斯氏艾美耳球虫泛素结合酶重组蛋白对家兔免疫保护效果的评价  

Evaluation of the immunoprotective effect of recombinant protein of Eimeria stiedai ubiquitin-conjugating enzyme on rabbits

作　　者：白鑫 肖洁 郑若愚 陈浩 蒲家艳 郝哥 熊常明 何维 任永军[2,3] 杨光友 BAI XinΔ;XIAO JieΔ;ZHENG Ruo-yu;CHEN Hao;PU Jia-yan;HAO Ge;XIONG Chang-ming;HE Wei;REN Yong-jun;YANG Guang-you(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Sichuan Animal Science Academy,Chengdu 610066,China;Animal Breeding and Genetics Key Laboratory of Sichuan Province,Chengdu 610066,China)

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]四川省畜牧科学研究院,四川成都610066 [3]动物遗传育种四川省重点实验室,四川成都610066

出　　处：《中国兽医科学》2023年第5期621-630,共10页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017YFD0501200);四川省科技计划项目(2022ZHYZ0003)。

摘　　要：观察斯氏艾美耳球虫(Eimeria stiedai)重组蛋白r Es-UCE、r Es-EFG和r Es-DG32对家兔的免疫保护效果。利用相对荧光定量PCR分析Es-UCE、Es-EFG和Es-DG323个基因在虫体不同发育时期的转录水平并进行原核表达与蛋白纯化。在进行了3个重组蛋白对家兔的免疫保护效果初步试验后,筛选出r Es-UCE进行进一步评价,将42只45日龄无球虫家兔分为空白对照组、攻虫对照组、空载蛋白对照组、佐剂对照组和r Es-UCE免疫组,各组分别颈部皮下注射1 m L无菌PBS,1 m L无菌PBS,1 m L(2 mg/m L)皂素,1 m L(100μg/m L)Trx-His-S tag蛋白,1 m L(100μg/m L)r Es-UCE,14 d后同等剂量进行二免。二免后第14天攻虫,除空白对照组外每只无球虫兔经口感染1×10^(4)个斯氏艾美耳球虫孢子化卵囊。感染后定期观察各组兔的临床症状、称重并采血,感染后第21天剖检并收集直肠粪样,测定并统计各组兔的死亡率、卵囊排出量、相对增重率、料肉比、肝指数、血清种特异性抗体与细胞因子等。结果显示,试验各组均未出现死亡,r Es-UCE免疫组平均相对增重显著大于攻虫对照组(P<0.05),肝指数显著低于攻虫对照组(P<0.05)。r Es-UCE免疫组相对增重率达90.69%,卵囊减少率达78.81%,细胞因子(IL-2、IL-10、IL-17和IFN-γ)水平和特异性抗体水平均与对照组存在显著差异(P<0.05)。结果表明,r Es-UCE可作为斯氏艾美耳球虫重组亚单位疫苗的候选抗原。The aim of this study was to observe the immunoprotective effect of Eimeria stiedai recombinant proteins rEs-UCE,rEs-EFG and rEs-DG32 on rabbits.The transcription levels of the Es-UCE,Es-EFG,and Es-DG32 genes in different developmental stages were analyzed using the relative quantitative realtime PCR(qPCR).The recombinant proteins rEs-UCE,rEs-EFG,and rEs-DG32 were obtained using a prokaryotic expression system.After a preliminary evaluation of the immunoprotective effect of the three recombinant proteins,rEs-UCE was selected for further evaluation.Forty-two 45-day-old coccidia-free rabbits were divided into 5 groups(blank control group,challenge control group,vector protein control group,adjuvant control group and rEs-UCE immunization group).1 mL sterile PBS,1 mL sterile PBS,1 mL PBS containing 2 mg Quil-A,1 mL PBS containing 100μg vector protein and 2 mg Quil-A,1 mL 100μg rEs-UCE and 2 mg Quil-A were respectively injected subcutaneously via the neck to the rabbits,and a booster immunization were conducted 14 d after the first immunization.The rabbits were infected with 1×10^(4) E.stiedai sporulated oocysts 14 d after the booster immunization.The clinical symptoms of each group were observed after the infection,blood and fecal samples were collected,the rabbits were weighted at regular intervals every week.21 d after the infection,mortality,oocyst output,relative weight gain rate,feed conversion ratio,liver index,specific antibody and cytokines level were detected for each group.No death occured in the experiment animals.The average relative weight gain rate and the liver index of the rEs-UCE immunization group were significantly different from the challenge control group(P<0.05).The relative weight gain rate of the rEs-UCE immunization group was 90.69%,the reduction rate of oocysts was 78.81%,and the levels of cytokines(IL-2,IL-10,IL-17 and IFN-γ)and specific antibody in the rEs-UCE immunized group were also higher than those in the control group(P<0.05).The results showed that rEs-UCE can be used as a candi

关 键 词：家兔 斯氏艾美耳球虫 泛素结合酶(UCE) 重组蛋白 免疫保护 

分 类 号：S852.723[农业科学—基础兽医学]