鸭肠炎病毒gB N端(60～110aa)抗原优势区的原核表达及抗血清的制备  

Prokaryotic expression and antiserum preparation of N-terminal antigenic dominant region in glycoprotein B(60-110aa) of duck enteritis virus

作　　者：曹春秋[1] 张树栋[1] 刘琪[1] 董井泉 高明春[1] 张文龙[1] 许丽[2] 于长泳[3] 马波[1] 王君伟[1] 

机构地区：[1]东北农业大学动物医学学院,辽宁沈阳110164 [2]东北农业大学动物科技学院,黑龙江哈尔滨150030 [3]辽宁省动物疫病预防控制中心,辽宁沈阳110164

出　　处：《中国兽医科学》2015年第9期888-895,共8页Chinese Veterinary Science

基　　金：2013年度黑龙江省博士后资助项目(LBH-Z13021);高等学校博士学科点专项科研基金资助项目(20102325110004);东北农业大学大学生创新创业训练项目(2013029)

摘　　要：以本实验室已克隆的鸭肠炎病毒（DEV）C-KCE株ul27基因序列为模板,设计特异性引物,PCR扩增获得编码鸭肠炎病毒gB N端第60～110aa的目的基因片段,大小为150bp。将目的基因亚克隆至原核表达载体pET-32a（＋）中,阳性质粒被命名为pET-32a（＋）-gB-1。经IPTG诱导,SDS-PAGE分析表明,重组蛋白以包涵体形式表达,大小约为27ku。用Ni-NTA柱亲和层析获得纯化的重组蛋白,鸭抗DEV阳性血清经间接ELISA检测证实其具有抗原性,并以此为免疫原免疫家兔制备抗血清,经间接ELISA检测,抗血清效价为1∶25 600。应用间接免疫荧光及Western-blot分析制备的抗血清的反应性,均证实抗血清可特异性识别DEV gB蛋白。分别以重组蛋白和DEV超离病毒为检测抗原进行间接ELISA,检测DEV免疫鸭的抗体消长规律,结果显示两者的抗体变化趋势一致。以上结果表明,制备的抗血清可作为DEV检测的候选试剂,表达的重组蛋白可作为DEV抗体检测的诊断抗原。Specific primers for duck enteritis virus（DEV）C-KCE strain ul27 gene partial sequences were designed according to the gene sequence cloned in our laboratory.The antigenic domain region of DEV gB gene（180-330bp）were obtained by PCR.The positive recombinant plasmid named pET-32a（＋）-gB-1was expressed in prokaryotic expression system pET-32a（＋）/Rosetta（DE3）pLysS.Then the recombinant protein was induced with IPTG and SDS-PAGE analysis showed that the target protein was mainly expressed as inclusion bodies and its molecular weight was about 27 ku.Recombinant protein was purified by Ni-NTA column affinity chromatography,and ELISA analysis of the recombinant protein with duck anti-DEV positive serum showed antigenicity.The purified recombinant protein was immunized with rabbit.Indirect ELISA analysis showed that the titer of the antiserum was 1∶25 600.Western-blot and indirect immunofluorescence analysis confirmed that the antiserum not only reacted with the recombinant gB-1but also with the gB,which was expressed in DEV infected CEF specifically.The similar serum dynamics were detected by the recombinant protein and DEV as the detection antigen with the indirect ELISA.All results revealed the antiserum can be used as a candidate reagent to detect DEV and the recombinant protein can be used as a diagnostic antigen to detect the serum of DEV.

关 键 词：鸭肠炎病毒 GB 原核表达 抗血清 

分 类 号：S852.659.1[农业科学—基础兽医学]