不可分型流感嗜血杆菌重组Hap蛋白的表达及其生物学活性分析  

Expression and biological activity identification of recombinant Hap protein of NTHi

作　　者：姚锋[1] 李婉宜[1] 邝玉[1] 李明远[1] 丰锋[1] 冯伟[1] 张强[1] 

机构地区：[1]四川大学华西基础医学与法医学院微生物学教研室,四川成都610041

出　　处：《南方医科大学学报》2010年第5期953-956,共4页Journal of Southern Medical University

基　　金：四川省卫生厅科研基金(070137)

摘　　要：目的在原核系统中表达并纯化不可分型流感嗜血杆菌(NTHi)的重要粘附因子Hap蛋白,并对其免疫原性和粘附活性进行初步研究。方法 IPTG诱导重组质粒pET32a(+)-Hap在E.coliBL21中高效表达,Ni2+亲和层析柱纯化表达产物。用纯化的Hap重组蛋白进行体外竞争黏附实验,SEM观察及菌落形成单位计数法分析该重组蛋白的黏附活性。纯化蛋白与黏膜免疫佐剂CT-B联合鼻腔免疫BALB/C小鼠,ELISA检测小鼠抗Hap的IgA及IgG抗体水平。结果纯化产物的SDS-PAGE分析显示获得单一的目的蛋白条带,Gelanalysis软件分析蛋白纯度可达85%,纯化产物超滤浓缩后,测得其蛋白浓度为3.2g/L。体外竞争黏附实验观察到Hap重组蛋白的加入可明显抑制NTHi对胞外基质的黏附,且细菌黏附量的减少与对照组相比差异具有统计学意义(P<0.01)。该重组蛋白与免疫佐剂CT-B联合免疫小鼠,可刺激机体产生较高水平的IgG或IgA抗体,与重组抗原单独免疫组相比,差异具有显著性(P<0.05)。结论 Hap蛋白在原核系统中获得较高浓度和纯度的表达,纯化Hap重组蛋白具有良好的免疫原性和粘附活性。Objective To express and purify Hap protein of nontypeable Haemophilus influenzae （NTHi） in prokaryotic system, and study its immunogenicity and adhesive activity. Method Hap protein was expressed in E.coli BL21 with pET32a （＋）-Hap and purified by affinity chromatography. The adhesive activity of the recombinant Hap protein was observed in competitive adhesion assay using scanning electron microscope and bacterial counting. BALB/C mice were immunized intranasally with the purified recombinant Hap protein and cholera toxin B subunit （CT-B）, and anti-Hap IgA and IgG were detected by enzyme-linked immunosorbent assay. Results SDS-PAGE analysis showed a single band of the target protein, whose purity reached 85% according to the result of Gel analysis software. The concentration of the protein was 3.2 g/L after ultrafiltration and condensation. Competitive adhesion assay showed that compared with control group, the recombinant Hap protein significantly inhibited the adhesion of NTHi to ECM （P0.01）. Compared with Hap immunization alone, immunization with Hap combined with CT-B resulted in significantly higher titers of anti-Hap IgG and IgA in mice （P0.05）. Conclusion Highly purified recombinant Hap protein has been obtained in a prokaryotic system and shows good immunogenic and adhesive activities. These results will establish the basis for further study of NTHi vaccine.

关 键 词：不可分型流感嗜血杆菌 Hap重组蛋白 免疫原性 粘附活性 

分 类 号：R378[医药卫生—病原生物学]