鸭坦布苏病毒E基因主要抗原域的原核表达及单克隆抗体的制备  

Preparation of monoclonal antibody against the main antigen domain of E protein from duck Tembusu virus

作　　者：孙涛[1] 王超[2] 徐彪[1] 王宏华 邓明俊[1] 郑小龙[1] 岳志芹[1] 

机构地区：[1]山东出入境检验检疫局,山东青岛266002 [2]泰安出入境检验检疫局,山东泰安271000 [3]青岛蔚蓝生物制品有限公司,山东青岛266000

出　　处：《中国预防兽医学报》2016年第6期496-499,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家质检总局科研项目(2013IK038)

摘　　要：为制备鸭坦布苏病毒(DTMUV)E基因主要抗原域的单克隆抗体(MAb),本研究经DNAStar软件分析预测E蛋白含有的优势抗原表位区,以DTMUV QD株基因组RNA为模板,通过RT-PCR扩增涵盖编码主要优势表位的E基因片段(943 bp^1 287 bp),并将其克隆至表达载体p ET-28a中,转化于大肠杆菌中进行诱导表达。对表达的重组蛋白进行western blot鉴定,将纯化后的重组蛋白免疫BALB/c小鼠,取其脾脏细胞与SP2/0细胞融合,经筛选和鉴定,获得了两株能够稳定分泌抗E蛋白的杂交瘤细胞株,命名为4G8和6B12,其亚型均为Ig G1型,并且识别于不同的抗原位点。经间接免疫荧光检测,MAb 4G8和6B12均能够与DTMUV发生特异性反应。本研究所制备的MAb为进一步鉴定DTMUV以及分析囊膜糖蛋白E的结构和功能奠定了基础。To prepare monoclonal antibodies （MAb） against duck Tembusu virus （DTMUV）, the sequence encoding main antigen region （943 bp to 1,287 bp） of DTMUV E gene was amplified by RT-PCR and cloned into the pET-28a for expression in Escherichia coil, and the truncated recombinant protein containing the domain epitopes of DTMUV E protein was expressed under the induction of IPTG, which was purified and immunized into BALB/c mice, and then the two MAbs 4G8 and 6B12 secreting anti-E protein antibodies were prepared from the fusion cells, which isotype was IgG1. Western blot and IFA showed that both MAbs 4G8 and 6B12 were able to react with DTMUV. The prepared MAbs would facilitate to further study the structure and function of the DTMUV glycoprotein E.

关 键 词：鸭坦布苏病毒 抗原域 原核表达 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]