摘要
目的原核表达肺炎球菌表面蛋白A(Pneumococcal surface protein A,PspA),并探讨其作为多糖结合疫苗候选载体的可行性。方法合成PspA基因,定向克隆至pET-30a(+)载体,构建重组表达质粒pET-30a-rPspA,转化E.coli Sta(rDE3)菌株,IPTG诱导表达。表达产物经Ni离子亲和层析纯化后,经Western blot鉴定。取破伤风类毒素(Tetanus toxoid,TT)及纯化的rPspA,分别与A群脑膜炎球菌多糖(Group A meningococcal capsular polysaccharide,GAMP)通过溴化氰活化法进行偶联,获得rPspA-GAMP与TT-GAMP多糖蛋白结合物,对其进行纯化及检定。多糖结合物分别以滴鼻和肌肉注射途径免疫BALB/c小鼠,评价其诱发的体液免疫和黏膜免疫水平。结果重组表达质粒经双酶切及测序鉴定构建正确;表达产物主要以可溶形式存在,表达量约占菌体总蛋白的20%,经纯化后纯度可达90%,可与His单抗特异性结合;肌肉注射途径显示,两种蛋白载体的结合物均能诱发较高水平的体液免疫,不同载体间差异无统计学意义(P>0.05);滴鼻途径显示,载体蛋白rPspA的结合物刺激产生sIgA的能力优于传统载体TT,差异有统计学意义(P<0.05)。结论原核表达并纯化了rPspA,其具有新型多糖蛋白载体的潜力,也可作为黏膜投递型多糖结合疫苗的候选载体。
Objective To express pneumococcal surface protein A(PspA) in prokaryotic cells and investigate its feasibility as a carrier of polysaccharide conjugate vaccine.Methods PspA was synthesized and cloned into vector pET-30a(+).The constructed recombinant plasmid pET-30a-rPspA was transformed to E.coli Star(DE3) and induced with IPTG.The expressed product was purified by nickel ion affinity chromatography and identified by Western blot.Tetanus toxoid(TT) and purified recombinant PspA(rPspA) were conjugated with group A meningococcal capsular polysaccharide(GAMP) by activation with cyanogen bromide respectively,and the obtained TT-GAMP and rPspA-GAMP conjugates were purified and identified.The conjugates were administered to mice by intranasal and intramuscular routes separately,and the induced humoral and mucosal immune responses were evaluated.Results Restriction analysis and sequencing proved that recombinant plasmid pET-30a-rPspA was constructed correctly.The expressed rPspA protein mainly existed in a soluble form,contained about 20% of total somatic protein,reached a purity of about 90% after purification and showed specific binding to anti-His monoclonal antibody.Both rPspA-GAMP and TT-GAMP conjugates administered by intramuscular route induced high humoral immune response levels which showed no significant difference(P 0.05).However,the sIgA titer induced with rPspA-GAMP by mucosal route was significantly higher than those with TT-GAMP(P 0.05).Conclusion Recombinant PspA was successfully expressed in prokaryotic cells and purified,which might be used as a potential novel carrier of polysaccharide protein and a candidate carrier of polysaccharide conjugate vaccine for mucosal administration.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第12期1381-1384,共4页
Chinese Journal of Biologicals
基金
国家十一五科技支撑计划资助项目(2008BAI66B03)
关键词
肺炎球菌表面蛋白A
多糖结合疫苗
载体
Pneumococcal surface protein A(PspA)
Polysaccharide conjugate vaccine
Carrier