马链球菌兽疫亚种重组表面蛋白rSeseC-02147免疫效力评价

Immune efficacy evaluation of recombinant surface protein rSeseC-02147 of Streptococcus equi subsp.zooepidimicus

【目的】通过原核表达系统制备马链球菌兽疫亚种(SEZ)重组表面蛋白rSeseC-02147,并系统评价其抵御SEZ感染的免疫效力,为SEZ亚单位疫苗的研发提供候选抗原。【方法】构建原核表达载体pCold I-SeseC-02147,转化大肠杆菌BL21(DE3)感受态细胞后进行异丙基硫代半乳糖苷(IPTG)诱导,纯化回收重组蛋白rSeseC-02147并免疫BALB/c小鼠,以SEZ灭活疫苗为阳性对照、PBS为阴性对照,二免后第14 d于小鼠颌下静脉采集血清,分别采用Western blotting和酶联免疫吸附试验(ELISA)检测重组蛋白rSeseC-02147反应原性及小鼠血清抗体效价和抗体亚型属性;并通过小鼠免疫保护试验、腹腔灌洗液细菌载量测定及脏器组织病理学观察,系统评估重组蛋白rSeseC-02147对小鼠的免疫保护力。【结果】构建的原核表达载体pCold I-SeseC-02147经IPTG诱导能成功表达获得重组蛋白rSeseC-02147,其纯化后的浓度为2.86 mg/mL。重组蛋白rSeseC-02147能与免疫组和阳性对照组小鼠血清发生特异性反应,而与阴性对照组小鼠血清未发生特异性反应,表明重组蛋白rSeseC-02147具有良好的反应原性与特异性;以重组蛋白rSeseC-02147免疫小鼠产生的血清抗体效价远高于SEZ灭活疫苗,且IgG1亚型极显著高于IgG2a亚型(P<0.01,下同)。重组蛋白rSeseC-02147可为小鼠提供抵御SEZ感染的免疫保护力,攻毒后14 d内的存活率60%;此外,重组蛋白rSeseC-02147免疫小鼠后的腹腔灌洗液细菌载量极显著低于阴性对照组小鼠,且肺脏、肾脏和脾脏等组织的病理损伤程度较轻。【结论】通过原核表达系统制备获得的重组蛋白rSeseC-02147具有良好的特异性与反应原性,通过抑制SEZ增殖及减轻SEZ对机体脏器的损伤而发挥免疫保护作用。可见,重组蛋白rSeseC-02147具有开发成SEZ亚单位疫苗的潜力。

【Objective】The aim of the study was to prepare the recombinant surface protein rSeseC-02147 of Strepto coccus equi subsp.zooepidimicus(SEZ)using a prokaryotic expression system,and to systematically evaluate its im mune efficacy against SEZ infections,providing a candidate antigen for the development of SEZ subunit vaccines.【Method】The prokaryotic expression vector pCold I-SeseC-02147 was constructed and transformed into Escherichia coli BL21(DE3)competent cells,followed by isopropyl thiogalacto side(IPTG)induction.The recombinant protein rSeseC-02147 was purified and recovered to immunize BALB/c mice,with the inactivated SEZ vaccine as the positive control and PBS as the negative control.Serum was collected from the submandibular vein of mice on the 14th day after the secondary immunization.The reactogenicity of the recombinant protein rSeseC-02147,as well as the antibody titers and anti body subtypes of the mouse serum were detected using Western blotting and enzyme-linked immunosorbent assay(ELISA),respectively.In addition,the immunoprotective efficacy of the recombinant protein rSeseC-02147 in mice was systematically evaluated by mouse immune protection tests,bacterial load detection in the peritoneal lavage fluid and his topathological observation of organs.【Result】The constructed prokaryotic expression vector pCold I-SeseC-02147,suc cessfully expressed the recombinant protein rSeseC-02147 after being induced by IPTG,achieving a purified concentra tion of 2.86 mg/mL.The recombinant protein rSeseC-02147 specifically reacted with the serum of mice in the immunized group and the positive control group,but did not react specifically with the serum of mice in the negative control group,indicating that rSeseC-02147 had good reactogenicity and specificity.The serum antibody titer produced by immunized mice with recombinant protein rSeseC-02147 was much higher than that from the inactivated SEZ vaccine,and the IgG1 subtype was extremely significantly higher than the IgG2a subtype(P<0.01,the same below).The recombinant protein rSeseC-02147 provided mice with immune protection against SEZ infection,with a survival rate of 60%within 14 d post challenge.Additionally,the bacterial load in the peritoneal lavage fluid of the mice immunized with the recombinant pro tein rSeseC-02147 was extremely significantly lower than that in the negative control group,and these mice exhibited milder pathological damage to tissues such as lungs,kidneys,and spleens.【Conclusion】The recombinant protein rSeseC-02147,prepared using the prokaryotic expression system,exhibits good reactogenicity and specificity.It provides im mune protection by inhibiting the SEZ proliferation and attenuating the damage of SEZ to bodily organs.Therefore,the re combinant protein rSeseC-02147 has the potential to be developed into an SEZ subunit vaccine.