牛流行热病毒山东分离株α3基因序列分析及原核表达与纯化  

Sequence Analysis,Prokaryotic Expression and Purification of α3 Protein of Bovine Epidemic Fever Virus Isolated from Shandong Province

作　　者：朱鸿超 何畅 侯佩莉 王洪梅 李雪漫 唐文娟 何洪彬 Zhu Hongchao;He Chang;Hou Peili;Wang Hongmei;Li Xueman;Tang Wenjuan;He Hongbin(College of Life Sciences,Shandong Normal University/Research Center for Ruminant Diseases,Jinan 250014,China;International Department,High School Attached to Shandong Normal University,Jinan 250014,China)

机构地区：[1]山东师范大学生命科学学院/反刍动物疾病研究中心,山东济南250014 [2]山东师范大学附属中学国际部,山东济南250014

出　　处：《山东农业科学》2021年第7期107-112,共6页Shandong Agricultural Sciences

基　　金：国家自然科学基金项目“牛流行热病毒α3基因负调控MAVS的分子机制研究”(31972665);大学生创新训练项目(S202010445057,S202010445196)。

摘　　要：为深入研究牛流行热病毒(BEFV)编码的α3非结构蛋白的功能及其在研发新型BEFV疫苗和诊断试剂上的应用,本研究对本实验室分离保存的BEFV采用RT-PCR法对其α3基因进行扩增;用DNAStar软件对序列进行核苷酸和氨基酸同源性分析;用ProtParam和抗原表位分析生物信息学软件研究α3蛋白的亲水性、优势抗原表位区;分别用限制性内切酶双酶切目的基因α3和pGEX-4T-1载体,构建pGEX-4T-1-α3原核表达载体,并进行诱导表达。结果表明,成功扩增出156bp的α3基因序列;该α3基因序列与参考毒株的核苷酸序列同源性为98.0%~100.0%,氨基酸序列的同源性也为98.0%~100.0%,表明α3蛋白具有较高的保守性;BEFV的α3蛋白抗原表位分析表明3~5、9~47aa是α3蛋白的优势抗原表位区段,总平均疏水性为-0.737,说明该蛋白以可溶性形式存在;构建的pGEX-4T-1-α3原核表达质粒在大肠杆菌中成功表达,应用可溶性蛋白纯化的方法获得较高纯度的α3蛋白。本研究为该蛋白的免疫原性验证以及新型牛流行热病毒疫苗研发提供了数据参考。To further study the function of nonstructural α3 protein encoded by bovine epidemic fever virus(BEFV) and its application in the development of new BEFV vaccine and diagnostic reagents, the α3 gene sequence of BEFV isolated and preserved in our laboratory was amplified. The nucleotide and amino acid homology of the sequences were analyzed by DNASTAR software. The ProtParam and epitope analysis software were used to study the hydrophilicity and dominant epitope region of α3 protein. The prokaryotic expression vector pGEX-4 T-1-α3 was constructed by restriction enzyme double digestion of target gene α3 and pGEX-4 T-1, respectively. The recombinant vector was transformed into Escherichia coli BL21 and induced by IPTG. The results showed that the 156 bp sequence of α3 gene was successfully amplified by PCR. The homology of nucleotide sequence and amino acid sequence of the α3 protein with the reference strain were both 98.0%~100.0%, indicating that the α3 protein was highly conserved. Epitope analysis of BEFV α3 protein showed that the dominant epitope regions were located at the 3~5 aa and 9~47 aa of α3 protein with a total average hydrophobicity of-0.737, indicating that the protein existed in soluble form. Then the pGEX-4 T-1-α3 prokaryotic expression plasmid was successfully constructed and expressed in Escherichia coli. The high-purity α3 protein was obtained by soluble protein purification method. In conclusion, this study provided data references for the detection of immunogenicity of the protein and the development of new bovine epidemic fever virus vaccine.

关 键 词：牛流行热病毒(BEFV) α3蛋白 序列分析 原核表达 纯化 

分 类 号：S858.23[农业科学—临床兽医学]