BLM解旋酶基因的克隆、表达载体构建及表达研究

doi:10.13560/j.cnki.biotech.bull.1985.2018-0461

生物技术通报 ›› 2018, Vol. 34 ›› Issue (11): 152-159.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0461

BLM解旋酶基因的克隆、表达载体构建及表达研究

赵佳福^1,2, 许厚强^1,2, 宋书弦^1,3, 段志强¹, 陈祥¹

1. 贵州大学高原山地动物遗传育种与繁殖教育部重点实验室贵州大学动物科学学院,贵阳 550025;
2. 贵州大学生命科学学院,贵阳 550025;
3. 贵州省大方县农牧局,大方 551600

收稿日期:2018-05-18 出版日期:2018-11-26 发布日期:2018-11-28
作者简介:赵佳福,男,博士研究生,研究方向：分子细胞遗传学;E-mail：zhaojiafu1984@126.com
基金资助:
教育部中国与美大、欧洲地区教育科研合作项目[教财司函（2017）860号],贵州大学培育项目[黔科合平台人才（2017）5788-20],贵州省优秀人才省长基金[黔省专合字（2012）60号]

Cloning and Expression Vector Construction of BLM Helicase Gene,and Its Expression Analysis

ZHAO Jia-fu^1,2, XU Hou-qiang^1,2, SONG Shu-xian^1,3, DUAN Zhi-qiang¹, CHEN Xiang¹

1. Key Laboratory of Animal Genetics,Breeding and Reproduction in the Plateau Mountainous Region,Ministry of Education,Guizhou University,College of Animal Science,Guizhou University,Guiyang 550025;
2. College of Life Science,Guizhou University,Guiyang 550025;
3. Agriculture and Animal Husbandry Bureau of Dafang County,Dafang 551600

Received:2018-05-18 Published:2018-11-26 Online:2018-11-28

摘要/Abstract

摘要： 克隆获得BLM基因全长CDS区,研究其在细胞中的表达情况。从人前列腺癌PC3细胞中提取总RNA,反转录成cDNA,采用分段克隆的方法获得BLM全长CDS区,将其定向插入到原核表达载体pET-32a和真核表达载体pEGFP-N3,构建pET-32a-BLM和pEGFP-N3-BLM。将获得的重组质粒转化BL21（DE3）感受态细胞或转染PC3细胞,通过SDS-PAGE、Western blotting、荧光定位观察等方法研究人BLM解旋酶在细胞中的表达或定位。BLM解旋酶的原核表达结果显示,不同IPTG浓度对BLM解旋酶的表达量影响不大,低温有助于其表达量的提高。Western blotting检测发现,构建的人BLM解旋酶重组原核和真核表达载体分别能在大肠杆菌细胞和PC3细胞中表达出分子量约为179 kD 的蛋白质,与预期分子量大小一致。荧光共定位结果显示,人BLM解旋酶主要定位在细胞核。成功克隆了人BLM解旋酶全长CDS区,且构建的重组表达载体pET-32a-BLM和pEGFP-N3-BLM能在相应的宿主细胞中正确表达。

关键词: BLM解旋酶, 基因克隆, 表达载体, 亚细胞定位, 蛋白印迹

Abstract: This work aims to clone the full-length CDS region of BLM gene and to study the expression of BLM in cells. The total RNA extracted from the PC3 cells of human prostatic carcinoma was transcribed into cDNA. Then the full-length CDS region of BLM obtained by subsection cloning was inserted into prokaryotic expression vector pET-32a and eukaryotic expression vector pEGFP-N3,and the recombinant expression vectors pET-32a-BLM and pEGFP-N3-BLM were constructed. The recombinant plasmids were transformed into competent cells BL21（DE3）or transfected into PC3 cells. Further the expressions and localizations of BLM helicase in the above two cells were studied by SDS-PAGE,Western blotting,and fluorescence localization analysis. The prokaryotic expression of BLM helicase showed that different IPTG concentration affected little BLM expression;low temperature was conducive to the increase of BLM expression level. It was further confirmed by Western blotting that the constructed recombinant prokaryotic and eukaryotic vectors of BLM helicase respectively expressed in BL21（DE3）and PC3 cells as 179 kD protein,which was consistent with the expected protein. The results of fluorescence localization indicated that human BLM helicase mainly was in the nucleus. In sum,the full-length CDS region of human BLM helicase is cloned successfully,and the constructed recombinant expression vector pET-32a-blm and pEGFP-N3-blm can be correctly expressed in corresponding host cells.

Key words: BLM helicase, gene cloning, expression vector, subcellular localization, Western blotting

赵佳福, 许厚强, 宋书弦, 段志强, 陈祥. BLM解旋酶基因的克隆、表达载体构建及表达研究[J]. 生物技术通报, 2018, 34(11): 152-159.

ZHAO Jia-fu, XU Hou-qiang, SONG Shu-xian, DUAN Zhi-qiang, CHEN Xiang. Cloning and Expression Vector Construction of BLM Helicase Gene,and Its Expression Analysis[J]. Biotechnology Bulletin, 2018, 34(11): 152-159.

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BLM解旋酶基因的克隆、表达载体构建及表达研究

Cloning and Expression Vector Construction of BLM Helicase Gene,and Its Expression Analysis

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