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

doi:10.11843/j.issn.0366-6964.2024.11.031

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (11): 5159-5172.doi: 10.11843/j.issn.0366-6964.2024.11.031

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

倪君丽¹^,²(), 刘欣超¹(), 孙栋¹^,², 方肆云³, 王定爱³, 申翰钦³, 严专强³, 戚南山², 孙铭飞²^,*(), 顾有方¹^,*()

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

收稿日期:2024-01-11 出版日期:2024-11-23 发布日期:2024-11-30
通讯作者: 孙铭飞,顾有方 E-mail:1565452246@qq.com;304368107@qq.com;smf7810@126.com;youfanggu@163.com
作者简介:倪君丽(1998-)，女，浙江平湖人，硕士生，主要从事鸡球虫病综合防控技术研究，E-mail：1565452246@qq.com
刘欣超(1987-)，女，吉林省白山人，副教授，主要从事动物寄生虫病研究，E-mail：304368107@qq.com
第一联系人：
倪君丽和刘欣超是同等贡献作者
基金资助:
“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);广东省科技计划项目(2023B1212060040);猪禽种业全国重点实验室开放课题(2023QZ-NK05);猪禽种业全国重点实验室开放课题(2022GZ07);广州市科技计划项目(2023B04J0137);广州市科技计划项目(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);科技创新战略专项资金(高水平农科院建设)(202122TD);科技创新战略专项资金(高水平农科院建设)(R2020PY-JC001);科技创新战略专项资金(高水平农科院建设)(R2019YJ-YB3010);科技创新战略专项资金(高水平农科院建设)(R2020PY-JG013);科技创新战略专项资金(高水平农科院建设)(R2020QD-048);科技创新战略专项资金(高水平农科院建设)(R2021PY-QY007);科技创新战略专项资金(高水平农科院建设)(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)

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

Junli NI¹^,²(), Xinchao LIU¹(), Dong SUN¹^,², Siyun FANG³, Dingai WANG³, Hanqin SHEN³, Zhuanqiang YAN³, Nanshan QI², Mingfei SUN²^,*(), Youfang GU¹^,*()

1. College of Animal Science and Technology, Anhui Science and Technology University, Fengyang 233100, China
2. 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
3. Wens Foodstuff Group Co. LTD, Xinxing 527400, China

Received:2024-01-11 Online:2024-11-23 Published:2024-11-30
Contact: Mingfei SUN, Youfang GU E-mail:1565452246@qq.com;304368107@qq.com;smf7810@126.com;youfanggu@163.com

摘要/Abstract

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。

关键词: 柔嫩艾美耳球虫, 多表位重组质粒, 真核表达, DNA疫苗

Abstract:

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-γ and 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.

Key words: Eimeria tenella, multi-epitope recombinant plasmids, eukaryotic expression, DNA vaccine

中图分类号:

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. 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[J]. 畜牧兽医学报, 2024, 55(11): 5159-5172.

Junli NI, Xinchao LIU, Dong SUN, Siyun FANG, Dingai WANG, Hanqin SHEN, Zhuanqiang YAN, Nanshan QI, Mingfei SUN, Youfang GU. Construction, Expression and Preliminary Functional Analysis of the Eukaryotic Plasmid ET Seven, a 7-valent Multiepitope Chimeric Recombinant Antigen of Chicken Coccidia[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5159-5172.

