徐颖华;张华捷;谭亚军;吴丽洁;王丽婵;侯启明;张庶民;

• 1:中国食品药品检定研究院百白破疫苗和毒素室卫生部生物技术产品检定方法及其标准化重点实验室

摘要(Abstract)：

目的原核表达百日咳黏附素(Pertactin,Prn),并制备抗Prn单克隆抗体。方法从无细胞百日咳疫苗生产菌株CS株基因组DNA中克隆Prn基因,插入表达载体pQE-30中,构建重组表达质粒pQE-30-Prn,转化E.coli M15,IPTG诱导表达。表达的重组Prn蛋白经阳、阴离子交换层析纯化后,采用Western blot和ELISA法鉴定重组Prn蛋白的反应原性。以纯化的重组Prn蛋白为免疫原,利用杂交瘤技术制备单克隆抗体,并对制备的单抗进行鉴定。结果重组表达质粒经双酶切和测序鉴定构建正确;表达的重组Prn蛋白相对分子质量约为69 000,主要以包涵体形式表达;纯化的重组Prn蛋白的纯度达95%以上,产量可达25 mg/L,能与不同来源的抗Prn-Ab血清特异性结合。获得2株分泌抗Prn单抗的杂交瘤细胞株,分泌的单抗均为IgG1类,轻链类型均为κ,效价均达1∶105以上,并能特异性识别Prn蛋白,而与百日咳杆菌其他抗原蛋白无交叉反应。结论原核表达、纯化了重组Prn蛋白,并制备了2株效价高、特异性强的单抗,为无细胞百日咳疫苗的质量控制及百日咳杆菌致病机制的研究提供了材料。

关键词(KeyWords)： 百日咳;黏附素;原核细胞;基因表达;单克隆抗体

Abstract:

Keywords:

基金项目(Foundation): 国家863计划“疫苗效果和质量评价新技术研究”(2012AA02A402)

作者(Authors): 徐颖华;张华捷;谭亚军;吴丽洁;王丽婵;侯启明;张庶民;

DOI: 10.13200/j.cjb.2012.10.105.xuyh.011

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