毒害艾美耳球虫配子体抗原基因DNA疫苗的构建与免疫保护效果评价

doi:10.11843/j.issn.0366-6964.2025.04.036

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (4): 1887-1896.doi: 10.11843/j.issn.0366-6964.2025.04.036

毒害艾美耳球虫配子体抗原基因DNA疫苗的构建与免疫保护效果评价

冯永翠¹^,²(), NaingHtet Aung¹^,², 张馨尹¹^,², 汪飞燕¹^,², 王乐乐¹^,²^,³, 张露¹^,², 朱玉¹^,², 许金俊¹^,², 陶建平¹^,², 刘丹丹¹^,²^,*()

1. 扬州大学兽医学院, 扬州 225009
2. 江苏高校动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009
3. 赣州职业技术学院, 赣州 341008

收稿日期:2024-06-12 出版日期:2025-04-23 发布日期:2025-04-28
通讯作者: 刘丹丹 E-mail:kkdfyc@163.com;ddliu@yzu.edu.cn
作者简介:冯永翠(1998-), 女, 侗族, 贵州铜仁人, 硕士生, 主要从事动物寄生虫与动物寄生虫病学研究, E-mail: kkdfyc@163.com
基金资助:
国家自然科学基金(31972698；31602039)；江苏省重点研发计划(现代农业，BE2021354)；江苏高校优势学科建设四期工程项目(2022-2025)；高等学校学科创新引智计划资助(D18007)

Construction and Evaluation of the Immunoprotection of a DNA Vaccine Targeting Eimeria necatrix Gametocyte Antigen Gene

FENG Yongcui¹^,²(), Naing Htet Aung¹^,², ZHANG Xinyin¹^,², WANG Feiyan¹^,², WANG Lele¹^,²^,³, ZHANG Lu¹^,², ZHU Yu¹^,², XU Jinjun¹^,², TAO Jianping¹^,², LIU Dandan¹^,²^,*()

1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China
3. Ganzhou Polytechnic, Ganzhou 341008, China

Received:2024-06-12 Online:2025-04-23 Published:2025-04-28
Contact: LIU Dandan E-mail:kkdfyc@163.com;ddliu@yzu.edu.cn

摘要/Abstract

摘要：

旨在评估毒害艾美耳球虫配子体抗原基因DNA疫苗的抗球虫免疫保护效果。本研究构建了配子体抗原基因的DNA疫苗pVAX-Engam59，并添加CpG ODN2006佐剂免疫雏鸡，通过间接ELISA和qRT-PCR方法分析雏鸡特异性抗体效价和细胞因子(IL-4、IFN-γ和TGF-β4)转录情况；最后，每只雏鸡经嗉囊感染2.5×10⁴个毒害艾美耳球虫孢子化卵囊，观察各试验组的临床症状、血便排出情况，感染8 d后检测各试验组的卵囊排出量、增重、病变记分等，并计算抗球虫指数。结果表明，与空载体pVAX-1组、未免疫攻虫组、未免疫未攻虫组相比，pVAX-Engam59、pVAX-Engam59+CpG ODN2006免疫雏鸡后血清特异性抗体水平显著升高(P < 0.05)，细胞因子IFN-γ、IL-4、TGF-β4表达水平显著升高(P < 0.05)；卵囊减少率分别为64.65%与64.48%，血便数以及病变记分显著降低、相对增重率显著增加(P < 0.05)；ACI值分别为156.02与159.72。综上，本研究成功构建了毒害艾美耳球虫配子体抗原基因的DNA疫苗，且与pVAX-Engam59相比，pVAX-Engam59+CpG ODN2006佐剂组免疫保护效果较好，二者均接近抗球虫中效水平，有望成为抗球虫病的候选DNA疫苗。

关键词: 毒害艾美耳球虫, 配子体抗原基因, DNA疫苗, 免疫保护

Abstract:

The purpose of this experiment was to investigate the immunoprotection of a DNA vaccine targeting Eimeria necatrix gametocyte antigen gene against coccidiosis. In this study, a DNA vaccine of gametocyte antigen gene, pVAX-Engam59, was constructed and combined with CpG ODN2006 adjuvant to immunize the chicks. The level of specific antibody in each group was detected by indirect ELISA. The mRNA transcription of cytokine (IL-4, IFN-γ, and TGF-β4) was analyzed by qRT-PCR. Finally, each chick was infected with 2.5×10⁴ E. necatrix sporulated oocysts through the crop, and the clinical symptoms and bloody stools of each group were observed. The oocysts output, weight gains and lesion scores of each group were detected after 8 days post-infection. The results showed that the serum specific antibody and cytokine (IL-4, IFN-γ, and TGF-β4) levels of pVAX-Engam59 and pVAX-Engam59+CpG ODN2006 were significantly increased (P < 0.05), and the oocyst reduction ratios were 64.65% and 64.48%, respectively. Compared to challenged and unchallenged control group, the number of bloody stools and the lesion scores in the pVAX-Engam59 and pVAX-Engam59+CpG ODN2006 groups were significantly lower (P < 0.05), and the relative weight gain rates were significantly higher (P < 0.05). The ACI values were 156.02 and 159.72, respectively. In summary, we successfully constructed DNA vaccine targeting E. necatrix gametocyte antigenic gene, and both pVAX-Engam59+CpG ODN2006 and pVAX-Engam59 showed good immunoprotective effects, which were close to the anti-coccidial intermediate efficiency level, and could be candidates for a DNA vaccine against coccidiosis.

Key words: Eimeria necatrix, gametocyte antigen gene, DNA vaccine, immunoprotection

中图分类号:

冯永翠, NaingHtet Aung, 张馨尹, 汪飞燕, 王乐乐, 张露, 朱玉, 许金俊, 陶建平, 刘丹丹. 毒害艾美耳球虫配子体抗原基因DNA疫苗的构建与免疫保护效果评价[J]. 畜牧兽医学报, 2025, 56(4): 1887-1896.

