羊口疮病毒ORF080基因的原核表达、纯化及鉴定  

Prokaryotic expression, purification and identification of ORF080 gene of orf virus

作　　者：陈珍珍[1] 曾显成[1] 池雪林[1] 白丁平[1] 刘云辉 胡可慧 罗树红[2] 黄小红[1] CHEN Zhenzhen;ZENG Xiancheng;CHI Xuelin;BAI Dingping;LIU Yunhui;HU Kehui;LUO Shuhong;HUANG Xiaohong(University Key Laboratory for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;School of Stomatology and Medicine,Foshan University,Foshan,Guangdong 528000,China)

机构地区：[1]福建农林大学中西兽医结合与动物保健福建省高校重点实验室,福建福州350002 [2]佛山科学技术学院口腔医学院,广东佛山528000

出　　处：《福建农林大学学报（自然科学版）》2019年第6期776-780,共5页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建农林大学科技创新专项基金项目(KFA17221A、KFA17222A)

摘　　要：本试验旨在原核表达、纯化羊口疮病毒ORF080蛋白并鉴定其反应性.根据GenBank已发表的羊口疮病毒OV-GO株ORF080基因序列(登录号:KP010354),将密码子优化后合成该序列并克隆至pET-32a(+)原核表达载体中,构建pET-32a(+)-ORF080重组质粒.经双酶切和测序鉴定后转化至大肠杆菌Rossetta感受态细胞中,用IPTG诱导,诱导产物经SDS-PAGE鉴定,重组蛋白以可溶性和包涵体两种形式获得表达.采用镍柱对可溶性重组蛋白进行纯化,再经SDS-PAGE和蛋白质免疫印迹双重鉴定.SDS-PAGE检测结果显示,在57 ku处出现单一重组蛋白条带;蛋白质免疫印迹鉴定证实该纯化的蛋白能与抗组氨酸标签的抗体和羊口疮阳性血清发生特异性反应,证明成功表达并纯化出具有良好反应性的ORF080蛋白.试验结果为进一步对ORF080蛋白结构、功能及基因工程疫苗的研究提供参考.To study the reactivity of the ORF080 protein encoded by orf virus,the ORF080 gene sequence of OV-GO strain was firstly downloaded(registration number:KP010354)from GenBank,then the gene sequence was synthesized with optimized codons and cloned into the prokaryotic expression vector pET-32a(+)to construct the recombinant plasmid pET-32a(+)-ORF080.After identified by double enzyme digestion and sequencing,the recombinant plasmids were transformed into E.coli Rossetta,with the expression of ORF080 gene being induced by IPTG.SDS-PAGE analysis verified that the target proteins were widely distributed in supernatant and precipitation.The recombinant protein was further purified by nickel column and identified by SDS-PAGE and Western blotting.A single 57 ku protein band was detected by SDS-PAGE analysis.Western blot analysis showed that the purified target protein was recognized by anti-histidine labeled antibody and orf virus antisera,proving good reactivity of the ORF080 protein.The results shed light upon structure,function and genetic engineering vaccine based on the ORF080 protein.

关 键 词：羊口疮病毒 ORF080 蛋白 原核表达 

分 类 号：S852.654[农业科学—基础兽医学]