马链球菌兽疫亚种SclE蛋白原核表达及其免疫效力评价
Prokaryotic expression of SclE protein from Streptococcus equi ssp.zooepidemicus and evaluation on its immune efficacy
徐宇萌 1郭政 1李亚娟 2邓静飞 1何颍欣 1黄云飞 2付强2
作者信息
- 1. 佛山科学技术学院,广东佛山 528225
- 2. 佛山科学技术学院,广东佛山 528225;佛山佛科院动物医院有限公司,广东佛山 528225
- 折叠
摘要
[目的]探究马链球菌兽疫亚种(Streptococcus equi ssp.zooepidemicus,SEZ)表面蛋白SclE的免疫效力,为预防SEZ引起的链球菌病提供理论依据.[方法]采用PCR扩增SEZ的SclE基因,再将该片段克隆至原核表达pCold I载体上,然后转化大肠杆菌BL21(DE3)感受态细胞,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后以SDS-PAGE进行分析鉴定,获得的重组蛋白SclE(rSclE)(100 μg/mL,0.5 mL)以腹腔注射形式免疫BALB/c小鼠(n=10,一免、二免的周期均为14d).一免、二免14d后收集血清,通过ELISA和Western blotting检测抗体水平和特异性.二免后进行免疫保护试验,并对攻毒后小鼠的肝、脾、肺、肾进行细菌载量检测和病理组织学观察.[结果]SDS-PAGE检测到大小约为93 kD的蛋白表达,与SclE的理论分子量大小一致;Western blotting检测显示,表达所得重组蛋白能与SEZ阳性对照组(腹腔注射0.5 mL SEZ灭活疫苗)小鼠血清发生特异性结合,表明其具有良好的反应原性.ELISA检测显示,二免后小鼠血清中特异性抗体水平与一免后相比均有显著提升(P<0.05,下同),且可诱导小鼠产生高滴度的IgG,其中IgG1抗体水平(0.436±0.031)显著高于IgG2a(0.161±0.009),表明rSclE诱导的抗体亚型以IgG1为主;rSclE免疫小鼠可显著抑制SEZ在各脏器中的增殖能力,肝脏、脾脏出血及炎性细胞浸润的症状得到改善,肺脏和肾脏出血等病理损伤减轻;在小鼠免疫攻毒保护试验中保护70%小鼠免受致死剂量SEZ毒株的攻击.[结论]成功表达获得的rSclE能诱导小鼠产生SclE特异性抗体,且具有较好的保护效果,可作为一种SEZ潜在的疫苗候选抗原.
Abstract
[Objective]To investigate the immune efficacy of the surface protein SclE of Streptococcus equi ssp.zooepidemicus(SEZ),to provide theoretical basis for prevention of streptococcal disease caused by SEZ.[Method]The SclE gene of SEZ was amplified by PCR and cloned into prokaryotic expression vector pCold I.The vector was trans-formed into the competent cells of Escherichia coli BL21(DE3),and finally induced by isopropyl-beta-D-thiogalactopy-ranoside(IPTG),after SDS-PAGE analysis,BALB/c mice were immunized with recombinant protein SclE(rSclE)(100 μg/mL,0.5 mL)by intraperitoneal injection(n=10,the cycle of first and second immunization was 14 d).Serum was collected 14 d after the first and second immunization,and antibody levels and specificity were detected by ELISA and Western blotting.After secondary immunization,the mice were tested for immune protection,and the bacterial load of liver,spleen,lung and kidney after challenge was detected and histopathology was observed.[Result]SDS-PAGE identi-fication showed that rSclE was successfully expressed,and the size of rSclE was about 93 kD,which was consistent with the theoretical value.Western blotting showed that the recombinant protein could bind to SEZ positive control group mice serum specifically(intraperitoneal injection of 0.5 mL of SEZ inactivated vaccine),indicating that rSclE had good immu-nogenicity.ELISA showed that the specific antibody levels in the serum increased significantly after the secondary immu-nization(P<0.05,the same below),and the mice could produce high IgG titers,the antibody level of IgG1(0.436±0.031)was significantly higher than that of IgG2a(0.161±0.009).These results indicated that IgG1 was the main subtype of rSclE induced antibody.rSclE immunized mice could significantly inhibit the colonization ability of SEZ in each organ,liver and spleen hemorrhage and inflammatory cell infiltration were alleviated,lung and kidney bleeding and other patho-logical injuries were alleviated;in the mouse immune challenge protection experiment,70%mice were protected from lethal dose of SEZ strain.[Conclusion]The successfully expressed rSclE can induce mice to produce SCLEspecific anti-bodies,and has a good protective effect,and can be used as a potential vaccine candidate antigen for SEZ.
关键词
马链球菌兽疫亚种(SEZ)/SclE/亚单位疫苗/免疫Key words
Streptococcus equissp.zooepidemicus(SEZ)/SclE/subunit vaccine/immunization引用本文复制引用
基金项目
国家自然科学基金(31872443)
广东省基础与应用基础研究基金(2022A1515140052)
佛山市高层次人才及岭南学者科研启动项目(CGZ07001)
佛山市科技创新项目(2020001004909)
出版年
2023
国家科技期刊平台
NSTL
万方数据