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 and immunization of Glutamyl tRNA Synthetase of Streptococcus suis

【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433μg/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。

[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] 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 58 kD 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 （1.2×10^9 CFU）. 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.