禽脑脊髓炎病毒外壳蛋白VP1的原核表达及活性鉴定  

Prokaryotic Expression and Activity Identification of Avian Encephalomyelitis Virus Capsid Protein VP1

作　　者：胡亚歌 吴海洋 翟庆贺 牛晓赛 董新荣 卢尧 杨灵芝 郭建友 朱杰 HU Yage;WU Haiyang;ZHAI Qinghe;NIU Xiaosai;DONG Xinrong;LU Yao;YANG Lingzhi;GUO Jianyou;ZHU Jie(Shandong Binzhou Wohua Biotech Co.,Ltd.,BinZhou,Shandong 256600;Shandong New Hope Liuhe Group Co.,Ltd.,Qingdao,Shandong 266100;Agricultural and Rural Bureau of Fu′an County,Dongtai City,Yancheng,Jiangsu 224222)

机构地区：[1]山东滨州沃华生物工程有限公司,山东滨州256600 [2]山东新希望六和集团有限公司,山东青岛266100 [3]东台市富安镇农业农村局,江苏盐城224222

出　　处：《中国家禽》2022年第10期38-43,共6页China Poultry

基　　金：山东滨州沃华生物工程有限公司科研基金(202102QB02-01)。

摘　　要：为研究禽脑脊髓炎病毒(Avian encephalomyelitis virus,AEV)外壳蛋白VP1的免疫原性,研究根据AEV-187株VP1基因序列,设计一对表达引物,利用RT-PCR扩增获得了828 bp片段,成功构建表达质粒pET-32a-VP1,将质粒转化大肠杆菌BL21(DE3)感受态细胞,获得工程菌株pET-32a-VP1-BL21(DE3),经IPTG诱导后,采用SDS-PAGE、Western blot检测VP1蛋白的表达形式及反应活性;采用表达的VP1蛋白制备疫苗免疫SPF鸡,检测免疫后血清中AEV抗体水平。结果显示:VP1蛋白为部分可溶性表达,分子量为50 ku;与AEV阳性鸡血清能够发生反应;采用VP1蛋白制备的疫苗安全性良好,免疫SPF鸡后14 d即可检测到AEV抗体,免疫后21 d和28 d的抗体水平有大幅度增长,阳性率达到80%,证明表达的VP1蛋白具有免疫原性。综上所述,原核表达的VP1蛋白具有良好的抗原活性,为AEV亚单位疫苗的研制奠定了基础。In order to study the immunogenicity of avian encephalomyelitis virus(AEV) coat protein VP1, a pair of expression primers were designed according to the VP1 gene sequence of AEV-187 strain and a gene fragment of 828 bp was obtained by RT-PCR. The expression plasmid p ET-32a-VP1 was successfully constructed. The plasmid was transformed into Eescherichia coli BL21(DE3) competent cells to obtain the engineering strain pET-32a-VP1-BL21(DE3). After induction with IPTG, the expression and reactivity of VP1 protein were detected by SDS-PAGE and Western blot. The expressed VP1 protein was prepared the vaccine for SPF chickens, the antobody against AEV in serum after immunization was detected. The results showed that the VP1 protein achieved partial soluble expression, and its molecular weight was approximately 50 ku;It could react with AEVpositive chicken serum. The antibodies against AEV could be detected 14 days after immunization of SPF chickens. The antibody levels on 21 and 28 days after immunization increased significantly, and the positive rate reached 80%, indicating that VP1 protein had immunogenicity. In conclusion, the prokaryotic-expressed VP1 protein had good antigenic activity, laying the foundation for the development of AEV subunit vaccine.

关 键 词：禽脑脊髓炎病毒 VP1 原核表达 活性分析 

分 类 号：S855.3[农业科学—临床兽医学]