非洲猪瘟病毒多表位融合蛋白P72的构建、表达及免疫学特性研究  

Research on Construction, Expression and Immunological Characteristics of Multi-epitope Fusion Antigen P72 of African Swine Fever Virus

作　　者：郭晶 李重阳[1] 孟庆玲[1] 乔军[1] 伍晔晖 王立霞[1] 马帅 才学鹏[2] GUOJing;LIChongyang;MENGQingling;QIAOJun;WU Yehui;WANGLixia;MAShuai;CAIXuepeng(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou Gansu 730000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]中国农业科学院兰州兽医研究所,甘肃兰州730000

出　　处：《家畜生态学报》2019年第6期60-65,共6页Journal of Domestic Animal Ecology

基　　金：国家十三五重点研发计划子课题(2017YFD0502300);兵团中青年科技创新领军人才计划(2016BC001)

摘　　要：非洲猪瘟是由非洲猪瘟病毒(ASFV)引起的一种烈性传染病,对中国猪养殖产业的发展存在巨大的潜在威胁。为了建立有效的检测手段来防控ASFV的流行,本试验以GenBank中ASFV基因组序列为基础,运用在线软件对ASFV结构蛋白P72的优势线性抗原表位进行预测分析,利用预测结果分析合成多表位融合基因P72 (MeP72)。再将MeP72经pMD19-T克隆后插入到原核表达载体pET-32a(+)中得到重组质粒pET-MeP72,双酶切验证在402 bp及5 000 bp以上有条带,证明重组质粒构建成功。转化重组质粒pET-MeP72至大肠杆菌感受态细胞E.coli BL21 (DE3),用IPTG诱导表达并优化诱导条件,分析重组蛋白反应原性。SDS-PAGE检测结果显示,MeP72重组蛋白的分子质量为30.74 kDa;Western blot分析显示MeP72重组蛋白可被ASFV阳性血清特异性识别,表明其反应原性良好;小鼠免疫试验证实MeP72蛋白可诱导机体产生特异性抗体,具有良好的免疫原性。研究表明MeP72重组蛋白可在大肠杆菌中高效表达,并诱导机体产生特异性抗体,为诊断抗原的研发奠定了前期基础。African swine fever, caused by African swine fever virus(ASFV), is a fulminating infectious disease of pigs, which results in a great threat to the development of pig breeding industry. In this study, based on the genomic sequence of ASFV in Gen Bank, the dominant linear epitope of the structural protein P72 of ASFV was predicted and analyzed by using online software. Then multi-epitope fusion gene P72(MeP72) was constructed, artificially synthesized and inserted in prokaryotic expression vector pET-32 a(+) to generate recombinant plasmid pET-MeP72 and double enzyme digestion proved that the recombinant plasmid was constructed successfully. pET-MeP72 was transformed into competent cell E.coli BL21(DE3) for expression under the induction of IPTG, reactivity of recombinant protein was analyzed. SDS-PAGE showed that the molecular weight of recombinant protein MeP72 was 30.74 kDa. Western blot demonstrated that pET-MeP72 of recombinant protein could be identified by the positive serum of ASFV, which indicated the reaction was good. The mouse immune test confirmed that the recombinant protein could induce specific antibodies.The study proves MeP72 recombinant protein can be highly efficiently expressed in E.coli and induce specific antibodies,which lays a foundation for the research and development of ASFV of the diagnostic antigens based on MeP72 fusion proteins.

关 键 词：非洲猪瘟病毒 MeP72 多表位融合抗原基因 原核表达 

分 类 号：S811.5[农业科学—畜牧学]