摘要
本研究旨在评价表达柔嫩艾美耳球虫(Eimeria tenella)顶膜抗原1(Apical membrane antigen 1,Et AMA1)基因的DNA疫苗对雏鸡柔嫩艾美耳球虫人工感染的免疫保护效果。将Et AMA1基因和鸡IFN-γ基因插入真核表达载体p CAGGS中,分别构建了真核表达重组质粒p CAGGS-Et AMA1和p CAGGS-Et AMA1-IFN-γ。将重组质粒p CAGGS-Et AMA1转染进293T细胞中,经间接免疫荧光和Western blot实验鉴定,观察其在体外表达情况。将p CAGGS-Et AMA1和p CAGGS-Et AMA1-IFN-γ于7日龄和14日龄腿部肌肉注射免疫雏鸡,21日龄人工感染1×10^4个柔嫩艾美耳球虫孢子化卵囊,第29日龄时处死试验鸡,以平均增重、卵囊产量和病变记分来评价重组质粒的免疫保护效果。结果显示,p CAGGS-Et AMA1转染的293T细胞出现明显的红色荧光;两种真核表达重组质粒免疫组鸡的平均增重与未攻虫组的平均增重差异不显著,不具有统计学意义,而未免疫攻虫组则与未攻虫组在增重方面差异具有显著统计学意义;免疫组鸡相较于未免疫攻虫组在病变记分方面显著下降;免疫组的卵囊减少率分别为67.43%和72.89%;p CAGGSEt AMA1-IFN-γ组在增重、盲肠病变记分和卵囊减少率方面都优于p CAGGS-Et AMA1组,但差异不具有统计学意义。表明构建的2种重组质粒对雏鸡柔嫩艾美耳球虫感染有一定的免疫保护效果。
In order to evaluate the immunogenicity of the DNA vaccine expressing,the gene of Eimeria tenella apical membrane antigen-1(Et AMA1)and chicken IFN-γgene were ligated to the eukaryotic expression vector pCAGGS to construct recombinant plasmids pCAGGS-Et AMA1 and pCAGGS-Et AMA1-IFN-γfor use as DNA vaccine.These two recombinant plasmids were transfected into 293 T cells for protein expression,which was confirmed in indirect immunofluorescence assay and Western blot.Then,7-day-old chickens were immunized with 100μg of pCAGGS-Et AMA1 or pCAGGS-Et AMA1-IFN-γand boosted 7 days later.Chickens were challenged via oral infection with 10,000 sporulated oocysts of E.tenella at 7 days post the second immunization.All chickens were euthanized at 8 days later to examine body weight gains,cecum lesion scores and oocysts reduction rates.The results showed no significant difference in body weights between vaccinated chickens and non-vaccinated control chickens prior to challenge.However,significant difference in body weight was noted between challenged chickens and unchallenged control group.As compared with non-vaccinated chickens,vaccinated chickens with pCAGGS-Et AMA1 and pCAGGS-Et AMA1-IFN-γDNA vaccines obviously showed less cecal lesions and oocyst reduction ratios at 67.43%and 72.89%,respectively.Moreover,vaccination with pCAGGS-EtAMA1-IFN-γprovided better protection than with pCAGGS-EtAMA1 regarding body weight gains,cecum lesion scores and oocyst reduction rates.These results suggested that the DNA vaccine induced some protective immunity against E.tenella infection.
作者
王晔
赵其平
朱顺海
董辉
姜连连
薛璞
舒凡帆
黄兵
韩红玉
WANG Ye;ZHAO Qi-ping;ZHU Shun-hai;DONG Hui;JIANG Lian-lian;XUE Pu;SHU Fan-fan;HUANG Bing;HAN Hong-yu(Key Laboratory of Animal Parasitology of Ministry of Agriculture,Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China)
出处
《中国动物传染病学报》
CAS
北大核心
2019年第6期1-9,共9页
Chinese Journal of Animal Infectious Diseases
基金
国家自然科学基金(31672551)
国家寄生虫种质资源共享服务平台(平台-TDRC-22)
