鹅细小病毒野毒株VP3基因的原核表达及抗原性检测  

Prokaryotic expression of VP3 gene of goose parvovirus wild strain and detection of antigenicity of the expressed protein

作　　者：鞠环宇[1] 卫娜[1] 尚绪增[1] 荆志强[1] 郭鹭[1] 马波[1] 王君伟[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2009年第6期492-497,共6页Chinese Veterinary Science

基　　金：黑龙江省教育厅重大科技项目(10541Z004);黑龙江省科技攻关项目(GB01B503-02;GB04B504)

摘　　要：根据发表的鹅细小病毒(GPV)基因序列设计合成了2对检测GPV的特异性引物,用其对疑似小鹅瘟的病料进行PCR检测,经序列比对后确定为GPV感染;利用引物Gs/Ga扩增获得的GPV野毒株VP3基因,测序后将VP3基因连接到原核表达载体pET-30a上,获得重组质粒pET-30a-VP3,将其转化感受态菌Rosetta(DE3)pLysS中,经IPTG诱导,SDS-PAGE分析。结果显示,重组菌可表达出分子质量约为66 000 u的重组融合蛋白,表达的蛋白以包涵体形式存在于菌体中。表达产物经Ni2+亲和层析柱纯化,获得了纯度较高的目的蛋白。Western-blot分析结果显示,纯化的蛋白能与GPV阳性血清发生特异性反应,证实表达的蛋白具有较好的抗原性。Two pairs of primers were designed according to the published VP3 gene sequences of goose parvovirus（GPV）. The samples suspected with gosling plague were examined by PCR using the two pairs of primers. The PCR products were sequenced and analyzed, and the results indicated that the samples were infected by GPV. Then the VP3 gene of GPV was amplified by primers Gs and Ga from the samples and sequenced. The VP3 gene was cloned into the expression vector pET-30a. The recombinant plasmid was transformed into competent cells of Escherichia coli Rosetta（DE3） pLysS strain. The transformed bacteria were induced by IPTG and their expressed product was analyzed by SDS-PAGE and Western-blot. The results showed that a fusion protein of 66 ku in molecular mass existed in inclusion bodies. The protein purified by Ni^2＋ affinity chromatography purification system could react specifically with GPV positive serum by Western-blot,which revealed that the expressed protein had good immunoreactivity.

关 键 词：鹅细小病毒 VP3基因 克隆 原核表达 纯化 抗原性 

分 类 号：S852.659.2[农业科学—基础兽医学]