绵羊痘病毒多表位基因的构建及原核表达
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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)


Construction and prokaryotic expression of multi-epitope chimeric gene of sheeppox virus
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    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41 ku的融合蛋白。在IPTG浓度为0.5 mmol/L,温度为37℃、诱导4 h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。

    Abstract:

    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 ORF134 gene 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 ℃,0.5 mmol/L and 4 h,respectively.The fusion protein was identified correctly by positive serum against capripoxvirus by Western-blotting,suggesting potential value of genetically engineering vaccine.

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陈轶霞,王明明,龙玲,邵忠伟,刘俊林.绵羊痘病毒多表位基因的构建及原核表达[J].华中农业大学学报,2015,34(2):86-90

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  • 收稿日期:2014-04-22
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  • 在线发布日期: 2015-01-30
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