摘要
为制备抗小反刍兽疫病毒(PPRV)Nigeria75/1株P蛋白的单克隆抗体并获得其单链抗体(ScFv)基因,利用纯化的PPRV和带有GST标签的重组P蛋白免疫BALB/c小鼠,制备抗PPRV P蛋白的单克隆抗体(m Ab),从分泌抗PPRV P单克隆抗体的杂交瘤细胞中提取总RNA,扩增VH基因和VL基因,采用重叠延伸PCR(SOE-PCR)法获得单链抗体全长基因,并克隆至原核表达载体p ET32a(+)中,再将构建的重组质粒p ET32a-ScFv-3A6转化大肠杆菌BL21(DE3),诱导单链抗体重组蛋白表达。结果,获得了4株能稳定分泌抗PPRV P蛋白的单克隆抗体杂交瘤细胞株3A6、1P4、3F8和1P1,对m Ab 3A6进行了Western-blot鉴定和间接免疫荧光试验(IFA)。成功扩增了杂交瘤细胞株3A6的ScFv基因,获得了ScFv-3A6重组蛋白,大小为47 ku,证实该重组蛋白具有较强的抗原结合活性。上述研究结果表明,本研究获得了4株能稳定分泌抗PPRV P蛋白的单克隆抗体杂交瘤细胞株,并成功扩增到杂交瘤细胞株3A6的ScFv基因,实现了原核表达,为PPR防控新措施的研究奠定了基础。
The present study aimed at preparation of monoclonal antibody against peste des petits ruminants virus（PPRV） P protein and obtained the relative single chain variable fragment（ScFv） genes,providing a possibility to better understand the pathogenesis mechanism of PPRV disease and to develop the diagnostic methods and therapeutic ScFv antibodies laying the foundation in the future.The purified PPRV and recombinant P protein were injected into BALB/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3A6 which secreted anti-PPRV P protein m Ab with Trizol reagent,and then the ScFv gene was constructed by SOE-PCR（splicing by overlap extension）. The recombinant ScFv gene was cloned into p ET32a（＋） vector.The p ET32a-ScFv-3A6 plasmid was transferred into E.coli BL21（DE3） and the expression of the recombinant protein was induced. In result,four cell lines producing monoclonal antibody against PPRV P protein were acquired and named as 3A6,1P4,3F8 and 1P1.The m Ab 3A6 was identified by Western-blotting and IFA.The recombinant ScFv gene was constructed. The expression of recombinant fusion ScFv protein p ET32a-ScFv-3A6 was about 47 ku. In conclusion,the four cell lines stably producing monoclonal antibody specifically bound to PPRV P protein were obtained,and the relative ScFv gene was constructed and successfully expressed in E.coli.These results may play an important role to explore the basis for the prevention and control of PPR in future.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2016年第10期1226-1232,共7页
Chinese Veterinary Science
基金
国家自然科学基金资助项目(31172342)(31472203)
国家科技支撑计划项目(2013BAD12B05)
关键词
小反刍兽疫病毒
P蛋白
单链抗体
原核表达
peste des petits ruminants virus
P protein
single chain variable fragment
prokoryotic expression
