诱导表达猪生长激素pGH载体系统构建及其在PK15细胞中表达验证  

Conditional Expression Vector of Pig Growth Hormone Leads to Regulatable Expression in PK15 Cells by Tetracycline(TET-ON) System

作　　者：白立景[1,2] 周荣[1] 敖红[1] 鞠辉明[3] 王楚端[2] 李奎[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]中国农业大学动物科技学院,北京100193 [3]扬州大学兽医学院,扬州225009

出　　处：《农业生物技术学报》2013年第4期483-488,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31000993;No.31272405);转基因新品种培育重大专项(No.20112X08008-001);中国农业科学院北京畜牧兽医研究所创新团队基金项目

摘　　要：为实现外源生长激素(GH)基因在特定时间、空间的可控表达,本研究构建四环素(TET-ON)表达载体系统,并在细胞水平对该载体系统进行诱导活性验证。结果表明:(1)成功构建了重组载体pTRE-GH12;(2)筛选建立了可诱导表达GH的细胞系;(3)利用强力霉素(doxycycline,DOX)诱导阳性细胞克隆并检测外源GH的表达,QRT-PCR结果表明,实验组细胞在DOX诱导后表达活性是诱导前264.481倍;对照组细胞在DOX诱导前诱导后表达无明显变化,实验组和对照组细胞间差异极显著(P<0.01),实验组DOX诱导前后差异极显著(P<0.01);(4)Western杂交后灰度分析结果和QRT-PCR趋势一致,实验组和对照组细胞间GH蛋白表达量差异极显著(P<0.01),实验组DOX诱导前后GH蛋白表达量差异极显著(P<0.01)。研究结果提示,四环素(TET-ON)诱导猪生长激素(GH)表达系统实现了可控表达,为以后制备可诱导表达GH的转基因动物提供了技术基础。Doxycyline（DOX）-inducible expression systems provide strict control over transgene expression spatially as well as temporally to avoid non-specific expression. Moreover DOX has no cytotoxic effects to the cells and animals, so it is used widely in transgenic research. Ptet-on system is more sensitive to DOX and yields lower background expression than that of the other inducible systems. For realizing the controllable expression of exogenous growth hormone （GH） gene at a specific time and space, we constructed inducible expression vector about tetracycline （TET-ON） system, and did a further study for testing its inducible activity at cellular level. The results showed that： （1） we have successfully constructed vector about pTRE-GH12; （2） we have harvested GH cells which had high inducible expression efficiency; （3）after induced by using DOX in the positive cells, we could get detection of the expression of exogenous GH, QRT-PCR results showed that the induction activity after using DOX was 264.481 times as before; there were no change in control group cells using or no using DOX , the differences between experimental group and control group were significantly different （P〈0.01）, so as the induction of DOX before and after in experimental group （P〈0.01）; （4）the results of Western blot were consisted with the result of QRT-PCR, the quantity of GH protein between the experimental group and control group cell showed significant differences （P〈0.01）, so as the experimental group before and after using DOX （P〈0.01）. All of our research results indicated： we have realized controllable expression of targeted gene pig growth hormone （pGH） by using tetracycline （TET-ON） inducible expression system, so as to provide a technological basis for preparing transgenic animals which can express GH inducible later.

关 键 词：猪生长激素(pGH) 四环素(TET-ON) 诱导表达 

分 类 号：S852.651[农业科学—基础兽医学]