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抗原片段 Antigen fragments	基因序列 Gene sequence	序列长度/ bp Sequence length
AMA1	CAAATAACCGTCCCCAACCCCACGGAGTGCGGCAAAGCTGTCTTCA- AAGTTTCTTCTTCAGACAACCCCACGCAGTACACGAAGCCACCCAC- CACTGAGGCCAGCAGCAGCACCAGCTCCAGCAACGCAGTGGCCACC- ATGTGGCCTGTGGGCGCCTTTTCCAAGGACGAACCCCGCATGCAGG- GGGTGGGCACCAACTATGCCAACTGGTACACCAATGGTACTTGTG- AGATGTATGACATGGTCCCCACTTGCTTCACCCTCGCCCCTAAC	273
AMA2	CCAGGGGGGACTTGTGTTATTTTTGCTGCTGTACCCTCTTGCTTCCT- GAGGGCCCCCGGGCACACAGCCTACACCTCAGTGGGTTCTTTGGCGG- AGGAGGCCCTCCCGGACTCCGCGCCGGGGGGCCCCCCCTCTTCTGGG- GGCGGGGGGGGCCCCCAGGAGCCCCAGGGGCCCCAGGAGCCCCAGCC- GGGGGGCCCCGAACCCGAACCCAGCCCCGAACCCAGCCCCGAACCCG- GAGAGCCCGGCAGCGGCGAAGGAGGGGGGGAAGAGGGGGAAACAA- ATCCTCCAAAAGAGCCTCAAGAAGAGCCTTCCTTGCCGCCTGAAGA- GGGGGGAGGTCAAGAAGAA	345
MIC3	GAACAGGGAAAAATAGGAAACTGGTGTTCCCAGAGTTGGGTTTTTT- GCTCAACTGCCATCATCTACGACCCCGCCTACTCCTACGGCACCTGCA- AGTGCGAGTCGTACAGAAACCGCTGCCCCATAGGCAGCAGCTGCCTC- GACTCCAGCTTCGGGGCTTTTTGCAAATGCAATGAAGACTTAGAAG- AGCGAAACGGGGGCTGCCACTTCACCACAACGACCACAACCACCACC- ACCACCACCACCACCACAACCACCACCACCACAACAACCACCACCACC- ACGCCCCCCGCGCGATCTCTCTGCAAA	309
MIC4	TTGCTCAAGGGCAAGAGCCTTGAGGATTGCAGAAAGGCTTGCTCAG- CAGACCAGAAGTGCACCAACTTCACGAGCTGGCTAAATAAGGAGTG- CTATATCAAGGATGACCAGGACCACCGGATGCAGGAACCAATACCG- GAGGCTACATCCGGATGGGTTTCGTGCTCAACG	171
MIC13	GGCGCCCTCGGAGTCTGGCGCTGCTACGCAGGGGCCACCATCAACCTC- AGTGCCCACAGCCTAGCTTGCGTCACCAACTGTGGGGACCCCATGAA- CTGCTCCGGACAGCTCACCCACATCTCTCCAATCTATTTCGATAGCA- ACTCCGACATGGTGGCAATCATCAAGCAGGAAAAGGTCAGGTACTG- CGGAAACTCGCGCTCTCGGCTGTCCACC	216
ROP5	CTAACGATTCCGTCGACCCCTCAAGTTGACATGAGTGGAAAAAGCC- CCATCGTCGTGCCACCTGTTCCAGCCGTTTTAGTCACTCCTGGGTCC- ACACGTATTGCGGTTGAGAGCCGAGAGACGGAAAGTGCAGGACAT- GGCACAGCAAGTGGAGTAAGCGCCACTGCCGGCGATGCTGCCAGGA- TCTCAGCAGGTACTAGC	201
SAG1	GACAGGGCTGTTTCCTTTGTCGCCCTATACAACCCCAAAACCAGCCC- CGTTGTCAGTTGCGTGCTCCTCCAGTGCCCTAATGCA	84

引物名称 Primer name	引物序列(5′→3′) Primer sequence	片段大小/bp Fragment length	退火温度/℃ Annealing temperature
AMA1-F	CAAATAACCGTCCCCAACC	273	54
AMA1-R	GTTAGGGGCGAGGGTGA		56
AMA2-F	CCAGGGGGGACTTGTGTTAT	345	57
AMA2-R	TTCTTCTTGACCTCCCCCC		56
MIC3-F	GAACAGGGAAAAATAGGAAACT	348	51
MIC3-R	ATCAATAACATCGCAGAAGCC		54
MIC4-F	GGCAGCGGAGAGAGCAAG	285	59
MIC4-R	CGTTGAGCACGAAACCCAT		57
MIC13-F	AATGATTCTCTGTGCCAAAACC	361	55
MIC13-R	ACTGCTGTTGCCCTTCCAT		58
ROP5-F	AACAGTGGTAAAATACACAAGGAC	501	54
ROP5-R	TTTTGTGCTCCTTGAGGTGCT		58
SAG1-F	ATGCCCCAAAGTCTTAGGAG	401	55
SAG1-R	TTTTCTTCCATTGCTCGTCGT		56

分组 Group	处理方式(肌肉多点位注射) Treatment (intramuscular multi-point injection)
空载体质粒组 Empty plasmid group	100 μg·羽^-1 pcDNA3.1质粒+磷酸钙纳米颗粒+壳聚糖
磷酸钙纳米颗粒佐剂对照组 Calcium phosphate nanoparticles adjuvant control group	磷酸钙纳米颗粒+壳聚糖(每羽剂量与免疫组相同)
空白对照组 Blank control group	不做任何处理
磷酸钙纳米颗粒免疫质粒组 Calcium phosphate nanoparticles immune plasmid group	100 μg·羽^-1 pcDNA3.1-ET seven质粒+磷酸钙纳米颗粒+壳聚糖

引物名称 Primer name	引物序列(5′→3′) Primer sequences	退火温度/℃ Annealing temperature
IL-2-F	TTGGAAAATATCAAGAACAAGATTCATC	50.9
IL-2-R	TCCCAGGTAACACTGCAGAGTTT	58.3
IL-6-F	CGTGTGCGAACAGCATGGAGA	60.9
IL-6-R	TCAGGCATTTCTCCTCGTCGAAGC	61.7
IL-10-F	AGCAGATCAAGGAGACGTTC	54.4
IL-10-R	ATCAGCAGGTACTCCTCGAT	55
IL-12-F	CCAAGACCTGGAGCACACACCGAAG	64.8
IL-12-R	CGATCCCTGGCCTGCACAGAGA	64.7
IFN-γ-F	ACACTGACAAGTCAAAGCCGCACA	61.7
IFN-γ-R	AGTCGTTCATCGGGAGCTTGGC	62.7
CD-40-F	CCTGGTGATGCTGTGAATTG	54.1
CD-40-R	CTTCTGTGTCGTTGCATTCAG	54.1
β-actin-F	CAACACAGTGCTGTCTGGTGGTA	59.6
β-actin-R	ATCGTACTCCTGCTTGCTGATCC	59.2

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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

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