猪结合珠蛋白基因的克隆、原核表达及单克隆抗体制备

Gene cloning and prokaryotic expression of porcine haptoglobin and preparation of monoclonal antibodies

本研究的目的在于利用重组猪结合珠蛋白(Haptoglobin,Hp)制备特异性单克隆抗体。以梅山猪肝脏总RNA为模板,利用RT-PCR方法扩增猪hp基因,将其克隆到原核表达载体pGEX-KG和pET-32a上,转化至E.coliBL21中,IPTG诱导下高效表达重组GST-Hp和HIS-Hp蛋白。用纯化的重组GST-Hp免疫BALB/c小鼠,用纯化的HIS-Hp作为包被抗原进行间接ELISA检测,通过淋巴细胞杂交瘤技术筛选阳性细胞株,制备单克隆抗体。结果表明筛选到2株高效分泌抗猪Hp单克隆抗体的杂交瘤细胞株,命名为3B4和3C8。Western-blot和间接ELISA检测均表明制备的单克隆抗体具有良好的特异性。

The aim of this study was to prepare the high-specific anti-porcine haptoglobin（Hp）monoclonal antibodies using the recombinant expressive porcine Hp protein.The total RNA of Meishan porcine liver was extracted and the porcine Hp gene was amplified by RT-PCR.Then Hp gene was cloned into the prokaryotic expression vector pGEX- KG and pET-32a.The recombinant expression vectors were transformed into E.coli BL-21and induced by IPTG for the expression of the recombinant GST-Hp and HIS-Hp.The purified recombinant GST-Hp protein was used to immunize the Balb/c mice.The purified recombinant HIS-Hp protein was used to detect the anti-Hp antibody.The anti-Hp monoclonal antibody was obtained by screening the positive hybridoma cell lines using the hybridoma tech- nique.Results showed that two hybridoma cell lines efficiently secreting monoclonal antibody against porcine hp were established,named as 3B4and 3C8.And high specificity of monoclonal antibodies were detected by Western-blot and indirect ELISA.