Gao Shan-dian, Chang Hui-yun, Du Jun-zheng, Shao Jun-jun, Cong Guo-zheng, Lin Tong, Han Xue-qing, Xie Qing-ge
Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, National FMD Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2010 Jul;26(7):631-4.
To induce the expression of structure protein VP1 and its C terminus of foot-and-mouth disease virus (FMDV) serotype SAT2 in E.coli and analyze their reactivities with FMDV positive antiserum.
The plasmid pGEM-SAT2P1 carrying the VP1 coding region of FMDV serotype SAT2 isolated from South African was used as template for RT-PCR to get the coding fragment of VP1 and its C terminus. The fragments were then cloned into expression vector pET-30a(+) to get recombinant plasmids pET-SAT2VP1 and pET-SAT2VP1C. The recombinant plasmids were transformed into E.coli BL21(DE3)pLysS and induced by IPTG to express VP1 and its C terminus protein. The expressed VP1 and its C terminus were then purified by Ni-NTA His Bind Resin affinity chromatography and analyzed by Western blot. New Zealand rabbits were immunized to prepare polyclonal antibodies against VP1 and VP1C. The antisera were obtained and polyclonal antibody was characterized by ELISA.
SDS-PAGE demonstrated that VP1and its C terminus expressed in the E.coli transformants had a molecular weight of 33000 and 19000 and contained in the inclusion body. Purified VP1 and its C terminus was obtained by Ni-NTA His Bind Resin affinity chromatography and a single clear band appeared in the SDS-PAGE gel. Western blot analysis showed that the purified VP1 and VP1 C terminus could react with bovine antiserum against the same serotype FMDV without cross-reactivity with the negative bovine serum.
Rabbit polyclonal antibodies against VP1 and VP1C were successfully prepared, the titers of which were above 1:12800 and had obvious specificity.
诱导口蹄疫病毒（FMDV）南非SAT2型结构蛋白VP1及其C端在大肠杆菌中表达，并分析其与FMDV阳性抗血清的反应性。
以携带从南非分离的FMDV SAT2型VP1编码区的质粒pGEM-SAT2P1为模板进行RT-PCR，获得VP1及其C端的编码片段。然后将片段克隆到表达载体pET-30a(+)中，得到重组质粒pET-SAT2VP1和pET-SAT2VP1C。将重组质粒转化到大肠杆菌BL21(DE3)pLysS中，用IPTG诱导表达VP1及其C端蛋白。表达的VP1及其C端经Ni-NTA His Bind Resin亲和层析纯化后，进行Western blot分析。免疫新西兰兔制备针对VP1和VP1C的多克隆抗体。获得抗血清并用ELISA对多克隆抗体进行鉴定。
SDS-PAGE显示，在大肠杆菌转化体中表达的VP1及其C端分子量分别为33000和19000，且包含在包涵体中。通过Ni-NTA His Bind Resin亲和层析获得纯化的VP1及其C端，SDS-PAGE凝胶上出现单一清晰条带。Western blot分析表明，纯化的VP1和VP1 C端能与针对同一血清型FMDV的牛抗血清反应，与阴性牛血清无交叉反应。
成功制备了兔抗VP1和VP1C多克隆抗体，其效价均高于1:12800，且具有明显特异性。
