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| 口蹄疫病毒非结构蛋白3D原核表达、纯化和反应原性分析 | |
| 引用本文： | 骆继怀,杨利恒,高闪电,独军政,常惠芸,薛慧文. 口蹄疫病毒非结构蛋白3D原核表达、纯化和反应原性分析[J]. 甘肃农业大学学报, 2012, 47(3): 7-12 |
| 作者姓名： | 骆继怀 杨利恒 高闪电 独军政 常惠芸 薛慧文 |
| 作者单位： | 1. 甘肃农业大学动物医学院,甘肃兰州730070;中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,国家口蹄疫参考实验室,甘肃兰州730046 2. 甘肃农业大学动物医学院,甘肃兰州,730070 3. 中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,国家口蹄疫参考实验室,甘肃兰州730046 |
| 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. |
| 关 键 词： | 口蹄疫病毒 3D基因 非结构蛋白 反应原性分析 |
Prokaryotic expression,protein purification and reactivity analysis of non-structural protein 3D from foot-and-mouth disease virus |
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| LUO Ji-huai , YANG Li-heng , GAO Shan-dian , DU Jun-zheng , CHANG Hui-yun , XUE Hui-wen. Prokaryotic expression,protein purification and reactivity analysis of non-structural protein 3D from foot-and-mouth disease virus[J]. Journal of Gansu Agricultural University, 2012, 47(3): 7-12 | |
| Authors: | LUO Ji-huai YANG Li-heng GAO Shan-dian DU Jun-zheng CHANG Hui-yun XUE Hui-wen |
| Affiliation: | 1(1.College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;2.State Key Laboratory of Veterinary Etiological Biology,National FMD Reference Laboratory,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China) |
| Abstract: | 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 3D 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 FMDV cultured in cells was detected by indirect immunofluorescence techniques.The results showed that the 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-serum prepared with the same serotype FMDV.Indirect immunofluorescence detection showed that polyclonal antibody could reacted with natural antigens from FMDV cultured in cells.By contrast,there was no reaction in the negative control. |
| Keywords: | foot-and-mouth disease virus(FMDV) 3D gene non-structural protein reactivity analysis |
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