• 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

山羊地方性鼻内肿瘤病毒SU蛋白的表达及其多克隆抗体的制备和特性分析

Expression of SU protein of ENTV-2FJ strain, preparation and characterstic analylsis of its polyclonal antibody

  • 摘要:
      目的  明确山羊地方性鼻内肿瘤病毒(ENTV-2)SU蛋白的功能。
      方法  采用RT-PCR 方法从 ENTV-2FJ分离株中扩增获得SU基因片段,将其克隆到pMD-19T载体;测序验证后,再亚克隆到PET-32a(+)上,在RosettagamiB(DE3) 感受态细胞进行表达,采用SDS-PAGE 、Western-blot和ELISA对重组蛋白进行鉴定及抗原性分析。
      结果  结果显示,表达的重组菌融合蛋白大小约64.38 kD,在IPTG终浓度0.4 mmol·L−1、37 ℃诱导4 h表达效果最好。用纯化的ENTV-2进行SDS-PAGE,SU重组蛋白免疫小鼠制备的多克隆抗体作为一抗,进行 Western-blot,结果显示鼠抗SU蛋白多克隆抗体能与ENTV-2抗原进行特异性的反应。
      结论  表达的SU蛋白有较好的抗原性,为制备ENTV-2特异性单克隆抗体及建立ENTV-2特异性血清学方法奠定了基础。

     

    Abstract:
      Objective  In order to determine the function of SU protein of enzootic nasal tumor virus of goats (ENTV-2),
      Method  RT-PCR method was used to amplify the SU gene fragment from ENTV-2 FJ and then the SU gene was cloned into pMD-19T Simple Vector; After sequencing, the cloning vector was subcloned into PET-32a (+), the recombinant plasmid was transformed into RosettagamiB (DE3) competent cells. SDS-PAGE, Western-blot and ELISA were used for identification and antigenicity analysis of recombinant proteins.
      Result  The result showed that the expressed recombinant protein was about 64.38 kD, and the best expression condition was induced at 37 ℃ for 4 h at a final IPTG concentration of 0.4 mmol/L. The purified ENTV-2 virus was used for SDS-PAGE, and the mice anti-SU polyclonal antibody was used as primary antibody for Western-blot. The Western-blot analysis showed the mice anti-SU polyclonal antibody could react with ENTV-2 antigen specifically. It is proved that the expressed SU recombinant protein has better antigenicity.
      Conclusion  The results proved that the expressed SU recombinant protein has better antigenicity, and provided a basis for the preparation of ENTV-2 specific monoclonal antibody and the establishment of ENTV-2 specific serological method.

     

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