CAJ | 학술논문

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。
목적：재대장간균중표체Clusterin단백，병제비기토항Clusterin단백적다극륭항체。방법：종MCF-7세포제취mRNA ，역전록위cDNA ，이차위모판PCR확증Clusterin편마기인；장Clusterin편마구cDNA삽입적pRSET-A원핵표체재체，구건pRSET-A-clusterin ；장중조질립전화BL21（DE3）감수태세균，이IPTG유도6× His-Clusterin융합단백적표체，경친화순화주여응효주진행층석순화。경SDS-PAGE화Western-blot감정후，장순화적융합단백여불씨좌제혼합제비성유포수현액，상규면역신서란대백토제비다항，Western-blot검측해항체식별내원성clusterin적특이성。결과：성공구건료Clusterin단백적원핵표체재체pRSETA-clusterin ；경표체병순화적Clusterin단백순도체도98％이상，면역신서란대백토후제비득도료특이성적항Clusterin적다항。결론：성공지표체병순화료Clusterin단백，병제비료특이성항Clusterin다항。
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 .