摘要
为获得具有天然构象及良好抗原性的犬瘟热病毒(CDV)血凝蛋白(H),本研究以HLJ2-07毒株为模板,利用RTPCR方法扩增出H基因,将其克隆到pFastBacTMHTA载体中,利用大肠杆菌DH10BacTM同源重组构建穿梭质粒reBACmidrH,将重组穿梭质粒转染Sf9昆虫细胞,进行杆状病毒表达。利用Western blotting对获得的重组蛋白进行鉴定及抗原性分析。同时,利用在线网站与软件对CDV H蛋白进行B细胞表位分析预测。结果显示,在杆状病毒系统中表达的H蛋白能与His-tag单克隆抗体及CDV阳性血清发生特异性反应,表明该蛋白成功表达,且具有良好的反应原性;用Swiss-model同源建模软件及B细胞表位预测软件,预测H蛋白可能具有的B细胞表位有5处,分别是175—195、240—250、365—380、480—508及526—555。本研究获得了具有天然构象及良好抗原性的CDV重组H蛋白,可为诊断试剂开发、单克隆抗体制备及表位筛选等工作奠定基础。
To maintain the native conformation and antigenicity of hemagglutinin（H）of canine distemper virus,the H gene which referred to HLJ2-07 strains was accurately amplified by RT-PCR and cloned into pFastBacTMHTA vector.The recombinant plasmid was successfully transformed into E.coli DH10 BacTM competent cell and constructed shuttle vector,which was transfected into Sf9 cell to save baculovirus expressing destined protein.To determine the antigenicity of recombinant protein,Western blotting analysis was used to detect the interesting protein.Furthermore,the identification and characterization of B cell epitopes were predicted and analyzed by related software and online websites.The result demonstrated that the fusion protein generated by recombinant baculovirus was reacted with both anti-His tag monoclonal antibodies and canine distemper virus positive serum by Western blotting test,which showed that this protein had sufficient antigenicity.In addition,B cell epitopes were selected by between Swiss-model and online websites for consistent sequences to improve predictive accuracy,whereas the possible sites were 175 to 195,240 to 250,365 to 380,480 to 508 and 526 to 555.These efforts served as a strong foundation for the development of diagnostic kits,the production of monoclonal antibodies and selection of protein epitopes.
出处
《中国畜牧兽医》
CAS
北大核心
2014年第8期25-29,共5页
China Animal Husbandry & Veterinary Medicine
基金
国家科技支撑计划(2013BAK11B01-31)
关键词
犬瘟热病毒
血凝蛋白
杆状表达系统
B细胞表位
canine distemper virus hemagglutinin protein baculovirus expression system B cell epitopes
