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目的原核表达并纯化肺炎链球菌(Streptococcus pneumonia,S.pn)热休克蛋白ClpL,并评价其作为S.pn疫苗候选蛋白的可行性。方法PCR扩增S.pn D39全长ClpL基因,克隆至原核表达载体pET-28a(+)中,转化E.coli BL21(DE3),IPTG诱导表达,表达的重组ClpL蛋白经Ni-NTA亲和层析柱纯化后,免疫BALB/c小鼠,ELISA检测抗ClpL多克隆抗体的效价及亚型;Western blot分析ClpL蛋白在5种常见S.pn中的保守性;流式细胞术及Western blot分析ClpL蛋白在S.pn中的亚细胞定位。结果重组表达质粒pET-28a(+)-ClpL经双酶切及测序证实构建正确;ClpL蛋白在E.coli BL21(DE3)中获得了可溶性表达,纯化后纯度达90%,浓度为4.97 mg/ml;免疫小鼠可产生高滴度、高特异性的IgG抗体,以IgG1和IgG2b亚型为主;ClpL蛋白在5株常见S.pn中均有表达;ClpL蛋白为分泌型蛋白,不表达于S.pn表面。结论原核表达并纯化了S.pn ClpL蛋白,其作为一种在S.pn中保守存在的分泌型蛋白,可能是一种较好的疫苗候选蛋白。
Objective To express and purify the heat shock protein ClpL of Streptococcus pneumoniae (S.pn) and evaluate its feasibility as a candidate protein of S.pn vaccine. Methods ClpL gene of S.pn D39 was amplified by PCR and cloned into prokaryotic expression vector pET-28a (+). The recombinant plasmid was transformed into E.coli BL21 (DE3) and induced by IPTG. The recombinant ClpL protein was identified by Ni-NTA affinity After purification, the BALB / c mice were immunized and the titer and subtype of the anti-ClpL polyclonal antibody were detected by ELISA. The conservatism of ClpL protein in five common S.pn strains was analyzed by Western blot. Flow cytometry and Western blot analysis of subcellular localization of ClpL protein in S. pn. Results The recombinant plasmid pET-28a (+) - ClpL was confirmed by double enzyme digestion and sequencing. ClpL protein was successfully expressed in E. coli BL21 (DE3). The purity of ClpL protein was 90% after purification and the concentration was 4.97 mg / ml; Immunized mice produce high titer, high specificity of IgG antibodies, mainly IgG1 and IgG2b subtypes; ClpL protein in five common S.pn are expressed; ClpL protein is a secreted protein, not expressed On S.pn surface. Conclusions The prokaryotic expression and purification of S.pn ClpL protein, which is a secreted protein that is conserved in S.pn, may be a good vaccine candidate protein.