百日咳丝状血凝素分子中强免疫原性片段的筛选、克隆与表达

目的筛选百日咳丝状血凝素分子中强免疫原性的片段,并对其进行克隆及表达。方法采用生物信息学软件对丝状血凝素分子的结构进行预测,从中筛选出具有强免疫原性的片段FHAs。经PCR扩增,T-A克隆后,构建表达质粒pET-22b(+)-FHAs,酶切鉴定正确后测序。将测序正确的质粒转化E.coliBL21(DE3),IPTG诱导目的蛋白的表达,并对表达产物进行Tricine-SDS-PAGE分析及Western blot鉴定。结果从丝状血凝素分子中筛选出具有138个氨基酸的肽段FHAs,经PCR扩增,得到约400bp的目的基因片段,构建的重组质粒经双酶切和测序鉴定,证明质粒构建正确,重组工程菌pET-22b(+)-FHAs/BL21经IPTG诱导,目的蛋白的表达量在50%以上,主要以包涵体形式存在。Western blot分析证实该蛋白能与抗FHA单抗发生反应。结论筛选出的肽段FHAs具有强的免疫原性和免疫反应特异性,为百日咳基因工程疫苗的研究奠定了基础。

Screening, Gene Cloning and Expression of A Highly Immunogenic Fragment of Filamentous Hemagglutinin of Bordetella pertussis

Objective To screen,clone and express a highly immunogenic fragment of filamentous hemagglutinin of Bordetella pertussis.Methods The molecular structure of filamentous hemagglutinin of Bordetella pertussis was predicted by bioinformatic software,from which a highly immunogenic fragment FHAs was screened and amplified by PCR.After T-A cloning,the amplified fragment was inserted into plasmid pET-22b,and the constructed recombinant plasmid pET-22b (+)-FHAs was identified by sequencing and transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was identified by Tricine-SDS-PAGE and Western blot.Results The FHAs fragment encoding 138 amino acids was screened,the target gene fragment at a length of about 400 bp was amplified,and recombinant E.coli strain pET-22b (+)-FHAs/BL21 was successfully established.The expressed product,mainly existing in a form of inclusion body,contained more than 50% of total somatic protein and showed specific reaction with McAb against FHA as proved by Western blot.Conclusion The screen FHAs fragment showed good immunogenicity and immunoreactivity,which laid a foundation of development of recombinant pertussis vaccine.