肺炎链球菌DnaJ蛋白的原核表达及其免疫保护效果

目的原核表达肺炎链球菌(Streptococcus pneumoniae)热休克蛋白40(heat shock protein 40,HSP40,DnaJ),并探讨其免疫保护效果。方法从2、3、6B、14、19F、R6型肺炎链球菌基因组DNA中扩增DnaJ基因,插入原核表达载体pET-32a中,构建重组表达质粒pET-32a-DnaJ,转化大肠埃西菌(E.coli)BL21(DE3),IPTG诱导表达。表达的重组DnaJ蛋白经Ni-NTA树脂纯化后,分别经腹腔和鼻黏膜免疫小鼠,采用ELISA法检测小鼠血清和唾液中特异性IgG和IgA抗体滴度;用重组蛋白刺激小鼠脾细胞,ELISA法检测细胞因子水平的变化;流式细胞术检测特异性抗DnaJ血清结合到肺炎链球菌表面的能力;检测抗DnaJ血清抑制R6型肺炎链球菌黏附A549细胞的能力。结果 6株肺炎链球菌的PCR扩增产物均可见1 119 bp的DnaJ基因片段,测序结果与GenBank中登录的序列一致;重组表达质粒经双酶切鉴定构建正确;纯化的重组DnaJ蛋白相对分子质量约为38 000,纯度达90%以上,可与DnaJ小鼠免疫血清发生特异性反应。腹腔免疫DnaJ可使小鼠血清产生高滴度的特异性抗DnaJ抗体,鼻黏膜免疫DnaJ可增加小鼠血清和黏膜中抗DnaJ IgG和IgA抗体水平(P<0.01);鼻黏膜免疫DnaJ可使小鼠脾细胞释放高水平的细胞因子IL-10、IFNγ和IL-17A;抗DnaJ血清对肺炎链球菌具有更强的结合能力,且可抑制肺炎链球菌黏附肺癌上皮细胞A549。结论 DnaJ可诱导小鼠产生保护性免疫应答,提示其具有作为肺炎链球菌蛋白疫苗抗原的潜力。

Prokaryotic expression and immune protective effect of DnaJ protein of Streptococcus pneumoniae

Objective To express the heat shock protein 40(HSP40,DnaJ)of Streptococcus pneumonia and investigate its protective effect.Methods DnaJ gene was amplified from S.pneumonia of types 2,3,6B,14,19F and R6,and inserted into prokaryotic expression vector pET-32a.The constructed recombinant plasmid pET-32a-DnaJ was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed recombinant DnaJ protein was purified NiNTA chromatography,with which mice were immunized by i.p.and i.n.routes and determined for specific IgG and IgA titers in sera and saliva by ELISA.Murine splenocytes were stimulated with recombinant DnaJ protein and determined for cytokine level by ELISA.The binding ability of specific antiserum against DnaJ onto the surface of S.pneumonia was determined by flow cytometry.The ability of antiserum against DnaJ in inhibiting the adherence of S.pneumonia of type R6 to A549 cells was determined.Results DnaJ gene fragments each at a length of 1 119 bp was amplified from the six S.pneumoniae strains,of which the sequencing results were consistent with those reported in GenBank.Restriction analysis proved that recombinant expression plasmid was constructed correctly.Purified recombinant DnaJ protein,with a relative molecular mass of about 38 000,reached a purity of more than 90% and showed specific reaction with mouse anitserum against DnaJ.Immunization with DnaJ by i.p.route induced high titer specific antibody against DnaJ,which that by i.n.route increased the IgG level in sera and IgA level in mucosa(P < 0.01).However,immunization with DnaJ by i.n.route promoted the secretion of IL-10,IFNγ and IL-17A by murine splenocyte.Antiserum against DnaJ showed increased binding ab ility to S.pneumonia and inhibited the adherence of S.pneumonia to A549 cells.Conclusion DnaJ induced protective immune response in mice,indicating its potential as an antigen of S.pneumoniae protein vaccine.