小鼠肺炎链球菌脂蛋白SPD1609多克隆抗体的制备

Preparation of mouse polyclonal antibodies against lipoprotein SPD1609 in Streptococcus pneumoniae

目的制备肺炎链球菌脂蛋白SPD1609的小鼠多克隆抗体。方法通过PCR扩增肺炎链球菌D39菌株中的spd1609基因,连接到原核表达载体pGEX-4T-1上,构建重组质粒pGEX-4T-1609。将重组质粒转化大肠杆菌BL21(DE3),用异丙基β-D-硫代半乳糖苷(IPTG)诱导表达谷胱甘肽巯基转移酶-SPD1609(GST-1609)融合蛋白。利用GST亲和层析柱纯化GST-1609融合蛋白,纯化后的融合蛋白用凝血酶(thrombin)外切酶切掉GST标签,进一步通过GST亲和层析得到SPD1609蛋白。用纯化的不含GST标签的SPD1609蛋白免疫小鼠,制备多克隆抗体,用ELISA检测抗体的效价,Western blot法检测抗体的特异性。结果成功构建了原核表达载体pGEX-4T-1609,经GST亲和层析柱分离纯化后可得到相对分子质量(Mr)35000的SPD1609蛋白,蛋白纯度在95%以上。ELISA检测结果显示纯化后的SPD1609蛋白可诱导小鼠产生特异性免疫应答,免疫小鼠血清抗体的效价达1∶40960,Western blot法检测显示此多克隆抗体可以特异地识别原核表达和肺炎链球菌细胞内表达的SPD1609蛋白。结论成功制备具有较好特异性的SPD1609蛋白的小鼠多克隆抗体。

Objective To prepare mouse polyclonal antibodies against lipoprotein SPD1609 in Streptococcus pneumoniae. Methods Firstly, the spd1609 gene in Streptococcus pneumoniae(S. pneumoniae) D39 strain was amplified and ligated into the prokaryotic expression vector of pGEX-4T-1 to generate recombinant plasmid pGEX-4T-1609. Secondly, the fusion protein of GST-1609 was expressed in Escherichia coli BL21(DE3), and purified using GST affinity chromatography. After cleaving the GST-tag of the fusion protein, the SPD1609 protein was obtained and purified using GST affinity chromatography. Finally, BALB/c mice were immunized with the purified SPD1609 protein to produce polyclonal antibodies. The titer and specificity of polyclonal antibodies were detected using ELISA and Western blotting. Results In this study, the recombinant vector of pGEX-4T-1609 was constructed, and the SPD1609 protein(Mr is approximately 35 000) was expressed and purified with a higher 95% purity. The ELISA results suggested that the SPD1609 protein could induce specific immune responses in mice, and the titer of antibody in the immunized mice was 1∶40 960. Western blotting indicated that the polyclonal antibody could specifically recognize the SPD1609 protein expressed in prokaryotic expression vector and in S. pneumoniae. Conclusion The mouse polyclonal antibody with good specificity against SPD1609 protein has been successfully obtained.