百日咳丝状血凝素与志贺毒素B亚单位的融合表达及其免疫保护效果评价

Fusion expression and immune protective effect of pertussis filamentous hemagglutinin and StxB

目的融合表达百日咳丝状血凝素(filamentous haemagglutinin,FHA)片段FHA1877-2250与志贺毒素B亚单位(Shiga toxin B subunit,StxB)蛋白,并对融合蛋白StxB-Fha1877-2250的免疫保护效果进行评价。方法设计引物将StxB片段连接至FHA1877-2250N-端,构建重组表达质粒pET-28a-FHA1877-2250和pET-28a-StxB-Fha1877-2250,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经镍柱亲和层析和复性后,通过皮下免疫小鼠,共3次,间隔2周。末次免疫后10 d,尾静脉采血,间接ELISA法检测血清抗体效价;血清体外杀菌试验检测杀菌效率;末次免疫后第14天,经腹腔接种2倍半数致死剂量(1.21×108CFU)的百日咳Bp148株菌液,评价动物攻毒保护效果。结果重组表达质粒经菌落PCR和测序证实构建正确。表达的重组蛋白FHA1877-2250、StxB-Fha1877-2250经SDS-PAGE和Western blot分析显示,相对分子质量分别约为43000和51000。StxB-Fha1877-2250免疫小鼠血清抗体效价可达1∶3200。StxB-Fha1877-2250组的血清杀菌效率比FHA1877-2250组略好。以百日咳Bp148菌株攻毒,FHA1877-2250、StxB-Fha1877-2250蛋白对免疫小鼠具有保护力,保护率分别为40%和60%。结论重组融合蛋白StxB-Fha1877-2250相比于FHA1877-2250具有更好的免疫原性和保护效果,为百日咳亚单位疫苗的研究提供了新的思路。

Objective To express pertussis filamentous hemagglutinin (FHA)fragment FHA1877-2250fused with Shiga toxin B unit(StxB)protein and evaluate the immune protiective effect of StxB-Fha1877-2250fusion protein.Methods Primers were designed to link StxB to the N-terminus of FHA1877-2250.The constructed recombinant plasmids pET-28a-FHA1877-2250and pET-28a-StxB-Fha1877-2250were transformed to E.coli BL21(DE3) and induced with IPTG.The expressed recombinant protein was purified by nickel ion affinity chromatography and re-naturalized,with which BALB/c mice were immunized s.c.for 3 times each at a interval of 2 weeks.Serum samples were collected 10 d after the last immunization and determined for antibody titer by indirect ELISA.The bactericidal efficiency was evaluated by serum bactericidal assay(SBA)in vitro.The mice were challenged i.p.with Bordetella pertussis Bp148 strain at twice median lethal dose(1.21×108CFU) on day 14 after the last immunization to evaluate the protective effect.Results Colony PCR and sequencing proved that recombinant plasmids pET-28a-FHA1877-2250and pET-28a-StxB-Fha1877-2250were constructed correctly.SDS-PAGE and Western blot showed that the relative molecular masses of expressed proteins FHA1877-2250and StxB-Fha1877-2250were about 43 000 and about 51 000 respectively.The serum antibody titers induced by StxB-Fha1877-2250in mice was 1∶3 200.The bactericidal efficiency of serum of mice immunized with StxB-Fha1877-2250was slightly higher than that with FHA1877-2250.Both FHA1877-2250and StxB-Fha1877-2250proteins showed protective effect against the challenge with Bp148 strain,of which the protection rates were 40%and 60%respectively.Conclusion Recombinant protein StxB-Fha1877-2250showed better immunogenicity and protective effect than FHA1877-2250,which provided a new idea for the research on pertussis subunit vaccine.