人白细胞介素12双亚基共表达载体的构建及表达  

Construction and expression of human interleukin 12 double-subunit co-expressing vector

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

机构地区：[1]湖南长沙中南大学湘雅二医院消化内科和心血管内科,长沙410011

出　　处：《中国免疫学杂志》2004年第2期79-82,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金项目 (批准号 :3 95 70 3 3 5 ) ;医学遗传学国家重点实验室开放课题基金资助

摘　　要：目的 :为了基因治疗研究的需要构建人白细胞介素 12 (hIL 12 )双亚基基因共表达质粒 [(P(+) IL 12 ) ],比较它与单个亚基基因表达质粒 [P(+) P4 0 和P(- ) P35]1∶1共同转染肝癌细胞HepG2后 4 8小时表达结果。方法 :分别克隆hIL 12P4 0 、P35亚基cDNA全长至pcDNA3 1(± )中构建P(+) P4 0 和P(- ) P35,再将两者串联克隆至pcDNA3 1(+)中构建P(+) IL 12。脂质体转染 3种质粒至肝癌细胞HepG2 ,4 8小时后抽取细胞总RNA做半定量RT PCR ,同时取细胞上清做ELISA及Western杂交 ,结果进行比较分析。结果 :两种转染方式均可以表达hIL 12蛋白质且半定量RT PCR显示较未转染组hIL 12的mRNA水平均增加 ;转染后 4 8小时P(+) IL 12组细胞培养上清hIL 12表达量较 [P(+) P4 0 和P(- ) P35]按 1∶1比例共同转染组显著增高(P <0 0 5 )。结论 :人白细胞介素 12双亚基基因串联共表达质粒 [P(+) IL 12 ]能够转入HepG2并表达hIL 12蛋白质 ,并且转染后 4 8小时hIL 12表达量较单个亚基基因表达质粒 [P(+) P4 0 、P(- ) P35]1∶1共转染者显著高。Objective:In order to serve for study of gene therapy human interleukin 12 double-subunit co-expression eukaryotic plasmid was constructed and compared its transient expression with that of single-subunit expressing ones 48 hours after they were transfected into HepG2 cells.Methods:After the full length cDNAs of both P 40 and P 35 subunit genes were cloned respectively into pcDNA3.1(±) to get P(+)/P 40 and P(-)/P 35 ,they were linked tandem and cloned into pcDNA3.1(+) to get P(+)/IL-12. P(+)/IL-12 was transfected into HepG2 cells while P(+)/P 40 and P(-)/P 35 (1∶1)were co-transfected into HepG2 by using liposome. 48 hours later, expression of hIL-12 was detected by both using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and conducting enzyme-linked immunoabsorbent assay (ELISA) and Western blotting.Results:Both transfecting groups could express hIL-12 protein; Compared with control group, the result of semi-quantitative RT-PCR showed both P 40 and P 35 mRNA increased.The result of ELISA showed hIL-12 production of double-subunit co-expressing plasmid was significantly higher than that of single-subunit expressing ones (P<0.05) in the supernate 48 hours after transfected HepG2 cells.Conclusion:Double-subunit co-expressing plasmid could be transfected into HepG2 cells, furthermore the transient expression of hIL-12 was significantly higher than that of single-subunit expressing ones.

关 键 词：人白细胞介素12 真核表达载体pcDNA3.1(±) HEPG2细胞 表达 

分 类 号：R392.11[医药卫生—免疫学]