鸡传染性贫血病毒VP1基因的克隆表达及抗原性分析  

Cloning and expression of VP1 genes of chicken infectious anemia virus and the antigenicity of the expressed products

作　　者：杨勇[1] 任玉红[1] 梁宝怀 朱雅宁[3] 梁智选[3] 

机构地区：[1]山西农业大学动物科技学院,山西太谷030801 [2]山西省大同市南郊区畜牧局,山西大同037001 [3]天津市禽病诊断培训中心,天津300402

出　　处：《中国兽医科学》2007年第2期140-144,共5页Chinese Veterinary Science

基　　金：天津市科技培育项目(043122011)

摘　　要：参考已发表的鸡传染性贫血病毒(CIAV)VP1基因序列,设计并合成了2对引物,经PCR扩增获得了2个基因片段。将PCR产物克隆至pGEM-T Easy载体中,成功构建了克隆载体pGEM-CIAVVP1A和pGEM-CIAVVP1B。将上述重组质粒和原核表达载体pMXB10分别用NdeⅠ+EcoRⅠ、NdeⅠ+XhoⅠ双酶切,并将纯化的2个基因亚克隆至pMXB10中,构建出原核表达载体pMXB10-VP1A和pMXB10-VP1B。在0.3 mmol/L IPTG诱导下,目的基因在大肠杆菌ER2566中以分泌型得到了大量表达。Western-blot分析发现,表达蛋白具有CIAV抗原性。表达蛋白经纯化后作为包被抗原,用间接ELISA鉴定,与CIAV阳性血清均能发生特异性反应。2组蛋白免疫SPF鸡后,用全病毒ELISA试剂盒检测血清呈阳性,表明2组蛋白均可诱发机体产生抗CIAV的抗体。Based on the published VP1 gene sequence of chicken infectious anemia virus（CIAV）, two pairs of primers were designed and synthesized,and then two genes of VP1 were amplified by PCR. PCR products were cloned into pGEM-T Easy Vector to construct recombinant vectors pGEM-CIAVVP1A and pGEM-CAIVVP1B, pGEM-CIAVVP1A and pGEM-CIAVVP1B were digested by Nde Ⅰ ＋ EcoR Ⅰ , and pMXB10 was digested by Nde Ⅰ ＋ Xho Ⅰ , Then the prokaryotic expression vectors pMXB10-VP1A and pMXB10-VP1B were constructed by subcloning the purified VP1 genes into the pMXB10. The recombinant plasmids were expressed efficiently in the secreted form in Escherichia coli ER2566 by induction with 0. 3 mmol/L IPTG. Western-blot analysis showed that the expressed proteins had antigenic activity. Using the purified proteins as antigen, the expressed proteins could be recognized by the antibody against CIAV in indirect ELISA. The sera from SPF chicken immunized by the two proteins were positive using the entire viral ELISA kit. The results showed that the two proteins could induce the animal to generate antibodies against CIAV.

关 键 词：鸡传染性贫血病毒 VP1基因 原核表达 酶联免疫吸附试验 

分 类 号：S852.659.2[农业科学—基础兽医学] Q786[农业科学—兽医学]