猪流行性腹泻病毒S基因片段的原核表达及其表达产物的反应原性  

Prokaryotic expression of four S gene segments of porcine epidemic diarrhea virus and reactivity analysis of their expressed products

作　　者：姜艳平[1] 葛俊伟[1] 汪淼[1] 乔薪瑗[1] 唐丽杰[1] 刘兆磊[1] 李一经[1] 

机构地区：[1]东北农业大学动物医学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2009年第7期602-607,共6页Chinese Veterinary Science

基　　金：黑龙江省"十一五"科技攻关项目(GA06B202-4)

摘　　要：为了筛选反应原性较好的猪流行性腹泻病毒(PEDV)S基因片段,利用生物学软件对PEDV S基因进行了抗原位点分析,在不破坏线性抗原位点的前提下将S基因分为连续的4段进行表达,Western-blot检测表明,S1、Ps420、S2、S3蛋白都有很好的抗原性。纯化这4种蛋白,并免疫家兔,分别制备了S1、Ps420、S2、S3抗血清,间接ELISA检测结果显示,这4种抗血清都能识别细胞培养的PEDV,其中S1抗血清的反应性最好,其P/N值达到8.01,其次为Ps420抗血清。免疫荧光试验表明,S1、Ps420抗血清能识别天然的PEDV病毒粒子。提示,S1、Ps420抗血清在病原检测诊断中有一定的潜在应用价值。To screen S gene segments encoding protein with good reactivity of porcine epidemic diarrhea virus（PEDV）,the S gene was analyzed on antigenic site by bioinformatics softwares. The S gene was segmented into four overlapping sections, named S1, Ps420, S2 and S3 gene, then the four sections were cloned into the prokaryotic expression vector pGEX-6P 1 to generate recombinant plasmids pGEX-6P-S1, pGEX 6P-Ps420, pGEX-6P-S3, and pProHTa-S2, respectively. The four plasmids were transformed into Escherichia coli BL21（DE3）pLysS and induced with IPTG, respectively. Western-blotting showed that expressed proteins had good antigenicity. To detect the reactivity of the four purified proteins,they were utilized to immune rabbits to produce anti-serum against single factor. The indirect ELISA results demonstrated that the four anti-sera were able to recognize the natural virus propagated in tissue culture. Of these, S1-produced anti-serum was the best,and the P/N value reached 8.01. The immunofluorescence assay showed that the anti-sera produced by protein S1 and Ps420 could also identify natural PEDV particle. These results showed that the antisera produced by proteins S1 and Ps420 were potentially useful as diagnostic antibody against PEDV.

关 键 词：猪流行性腹泻病毒 重组蛋白 病毒检测 诊断试剂 

分 类 号：S852.659.6[农业科学—基础兽医学]