犬细小病毒VP2主要抗原表位基因的密码子优化及原核表达  

Codon optimization and prokaryotic expression of the major antigenic epitope genes of Canine parvovirus VP2

作　　者：潘素敏[1] 仲飞[2] 贺英[1] 史秋梅[1] 潘素娜 崔丹[2] 王晓燕 PAN Sumin;ZHONG Fei;HE Ying;SHI Qiumei;PAN Suna;CUI Dan;WANG Xiaoyan(Animal Science Department,Hebei Normal University of Science ＆ Technology,Qinhuangdao 066600,China;College of Veterinary Medicine,Agricultural University of Hebei,Baeding 071001,China;Animal Health Supervision Station of Liu Shou-ying Funing District Agricultural Livestock and Fisheries Bureau,Funing 066301,China;Animal Health Supervision Institute of Zhangjiakou,Zhangjiakou 075000,China)

机构地区：[1]河北科技师范学院动物科技学院,河北秦皇岛066600 [2]河北农业大学动物医学院,河北保定071001 [3]河北省秦皇岛市抚宁区农牧水产局留守营动物卫生监督站,河北抚宁066301 [4]张家口市动物卫生监督所,河北张家口075000

出　　处：《黑龙江畜牧兽医》2018年第19期118-121,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家星火计划项目(2015GA620002);河北省高等学校科学技术研究项目(ZD2017234);秦皇岛市科学技术研究项目(201602A046)

摘　　要：为了制备大量廉价的可溶性犬细小病毒（CPV）VP2蛋白抗原,试验截选了VP2抗原表位集中的基因片段VP2-70进行密码子优化及合成,将合成基因克隆到pET-32a（＋）载体中,构建原核表达载体pET-32-VP2-70,转化BL21（DE3）工程菌,IPTG诱导表达后进行Ni-NTA亲和层析纯化,Western-blot检测验证其生物学活性。结果表明：试验构建的VP2-70表达载体能够介导VP2-70融合蛋白在大肠杆菌中高效表达,表达量高出野生型VP2-70融合蛋白16 mg/L;Western-blot检测显示,该蛋白具有很好的免疫反应原性,可为CPV亚单位疫苗和免疫学诊断方法的研究提供候选抗原。The aim of the present study was to prepare a large number of cheap soluble canine parvovirus（CPV）VP2 protein antigen. The VP2-70 fragment containing more VP2 antigen epitopes was selected for codon optimization and synthesis, The codon-optimized VP2-70 gene was cloned into pET-32 a（＋） plasmid to construct the prokaryotic expression vector pET-32-VP2-70. The expression vector was transformed into E.coli BL21（DE3） to express VP2-70 protein. After induced by IPTG, Ni-NTA affinity chromatography and Western-blot assay were carried out to verify its biological activity. The results showed that the VP2-70 fusion protein was expressed in E.coli at the high level, and the expression level of the fusion protein was 16 mg/L higher than that of the wild type VP2-70 protein. The results of Western-blot showed that the protein had good immunoreactivity and could provide candidate antigen for the research of CPV subunit vaccine and immunological diagnosis.

关 键 词：犬细小病毒 抗原表位基因 密码子优化 克隆 原核表达 融合蛋白 

分 类 号：S852.655[农业科学—基础兽医学] Q755[农业科学—兽医学]