人白细胞介素12双亚基共表达真核载体的构建  

Construction of double-subunit co-expression eukaryotic vector of human interleukin 12

作　　者：羊东晔[1] 卢放根[1] 赵水平[2] 汤熙翔 刘小伟[1] 吴小平[1] 

机构地区：[1]中南大学湘雅二医院消化内科,长沙410011 [2]中南大学湘雅二医院心内科,长沙410011 [3]生生基因药物研究所,长沙410008

出　　处：《湖南医科大学学报》2003年第4期338-342,共5页Bulletin of Hunan Medical University

基　　金：国家自然科学基金 (编号 395 70 335 ) ;科技部博士新药创新基金 ;医学遗传学国家重点实验室开放课题基金

摘　　要：目的 :为满足基因治疗的需要构建人白细胞介素 12 (hIL 12 )双亚基共表达质粒。方法 :先从人胚肾组织的逆转录产物中用PCR方法扩增出它的两个亚基P4 0 和P3 5的cDNA全长 ,分别克隆入真核表达载体pcDNA3.1(+/ )中构建单亚基质粒———P(+) /P4 0 和P( ) /P3 5,然后再将二者串联克隆入真核表达载体 pcDNA3.1(+)中得到P(+) /IL 12质粒。脂质体转染HepG2后 2 4,48hELISA检测细胞培养上清内hIL 12蛋白质表达。结果 :P(+) /IL 12质粒经酶切和测序证明两亚基连接方向正确 ,序列无突变 ;ELISA检测细胞上清结果证实可表达hIL 12蛋白质。结论 :成功构建P4 0 和P3 5双亚基真核共表达质粒———P(+) /IL 12 ,为模拟hIL 12生理表达方式 ,简化hIL 12基因治疗操作奠定了基础。Objective To construct the double-subunit co-expression plasmid P(+)/IL-12 to fulfill the gene therapy. Methods The full length cDNAs of P 40 and P 35, two subunits of hIL12, were amplified from the reverse transcription production of human embryo kidney using polymerase chain reaction (PCR) and respectively cloned into eukaryotic expression vector pcDNA3.1(+/-) to construct the single-subunit plasmids——P(+)/P 40 and P(-)/P 35. Then we linked cDNAs of P 40 and P 35 tandem and cloned them into pcDNA3.1(+) to get P(+)/IL-12 plasmid. The hIL-12 protein in supernate was detected with enzyme-linked immunoabsorbent assay (ELISA) after P(+)/IL-12 was transfected into HepG2 through liposome. Results The plasmid of P(+)/IL-12 was testified using enzyme cutting and sequencing analysis.ELISA showed that hIL-12 protein could be expressed after transfecting into HepG2. Conclusion The successful construction of P(+)/IL-12 plasmid can not only simplify the operation steps but also mimic hIL-12 physiological expression style in the gene therapy, thus laying the foundation for the use of hIL-12 in the gene therapy.

关 键 词：人白细胞介素12 双亚基 表达 真核载体 克隆 

分 类 号：R392.11[医药卫生—免疫学]