牛源多杀性巴氏杆菌PlpE蛋白的原核表达及多克隆抗体制备
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Prokaryotic Expression of PlpE Protein of Bovine Pasteurella multocida and Preparation of Polyclonal Antibodies
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    摘要:

    为体外表达牛源多杀性巴氏杆菌(Pasteurella multocida)PlpE基因及制备抗PlpE蛋白多克隆抗体,根据PlpE基因核酸序列设计了1对特异性引物,通过PCR扩增PlpE基因,构建重组质粒pET-32a-PlpE;摇菌提取质粒,进行双酶切鉴定,鉴定正确后将重组质粒pET-32a-PlpE转化至BL21(DE3)感受态细胞,加入IPTG进行诱导,对诱导蛋白进行SDS-PAGE和Western Blot鉴定;使用纯化蛋白PlpE免疫北京大耳白兔,制备多克隆抗体,并进行间接ELISA和Western Blot分析。结果显示,扩增的PlpE基因大小为1 050 bp,在原核细胞中诱导表达的PlpE蛋白约56 kDa。间接ELISA结果显示,所制备多克隆抗体效价可达1:512 000;Western Blot结果显示,所制备多克隆抗体反应性良好。PlpE蛋白的成功表达和多克隆抗体的成功制备,为下一步多杀性巴氏杆菌诊断方法的建立和疫苗研发奠定了基础。

    Abstract:

    In order to express the PlpE gene of bovine Pasteurella multocida in vitro and prepare corresponding polyclonal antibodies,a pair of specific primers were designed according to the nucleic acid sequence of the gene,then pET-32a-PlpE,the recombinant plasmid,was constructed through PCR amplification,after plasmid extraction and identification by double enzyme digestion,the recombinant plasmid was transformed into BL21(DE3)cells and the bacteria was induced by IPTG and the induced protein was identified by SDS-PAGE and Western Blot;Beijing White Rabbits were vaccinated with purified PlpE protein to prepare polyclonal antibodies,which were analyzed by indirect ELISA and Western Blot. The results showed that the amplified PlpE gene was 1 050 bp,and the induced PlpE protein was about 56 kDa. It was shown that,by indirect ELISA,the titer of the polyclonal antibodies could be up to 1:512 000;and by Western Blot,the antibody could react well with the positive serum. It was concluded that a foundation was laid for the establishment of the method for identifying Pasteurella multocida and development of relevant vaccines in the future.

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张智,刘泽,乌吉斯古楞,刘重阳,权璞宏,任旭荣,宋庆庆,关平原.牛源多杀性巴氏杆菌PlpE蛋白的原核表达及多克隆抗体制备[J].《中国动物检疫》编辑部,2021,38(1):94-99.

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  • 在线发布日期: 2020-12-30
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