摘要
采用RT-PCR技术扩增获得口蹄疫病毒(FMDV)细胞毒O/Akesu/58的非结构蛋白3D(NSP 3D)基因编码区,并定向克隆到pProexHTb原核表达载体上,再将重组pProex-3D转化至大肠杆菌BL21,经IPTG诱导,表达产物用亲和层析法纯化,经SDS-PAGE鉴定和Western-blot分析抗原性;以纯化的表达蛋白免疫豚鼠制备其多克隆抗体,ELISA测定抗体效价,间接免疫荧光法检测制备的豚鼠抗NSP 3D多克隆抗体与细胞毒天然抗原的反应原性.结果表明:表达的目的蛋白大小为53ku左右;ELISA结果表明,制备的多克隆抗体效价达1∶1 024以上;Western-blot分析表明,纯化后的蛋白能与FMDV感染动物血清发生特异性反应;间接免疫荧光检测发现,豚鼠抗3D蛋白多克隆抗体可与细胞毒天然抗原反应,与阴性对照未见反应.
NSP 3D gene coding region was obtained from the cytotoxin O/Akesu/58 by RT-PCR and cloned into prokaryotic expression vector pProexHTb. The recombinant expression plasmid pProex-3D was transformed into E. coli BL21 (DE3). The recombinant NSP 3 D was induced by IPTG, and purified by Ni-NTA His Bind Resin affinity chromatography. Then the size was identified by SDS-PAGE and the antigenicity was analyzed by western-blot. Guinea pigs were immunized to prepare polyclonal antibodies with expressed NSP 3D. The antisera was obtained and polyclonal antibody was characterized by ELISA. The reactiongenicity between polyclonal antibody from guinea-pig and natural antigens from FM- DV cultured in ceils was detected by indirect immunofluorescenee techniques. The results showed thatthe recombinant NSP 3D size was about 53 ku and the titers of polyclonal antibody was more than 1 " 1 024. The Western-blot result verified that purified NSP 3D could specifically react with bovine anti-ser- um prepared with the same serotype FMDV. Indirect immunofluorescence detection showed that poly- clonal antibody could reacted with natural antigens from FMDV cultured in cells. By contrast, there was no reaction in the negative control.
出处
《甘肃农业大学学报》
CAS
CSCD
北大核心
2012年第3期7-12,共6页
Journal of Gansu Agricultural University
基金
农业部转基因重大专项(2008ZX08011-004
2009ZX08007-008B
2009ZX08006-002B)
关键词
口蹄疫病毒
3D基因
非结构蛋白
反应原性分析
foot-and-mouth disease virus (FMDV)
3D gene
non-structural proteim reactivity analysis