| 摘要: |
| [目的]表达猪瘟保护性抗原E0蛋白,用以建立猪瘟抗体检测方法,为进一步开发诊断试剂盒和研究E0蛋白的免疫学功能奠定基础.[方法]将猪瘟野毒株FJ237-E0基因插入原核表达载体pET32a中,以BL21(DE3)为表达菌株进行诱导表达,将表达的重组蛋白通过His亲和层析柱进行纯化,以纯化的His-E0融合蛋白作为诊断抗原,通过探索抗原包被量和抗体血清稀释倍数.建立检测猪瘟EO抗体的ELISA方法.[结果]成功克隆了CSFV E0基因,其核苷酸序列为687 bp,编码229个氨基酸.经His亲和层析柱纯化检测纯度为0.587 mg/mL；SDS-PAGE电泳结果显示,表达的E0蛋白分子质量约为50.3 ku。表达的重组蛋白为可溶性蛋白,表达量占菌体总蛋白的30%,ELISA方阵确定的最佳抗原包被量为100 ng,血清稀释倍数为1:30,建立的ELISA方法与IDEXX公司猪瘟病毒抗体检测试剂盒的阳性符合率为92.3%,阴性符合率为85.7%.[结论]建立的ELISA检测方法,不但为猪瘟抗体监测提供了一种比较实用的血清学监测手段,也为进一步开发猪瘟抗体检测试剂盒奠定了基础. |
| 关键词: E0基因 原核表达 间接ELISA |
| DOI： |
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| 基金项目:农业部动物疫情防制与监测项目(NY200609-03) |
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| Prokaryotic expression of CSFV E0 protein and establishment of an indirect ELISA for detection of antibody |
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| Abstract: |
| 【Objective】 The study obtained expression of E0 protein of CSFV to establish CSFV antibody serological diagnostic method,to lay a foundation for the development of diagnosis kit and research on the immunology function of E0 protein.【Method】 The wild virus FJ237 E0 gene was cloned into prokaryotic expression vector pET32a.After the recombinant expression plasmids were transformed into BL21(DE3),a pure recombinant His-E0 protein by His affinity columns using the purified protein to coate antigen.【Result】 E0 gene of was successfully cloned and sequenced,which contained 687 bp,SDS-PAGE electrophoresis analysis detected a band of protein about 50.3 ku in the expression product of E0 gene in insect cells.The results revealed that the E0 protein could be expressed in the systems as soluble protein accounted for 30% of whole bacterial protein.The concentration of coated antigen was 100ng and the dilution of serum samples was 1∶30.Positive coincidence rate is 92.3% and negative coincidence rate is 85.7% in the method of our experiment with the kit of IDEXX.【Conclusion】 The development of the CSFV indirect ELISA offered a simple and practical way for monitoring antibody of CSF ,which provides much information for a diagnosis kit. |
| Key words: E0 gene prokaryotic expression indirect ELISA |
