副溶血弧菌T3SS1 VscF蛋白多肽抗体的制备与应用
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S851.1

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国家自然科学基金面上项目(31871893)


Preparation and Application of Polyclonal Antibodies against T3SS1 VscF Protein of Vibrio parahaemolyticus
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    摘要:

    副溶血弧菌(Vibrio parahaemolyticus)是一种世界范围内广泛存在的革兰氏阴性食源性致病菌,其vscF基因编码的T3SS1针状蛋白VscF,在侵袭宿主细胞过程中扮演重要角色。为深入研究VscF蛋白在T3SS1针状结构组装过程中的功能,并制备VscF特异性多肽抗体,利用OptimumAntigenTM抗原多肽设计软件,筛选最佳抗原目标肽段,然后将肽段与KLH载体蛋白偶联后免疫新西兰大白兔,收集兔血清制备多克隆抗体,并利用间接ELISA、Western Blot、间接免疫荧光检测方法,鉴定多抗效价和特异性。间接ELISA检测结果显示,该多肽抗体效价达1:512 000,显示出该多肽抗体的高敏感度;Western Blot与间接免疫荧光检测结果显示,该多抗能与原核表达的VscF蛋白发生特异性反应。结果表明,筛选的VscF短肽制备的多克隆抗体敏感度高,可作为VscF验证试验中具有特异性结合作用的抗体,能够为后续生物试剂机理研究奠定基础。

    Abstract:

    Vibrio parahaemolyticus,a gram-negative foodborne pathogenic bacteria,is widely distributed all over the world,and its VscF protein of type III secretion system(T3SS)encoded by its vscF gene plays an important role in invading host cells by the bacteria. In order to further study the function of VscF protein in the assembly process of T3SS1 needle-like structure,and prepare VscF-specific polypeptide antibodies,the best antigen target peptides were screened by OptimumAntigenTM,a software for designing antigen peptides,after the peptides were coupled with KLH carrier protein,New Zealand white rabbits were vaccinated to collect rabbit serum to prepare polyclonal antibodies,then the titer and specificity of the antibodies were identified by indirect ELISA,Western Blot and indirect immunofluorescence. It was showed that,by indirect ELISA,the titer of the polyclonal antibodies was up to 1:512 000,indicating high sensitivity. It was found that,by Western blot and indirect immunofluorescence,the polyclonal antibodies could specifically react with the prokaryotic VscF protein. It was concluded that polyclonal antibodyies prepared by the screened VscF peptides were with high sensitivity,which could be used as the antibodies with specific binding function in VscF validation test,and would lay a foundation for further study on the mechanism of biological reagents.

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薛婷月,廉乐乐,李婉君,薛峰.副溶血弧菌T3SS1 VscF蛋白多肽抗体的制备与应用[J].《中国动物检疫》编辑部,2021,38(9):93-98.

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