【摘要】 目的利用昆虫细胞/杆状病毒系统表达猪瘟病毒(CSFV)E2蛋白,用于E2蛋白功能、开发CSF新型疫苗以及建立相关血清学诊断方法等研究。方法采用RT-PCR扩增CSFV E2基因,将PCR产物克隆到pGEM-T-Easy载体,将该基因插入到pFast-BacHT A载体中,构建重组转座载体后转化DH10Bac感受态细胞,获得重组Bacmid质粒后转染sf9昆虫细胞,传毒3代,对表达蛋白进行Western-blot及免疫组化鉴定。结果成功克隆CSFVE2基因,其核苷酸序列为1119 bp。SDS-PAGE电泳结果显示表达E2蛋白相对分子质量约为43×103,Western-blot和免疫组化结果证实表达蛋白能够被CSFV标准阳性血清识别。结论在Bac-to-Bac杆状病毒系统中的成功表达了CSFV E2蛋白,与CSFV标准阳性血清具有较好的反应性。更多还原

【Abstract】 Objective To obtain expression of E2 gene of CSFV in Bac-to-Bac expression system and provide materials for research of the function of E2 protein,as well as to establish CSFV serological diagnostic method and develop CFSV subunit vaccine.Method CSFV E2 gene was amplified by RT-PCR to be cloned into pGEM-T-Easy vetcor.The E2 gene was inserted into pFastBacHTa vector,then transferred the recombinant plasmid pFastBacHT A-E2 into DH10Bac to get recombinant Bacmid.Subsequently,the recombinant Bacmid was transfected into Sf9 cells.The expressed recombinant protein was identified by western-blot and immunohistochemistry analysis after three cell passages.Results E-2 gene of CSFV was successfully cloned and sequenced which contains 1119 bp SDS-PAGE electrophoresis analysis detected a band of protein about 43×10~3 in the expression product of E2 gene in insect cells.The obtained recombinant E2 protein reacted with CSFV-positive serum that was substantiated by Western-blot and immunohistochemical analysis.Conclusion The E2 gene of CSFV is successfully expressed in Bac-to-Bac expression system and shows reactivity with CSFV positive serum.更多还原