GST-hZimp10融合蛋白的重组及抗体制备  

Recombination of GST-hZimp fusion protein and preparation of anti-hZimp 10 antibody

作　　者：杨柳[1] 汪小菀 王冰瑜 宋润敏 李江[1] 

机构地区：[1]吉林大学口腔医院口腔修复科,吉林长春130021 [2]东北师范大学生命科学学院肿瘤信号转导实验室,吉林长春130024

出　　处：《吉林大学学报（医学版）》2015年第3期656-661,共6页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划项目资助课题(YYZX201241);吉林省科技厅科研基金资助课题(201115106;20150414007);吉林省长春市科技局科研基金资助课题(2011122)

摘　　要：目的:构建pGEX-4T-1/hZimp10重组质粒,并在大肠杆菌中表达,纯化出GST-hZimp10融合蛋白,用以制备抗hZimp10抗体。方法:采用PCR技术以pcCDNA3.1-Flag-hZimp10为模板,扩增出hZimp10蛋白N端128个氨基酸对应DNA片段,并将其与谷胱甘肽硫转移酶(GST)融合蛋白表达质粒pGEX-4T-1进行重组,酶切鉴定后获得重组质粒,并转化到大肠杆菌BL21中,经IPTG诱导表达,获得GST-hZimp10融合蛋白,纯化后将其作为抗原免疫家兔制备多克隆抗体,用间接ELISA法检测抗体效价,Western blotting法检测抗体特异性。结果:经BamHⅠ和XhoⅠ双酶切鉴定,确定PGEX-4T-1/hZimp10重组质粒中包含384bp的片段,与测序结果一致,表明pGEX-4T-1/hZimp10重组质粒构建成功。SDS-PAGE电泳,融合蛋白成功表达,蛋白相对分子质量大小与预期相符。间接ELISA法检测抗hZimp10抗体效价可达1∶100 000以上。Western blotting法,hZimp10在LNCaP细胞中具有很高的特异性。结论:成功获得GST-hZimp10融合蛋白,且制备了抗hZimp10抗体。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 a384 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 antihZimp10 antibody showed high titer（1∶100 000）and high spencificity in the LNCaP cells.Conclusion GSThZimp10 fusion protein is obtained successfully and the anti-hZimp10 antibody is prepared.

关 键 词：hZimp10 谷胱甘肽硫转移酶融合蛋白 多克隆抗体 

分 类 号：Q78[生物学—分子生物学] R392[医药卫生—免疫学]