HBV前S2蛋白单克隆抗体的制备和生物学特性的鉴定  

Preparation and Biology Characterization of Monoclonal Antibody Against Pre S2 Protein of HBV

作　　者：赖梅梅[1] 王雪梅[1] 张君[1] 蔡雪飞[1] 郑健[1] 黄爱龙[1] 

机构地区：[1]重庆医科大学附属第二医院病毒性肝炎研究所感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《生物技术通报》2008年第3期76-80,共5页Biotechnology Bulletin

基　　金：国家自然科学基金资助(No.30400374)

摘　　要：目的克隆表达HBV前S2蛋白,用纯化的重组蛋白GST-S2免疫BALB/C小鼠,通过杂交瘤技术制备抗HBV前S2蛋白的单克隆抗体(mAb),并鉴定其特性。方法利用含双拷贝乙肝病毒adw2亚型基因全序列的质粒pEcob6,经PCR方法扩增出HBV前S2片段,在GST表达系统中表达,表达产物用谷胱甘肽亲和层析纯化后,用于免疫BALB/c小鼠,采用杂交瘤技术制备抗前S2蛋白的mAb,采用间接ELISA方法和Western blot进行mAb特异性鉴定;同时采用间接ELISA方法鉴定mAb相对亲和力。结果获得一株可分泌特异性mAb的杂交瘤细胞7A6;相对亲和力均在107以上。Western blot显示该株mAb能特异识别重组前S2蛋白。结论成功地制备出抗HBV前S2蛋白的一株mAb。Objective To prepare monoclonal antibody （mAb）against Pre S2 protein of HBV. Method：The pre S2 was amplified with PCP,. from the pEcob6 plasmid contained the genomic-length cDNA of the subtype adw2 of the double copies HBV,and was inserted to the multi-cloning sites of plasmid pGST vector and expressed with induction of IPTG. After purification used Glutathione-Sepharose-4B,BALB/c mice were immunized with the recombinant Pre S2 protein. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line SP2/0 cells. The hybridoma cells that secreted anti-Pre S2 protein mAbs were cloned with limited dilution method. The affinity of the obtained mAbs were determined by indirect ELISA. The specificity of mAbs was tested by ELISA and Western blot analysis. Results From over 180 positive hybridomas which secreted anti-PreS2 protein mAbs,a pair of hybridomas were screened out,designated 7A6 and 4B8. chromosome analysis revealed that the obtained hybridomas were with the universal characteristics of the monoclonal hybridoma cells which secreted mAb. The relative affinity constants of 7A6 and 4B8 were both determined as 107. Conclusions：A pair of high titer,specific mAbs against Pre S2 protein have been successfully prepared and primarily identified.

关 键 词：乙肝病毒 前S2蛋白 单克隆抗体 生物学特性 

分 类 号：Q78[生物学—分子生物学]