【摘要】 目的利用原核表达系统表达重组肿瘤型丙酮酸激酶(TU M2-PK),并制备TU M2-PK的单克隆抗体(mAb)。方法提取卵巢癌细胞株SKOV3的RNA,通过逆转录、PCR扩增TU M2-PK基因,将其分别与带有His标签的pET-32a及GST标签PGEX-4T-1载体连接,转化DH5α菌进行基因克隆并测序鉴定。进一步构建表达体系,用异丙基β-D-硫代半乳糖苷(IPTG)诱导,最终在BL21菌中表达TU M2-PK融合蛋白(分别含有His、GST-Tag),并进行SDS-PAGE及Western blot鉴定。获得的蛋白经纯化后作为免疫原及检测原,进行杂交瘤细胞的筛选。采用间接ELISA法测小鼠血清及细胞上清TU M2-PK抗体效价,Western blot分析mAb特异性。结果成功构建pET-32a-TU M2-PK、pGEX-4t-1-TU M2-PK工程质粒,获得两种标签的融合蛋白。将重组蛋白纯化后作为免疫原免疫小鼠,筛选获得两株稳定分泌TUM2-PK抗体的杂交瘤细胞株。分别命名为6A11、7C11,Western blot实验显示,两株均可以与重组蛋白特异性结合,并且能识别子宫颈癌细胞株(Hela)、胃腺癌细胞(SGC-7901)、肝癌细胞(HepG2)中的天然蛋白。结论成功制备了TU M2-PK蛋白及其单克隆抗体,为进一步开发TU M2-PK诊断试剂打下基础。更多还原

【Abstract】 Objective To express recombinant tumor M2-pyruvate kinase protein(TU M2-PK)using prokaryotic expression system,and to prepare monoclonal antibodies(mAb) against TU M2-PK.Methods RNA was extracted from human ovarian carcinoma cell strain(SKOV3) and reverse transcribed by PCR amplification.TU M2-PK gene was inserted into vectors pET-32a of His tag and PGEX-4T-1 of GST Tag,and cloned into DH5α.The recombinant plasmids were transformed into E.coli BL21 cells,and protein expression(including His-Tag and GST-Tag respectively) was induced by IPTG and the expressed proteins were identified by SDS-PAGE and Western blot.BALB/c mice were immunized with these purified recombinant proteins and hybridomas were got by cell fusion.Antibodies in the serum of mice and the cell culture supernatant were detected using ELISA.Results Recombinant plasmids pET-32a-TU M2-PK and pGEX-4t-1-TU M2-PK were constructed successfully.After expression,purification,immunization of BALB/c mice and cell fusion,two hybridoma cell strains,6A11 and 7C11,were obtained.Western blot showed that antibodies secreted by the two strains bound to the recombinant protein,as well as proteins extracted from cell strains of cervical cancer cell(Hela),gastric carcinoma cell(SGC-7901),and hepatoma(HepG2).Conclusion Monoclonal antibodies against recombinant TU M2-PK protein are prepared successfully,which lays a foundation for further development of TU M2-PK diagnostic reagents.更多还原