Clusterin蛋白在大肠杆菌中的表达及其多抗制备

Expression of Clusterin Protein in the E.coli and Preparation of Its Polyclonal Antibody

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. DH5a, 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 SuperdexTM 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- suits：The prokaryotic expression vector pRSETA-clusterin was successfully constructed. The protein purity of the puri- fied clusterin was over 98~. The purified elusterin is inoculated to New Zealand rabbits three times and the polyclonal antibody against clusterin with high titer was prepared. Furthermore, the polyclonal antibody can reeongnized the en- dogenous clusterin protein in MCF-7 cells. Conclusion： The recombinant elusterin protein was successfully expressed and purified. The specific polyclonal antibodies against clusterin was prepared.