整合型诱导表达猪生长激素（pGH）载体的构建及表达研究

doi:10.11843/j.issn.0366-6964.2013.07.003

畜牧兽医学报 ›› 2013, Vol. 44 ›› Issue (7): 1008-1013.doi: 10.11843/j.issn.0366-6964.2013.07.003

整合型诱导表达猪生长激素（pGH）载体的构建及表达研究

鞠辉明^1，2，白立景²，姜星¹，李奎^2*

（1. 扬州大学兽医学院，扬州 225009; 2. 中国农业科学院北京畜牧兽医研究所/农业部畜禽遗传资源与利用重点开放实验室，北京 100193）

收稿日期:2012-07-08 出版日期:2013-07-23 发布日期:2013-07-23
通讯作者: 李奎，E-mail：likuihau@yahoo.com
作者简介:鞠辉明(1975-)，男，江苏泰兴人，副教授，博士，主要从事家畜胚胎及基因工程的研究，Tel：010-62833312，E-mail: hymanju@gmail.com
基金资助:
国家转基因新品种培育重大专项（2011ZX08006-003）；国家自然科学基金资助项目（31272405；31000993）；江苏高校优势学科建设工程资助项目

Construction and Expression Study of Integrated pGH Inducible Expression Vector

JU Hui-ming^{1, 2}, BAI Li-jing², JIANG Xing¹, LI Kui^2*

(1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009， China; 2. Key Laboratory of Farm Animal Genetic Resources and Utilization of Ministry of Agriculture,Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China)

Received:2012-07-08 Online:2013-07-23 Published:2013-07-23

摘要/Abstract

摘要：

旨在构建整合型诱导表达猪生长激素（Porcine growth hormone，pGH）载体，在细胞水平验证其诱导效率及表达效率。从pTRE-GH12载体上扩增TRE-GH片段，通过Sal I酶切位点连接到pCAGGS-rtTA载体中，构建重组载体pCAGGS-rtTA-TRE-GH12（下称pTTGH），将pTTGH质粒转染猪PK15细胞，经G418筛选后，在培养液中添加诱导底物强力霉素（Doxycycline，DOX）后不同时间段通过实时荧光定量PCR（QRT-PCR）测定细胞内pGH mRNA的表达；在培养液中添加不同浓度的的DOX，通过QRT-PCR及Western blot测定细胞内pGH的表达变化。酶切及测序结果表明成功构建了pTTGH载体；QRT-PCR结果表明，和对照组相比，试验组细胞培养液中添加诱导底物后外源GH基因表达在36 h时最高；在一定浓度范围内，外源GH表达水平和添加DOX的浓度呈现出正相关性，而正常细胞组中GH的表达水平不受DOX添加与否及量的影响。通过条带灰度分析Western杂交结果验证了培养基中一定范围内DOX浓度和pGH表达量呈现出正相关。试验结果表明，本研究成功构建了整合型诱导表达GH载体并能实现GH的可控表达，本研究为以后制备可控表达GH转基因动物、进一步研究GH对机体影响奠定基础。

Abstract:

This study aimed to construct integrated porcine growth hormone (pGH) inducible expression vector and to validate the induction and expression efficiency at the cellular level. pTRE-GH fragment was amplified by PCR from pTRE-GH12 vector, then the ligation of the Sal I digestion fragment to the Sal I digestion vector pCAGGS-rtTA, the recombinant vector pCAGGS-rtTA-TRE-GH12（pTTGH）was constructed. The pTTGH vector DNA transfected porcine PK15 cells, and enriched with G418, pGH mRNA expression level was determined by QRT-PCR after adding induction substrate, with doxycycline (DOX) in culture medium. The expression change was observed by QRT-PCR and Western blot assay for pGH after different concentration of DOX inducing. The results of sequencing and restriction enzyme digestion showed that the pTTGH vector was constructed successfully. Compared with control cell groups, QRT-PCR results showed that pGH mRNA expression level was the highest at 36 h after DOX adding; within a certain range of the DOX concentration, the exogenous pGH mRNA expression level showed positive relationship with DOX concentration. Nevertheless, the pGH expression level showed no obvious relationship with DOX concentration and adding or not in normal cell groups. Results of gray intensity analysis showed that pGH protein level and a certain range of the DOX concentration in culture media had positive relationship. The results showed that the vector had been successfully constructed and which could realize pGH controllable expression. This study established a foundation for preparing pGH controllable expression transgenic animal and further studying on GH affection.

中图分类号:

S828
S813.3

鞠辉明，白立景，姜星，李奎. 整合型诱导表达猪生长激素（pGH）载体的构建及表达研究[J]. 畜牧兽医学报, 2013, 44(7): 1008-1013.

JU Hui-ming, BAI Li-jing, JIANG Xing, LI Kui. Construction and Expression Study of Integrated pGH Inducible Expression Vector[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(7): 1008-1013.

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整合型诱导表达猪生长激素（pGH）载体的构建及表达研究

Construction and Expression Study of Integrated pGH Inducible Expression Vector

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