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| 人胰岛素基因杆状病毒表达载体的构建及其在Sf9细胞中的表达 | |
| 引用本文： | 李华,刘维全,江禹,王本旭,殷震,王吉贵,齐顺章,王萍.人胰岛素基因杆状病毒表达载体的构建及其在Sf9细胞中的表达[J].中国兽医学报,2001,21(3):258-262. |
| 作者姓名： | 李华 刘维全 江禹 王本旭 殷震 王吉贵 齐顺章 王萍 |
| 作者单位： | 1. 中国医科大学实验动物部, 2. 解放军军需大学军事兽医研究所, 3. 中国农业大学生物学院, 4. 吉林大学耳鼻喉研究所, |
| 基金项目： | 吉林省科委基金资助项目(98579) |
| 摘 要： | 对人胰岛素基因真核表达载体（pCMA／mINS）进行Xho I酶切，回收的含人胰岛素基因组基因的1608bp片段与线性化的杆状病载体pFast Bac I进行连接，获得重组载体pFast/mINS。将该重组载体转化DH10Bac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/mINS。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，而细胞培养上清中未检测到胰岛素和胰岛素原。 |
| 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 |
| 文章编号： | 1005-4545(2001)03-0258-05 |
| 修稿时间： | 2000年11月30 |
Construction of Baculovirus Expression Vector of Human Insulin Gene and Its Expression in Sf9 Cells |
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| LI Hua ,LIU Wei quan ,WANG Ji gui ,JIANG Yu ,WANG Ben xu ,WANG Ping ,QI Shun zhang ,YIN Zhen.Construction of Baculovirus Expression Vector of Human Insulin Gene and Its Expression in Sf9 Cells[J].Chinese Journal of Veterinary Science,2001,21(3):258-262. | |
| Authors: | LI Hua LIU Wei quan WANG Ji gui JIANG Yu WANG Ben xu WANG Ping QI Shun zhang YIN Zhen |
| Institution: | LI Hua 2,LIU Wei quan 1*,WANG Ji gui 3,JIANG Yu 1,WANG Ben xu 1,WANG Ping 4,QI Shun zhang 3,YIN Zhen 1 |
| Abstract: | 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. |
| Keywords: | human insulin gene vector construction baculovirus expression vector system Sf 9 |
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