肺炎链球菌假想蛋白SPD0414的表达纯化及保守性分析

Expression and purification of putative protein SPD0414 from Streptococcus pneumoniae and analysis of its conservation

目的:获得纯化的肺炎链球菌(S.pn)重组假想蛋白SPD0414,并分析其在常见S.pn菌株中的保守性。方法:利用PCR方法扩增SPD0414蛋白胞外区核酸序列,将其克隆到原核表达载体ppSUMO内,转化到E.coliBL21(DE3)中,经IPTG诱导表达后Ni-NTA树脂纯化重组蛋白。用SDS-PAGE和Western blot鉴定蛋白特异性及纯度。通过纯化蛋白免疫BALB/c小鼠制备其多克隆抗体,并用间接ELISA检测多克隆抗体的效价,Western blot方法分析多克隆抗体的特异性,同时,鉴定该蛋白在6种常见肺炎链球菌分离株的保守性。结果:克隆的SPD0414序列与Gene Bank中的数据相符,并实现了重组SPD0414蛋白高水平的可溶表达纯化蛋白免疫BALB/c小鼠获得高滴度、高特异性的的多克隆抗体,Western blot验证SPD0414蛋白在6株常见肺炎链球菌菌株中均有表达。结论:成功制备了高滴度、高特异性的SPD0414多克隆抗体,证实了SPD0414在肺炎链球菌不同菌株间保守性较高,为肺炎链球菌多肽联合疫苗的研制奠定了基础。

Objective：To obtain purified pneumococcal SPD0414 recombinant protein,and further to investigate the expressing of SPD0414 in 5 different serotypes of S.pneumoniae.Methods：Truncated SPD0414 gene was PCR amplified,and the fragments were then cloned into ppSUMO expressing vector.Recombinant protein SPD0414 was over-expressed and purified from the host bacteria of BL21（DE3）,and was then identified by SDS-PAGE and Western blot.Purified protein was introduced into BALB/c mice to produce polyclonal antibodies,and antibody titers were determined by indirect ELISA,then the polyclonal antibodies were used to analyze the expression and conservation of SPD0414 in 6 different S.pneumonia strains.Results：The SPD0414 gene was successfully cloned into ppSUMO vector which was confirmed by gene sequencing.The soluble recombinant protein SPD0414 was successfully over-expressed in E.coli BL21 manner,which was confirmed by SDS-PAGE and Western blot,respectively.Specific polyclonal antibodies were obtained from BALB/c mice and were used to confirm the conservatism of SPD0414 protein in 6 different strains of S.pneumoniae.Conclusion：Specific polyclonal antibodies of high titer against SPD0414 are obtained from the immunized BALB/c mice.Furthermore,SPD0414 protein is demonstrated to be exceptionally conserved in 6 different serotypes of S.pneumoniae.It is suggested that SPD0414 is a relatively highly-conserved protein,and that SPD0414 may be a promising candidate target antigen for developing pneumococcal protein vaccine.