人RhoB基因真核表达载体的构建和表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2019-0213

摘要/Abstract

摘要： 旨在构建带Flag标签的人RhoB基因真核表达载体并检测其表达。提取人胚肾细胞HEK-293T总RNA并反转录获得cDNA;设计引物并利用PCR技术扩增RhoB基因的ORF序列;将所扩增序列插入到带Flag标签的pCMV5真核表达载体,插入位点位于限制性内切酶SalⅠ和XbaⅠ之间,得到真核表达载体Flag-RhoB。酶切并测序验证该质粒的准确性。将成功构建的Flag-RhoB质粒转染乳腺癌MCF7细胞、宫颈癌HeLa细胞以及结直肠癌HCT116细胞,Western Blotting检测RhoB蛋白的表达情况;转染Mv.1.Lu细胞,免疫荧光技术检测该蛋白在细胞中的定位情况。成功扩增出RhoB基因的ORF序列;连入pCMV5中获得真核表达质粒Flag-RhoB;酶切鉴定得到590 bp大小的片段,测序结果显示连入的是RhoB基因的cDNA序列与GenBank相符。Western Blotting结果显示,该质粒可在MCF7细胞、HeLa细胞及HCT116细胞中正确表达。免疫荧光实验显示过表达的RhoB蛋白主要定位于细胞膜上,细胞质中也有分布。成功构建了带Flag标签的人RhoB基因的真核表达载体,该质粒可在细胞中顺利表达。

关键词: RhoB, 真核表达, 免疫荧光

Abstract: This work aims to construct a eukaryotic expression vector carrying Flag-taged human RhoB gene and to validate its expression in cells. First,total RNA from human embryonic kidney cell HEK-293T was extracted and its cDNA was obtained by reverse transcription. Then,primers were designed and ORF sequence of RhoB gene was amplified by PCR. The amplified sequence was then inserted into a modified eukaryotic expression vector pCMV5 containing Flag tag at the loci between endonuclease SalⅠand Xba Ⅰ,thus the eukaryotic expression vector Flag-RhoB was obtained,and its accuracy was sequenced and validated by enzymatic digestion. The constructed plasmid Flag-RhoB was transfected into breast cancer MCF7 cells,cervical cancer HeLa cells or colorectal cancer HCT116 cells. Western Blotting was employed to determine RhoB protein expression. Flag-RhoB was transfected into Mv.1.Lu cells,immunofluorescence were employed to determine its sub-cellular localization. As results,the ORF region of RhoB gene was successfully amplified,then linked into vector pCMV5 and the recombinant plasmid Flag-RhoB was acquired. A 590 bp fragment was obtained by double digestion,and sequencing results showed that the cDNA sequence of RhoB gene was consistent with that in GenBank. Results from Western Blotting demonstrated this Flag-RhoB plasmid expressed well in the MCF7 cells,HeLa cells or HCT116 cells. Immunofluorescence revealed that over-expressed RhoB protein was mainly located in the plasma membrane,and little in cytosol. In sum,human RhoB eukaryotic expression vector Flag-RhoB is successfully constructed and expresses well in cells.

Key words: RhoB, eukaryotic expression, immunofluorescence

王梅林, 牛精铃, 金若其, 徐莹莹, 蔡晶晶, 陈群力. 人RhoB基因真核表达载体的构建和表达分析[J]. 生物技术通报, 2019, 35(10): 174-179.

WANG Mei-lin, NIU Jing-ling, JIN Ruo-qi, XU Ying-ying, CAI Jing-jing, CHEN Qun-li. Eukaryotic Expression Vector Construction of Human RhoB Gene and Its Expression Analysis[J]. Biotechnology Bulletin, 2019, 35(10): 174-179.

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