猪链球菌2型溶菌酶释放蛋白功能性片段的原核表达及其免疫保护性

目的表达猪链球菌2型(SS2)溶菌酶释放蛋白(MRP)的功能性片段(tmrp),并检测其对小鼠的免疫保护性。方法将构建的重组克隆载体pMD-tmrp经BamHⅠ、EcoRⅠ双酶切,将切下的目的基因tmrp亚克隆至原核表达载体pGEX-3X中,转化E.coliBL21(DE3),鉴定正确后,用IPTG诱导,谷胱甘肽亲和层析纯化GST-tmrp,免疫BALB/c小鼠,测定其免疫保护率。结果阳性工程菌经IPTG诱导,表达了可溶性目的蛋白,相对分子质量约为73000。0.8mmol/LIPTG,37℃,pH7.2诱导4h,表达量最高,为25.36%。经亲和层析纯化,融合蛋白纯度达93.7%。免疫BALB/c小鼠后,免疫保护率可达60%。结论已成功获得了具有免疫活性的GST-tmrp。

Prokaryotic Expression and Immunoprotecfion of Functional Fragment of Muramidase-released Protein of Streptococcus suis Serotype 2

Objective To express a functional fragment of muramidase-released protein(MRP) of Streptococcus suis serotype 2 (SS2),TMRP,and study its immunoprotection in mice.Methods Digest the recombinant plasmid pMD-tmrp with BamH I and EcoR I,and subclone the obtained tmrp gene to prokaryotic expression vector pGEX-3X.Transform the constructed recombinant plasmid pGEX-tmrp to E.coli BL21(DE3) for expression under induction of IPTG.Purify the expressed GST-tmrp by GST affinity chromatography.Immunize BALB/c mice with the purified GST-tmrp and determine the protective rate.Results Soluble GST-tmrp with a relative molecular mass of about 73 000 was expressed and reached a purity of 93.7% after purification.The expression level reached 93.7% after induction with 0.8 mmol/L IPTG at 37℃,pH7.2 for 4 h.The protective rate of mice immunized with the purified fusion protein was 60%.Conclusion The GST-tmrp with immunological activity was successfully expressed.