牛边缘无浆体膜表面蛋白5的原核表达及其免疫原性

Prokaryotic Expression and Immunogenicity of Membrane Surface Protein 5 of Anaplasma marginale

目的原核表达牛边缘无浆体(Anaplasma marginale)膜表面蛋白5(Membrane surface protein 5,MSP5),并分析其免疫原性。方法 pQE-MSP5/BL21(DE3)阳性重组菌经IPTG诱导表达,表达产物经MagneHisTM蛋白纯化系统纯化后,进行SDS-PAGE分析。将纯化的MSP5蛋白免疫小鼠,以生理盐水免疫作为阴性对照,ELISA法检测血清抗体水平,淋巴细胞增殖试验检测脾淋巴细胞增殖水平,流式细胞术检测T淋巴细胞亚群分类。结果表达的重组MSP5蛋白相对分子质量约为23 000;纯化蛋白的纯度为83.2%,浓度为1.087 mg/ml;纯化的MSP5蛋白可刺激小鼠产生特异性抗体;免疫组小鼠淋巴细胞增殖水平明显高于阴性对照组(P<0.05);免疫组小鼠CD3+、CD4+和CD4+/CD8+比例高于阴性对照组,CD8+比例低于阴性对照组。结论已成功表达并纯化了牛边缘无浆体MSP5,其免疫原性良好,为制备单克隆抗体及建立准确可靠的诊断方法奠定了基础。

Objective To express the membrane surface protein 5（MSP5） of Anaplasma marginale in prokaryotic cells and analyze its immunogenicity.Methods Recombinant E.coli pQE-MSP5/BL21（DE3） was induced with IPTG,and expressed product was purified by MagneHisTM protein purification system then analyzed by SDS-PAGE.BALB/c mice were immunized with the purified MSP5,using physiological saline as negative control.The serum antibody level of immunized mice was determined by ELISA,while the splenic lymphocyte proliferation level by lymphocyte proliferation test,and T lymphocyte subgroup by flow cytometry.Results The expressed recombinant MSP5,with a relative molecular mass of about 23 000,reached a purity of 83.2% and a concentration of 1.087 mg/ml after purification.Purified MSP5 induced specific antibody in mice.The lymphocyte proliferation level,CD3＋ and CD4＋ lymphocyte counts as well as CD4＋/CD8＋ ratio of immunized mice were significantly higher,while the CD8＋ lymphocyte count was significantly lower,than those in negative control group（P 〈 0.05）.Conclusion The MSP5 of A.marginale was successfully expressed and purified,which showed high immunogenicity.It laid a foundation of preparing monoclonal antibody and developing an accurate and reliable diagnostic method for A.marginale infection.