PPRV Nigeria75/1 H蛋白原核表达及抗原表位预测  

Prokaryotic Expression of PPRV Nigeria75/1 H Protein and Prediction of Antigen Epitope

作　　者：李林杰[1,2,3] 常秋燕 马鹏[1,2,3] 王悦萦 马晓霞[1,2,3] 柏家林[1,2,3] LI Linjie;CHANG Qiuyan;MA Peng;MA Xiaoxia;BAI Jialin(Gansu Engineering and Technology Research Center for Animal Cell,Northwest Minzu University, Lanzhou 730030,China;Pennington Biomedical Research Center,Northwest Minzu University, Lanzhou 730030,China;Life Science and Engineering College,Northwest Minzu University, Lanzhou 730030,China)

机构地区：[1]西北民族大学甘肃省动物细胞工程技术研究中心,甘肃兰州730030 [2]西北民族大学生物医学研究中心,甘肃兰州730030 [3]西北民族大学生命科学与工程学院,甘肃兰州730030

出　　处：《华北农学报》2018年第5期111-116,共6页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31860696);动物医学生物工程创新团队发展计划(IRT_17R88);甘肃省科技计划资助(17YF1WA166)

摘　　要：克隆并原核表达小反刍兽疫病毒弱毒疫苗株血凝素蛋白(H)全长基因,并通过生物信息学的方法预测其B抗原表位。根据NCBI中PPRV基因组序列中H基因序列(GenBank X74443)设计一对引物,采用RT-PCR方法扩增其全长;将扩增产物克隆至原核表达载体pET-32a后,转化至大肠杆菌E. coli Rosetta,经IPTG 37℃诱导表达目的蛋白;表达的目的蛋白经带His标签的镍离子蛋白纯化柱纯化后,进行Western Blot鉴定。利用DNAStar软件采用生物信息学的方法预测其H蛋白潜在的抗原表位。结果显示,质粒pET-32a-H经双酶切鉴定及测序后可证明构建正确;表达的目的蛋白相对分子质量约67 ku,能被抗His标签抗体识别,主要以包涵体形式存在,有少量可溶性表达。通过生物信息学软件DNAStar成功找到H蛋白的潜在抗原表位5-8,14-16,73-75,83-90,125-131,142-147,170-177,236-245,281-285,312-317,360-363,370-379,388-391,445-449,487-489,503-505,520-522,532-535,544-551,592-595。试验成功构建阳性质粒pET-32a-H,表达目的蛋白,并成功预测出H蛋白潜在的抗原表位。为早日消除PPRV研制新型亚单位疫苗提供参考。To clone the gene encoding hemagglutinin(H)protein of Nigeria 75/1 strain of attenuated Peste des petits ruminants virus(PPRV),express in prokaryotic cells and prepare its polyclonal antibody by bioinformatics.According to the encoding sequence of PPRV H gene(GenBank X74443),a pair of primers were designed,with which the overall length were amplified by RT-PCR and inserted into vector pET-32a.The constructed recombinant plasmid was transformed to E.coli Rosetta for expression under the induction of 1 mmol/L IPTG at 37℃.The expressed products were purified by affinity chromatography using the nickel ion protein purification volume with His tag,and identified by Western Blot.Preparing its polyclonal antibody by bioinformatics.Restriction analysis proved that recombinant plasmid pET-32a-H was constructed correctly.The expressed PPRV H protein,with a relative molecular mass of about 67 ku.The prepared polyclonal antibody recognized the whole viral antigen of PPRV and recombinant PPRV H protein expressed in baculovirus.Recombinant plasmid pET-32a-H was constructed successfully,and PPRV H protein was successfully expressed.Useing bioinformatics could find polyclonal antibody 5-8,14-16,73-75,83-90,125-131,142-147,170-177,236-245,281-285,312-317,360-363,370-379,388-391,445-449,487-489,503-505,520-522,532-535,544-551,592-595.The positive plasmid pET-32a-H was successfully constructed,and the target protein was expressed.The potential epitope of H protein was successfully predicted.It is important for elimination of PPRV earlyer and preparation of novel subunit vaccines.

关 键 词：小反刍兽疫病毒 H蛋白 原核表达 B细胞抗原表位 

分 类 号：Q78[生物学—分子生物学]