that the prepared recombinant adnovirus vector Ad mIL 12 can express biologically active mIL 12 and can be further used for in vivo experiments.

关 键 词：腺病毒 白介素12 同源重组 细菌 载体 表达 

分 类 号：Q782[生物学—分子生物学]