鸡球虫病7价多表位嵌合重组抗原ET seven真核质粒的构建、表达及其初步功能分析  

Construction,Expression and Preliminary Functional Analysis of the Eukaryotic Plasmid ET Seven,a 7-valent Multiepitope Chimeric Recombinant Antigen of Chicken Coccidia

作　　者：倪君丽 刘欣超 孙栋 方肆云 王定爱 申翰钦 严专强 戚南山 孙铭飞 顾有方[1] NI Junli;LIU Xinchao;SUN Dong;FANG Siyun;WANG Dingai;SHEN Hanqin;YAN Zhuanqiang;QI Nanshan;SUN Mingfei;GU Youfang(College of Animal Science and Technology,Anhui Science and Technology University,Fengyang 233100,China;Key Laboratory of Livestock Disease Prevention of Guangdong Province,Key Laboratory of Avian Influenza and Other Major Poultry Diseases Prevention and Control of Ministry of Agriculture and Rural Affairs,Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;Wens Foodstuff Group Co.LTD,Xinxing 527400,China)

机构地区：[1]安徽科技学院动物科技学院,凤阳233100 [2]广东省农业科学院动物卫生研究所,广东省畜禽疫病防治研究重点实验室,农业农村部禽流感等家禽重大疾病防控重点实验室,广州510640 [3]温氏食品集团股份有限公司,新兴527400

出　　处：《畜牧兽医学报》2024年第11期5159-5172,共14页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：“十四五”广东省农业科技创新十大主攻方向“揭榜挂帅”项目(2023SDZG02);广东省基础与应用基础研究基金项目(2021B1515120006);广东省科技计划项目(2021B1212050021,2023B1212060040);猪禽种业全国重点实验室开放课题(2023QZ-NK05,2022GZ07);广州市科技计划项目(2023B04J0137,2023A04J0789);云浮市科技计划项目(2022020202);科技创新战略专项资金(高水平农科院建设)(202110TD,202122TD,R2020PY-JC001,R2019YJ-YB3010,R2020PY-JG013,R2020QD-048,R2021PY-QY007,R2023PY-JG018);广东省现代农业产业技术体系创新团队建设专项(2022KJ119);广东省农业科学院协同创新中心项目(XTXM202202)。

摘　　要：为构建以鸡柔嫩艾美耳球虫EtAMA1、EtAMA2、EtMIC3、EtMIC4、EtMIC13、EtROP5和EtSAG1等入侵相关蛋白分子为抗原基础的多表位嵌合重组抗原ET seven真核表达质粒,同时验证其在DF-1细胞中的表达和通过不同辅助递送方式在鸡体内的表达及其免疫功能。本研究利用生信分析获得7个抗原表位,分别提取柔嫩艾美耳球虫不同发育阶段虫体总RNA,通过RT-PCR扩增获得EtAMA1、EtAMA2、EtMIC3、EtMIC4、EtMIC13、EtROP5和EtSAG1等7个抗原的表位区域编码基因,构建基因图谱由公司合成以pcDNA3.1(+)为真核表达载体,构建真核表达质粒pcDNA3.1-ET seven;将鉴定阳性、测序正确的质粒pcDNA3.1-ET seven转染至DF-1细胞,利用RT-PCR和Western blot方法检测ET seven重组基因片段在DF-1细胞中的表达;以磷酸钙纳米颗粒作为辅助质粒递送佐剂检测pcDNA3.1-ET seven在鸡体内引起的细胞因子水平变化,间接ELISA检测特异性IgG抗体水平。结果显示:成功构建真核表达质粒pcDNA3.1-ET seven,RT-PCR和Western blot检测到ET seven的特异性表达,质粒通过磷酸钙纳米佐剂免疫鸡体后,检测细胞因子IL-2、IL-6、IL-10、IL-12、IFN-γ、CD-40均在三次免疫后7 d高水平表达,其中CD-40与IL-6的表达水平最高。pcDNA3.1-ET seven重组质粒结合磷酸钙佐剂肌肉注射二次免疫后7 d间接ELISA检测特异性IgG抗体呈阳性。结果表明,pcDNA3.1-ET seven重组质粒构建成功,在鸡体内有良好的免疫原性,为鸡球虫病的疫苗防控提供新的思路和技术支撑。The aim of this study was to construct multi-epitope recombinant plasmids of Eimeria tenella EtAMA1,EtAMA2,EtMIC3,EtMIC4,EtMIC13,EtROP5,and EtSAG1 antigens and verify their expression in DF-1 cells and chickens through various assisted delivery methods,we employed pcDNA3.1(+)as the eukaryotic expression vector.The eukaryotic expression plasmid pcDNA3.1-ET Seven was constructed through bioinformatics analysis,obtaining seven epitopes and reverse transcriptional amplification of gene fragments of seven antigen epitope regions in E.tenella mRNA templates at different time points.The company synthesized pcDNA3.1(+)as the eukaryotic expression vector,and constructed the eukaryotic expression plasmid pcDNA3.1-ET seven.Subsequently,the constructed positive plasmid,pcDNA3.1-ET Seven,was transfected into DF-1 cells.RT-PCR and Western blot analysis was employed to detect the expression of the ET Seven recombinant gene in DF-1 cells.The cytokine levels of pcDNA3.1-ET seven in chickens were detected by calcium phosphate nanoparticles as adjuvant plasmid delivery adjuvant,and specific IgG antibody levels were detected by indirect ELISA.The eukaryotic expression plasmid pcDNA3.1-ET Seven was successfully constructed,and the specific expression of ET Seven was detected through Western blot analysis and RT-PCR.After the plasmid was immunized with calcium phosphate nano-adjuvant,the detected cytokines IL-2,IL-6,IL-10,IL-12,IFN-γand CD-40 were all expressed at high levels seven days after three times of immunization,and the expression levels of CD-40 and IL-6 were the highest.The pcDNA3.1-ET Seven recombinant plasmid,when conjugated with calcium phosphate adjuvant,was administered intramuscularly,resulting in a positive detection of specific IgG antibodies through indirect ELISA.The results showed that the pcDNA3.1-ET seven recombinant plasmid was successfully constructed and had good immunogenicity in chickens.This finding provides novel insights and technical support for vaccine prevention and control of chicken coccidiosis.

关 键 词：柔嫩艾美耳球虫 多表位重组质粒 真核表达 DNA疫苗 

分 类 号：S855.9[农业科学—临床兽医学] S852.723[农业科学—兽医学]