Eukaryotic Expression Vector Construction of Human RhoB Gene and Its Expression Analysis

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

Abstract

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

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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