肺炎链球菌SPD1672蛋白EL4功能环的原核表达及多克隆抗血清制备

Prokaryotic expression of external loop 4 of Streptococcus pneumoniae SPD1672 protein and preparation of polyclonal antibody

目的原核表达肺炎链球菌SPD1672蛋白膜外EL4功能环,制备并鉴定其多克隆抗血清。方法生物信息学分析SPD1672蛋白结构功能区,用p Cold TF质粒构建含EL4功能环的原核表达载体,IPTG诱导蛋白质表达后行镍柱纯化。经HRV 3C蛋白酶酶切、鉴定后,免疫新西兰大白兔以制备多克隆抗血清,间接ELISA鉴定效价,western blot鉴定抗血清与肺炎链球菌D39菌株天然蛋白质的亲合力。结果 EL4环可能为SPD1672蛋白酶活性中心。成功构建p Cold TF-SPD1672EL4原核表达载体,诱导表达后获得纯度大于95%的重组融合目的蛋白质。该蛋白质免疫新西兰大白兔后获得效价达到5.12×106的多克隆抗血清,此抗血清与重组的SPD1672EL4蛋白及肺炎链球菌中天然SPD1672蛋白有较好的亲合力。结论成功获得高纯度的肺炎链球菌SPD1672EL4蛋白及其特异的多克隆抗血清,为进一步对SPD1672蛋白的结构与功能研究奠定了基础。

Objective To express and purify the external loop 4 of Streptococcus pneumoniae SPD1672 protein by prokaryotic expres- sion system, and prepare and identify its polyclonal antibody. Methods The function region in molecular structure of SPD1672 pro- tein was analyzed by bioinformatics. The DNA fragment encoding the external loop 4 of SPD1672 protein was inserted into prokaryotic expression vector pCold TF which was transfected to E. coli BL21 （DE3） for protein expression induced by IPTG at low temperature. The expressed recombinant protein was purified by Ni-Sepharose 6 FF affinity chromatography and identified by HRV 3C protease diges- tion. New Zealand rabbits were immunized with the purified protein to prepare polyclonal antibody. The antibody titer was determined by indirect ELISA, and the affinity of antibody yo natural SPD1672EL4 proteins from Streptococcus pneumoniae D39 was verified by western blot. Results The external loop 4 may be the center of protease activity of SPD1672. The prokaryotic expression vector pCold- TF-SPD1672EIA was successfully constructed and the induced recombinant protein was prepared with high purity of more than 95 per- cent. The antiserum with high titer of 5.12 × 10^6 was obtained, which showed good affinity to both induced SPD1672EL4 and natural SPD1672 protein of Streptococcus pneumoniae. Conclusion The recombinant SPDI672EL4 protein with high purity and specific poly- clonal antibody were successfully prepared for the further study on the structure and function of SPD1672.