肺炎链球菌神经氨酸酶nanA蛋白的原核表达、纯化及其免疫保护效果

Prokaryotic expression, purification and protective effects of nan A protein of Streptococcus pneumonia

目的原核表达并纯化肺炎链球菌(Streptococcus pneumonia,S.pn)神经氨酸酶nan A蛋白,并在小鼠模型中检测其保护效果,评价其作为S.pn疫苗候选蛋白的可行性。方法构建重组原核表达质粒p ET28a(+)-nan A,重组nan A蛋白经IPTG诱导及Ni-NTA亲和层析柱纯化后,采用黏膜(与CT佐剂混合)和腹腔(与Alum佐剂混合)给药途径免疫BALB/c小鼠,建立相应的S.pn感染模型,同时设相应对照组(佐剂+PBS),ELISA法检测特异性抗体及亚型;小鼠鼻腔滴定试验检测主动免疫保护效果;体内抗定植试验检测nan A蛋白对19F型S.pn在鼻咽部定植的保护作用。结果重组表达质粒p ET28a(+)-nan A经双酶切(NdeⅠ/XhoⅠ)及测序鉴定证明构建正确;重组蛋白的相对分子质量为55 000,主要以可溶性形式表达,约占菌体总蛋白的55%,纯化后纯度达90%;黏膜免疫组小鼠唾液中Ig A及Ig G效价分别为1.6×103和3.2×102,血清中Ig G效价为2.0×106,亚型主要为Ig G2a;腹腔免疫组血清中Ig G效价为0.5×106,亚型主要为Ig G1;黏膜和腹腔免疫小鼠生存时间较相应对照组显著延长(P<0.05),黏膜免疫组小鼠生存率较其对照组显著增加;黏膜免疫可显著降低S.pn 19F在宿主鼻咽部和肺部的定植。结论原核表达并纯化了nan A蛋白,诱导的小鼠免疫反应可有效抵抗S.pn的感染,并可显著降低S.pn在宿主鼻咽部及肺部的定植,表明nan A蛋白是较理想的S.pn疫苗候选蛋白。

Objective To express the neuraminidase nan A protein of Streptococcus pneumoniae（S. pn） in prokaryotic cells, purify the expressed product and determine its protective effect in mouse model so as to evaluate its feasibility as a candidate protein of S. pn vaccine. Methods Recombinant plasmid p ET28a（＋）- nan A was constructed and trans-formed to E. coli BL21（DE3） for expression under induction of IPTG. The expressed product was purified by Ni-NTA affinity chromatography. BALB / c mice were immunized with the purified protein mixed with CT（by intraperitoneal route）and aluminum（Alum） adjuvant（by mucosal route） respectively to establish the model of S. pn infection, using the corresponding adjuvant ＋ PBS as control. The specific antibody levels and subtype were determined by ELISA. The protective effect of active immunization was evaluated by nasal titration test, while that of nan A protein against the nasal col-onization of type 19 F S. pn by anti-colorization test in vivo. Results Restriction analysis（NdeⅠ/ XhoⅠ）and sequencing proved that the recombinant plasmid p ET28a（＋）-nan A was constructed correctly. The recombinant nan A protein, with a relative molecular mass of 55 000, mainly existed in a soluble form, contained about 55% of total somatic protein and reached a purity of 90%. The titers of Ig G and Ig A in saliva of mice immunized by mucosal route were 1. 6 × 103 and 3. 2 ×102respectively, while the Ig G in sera, which was mainly Ig G2 a, was 2. 0 × 10^6. However, the titer of Ig G in sera of mice immunized by intraperitoneal route, which was mainly Ig G1, was 0. 5 × 10^6. The survival time of mice immunized by mucosal and intraperitoneal routes as well as the survival rate of mice immunized by mucosal route increased significantly as compared with those in the corresponding control groups（P〈 0.05）. Mucosal immunization decreased the colonization of S. pn 19 F in nasopharyx and lung. Conclusion The nan A protein was expressed in prokaryotic cells and purified,which induced immune response against S. pn infection in mice and decreased the colonization of S. pn 19 F in nasopharyx and lung, indicating that nan A was an ideal candidate protein of S. pn vaccine.