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.
作者
潘素敏
仲飞
贺英
史秋梅
潘素娜
崔丹
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)
出处
《黑龙江畜牧兽医》
CAS
北大核心
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)
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
Canine parvovirus
antigen epitope gene
codon optimization
clone
prokaryotic expression
fusion protein
