牛病毒性腹泻病毒E0蛋白的表达与多克隆抗体制备  

Expression of the E0 protein of bovine viral diarrhea virus and preparation of its polyclonal antibody

作　　者：王雪枝[1] 朱远茂[1] 史鸿飞[1] 严昊[1] 蔡红[1] 王姝[1] 马磊[1] 薛飞[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/大动物传染病研究室,哈尔滨150001

出　　处：《黑龙江畜牧兽医》2014年第3期4-8,207,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金："十二五"国家科技支撑计划子课题(2012BAD12B0 3-3);公益性行业(农业)科研专项经费(201003060-04);哈尔滨市科技攻关计划(2012AA6BN020)

摘　　要：为了制备BVDV的E0蛋白兔源多克隆抗体,试验采用大肠杆菌对E0蛋白进行了表达,根据已发表的BVDV的VEDEVAC株的全基因组序列设计1对扩增E0的引物,经RT-PCR扩增出长约680 bp的E0基因片段,然后将目的基因克隆到pET-30a载体上,经酶切鉴定获得阳性重组质粒后进行测序,再将测序正确的阳性重组质粒转化BL21表达菌,经扩大培养及IPTG诱导后获得以包涵体形式存在的重组E0蛋白,经亲和层析法纯化后进行Western-blot检测,同时以纯化的E0蛋白免疫新西兰大白兔制备多克隆抗体,并对E0多克隆抗体进行了中和试验和免疫荧光检测。结果表明:E0蛋白具有良好的反应性;E0多克隆抗体的病毒中和试验测定其中和抗体效价达到1∶128,具有良好的反应性和特异性。说明制备的重组E0蛋白多克隆抗体可以用于BVDV的检测。To prepare a rabbit polyclonal antibody against the E0 protein of bovine viral diarrhea virus （ BVDV）, the E0 gene was expressed with E. coll. A pair of specific primers was designed for amplifying the E0, according to the published whole - genome sequence of VEDEVAC strain. A fragment of about 680 bp from the E0 gene was amplified by RT - PCR, and then the target gene was cloned into the pET - 30a vec- tor. The positive recombinant plasmids were obtained by restriction enzyme digestion, and then were sequenced. The identified recombinant plasmids were transformed into E. coli BL21. The recombinant E0 protein in inclusion body form was obtained after expanding culture and in- duction with IPTG. The recombinant E0 protein was analyzed by western blotting after purification with affinity chromatography. The purified re- combinant EO protein was used to immunize of New Zealand rabbits for preparing polyclonal antibody against the E0 protein. The neutralization and immunofluorescence assays were used to determine the polyclonal antibody. The result showed the recombinant E0 protein had good reactivi- ty. The results of the neutralization assay for the polyclonal antibody against the recombinant E0 protein showed that the neutralizing antibody ti- ter against BVDV reached 1：128. The results for immunofluorescence assay and Western blotting showed that the polyclonal antibody against the recombinant E0 protein also had good reactivity and specificity. The results indicate that the prepared polyclonal antibody against the recombi- nant E0 protein can be used for the detection of BVDV.

关 键 词：牛病毒性腹泻病毒 E0蛋白 家兔 多克隆抗体 RT—PCR Western—blot IFA 

分 类 号：S852.653[农业科学—基础兽医学]