GST-hZimp10融合蛋白的重组及抗体制备

doi:10.13481/j.1671-587x.20150342

Abstract

Abstract:

Objective To construct the pGEX-4T-1/hZimp10 recombinant plasmid and to express in E.coli, and to purify the GST-hZimp10 fusion protein to prepare anti-hZimp10 antibody. Methods The corresponding DNA fragment with the N-terminal 128 amino acids of hZimp10 protein was amplified by PCR using the template plasmid pcCDNA3.1-Flag-hZimp10 and then cloned into the expression vector pGEX-4T-1.After identified by enzyme digestion, the recombinant clone was transformed into the expression cells of E.coli BL21.The GST-hZimp10 fusion protein was induced by IPTG and then purified as an antigen into the rabbits to prepare the polyclonal antibody.At last, the titer and specificity of the antibody were analyzed with indirect ELISA and Western blotting method. Results The pGEX-4T-1/hZimp10 recombinant plasmid was constructed successfully.It contained a 384 bp insert fragment identified by BamHⅠ and XhoⅠ double digestion, and the result was consistent with expectations.The SDS-PAGE electrophoresis showed the fusion protein was well expressed and protein molecular weight was consistent with that expected.The indirect ELISA and Western blotting Results indicated that anti-hZimp10 antibody showed high titer (1:100 000) and high spencificity in the LNCaP cells. Conclusion GST-hZimp10 fusion protein is obtained successfully and the anti-hZimp10 antibody is prepared.

Key words: hZimp, glutathiones transferase fusion protein, polyclonal antibody

CLC Number:

YANG Liu, WANG Xiaowan, WANG Bingyu, SONG Runmin, LI Jiang. Recombination of GST-hZimp fusion protein and preparation of anti-hZimp10 antibody[J].Journal of Jilin University Medicine Edition, 2015, 41(03): 656-661.

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Recombination of GST-hZimp fusion protein and preparation of anti-hZimp10 antibody

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