FENG Yongcui, Naing Htet Aung, ZHANG Xinyin, WANG Feiyan, WANG Lele, ZHANG Lu, ZHU Yu, XU Jinjun, TAO Jianping, LIU Dandan. Construction and Evaluation of the Immunoprotection of a DNA Vaccine Targeting Eimeria necatrix Gametocyte Antigen Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1887-1896.

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组别 Group	免疫剂量/(μg·只^-1) Immunization dose	免疫日龄/d Immunization date	免疫途径 Immunization route	攻虫日龄/d Challenge date	攻虫剂量(×10⁴) Challenge dose (×10⁴)
pVAX-Engam59	100 μg	5、12	肌肉注射 Intramusclar injection	19	2.5
pVAX-Engam59+CpG ODN2006	100 μg+20 μg	5、12	肌肉注射 Intramusclar injection	19	2.5
pVAX-1	100 μg	5、12	肌肉注射 Intramusclar injection	19	2.5
CpG ODN2006	20 μg	5、12	肌肉注射 Intramusclar injection	19	2.5
未免疫攻虫组 Unimmunized-challenged control group	/	/		19	2.5
未免疫未攻虫组 Unimmunized-unchallenged control group	/	/		/	/

引物名称 Primer name	序列(5′→3′) Sequence
引物名称 Primer name	正向引物Forward primer	反向引物Reverse primer
IFN-γ	AGCTGACGGTGGACCTATTATT	GGCTTTGCGCTGGATTC
TGF-β4	GTGCTGGCGCTGTACAACCAA	ACATTCCGGCCCACGTAG
IL-4	ACGGCAGCAGGCAACACTAC	AGCTAGTTGGTGGAAGAAGGTACG
GAPDH	TCTGGCAAAGTCCAAGTGGT	ATCACAAGTTTCCCGTTCTCAG

组别 Group	粪便卵囊产量/(10⁶·只^-1) Fecal oocyst production	盲肠卵囊产量/(10⁶·只^-1) Cecum oocyst production	卵囊产量/(10⁶·只^-1) oocyst production	卵囊减少率/% Oocyst reduction rate	平均病变记分 Mean lesion score
pVAX-Engam59	0.09	1.90	1.99	64.65	2.00±0.00^b
pVAX-Engam59+CpG ODN2006	0.05	1.95	2.00	64.48	1.90±0.21^b
pVAX-1	0.23	5.01	5.24	6.90	2.25±0.68^ab
CpG ODN2006	0.10	3.47	3.57	36.59	2.50±0.67^a
未免疫攻虫组 Unimmunized-challenged control group	0.43	5.20	5.63	0.00	2.60±0.46^a
未免疫未攻虫组 Unimmunized-unchallenged control group	0.00	0.00	0.00	100.00	0.00±0.00^c

组别 Group	首免体重/g FIW	攻虫体重/g CW	剖杀体重/g DW	平均增重1 ($\bar x \pm s$)/g AWG 1	相对增重率1/% RWGR	平均增重2($\bar x \pm s$)/g AWG 2	相对增重率2/% RWGR 2
pVAX-Engam59	62.16	313.4	594.94	251.24±11.94^a	98.26	281.54±6.91^c	86.02
pVAX-Engam59+CpG ODN2006	60.30	315.6	605.97	255.30±14.94^a	99.84	290.37±13.51^b	88.72
pVAX-1	60.25	317.2	573.45	256.95±13.92^a	100.49	256.25±14.24^d	78.30
CpG ODN2006	60.40	315.2	568.89	254.80±7.30^a	99.65	253.69±12.08^d	77.51
未免疫攻虫组 Unimmunized-challenged control group	60.22	310.0	569.09	249.78±7.16^a	97.68	259.09±4.30^d	79.16
未免疫未攻虫组 Unimmunized-unchallenged control group	60.50	316.2	643.48	255.70±10.62^a	100.00	327.28±7.71^a	100.00

组别Group	5 d	12 d	19 d
pVAX-Engam59	0.26±0.02^a	0.53±0.01^a	0.71±0.00^b
pVAX-Engam59+CpG ODN2006	0.29±0.02^a	0.55±0.00^a	0.86±0.04^a
pVAX-1	0.26±0.01^a	0.22±0.02^b	0.32±0.01^d
CpG ODN2006	0.27±0.03^a	0.20±0.01^b	0.41±0.01^c
未免疫攻虫组 Unimmunized-challenged control group	0.26±0.00^a	0.22±0.00^b	0.32±0.03^d
未免疫未攻虫组 Unimmunized-unchallenged control group	0.28±0.01^a	0.21±0.01^b	0.35±0.01^d

毒害艾美耳球虫配子体抗原基因DNA疫苗的构建与免疫保护效果评价

Construction and Evaluation of the Immunoprotection of a DNA Vaccine Targeting Eimeria necatrix Gametocyte Antigen Gene

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组别 Group	存活率/% Survival rate	相对增重/% Relative weight gain rate	病变值(0~40) Lesion value(0-40)	卵囊值(0~40) Oocyst value(0-40)	ACI值 ACI value
pVAX-Engam59	100	86.02	20	10	156.02
Engam59+CpG ODN2006	100	88.72	19	10	159.72
pVAX-1	100	78.30	22.5	40	115.8
CpG ODN2006	100	77.51	25	20	132.51
未免疫攻虫组 Unimmunized-challenged control group	100	79.16	26	40	113.16
未免疫未攻虫组 Unimmunized-unchallenged control group	100	100.00	0	0	200

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