猪瘟病毒N^(pro)蛋白的原核表达与多抗制备  

PROKARYOTIC EXPRESSION OF N^(pro) PROTEIN OF CLASSICAL SWINE FEVER VIRUS AND PREPARATION OF POLYCLONAL ANTIBODIES

作　　者：王鑫[1,2] 武专昌[2] 魏建超[2] 赵秋华 史子学 刘珂[2] 邵东华[2] 李玉明[2] 邱亚峰[2] 马志永[2] 

机构地区：[1]临沂大学生命科学学院,临沂276000 [2]中国农业科学院上海兽医研究所,上海200241 [3]上海市闵行区动物疫病预防控制中心,上海201109 [4]上海市嘉定区农业委员会,上海201800

出　　处：《中国动物传染病学报》2015年第6期7-12,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学青年基金(31302132);上海市科技兴农重点攻关项目(沪农科攻字(2013)第5-6号);公益性行业(农业)科研专项(201303045)

摘　　要：为更好研究猪瘟病毒(Classical swine fever virus,CSFV)N^(pro)蛋白的生物学功能,RT-PCR扩增CSFV的N^(pro)基因,克隆至p Cold I质粒并转化BL21(DE3)表达菌。经IPTG诱导,成功表达了约24 k Da的重组蛋白His-N^(pro),用镍离子亲和层析柱进行纯化后,制备了兔源多克隆抗体。Western blot结果显示,制备的多克隆抗体能特异性识别猪瘟病毒感染PK15细胞内表达的约23k Da的N^(pro)蛋白,但此抗体不能用于N^(pro)蛋白的间接免疫荧光(indirect immunofluorescence assay,IFA)检测。纯化的重组蛋白His-N^(pro)和制备的N^(pro)多抗为N^(pro)蛋白功能研究奠定了基础。To better understand the biological function of N^pro protein of Classical swine fever virus（CSFV）,N^pro gene amplified in RTPCR was inserted into pCold I plasmid and then transformed into E.coli BL21（DE3）.Following induction with IPTG,approx 24 kDa recombinant protein His-N^pro was expressed and purified using Ni-NTZ resin.Afterwards,specific polyclonal antibodies were prepared by immunizing rabbit with purified His-N^pro protein.The N^pro protein was detected in CSFV-infected PK15 cells in Western blot,but not in indirect immunofluorescence assay.In conclusion,the availability of recombinant protein His-N^pro and polyclonal anti-N^pro antibodies has established foundation for further study of N^pro biological function.

关 键 词：猪瘟 N^pro 原核表达 多克隆抗体 

分 类 号：S852.659.5[农业科学—基础兽医学]