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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 and purify the neuraminidase nan A protein of Streptococcus pneumonia (S.pn) in prokaryotic cells and test its protective effect in mouse model to evaluate its feasibility as a candidate protein of S.pn vaccine. Methods Recombinant prokaryotic expression plasmid p ET28a (+) - nan A was constructed. The recombinant nan A protein was induced by IPTG and purified by Ni - NTA affinity chromatography. The recombinant adenovirus protein was purified by mucosal (mixed with CT adjuvant) and intraperitoneally BALB / c mice were immunized with different doses of S.pn, and the corresponding S.pn infection model was established. At the same time, the corresponding control group (adjuvant + PBS) was established. The specific antibodies and subtypes were detected by ELISA. The nasal titration test Immune protection effect; In vivo anti-colonization test to detect nan A protein on the 19F-type S.pn in nasopharyngeal colonization protection. Results The recombinant plasmid pET28a (+) - nan A was confirmed by Nde Ⅰ / Xho Ⅰ sequencing and sequencing. The recombinant protein had a molecular weight of 55 000 and was mainly expressed in soluble form, , And the purity was 90% after purification. The Ig A and Ig G titers in saliva of mucosal immunized mice were 1.6 × 103 and 3.2 × 102, respectively. The titer of Ig G in serum was 2.0 × 106, Ig G2a in the peritoneal immunization group, the IgG titer in the peritoneal immunization group was 0.5 × 106, and the subtype was mainly Ig G1. The survival time of mucosal and intraperitoneal immunized mice was significantly longer than that of the corresponding control group (P <0.05) Mucosal immunization can significantly reduce S.pn 19F colonization in the nasopharynx and lung of the host. Conclusion The prokaryotic expression and purification of nan A protein induced the mouse immune response is effective against S.pn infection and significantly reduced S.pn colonization in the nasopharynx and lung of the host, indicating that nan A protein is more ideal S.pn vaccine candidate protein.