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

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
白立景;周荣;敖红;鞠辉明;王楚端;李奎
【期刊名称】《农业生物技术学报》
【年(卷),期】2013(21)4
【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 grouprncells 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.
【总页数】6页(P483-488)
【作者】白立景;周荣;敖红;鞠辉明;王楚端;李奎
【作者单位】中国农业科学院北京畜牧兽医研究所,北京100193
【正文语种】中文
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