马腺疫链球菌GAPDH重组蛋白的表达及其免疫保护效果的研究

Expression and Immune Protection Effection Analysis of GAPDH Recombinant Protein of Streptococcus equi

为制备马腺疫链球菌(Streptococcus equi subspecies equi)GAPDH蛋白的多克隆抗体,研究其免疫保护作用。以S.equi分离株为研究对象,利用PCR技术扩增其GAPDH基因并构建原核表达质粒,转化至大肠杆菌。诱导表达重组蛋白,对纯化后的蛋白进行SDS-PAGE及Western blotting分析。以纯化后的GAPDH重组蛋白免疫小鼠,用酶联免疫吸附法、攻击保护试验检测其免疫效果。结果显示,成功构建了pET-28a-GAPDH原核表达载体,纯化获得30 kDa重组蛋白。该蛋白质能特异性识别该菌抗血清,诱导小鼠产生较高水平的特异性抗体IgG,在初次免疫后第45天,抗体水平效价可达1∶24 300,攻击保护率可达87.5%。该GAPDH重组蛋白具有良好的免疫原性和免疫保护效果。

To prepare a polyclonal antibody against GAPDH protein of Sterptococcus equi sub equi, and investigated its immunoprotection. In this study, S.equi isolate was used and GAPDH gene was amplified using PCR method the prokaryotic expression plasmids was constructed and transformed into Escherichia coli. The recombinant protein was induced and expressed. After purification the protein was analyzed by SDS-PAGE and Western blotting. The purified recombinant protein GAPDH were used to immunize mice,the immunogenicity of the recombinant protein was tested using ELISA and challenge protection methods.The result showed that the prokaryotic expression vector pET-28a-GAPDH was successfully constructed and the purified recombinant protein GAPDH was 30 kDa.The protein could specifically recognize antiserum of S. equi and could induce high levels of specific IgG antibody in mice. 45 d after the first immunization,the antibody titer could reach to 1∶24 300 and the protection rate was up to 87.5%.Our result indicated that the recombinant GAPDH had good immunogenicity and immune protection.