新型鸭细小病毒重组VP2蛋白的原核表达及其间接ELISA方法的建立  

Prokaryotic expression of recombinant VP2 protein of novel duck Parvovirus and the establishment of indirect ELISA method

作　　者：刘铭 袁万哲[1] 刘聚祥[1] LIU Ming;YUAN Wanzhe;LIU Juxiang(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北农业大学动物医学院,河北保定071000

出　　处：《黑龙江畜牧兽医》2021年第5期85-91,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：河北省农业关键共性技术攻关专项(18227517D);河北省现代农业产业技术体系蛋肉鸡产业创新团队项目(HBCT2018150210)。

摘　　要：为了建立新型鸭细小病毒(novel duck Parvovirus, NDPV)感染鸭群的抗体检测方法,试验采用PCR方法扩增新型鸭细小病毒的VP2基因,将其克隆至pET-32a(+)载体中,获得重组质粒pET-32a(+)-VP2;将该重组质粒转化入大肠杆菌BL21感受态细胞中,经优化诱导表达后通过SDS-PAGE电泳分析表达产物,并对表达的蛋白质进行纯化、复性;再利用阳性血清对表达的蛋白质进行Western-blot鉴定;以纯化的重组蛋白作为抗原包被ELISA酶标板,通过优化抗原包被量、一抗稀释度、抗原封闭条件、一抗和二抗的孵育条件等最终建立间接ELISA方法,并对该方法进行评定。结果表明:成功构建出NDPV重组VP2蛋白,在37℃、1.0 mmol/L IPTG条件下诱导5 h的表达量最大,蛋白质的分子质量约为49 ku,主要以不溶性的包涵体形式存在。纯化、复性后的重组VP2蛋白浓度为2.378 mg/mL,纯度为97%。Western-blot鉴定得到的条带单一,大小为49 ku。间接ELISA检测方法的最佳抗原包被量为200 ng/孔,一抗稀释度为1∶200;其他检测条件最优结果为4℃包被过夜,37℃封闭2 h,一抗37℃孵育1 h,二抗稀释10 000倍,二抗37℃孵育1 h。间接ELISA检测方法的检测临界值为0.445,该方法具有较好的特异性、敏感性和重复性,对临床样品的阳性检出率为93.3%。说明以重组VP2蛋白为包被抗原建立的间接ELISA检测方法效果良好,为临床NDPV的感染提供了一种新的检测方法。In order to establish a antibody detection method of novel duck Parvovirus(NDPV) which infected ducks, PCR method was used in this experiment to amplify the VP2 gene of the novel duck Parvovirus which was then cloned into the pET-32 a(+) vector to obtain the recombinant plasmid pET-32 a(+)-VP2. The recombinant plasmid was transformed into E. coli BL21 competent cells which were optimized and induced to express, and the expression products were analyzed by SDS-PAGE electrophoresis, and were purified and renatured. Western-blot identification of expressed protein was carried out using positive serum. The purified recombinant VP2 protein was used as the antigen, with coating an ELISA plate, optimizing antigen coating amount, primary antibody dilution, antigen blocking conditions, incubation conditions of primary antibody and secondary antibody. An indirect ELISA method was finally established, and was evaluated. The results showed that the NDPV recombinant VP2 protein was successfully constructed, and the maximum expression was induced under the conditions of 37 ℃ and 1.0 mmol/L IPTG for 5 h. The molecular weight of the protein was about 49 ku, mainly in the form of insoluble inclusion bodies. The concentration of recombinant VP2 protein after purification and renaturation was 2.378 mg/mL,and the purity was 97%. The Western-blot identification obtained a single band with a size of 49 ku. The optimal antigen coating volume of the indirect ELISA detection method was 200 ng/well, and the primary antibody dilution was 1∶200;the best results of other detection conditions were 4 ℃ coating overnight, 37 ℃ blocking for 2 h, primary antibody incubation at 37 ℃ for 1 h, and the secondary antibody with a dilution of 10 000 times. The critical value of the indirect ELISA detection method is 0.445. This method had good specificity, sensitivity and repeatability. The positive detection rate of clinical samples was 93.3%. The results suggested that the indirect ELISA detection method which was established with the recomb

关 键 词：新型鸭细小病毒 原核表达 重组VP2蛋白 WESTERN-BLOT 间接ELISA方法 

分 类 号：S852.65[农业科学—基础兽医学] Q816[农业科学—兽医学]