摘要
根据2005年从北京某牛场分离得到的一株牛肠道病毒2型(BEV-2)毒株序列,设计特异性引物,采用RT-PCR方法扩增BEV-2包含VP1(840 bp)蛋白基因片段的VP1+(1 275 bp)序列,扩增产物克隆到p MD-T19,进行测序验证。验证该序列为目的片段后,将其克隆到表达载体p ET-32a。重组质粒转化至Rosetta,经IPTG诱导后,SDS-PAGE显示重组蛋白在大肠杆菌中高效表达。将重组蛋白作检测抗原包被酶标板,成功建立了检测牛肠道病毒2型抗体的间接ELISA方法。上述工作,为下一步BEV-2单克隆抗体的制备打下了基础。
The specific primers were designed based on gene sequence of bovine enterovirus type 2 strain isolated from cows in Beijing in 2005.The gene fragment of VP1＋（1 275 bp）sequence induding the whole VP1（840 bp）of bovine enterovirus type 2 was amplified by RT-PCR,and the PCR products were cloned into p MD-T19. Confirmed by sequence analysis,the sequence was then cloned into the expression vector p ET-32 a. The results of SDS PAGE showed that the recombinant protein was highly expressed in E.Coli after the recombinant plasmid was transformed into host bacteria Rosetta and induced by IPTG. An indirect ELISA for testing antibodies against BEV-2 was established using the recombinant protein as coating antigen. The work above sets foundation for the preparation of monoclonal antibodies against BEV-2.
出处
《中国兽医杂志》
CAS
北大核心
2014年第11期40-43,共4页
Chinese Journal of Veterinary Medicine
基金
国家质检总局科研项目"牛肠道病毒2型抗体及抗原ELISA检测技术研究"(2013IK026)
