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| 稳定表达GHR基因和突变体的CHO细胞株的构建及其功能分析 | |
| 引用本文： | 吴丽华,陈晓丽,唐文琴,张霆,廖志勇.稳定表达GHR基因和突变体的CHO细胞株的构建及其功能分析[J].基础医学与临床,2010,30(1):13-18. |
| 作者姓名： | 吴丽华 陈晓丽 唐文琴 张霆 廖志勇 |
| 作者单位： | 1. 首都儿科研究所分子免疫实验室,北京,100020 2. 江西九江瑞昌二中,江西瑞昌,332200 3. 江西师范大学生命科学学院,江西南昌,330022 |
| 基金项目： | 北京市自然科学基金(5062014);;国家自然科学基金(30460036) |
| 摘 要： | 目的构建稳定表达先天性生长激素不敏感综合征相关基因-hGHR及突变体(hGHR-E42K、hGHR-H56R)的CHO细胞株,测定野生株和突变株的STAT5-磷酸化水平。方法利用已有的PUC-hGHR质粒,定点突变获得2个hGHR突变体(hGHR-E42K、hGHR-H56R),限制性酶切法将目的基因克隆到真核表达载体pcDNA3.1/(zeo+);然后用Lipofectamine2000将重组子转染至CHO细胞,通过Zeocin抗性筛选稳定表达hGHR及突变体的细胞群;并用RT-PCR和Western blot证实hGHR、STAT5-P表达水平。结果测序验证hGHR的E42K和H56R错义突变成功引入,并克隆到真核表达载体;转染后在CHO细胞株中成功检测到hGHR和突变体的mRNA和蛋白;突变细胞株(E42K、H56R)的磷酸化STAT5蛋白表达量低于野生株。结论成功构建携带稳定表达hGHR及突变体的CHO细胞株,E42K、H56R突变部分阻碍STAT5蛋白进行磷酸化。 |
| 关 键 词： | 人生长激素受体基因 突变体 CHO细胞 STAT5 |
| 收稿时间： | 2008-8-28 |
| 修稿时间： | 2009-4-21 |
Establishment and functional analysis of CHO cells with stable expression of hGHR gene and its mutants |
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| WU Li-hua,CHEN Xiao-li,TANG Wen-qin,ZHANG Ting,LIAO Zhi-yong.Establishment and functional analysis of CHO cells with stable expression of hGHR gene and its mutants[J].Basic Medical Sciences and Clinics,2010,30(1):13-18. | |
| Authors: | WU Li-hua CHEN Xiao-li TANG Wen-qin ZHANG Ting LIAO Zhi-yong |
| Institution: | 1.Department of Molecular Immunology;Capital Institute of Pediatrics;Beijing 100020;2.the Second Middle School of Ruichang;Ruichang 332200;3.College of Life Science;Jiangxi Normal University;Nanchang 330022;China |
| Abstract: | Objective To construct three eukaryotic expression vectors containing wilde-type hGHR gene and its mutants(hGHR-E42K and hGHR-H56R) related to congenital growth hormone insensitivity,then check their expression in CHO cells. Methods With the available PUC-hGHR vector,two mutate hGHR genes (hGHR-E42K and hGHR-H56R) were obtained through mutagenesis. Then three recombinants were cloned into eukaryotic expression vectors pcDNA3.1/zeo(+) with restriction enzymatic reactions. Then with Lipofectamine2000,we trans... |
| Keywords: | human growth hormone receptor gene mutant CHO cell STAT5 |
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