• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

羊口疮病毒115基因的克隆及原核表达

Cloning and Prokaryotic Expression of ORFV 115 Gene

  • 摘要:
        目的   获得羊口疮病毒(ORFV)115基因表达蛋白。
        方法   利用Oligo 7软件设计并筛选出115基因特异性扩增引物,以ORFV FJ-ND株基因组为模板,通过PCR技术扩增获得其基因序列,再将115基因克隆至pGEX-6p-1上,重组质粒pGEX-6p-115,并对其进行测序鉴定,鉴定正确后转化RosettaganmiB(DE3)感受态细胞,通过优化IPTG浓度和诱导时间获得重组融合蛋白最佳表达条件,用SDS-PAGE对表达的目的蛋白进行分析。
        结果   115基因全长450 bp,编码149个氨基酸;115基因可以在RosettaganmiB中表达,重组蛋白分子大小约42 kDa,主要以包涵体形式表达。
        结论   成功克隆了ORFV 115基因,构建了pGEX-6P-115原核表达质粒,优化了重组蛋白表达条件并进行纯化,为进一步探索ORFV 115蛋白在感染宿主过程中的机制和作用奠定基础。

     

    Abstract:
        Objective   ORFV 115 was cloned for a study on the gene.
        Method    Oligo 7 software was used to design and select primers for specific amplification of the gene. Using the genome of ORFV FJ-ND as template, the sequence was obtained by PCR. The gene was then cloned into pGEX-6p-1 to obtain the recombinant plasmid pGEX-6P-115 for sequencing and identification prior to transformation to RosettaganmiB (DE3) receptor cells. Expression conditions of the recombinant fusion protein were optimized with respect to IPTG concentration and induction time. SDS-PAGE was used to analyze the expressed target protein.
        Result   ORFV 115 was 450 bp encoded 149 amino acids and could be expressed in RosettaganmiB. It was approximately 42 kDa in molecular weight and mainly expressed as inclusion body.
        Conclusion   ORFV 115 was successfully cloned, the pGEX-6P-115 prokaryotic expression plasmid constructed, the recombinant protein purified, and the expression conditions optimized to facilitate further studies on the mechanism and role it plays in the infection on host animals.

     

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