绵羊痘病毒多表位基因的构建及原核表达  

Construction and prokaryotic expression of multi-epitope chimeric gene of sheeppox virus

作　　者：陈轶霞[1] 王明明[1] 龙玲[1] 邵忠伟[1] 刘俊林[1] 

机构地区：[1]西北民族大学生命科学与工程学院,兰州730124

出　　处：《华中农业大学学报》2015年第2期86-90,共5页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(31260609;31360533);西北民族大学中央高校基本科研业务费专项(ZYZ2012072)

摘　　要：通过计算机软件筛选出绵羊痘病毒基因组中心编码区的ORF90核蛋白、ORF112融合蛋白、ORF117糖蛋白、ORF55膜蛋白和基因组末端的ORF134宿主范围相关基因的6段T、B细胞优势抗原表位,以柔性氨基酸(GPGPG)作为接头,串联合成1条全新的多表位嵌合基因mE,将其克隆到原核表达载体pET-32中,采用酶切分析与序列测定方法筛选鉴定阳性重组质粒,构建pET32-mE质粒;用IPTG在不同条件下诱导表达,确定最佳表达条件,表达产物经SDS-PAGE分析。结果表明,重组蛋白是分子质量为41ku的融合蛋白。在IPTG浓度为0.5mmol/L,温度为37℃、诱导4h时目的蛋白的表达量最大,约占菌体的32%。Western-blotting试验表明,目的蛋白可被羊痘血清识别。To design a new gene encoding the multi-epitope chimeric antigen of sheeppox virus(SPPV)and express the chimeric gene,the dominant epitopes of ORF90,ORF112,ORF117,ORF55 and ORF134gene of SPPV were analyzed and selected by computer software and reported references.A recombinant multi-epitope chimeric gene including SPPV six multi-epitope genes was constructed and cloned into prokaryotic expression vector pET-32.The recombinant plasmids pET32-mE were induced by IPTG.The SDS-PAGE analysis showed that the concentration of the fusion protein in E.coli protein was about 32,with a molecular weight of 41 ku.The optimal temperature,concentration of IPTG and induced time for the recombinant protein were 37℃,0.5mmol/L and 4h,respectively.The fusion protein was identified correctly by positive serum against capripoxvirus by Western-blotting,suggesting potential value of genetically engineering vaccine.

关 键 词：绵羊痘病毒 抗原表位 多表位 嵌合基因 原核表达 

分 类 号：S852.654[农业科学—基础兽医学]