摘要
文章通过RT-PCR方法扩增传染性支气管炎病毒(IBV)HH06株M全长基因,经抗原性和亲水性分析,将M部分序列成功亚克隆于pET-30a(+)和PVAX1载体中。将阳性重组质粒pET30a-M转化E.coli Rosetta(DE3)感受态细胞,诱导表达获得20 ku重组M蛋白。Western blot检测表明,重组蛋白能够与IBV参考阳性血清反应。以纯化重组M蛋白免疫新西兰白兔制备多克隆抗体,其ELISA效价达到1:218;Western blot结果证实,其具有良好的反应性和特异性。间接免疫荧光试验表明,抗M蛋白多克隆抗体可检测到PVAX-M1转染BHK-21细胞表达的M蛋白,与IBV HH06毒株具有很好的特异性反应。病毒感染抑制试验表明,多抗血清对IBV Beaudette株的抑制率可达到25.9%。为IBV检测及M蛋白功能的研究奠定基础。
In this study, the complete IBV HH06 M gene was cloned and its partial gene was subcloned into prokaryotic expression vector pET-30a （＋） and eukaryotic expression vector PVAX1 according to hydrophilicity plot and antigenic index analysis results. The recombinant plasmid of pET30a-M was transformed into E. coli Rosetta （DE3） and induced with IPTG, the molecular weight of recombinant M protein is 20 ku. The recombinant M protein could be recognized by positive IBV antisera in Western blot. Then the purified recombinant M protein was used as antigen for immunization of rabbit to prepare anti-M polyclonal antibody. Indirect ELISA showed that the titer of anti-M polyclonal antibody was 1？？218, and it had highly reactivity and specialty in Western blot. Furthermore, IFA test demonstrated that this polyclonal antibody could react with BHK-21 cells transfected with PVAX-M1 plasmid and IBV-infected Vero cells. The pathogenic effect of Vero cells infected with IBV Beaudette strain could be inhibited by anti-M polyclonal antibody and the inhibition rate was up to 25. 9%. The rabbit anti-M protein polyclonal antibody obtained in this study laid a foundation for further functional research for M protein and detection of IBV.
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2014年第10期57-63,共7页
Journal of Northeast Agricultural University
基金
国家自然科学基金项目(31172295
31272569)
