论文部分内容阅读
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 candidate vector for polysaccharide conjugate vaccine. Methods The PspA gene was synthesized and cloned into pET-30a (+) vector. The recombinant plasmid pET-30a-rPspA was constructed and transformed into E. coli Sta (rDE3) strain for expression under induction of IPTG. The expressed product was purified by Ni ion affinity chromatography and identified by Western blot. Tetanus toxoid (TT) and purified rPspA were respectively coupled with Group A meningococcal capsular polysaccharide (GAMP) by cyanogen bromide activation method to obtain rPspA-GAMP and TT- GAMP polysaccharide protein conjugate, which is purified and assayed. Polysaccharide conjugates were intranasally and intramuscularly immunized BALB / c mice to evaluate their humoral and mucosal immune responses. Results The recombinant plasmid was identified by double enzyme digestion and sequencing. The expressed product was mainly existed in soluble form and accounted for about 20% of the total bacterial proteins. The purity of recombinant plasmid was about 90% (P> 0.05). The intranasal route showed that the conjugate of carrier protein rPspA showed no significant difference (P> 0.05). The results of intramuscular injection showed that the combination of the two protein carriers induced a higher level of humoral immunity, The ability of sIgA stimulation to produce sIgA was superior to that of the traditional vector TT, the difference was statistically significant (P <0.05). Conclusion Prokaryotic expression and purification of rPspA, which has the potential of a novel polysaccharide protein carrier, can also be used as a candidate carrier for the mucosal delivery polysaccharide conjugate vaccine.