猪链球菌谷氨酰胺tRNA合成酶的原核表达产物的小鼠免疫试验

Prokaryotic expression of Glutamyl tRNA Synthetase of Streptococcus suis and its immunization experiment

摘要:

摘要：【目的】猪链球菌谷氨酰tRNA合成酶（Glutamyl tRNA Synthetase, GtS）是一种催化谷氨酰胺与对应的tRNA发生酯化反应形成谷氨酰tRNA的蛋白酶。本研究通过小鼠免疫保护力实验来评价Gts的免疫原性。【方法与结果】使用clustalX对Genbank中链球菌属的GtS进行同源性分析，结果显示GtS的氨基酸序列在链球菌属内同源性达到95%以上。利用原核表达系统在大肠杆菌BL21中表达(His)6-GtS融合蛋白。SDS-PAGE和Western blot结果表明该融合蛋白在BL21菌株中高效表达，并且具有良好的免疫原性。融合蛋白经过组氨酸标签纯化试剂盒 (Novagen) 纯化后获得纯度为93.3%、浓度为433 μg/mL的重组蛋白rGtS。在小鼠免疫保护试验中，使用rGtS混合弗氏佐剂免疫Balb/c小鼠。免疫过后用4倍LD50剂量的SC19菌株（1.2×109CFU）攻毒，rGtS免疫组小鼠的存活率达50% (4/8)，高于空载体对照（1/8）。【结论】本研究证实rGtS融合蛋白具有良好免疫原性，能够提供部分的保护，是一种非常有潜力的亚单位疫苗候选蛋白。

Abstract:

Abstract: 【Objective】 Glutamyl tRNA Synthetase (Gts) is a protease which catalyzes esterification between tRNA and glutamine. The immunogenicity of Gts was evaluated through immunization and challenge experiment.【methods and results】According to ClustalX analysis, GtS is conservative in Streptococcuci. In this study, we cloned gts from the genome of SC19, and inserted it into prokaryotic expression plasmid pET28a-gts. The recombinant vector was transformed into E. coli BL21. Induced by IPTG, one 58kD protein was expressed and purified by using Ni+–NTA column (Novagen). The purity of rGtS was 93.3% and the concentration of purified protein was 433 μg/mL. We proved the immunogenicity of recombinant protein rGtS by western blot analysis. We immunized Balb/c mice with rGtS and Freund’s adjuvant, and after two boost vaccinations, all mice were challenged with 4 times LD50 amount of SC19 (1.2×109CFU). The survive rate of vaccination group is 50% (4/8), significantly higher than blank vector control group (1/8). 【Conclusion】 These results proved that GtS has certain immunogenicity and can offer partial protection against high dose challenge. Therefore Gts could be a potential candidate of subunit vaccine against Streptococcus suis.