Clusterin 蛋白在大肠杆菌中的表达及其多抗制备

Clusterin 蛋白在大肠杆菌中的表达及其多抗制备
潘鹭翔;魏莉;杨敬;吕欣;董阳超;徐志凯;雷迎峰
【期刊名称】《医学理论与实践》
【年(卷),期】2013(000)023
【摘要】目的：在大肠杆菌中表达Clusterin蛋白，并制备其兔抗Clusterin蛋白的多克隆抗体。

方法：从MCF-7细胞提取mRNA ，逆转录为cDNA ，以此为模板PCR扩增Clusterin编码基因；将Clusterin编码区cDNA插入的pRSET-A原核表达载体，构建pRSET-A-clusterin ；将重组质粒转化BL21（DE3）感受态细菌，以IPTG诱导6× His-Clusterin融合蛋白的表达，经亲和纯化柱与凝胶柱进行层析纯化。

经SDS-PAGE和Western-blot鉴定后，将纯化的融合蛋白与弗氏佐
剂混合制备成油包水悬液，常规免疫新西兰大白兔制备多抗，Western-blot检测该抗体识别内源性clusterin的特异性。

结果：成功构建了Clusterin蛋白的原核
表达载体pRSETA-clusterin ；经表达并纯化的Clusterin蛋白纯度达到98％以上，免疫新西兰大白兔后制备得到了特异性的抗Clusterin的多抗。

结论：成功地表达并纯化了Clusterin蛋白，并制备了特异性抗Clusterin多抗。

%Objective :To express and purify clusterin protein in the E .coli ,prepare rabbit polyclonal antibody against clusterin protein .Methods :The full lenghth clusterin
gene was amplified from MCF-7 cell .This fragment was cloned in-to pRSET-A by T4 ligase and transformed into E .coli .DH5ɑ,then the recombinant plasmid named pRSETA-clusterin was constructed successfully .The E .coli strain BL21 (DE3) was transformed by expression plasmid pRSETA-clus-terin .IPTG was then added to a final concentration
of 1 mM to induce expression of 6 × His-clusterin fusion protei .The purified clusterin was got through affinity chromatographic resin and Superdex TM 75 resin .After identification of the purified protein by Western-blot analysis using anti 6-His tag antibody .The polyclonal antibody was prepared from the New Zealand rabbit immunized with the purified clusterin and freund’s adjuvant mixture .Finally ,the specific recogni-tion of endogenous clusterin in MCF-7 cells by rabbit ployclonal antibody was performed by western blot analysis .Re-sults :The prokaryotic expression vector pRSETA-clusterin was successfully constructed .The protein purity of the puri-fied clusterin was over 98% .The purified clusterin is inoculated to New Zealand rabbits three times and the polyclonal antibody against clusterin with high titer was
prepared .Furthermore ,the polyclonal antibody can recongnized the en-dogenous clusterin protein in MCF-7 cells .Conclusion:The recombinant clusterin protein was successfully expressed and purified .The specific polyclonal antibodies against clusterin was prepared .
【总页数】4页(P3081-3083,3097)
【作者】潘鹭翔;魏莉;杨敬;吕欣;董阳超;徐志凯;雷迎峰
【作者单位】第四军医大学基础部微生物学教研室，陕西省西安市 710032;第四军医大学西京医院妇产科;第四军医大学基础部微生物学教研室，陕西省西安市710032;第四军医大学基础部微生物学教研室，陕西省西安市 710032;第四军医大学基础部微生物学教研室，陕西省西安市 710032;第四军医大学基础部微生物学
教研室，陕西省西安市 710032;第四军医大学基础部微生物学教研室，陕西省西安市 710032
【正文语种】中文
【中图分类】R378.2+1
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