百日咳黏附素的原核表达及其单克隆抗体的制备

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。

Prokaryotic expression of pertactin of Bordetella pertussis and preparation of its monoclonal antibody

Objective To express the pertactin(Prn)of Bordetella pertussis in prokaryotic cells and prepare the monoclonal antibody(McAb)against Prn.Methods Prn gene was cloned from the genomic DNA of CS strain for production of acellular pertussis vaccine and inserted into expression vector pQE-30.The constructed recombinant plasmid pQE-30-Prn was transformed to E.coli M15 for expression under induction of IPTG.The expressed recombinant protein was purified by anion and cation exchange chromatography,and identified for reactogenicity by Western blot and ELISA.McAb was prepared by hybridoma technique using the purified Prn protein as immunogen and identified.Results Restriction analysis and sequencing proved that recombinant plasmid pQE30-Prn was constructed correctly.The expressed Prn protein,with a relative molecular mass of about 69 000,mainly existed in a form of inclusion body and reached a purity of more than 95% after purification.The purified Prn protein,with a yield of 25 mg/L,showed specific binding to Prn-Ab of various origins.Two hybridoma cell strains were obtained,by which the secreted McAbs were IgG1(κ)with titers of more than 1 ∶ 105 and recognized Prn protein specifically while showed no cross reaction with other proteins of B.pertussis.Conclusion Recombinant Prn protein was expressed in prokaryotic cells and purified,and two hybridoma cell strains secreting high titer specific McAbs were obtained,which provided materials for quality control of acellular pertussis vaccine and study on pathogenic mechanism of B.pertussis.