HCCR重组蛋白的表达及其单克隆抗体的制备

Recombinant protein expression and monoclonal antibody preparation of HCCR

目的:克隆人宫颈癌基因(human cervical cancer oncogene,HCCR)的C末端截短序列(膜外区序列),重组表达并纯化HCCR-C端蛋白,制备和初步鉴定针对HCCR-C端蛋白的单克隆抗体。方法:从人肝癌细胞株HepG2中提取总RNA,RT-PCR扩增出HCCR-C端,构建其原核表达质粒,表达HCCR-C融合蛋白,以纯化的融合蛋白免疫BALB/c小鼠,常规细胞融合和选择性培养,亚克隆筛选出分泌特异抗体的杂交瘤细胞株,制备单抗腹水,Western blot、细胞组化初步鉴定抗HCCR-C端单克隆抗体。结果:成功克隆了HCCR-C末端截短序列,原核表达并纯化了HCCR-C端重组蛋白;经ELISA双筛,获得3株能稳定分泌抗HCCR-C端单克隆抗体的杂交瘤细胞株;Western blot及细胞免疫荧光结果显示,其分泌抗体效价高且均能特异识别HCCR蛋白。结论:成功制备并初步鉴定了针对HCCR-C端的单克隆抗体,为深入研究HCCR蛋白的生物学功能和建立新的肝细胞癌早期诊断方法奠定了可靠基础。

Objective：To clone human cervical cancer gene（HCCR） of the C-terminal truncated sequece（HCCR-C）,the recombinant protein of HCCR-C expressed and purified,the anti-HCCR-C monoclonal antibody（McAbs） prepared and identified.Methods：The HCCR-C gene was cloned from the cDNA which was amplified from human liver cancer cell line HepG2 by RT-PCR,then subcloned into the prokaryotic expression vector.Fusion protein of HCCR-C was expressed and purified and used for immunizing BALB/c mouse.Conventional hybridoma technology was used to produce hybridoma cells for preparation of McAbs.Anti-HCCR-C McAbs was detected by Western blot and cytochemistry.Results：The recombinant protein of HCCR-C was expressed and purified.The specific anti-HCCR-C McAbs that secreted by three hybridoma cell lines were identified by ELISA,Immunofluorescence and Western blot,which revealed that the secreted McAbs can recognize HCCR-C-protein specifically with high valence.Conclusion：HCCR sequence of C-terminal truncated were cloned.The anti-HCCR-C McAbs are successfully prepared by hybridoma technology with HCCR-C fusion protein expressed in E.coli and detected by Western blot and cytochemistry,Our study lay the foundation for the further study of the HCCR functions and establishment a new method of early diagnosis of HCC.