人胰岛素基因杆状病毒表达载体的构建及其在Sf_9细胞中的表达  

Construction of Baculovirus Expression Vector of Human Insulin Gene and Its Expression in Sf_9 Cells

作　　者：李华[1] 刘维全[2] 王吉贵[3] 江禹[2] 王本旭[2] 王萍[4] 齐顺章[3] 殷震[2] 

机构地区：[1]中国医科大学实验动物部,辽宁沈阳110001 [2]解放军军需大学军事兽医研究所,吉林长春130062 [3]中国农业大学生物学院,北京100094 [4]吉林大学耳鼻喉研究所,吉林长春130021

出　　处：《中国兽医学报》2001年第3期258-262,共5页Chinese Journal of Veterinary Science

基　　金：吉林省科委基金!资助项目 ( 985 79)

摘　　要：对人胰岛素基因组基因真核表达载体 ( p CMA/m INS)进行 Xho 酶切 ,回收的含人胰岛素基因组基因的 1 60 8bp片段与线性化的杆状病毒载体 p Fast Bac 进行连接 ,获得重组载体 p Fast/m INS。将该重组载体转化 DH1 0 Bac感受态细菌 ,在体内进行重组 ,并经 2次抗性与蓝白斑筛选 ,得到杆状病毒重组载体 Bacmid/m INS。将该载体转染 Sf9细胞 ,获得了重组杆状病毒 ,经 Tricine-SDS-PAGE和 Western-blotting检测 ,重组杆状病毒在 Sf9细胞中的表达产物是胰岛素原 。The transpositional recombinant vector was constructed firstly. Then it was transformed in E. coli DH10Bac and cultured in LB plate which contained Gm, Kan, X gal and IPTG. The white bacterial colonies were positive recombinant Bacmid named as Bacmid/mINS.The recombinant baculoviruses were obtained after the transfection of the recombinant vector into Sf 9 lines. The techniques of Tricine SDS PAGE and Western blotting were used to detect and identify the products expressed in Sf 9 lines by recombinant baculovirus. The results indicated that the products expressed in Sf 9 lines by baculovirus vector system were most likely proinsulin.None of the proinsulin and insulin were detected in the supernatant of Sf 9 cell culture.

关 键 词：人胰岛素基因 杆状病毒表达系统 表达载体 SF9细胞 基因表达 

分 类 号：TQ467.32[化学工程—制药化工] Q78[生物学—分子生物学]