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

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

Abstract

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

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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