猪链球菌靶向DC重组蛋白DC3pep-SDZ对斑马鱼的免疫保护性分析  

Analysis of Immune Protection of Streptococcus suis -Targeted Dendritic Cells Recombinant Protein DC3pep-SDZ on Zebrafish

作　　者：于恩远 荣睿璠 曾君 赵瑞利 郭海悦 张东超 胡烨 李颖[2] 王心如 YU Enyuan;RONG Ruifan;ZENG Jun;ZHAO Ruili;GUO Haiyue;ZHANG Dongchao;HU Ye;LI Ying;WANG Xinru(Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Breeding,College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300392,China;Tianjin Animal Disease Prevention and Control Center,Tianjin 300402,China)

机构地区：[1]天津农学院动物科学与动物医学学院天津市农业动物繁育与健康养殖重点实验室,天津西青300392 [2]天津市动物疫病预防控制中心,天津北辰300402

出　　处：《中国兽医杂志》2024年第4期35-42,共8页Chinese Journal of Veterinary Medicine

基　　金：天津市应用基础与前沿技术研究计划一般项目(18JCYBJC30100,14JCYBJC30000);天津市高校“中青年骨干创新人才”培养计划;天津市“131”创新型人才培养计划;天津市奶牛(肉羊)、生猪产业技术体系创新团队建设项目(ITTCRS2021000,ITTPRS2021005);天津市高等学校大学生创新创业训练计划(202110061017);天津市研究生科研创新项目(2021YJSS140);甘肃省东西协作项目(22CX8NL215)。

摘　　要：为了探究本课题组前期构建的由猪链球菌(SS)的分泌型DNA核酸酶A(SsnA)、二氢硫辛酰胺脱氢酶(DLDH)和高亲和力锌摄取系统蛋白(ZnuA)3个毒力因子的优势细胞表位和树突状细胞靶向肽(DC3pep)串联而成的重组蛋白DC3pep-SDZ(SsnA-DLDH-ZnuA)对致病型SS攻击斑马鱼的免疫保护效果,本试验表达和纯化重组蛋白DC3pep-SDZ和SDZ,分别免疫AB系野生型斑马鱼,攻击致病型2、3和9型SS(SS2、SS3和SS9),计算存活率,采用苏木精-伊红(H.E.)染色法观察斑马鱼脑组织病理变化,实时荧光定量PCR(RT-qPCR)分析SS3感染斑马鱼后脑组织炎性细胞因子表达量变化。结果显示,诱导表达的重组蛋白DC3pep-SDZ和SDZ分子质量分别为44和42 kDa,2个蛋白均以可溶的形式表达。SS2、SS3和SS9攻击斑马鱼后,重组蛋白DC3pep-SDZ组和重组蛋白SDZ组斑马鱼存活率分别为50.0%、65.0%和60.0%,45.0%、47.3%和45.0%,两组间差异不显著(P>0.05),但与PBS对照组相比差异均极其显著(P<0.001)。H.E.染色镜检结果显示,重组蛋白DC3pep-SDZ组的斑马鱼被3种分型SS攻击后脑组织均无明显病理变化。RT-qPCR结果显示,重组蛋白DC3pep-SDZ组促炎细胞因子:与PBS对照组相比较IL-6、TNF-α和IFN-γmRNA表达量分别极显著(P<0.01)、极其显著(P<0.001)和极显著(P<0.01)下调,与重组蛋白SDZ组相比较IL-6和TNF-αmRNA表达量分别极显著(P<0.01)和显著下调(P<0.05);重组蛋白DC3pep-SDZ组抗炎性细胞因子:与PBS对照组相比较IL-10和TGF-β1 mRNA表达量分别极显著(P<0.01)和极其显著(P<0.001)上调,与重组蛋白SDZ组相比较TGF-β1 mRNA表达量显著上调(P<0.05)。结果表明,重组蛋白DC3pep-SDZ较SDZ具有更好的免疫原性,能产生SS2、SS3和SS9交叉免疫保护作用,同时还能够有效缓解斑马鱼感染SS3后的脑部炎症。To investigate the immunoprotective effect of the recombinant protein DC3pep-SDZ(SsnA-DLDH-ZnuA)constructed by linking the dominant cell epitopes of three virulence factors of Streptococcus suis(SS),namely secretory DNA nuclease A(SsnA),dihydrolipoamide dehydrogenase(DLDH),and high-affinity zinc uptake system protein(ZnuA),with dendritic cell targeting peptide(DC3pep)against pathogenic SS in zebrafish,the recombinant proteins DC3pep-SDZ and SDZ were expressed and purified.AB strain wild-type zebrafish were immunized separately with DC3pep-SDZ and SDZ,then challenged with pathogenic serotypes 2,3, and 9 SS (SS2, SS3, and SS9), and survival rates were calculated. Brain tissue pathological changes in zebrafish were observed using Hematoxylin and Eosin (H.E.) staining, and changes in expression levels of inflammatory cytokines in zebrafish brain tissues infected with SS3 were analyzed using real-time fluorescence quantitative PCR (RT-qPCR). The results showed that the induced expression molecular weights of the recombinant proteins DC3pep-SDZ and SDZ were 44 and 42 kDa, respectively, both expressed in soluble form. After challenge with SS2, SS3, and SS9, the survival rates of zebrafish in the DC3pep-SDZ group and the SDZ groupwere 50.0%, 65.0%, 60.0%, and 45.0%, 47.3%, 45.0%, respectively, with no significant difference between the two groups ( P >0.05), but significantly different from the PBS control group ( P <0.001). H.E. staining microscopy results showed no significant pathological changes in the brain tissues of zebrafish attacked by the three serotypes of SS in the DC3pep-SDZ group. RT-qPCR results showed that in the DC3pep-SDZ group, pro-inflammatory cytokines IL -6 ( P <0.01), TNF-α ( P <0.001), and IFN-γ ( P <0.01) mRNA expression levels were significantly downregulated compared with the PBS control group, and IL -6 ( P <0.01) and TNF-α ( P <0.05) mRNA expression levels were significantly downregulated compared with the SDZ group;anti-inflammatory cytokines IL -10 ( P <0.01) and TGF-β1 ( P <0.001) mRNA

关 键 词：猪链球菌 斑马鱼 表位蛋白 DC3pep-SDZ 免疫保护 

分 类 号：S852.61[农业科学—基础兽医学]