摘要
利用RT-PCR和SOE PCR技术扩增得到猪繁殖与呼吸综合征病毒(PRRSV)HH08株NSP2全长基因,经抗原性和亲水性分析,将NSP2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-NSP2转化E.coli Rosetta(DE3)感受态细胞,经诱导表达获得107ku的重组NSP2蛋白。Western-blot检测表明,重组蛋白能够与PRRSV参考阳性血清反应。以纯化的重组NSP2蛋白免疫新西兰白兔制备多克隆抗体,其ELISA效价达到1∶1015以上;Western-blot试验表明,其具有良好的反应性和特异性。间接免疫荧光试验显示,用抗NSP2多克隆抗体可以检测到PVAX-NSP2转染BHK-21细胞所表达的NSP2蛋白,且与经典型和高致病型PRRSV毒株均有很好的特异性反应。病毒感染抑制试验表明,多抗血清对PRRSV经典和高致病性毒株的抑制率可达到68%和53%。上述研究结果为PRRSV检测及NSP2蛋白功能的深入研究奠定了基础。
In this study,the complete PRRSV HH08 NSP2 gene was cloned and the partial gene subcloned into prokaryotic expression vector pET-30a（2c） and eukaryotic expression vector PVAX1 after hydrophilicity plot and antigenic index analysis. E. coli Rosetta （DE3） was transformed with the recombinant plasmid pET30a-NSP2. The recombinant NSP2 protein that molecular weight is 107 ku was expressed. It could be recognized by specific PRRSV antisera in western blot. Then the purified recombinant NSP2 protein as antigen was used to immunize rabbit for preparation of anti-NSP2 polyclonal antibody. An indirect ELISA assays showed that the titer of anti-NSP2 polyclonal antibody was 1 ： 101^15 ,and it had highly reactivity and specialty in Western-blot. Also,IFA test demonstrated that this polyclonal antibody could react with the BHK-21 cells which can express PRRSV NSP2 protein and the Marc-145 cells infected with PRRSV. Both attenuated and highly pathogenic PRRSV strains could be inhibited by the anti-NSP2 polyclonal antibody and the inhibition rates were 68% and 53% respectively. The rabbit anti-NSP2 protein polyclonal antibody obtained in this study laid a foundation for further functional research for NSP2 protein and detection of PRRSV.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2013年第4期377-383,共7页
Chinese Veterinary Science
基金
黑龙江省自然科学基金项目(ZJN0702-01)
国家自然科学基金项目(31172295
31272569)
作者简介
马玲（1988-），女，山东潍坊人，硕士生，E—mail：liuqing79135@126．com。
通信作者，教授，博士生导师，研究方向为动物病理学，Tel：0451-55190723，E-mail：ligx@neau．edu．cn，1iguangxingl968@hotmail．com
