肺炎链球菌假想蛋白SPD0873的表达纯化及保守性分析

Expression and Purification of Streptococcus Pneumoniae Putative Protein SPD0873 and Analysis of Its Conservative Nature

目的通过构建原核表达载体，获得纯化的肺炎链球菌S．pn重组假想蛋白SPD0873，并制备多克隆抗体，进一步分析其在常见S．pn菌株中的保守性。方法分离培养D39型肺炎链球菌，获取其基因组DNA。利用PCR方法扩增去除信号肽的spd0873序列，采用基因体外重组法将spd0873序列克隆到原核表达载体pET-32（a）内，测序鉴定。将重组质粒转化到E．coli Rossetta（DE3）中，经IPTG诱导大量表达融合6个组氨酸标签的SPD0873重组蛋白，经Ni—NTA树脂纯化后，获得的重组蛋白用SDS—PAGE和Western印迹鉴定；将鉴定后纯化的蛋白免疫BALB／C小鼠制备多克隆抗体，并用间接ELISA检测多克隆抗体的效价，Western印迹方法分析多克隆抗体的特异性，同时，鉴定该蛋白在5种常见肺炎链球菌分离株的保守性。结果克隆的spd0873序列与GenBank中的数据相符，并实现了SPD0873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印迹验证SPD0873蛋白在5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SPD0873蛋白多克隆抗体，同时，检测到SPD0873蛋白在5种常见的肺炎链球菌菌株中非常保守，为研究该蛋白的生物学功能及肺炎链球菌多肽联合疫苗的研制奠定了基础。

Objective To obtain purified SPD0873 protein produced by prokaryotic expression system, prepare polyclonal antibody by immunizing BALB/C mice, and further investigate if SPD0873 protein expresses in 5 most common serotypes of S. pneumoniae. Methods Template DNA was isolated from cultured S. pneumoniae D39, from which a truncated spd0873 gene with N-terminal removal of its signal peptide was amplified by PCR. The PCR fragments were then cloned into pET-32 （a） expression vector, followed by sequencing analysis. Recombinant protein SPD0873 was over-expressed and purified from the host of Rossetta （DE3）, and identified by SDS-PAGE and Western blot. Purified protein was introduced to BALB/C mice to obtain polyclonal antibody. Antibody titers were evaluated by indirect ELISA. The antiserum was used to analyze the expression and conservative features of SPD0873 protein in 5 different S. pneumonia strains. Results The spd0873 gene was successfully cloned into pET-32 （a） , as confirmed by gene sequencing. The recombinant SPD0873 protein was successfully over-expressed in soluble manner, as shown by SDSPAGE and Western blot. Specific antiserum obtained from BALB/C mice showed high titers and high specificity in confirming conservative expression of SPD0873 protein in 5 common strains of S. pneumoniae. Conclusion High titers and specific polyclonal antibodies against SPD0873 were obtained by immunizing BALB/C mice. Furthermore, SPD0873 protein was demonstrated to be exceptionally conserved among 5 different serotypes of S. pneumoniae, suggesting that SPD0873 is a highly-conserved protein antigen, which is beneficial for analysis of its biological function, thus making SPD0873 a promising vaccine protein candidate.