Prokaryotic Expression of Eimeria tenella AMA1 and Evaluation of Its Immune Protection Effect

doi:10.7668/hbnxb.20195307

Abstract

Abstract:

In order to investigate the immune protective effect of apical membrane antigen 1(AMA1)of Eimeria tenella on chickens infected with E.tenella, the AMA1 gene amplified was connected to expression vector pET-32a(+).The recombinant plasmid was transformed into E.coli BL21(DE3)to express recombinant protein.108 chickens were randomly divided into 6 groups with 3 replicates in each group and 6 chicks in each replicate,including non-immune and non-attacking group(negative control group),non-immune attack group(positive control group),Freund's adjuvant group and 12.5,25.0,50.0 μg rEtAMA1 immunized groups.All of the chickens were injected with different concentration of recombinant protein at the age of 14,21 days,and infected orally with 5×10⁴ sporulated oocysts of E.tenella at the age of 28 days and killed at the age of 35 days.The immune protection effect of rEtAMA1 was evaluated by body weight gain,lesion score,oocyst production,immune organ index and levels of cytokines and antibodies.There was no significant difference in average weight gain among the groups.Compared with the positive group,the scores of cecal lesions in 25.0 μg and 50.0 μg rEtAMA1 groups were significantly reduced by 53%,41%,the OPG in 12.5,25.0,50.0 μg rEtAMA1 groups were significantly reduced by 16%,27%,54% respectively,the immune organ index of bursa of Fabricius in 50 μg rEtAMA1 groups significantly increased by 27%,the immune organ index of thymus in 25.0 μg and 50.0 μg rEtAMA1 groups significantly increased by 25%,22% respectively.There was no significant difference in the immune organ index of spleen.Seven days after the second immunization,the levels of IL-2 and IFN-γ in 12.5 μg and 25.0 μg rEtAMA1 groups were significantly higher than the negative control group.The level of serum total IgG concentration in the 50.0 μg rEtAMA1 group significantly increased by 47% than that in the negative control group,and the concentration after the second immunization was obviously higher than that after the first immunization.The results showed that the rEtAMA1 could alleviate cecal lesions,decrease the oocyst production,promote the development of immune organs and increase the levels of cytokines and antibodies in serum,which had a certain immune protective effect on chickens infected with E.tenella.

Key words: Eimeria tenella, AMA1, Prokaryotic expression, Immune effect

LIU Yongning, SHAN Yige, PAN Chenfan, LIU Qianlin, LI Yilin, DUAN Sizhang, AN Jian, ZHANG Jianjun. Prokaryotic Expression of Eimeria tenella AMA1 and Evaluation of Its Immune Protection Effect[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(1): 232-238. doi: 10.7668/hbnxb.20195307.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20195307

http://www.hbnxb.net/EN/Y2025/V40/I1/232

Figures/Tables 8

Fig.1 PCR amplification of AMA1 gene M.DL-4500 DNA Marker; 1—2.PCR product of AMA1 gene.

Fig.2 Double restriction enzyme digestion identification M.DL-4500 DNA Marker; 1.pET-32a(+)vector;2.Recombinant expression vector of pET-32a(+)-AMA1.

Fig.3 Solubility assay of recombinant AMA1 protein M.Protein Marker(10—150 ku); 1.Recombinant bacteria without induction; 2.Recombinant bacteria with induction with 1 mmol/L IPTG; 3.Supernatant after ultrasonic lysis; 4.Precipitation after ultrasonic pyrolysis.

Fig.4 Purification of recombinant AMA1 protein M.Protein Marker(15—120 ku); 1.Purified protein;2.Protein before purification.

Tab.1 Immune protective effect of recombinant AMA1 protein

组别 Group	平均体增质量/g Average body weight gain	相对体增质量率/% Relative body weight gain	盲肠病变平均计分 Average score of cecal lesions	平均OPG/(×10⁶) Average OPG
阴性组Negative control group	44.16±7.36a	100.00a	0	0
阳性组Positive control group	37.50±6.89a	84.92±21.07a	2.83±0.41a	3.93±0.17a
弗氏佐剂对照组Freund's adjuvant group	38.33±9.87a	86.80±26.62a	2.50±0.55a	3.84±0.18a
12.5 μg rEtAMA1	45.00±10.49a	101.90±28.84a	2.50±0.55a	3.30±0.37b
25.0 μg rEtAMA1	36.66±7.53a	83.02±21.97a	1.33±0.52b	2.86±0.25c
50.0 μg rEtAMA1	39.16±3.77a	88.68±17.07a	1.67±0.52b	1.79±0.16d

Fig.5 The effect of immunity on immune organ index Different letters at the same index are significantly different (P<0.05).

Fig.6 Serum IL-2 and IFN-γ concentration at age of 21 and 28 d Groups marked with different letters at the same age are significantly different(P<0.05), the same letter means that the difference is not significant (P>0.05). The same as Fig.7.

Fig.7 Serum total IgG concentration at the age of 21 and 28 d

References 21

[1]	廖申权, 戚南山, 吕敏娜, 吴彩艳, 李娟, 蔡海明, 林栩慧, 胡俊菁, 于林增, 张健騑, 谢明权, 孙铭飞. 鸡球虫病流行病学、防治药物与疫苗研究进展[J]. 广东农业科学, 2020, 47(11):171-181.doi:10.16768/j.issn.1004-874x.2020.11.019.
	Liao S Q, Qi N S, Lyu M N, Wu C Y, Li J, Cai H M, Lin X H, Hu J J, Yu L Z, Zhang J F, Xie M Q, Sun M F. Research progress in the epidemiology,anticoccidial drugs and vaccines of avian coccidiosis[J]. Guangdong Agricultural Sciences, 2020, 47(11):171-181.
[2]	Peek H W, Landman W J M. Coccidiosis in poultry:anticoccidial products,vaccines and other prevention strategies[J]. The Veterinary Quarterly, 2011, 31(3):143-161.doi:10.1080/01652176.2011.605247. pmid: 22029884
[3]	Zhang M, Li X Y, Zhao Q P, She R F, Xia S H, Zhang K Y, Zhang L F, Wang X Y, Wang M, Liu Y C, Wang C M, Zhang J, Xue F Q, Fei C Z. Anticoccidial activity of novel triazine compounds in broiler chickens[J]. Veterinary Parasitology, 2019, 267:4-8.doi:10.1016/j.vetpar.2019.01.006. pmid: 30878083
[4]	李烨. 鸡球虫病诊断技术与防治方法的研究进展[J]. 现代畜牧兽医, 2023(6):75-78.doi:10.20154/j.cnki.issn1672-9692.2023.06.018.
	Li Y. Research progress in diagnosis and control of avian coccidiosis[J]. Modern Journal of Animal Husbandry and Veterinary Medicine, 2023(6):75-78.
[5]	戚南山, 孙铭飞, 廖申权, 吴彩艳, 吕敏娜, 袁建丰, 余劲术, 李祥瑞, 蔡建平. 顶复门原虫入侵相关因子的研究进展[J]. 畜牧兽医学报, 2012, 43(2):167-174.
	Qi N S, Sun M F, Liao S Q, Wu C Y, Lyu M N, Yuan J F, Yu J S, Li X R, Cai J P. Invasion-associated factors of Api complexa[J]. Chinese Journal of Animal and Veterinary Sciences, 2012, 43(2):167-174.
[6]	Krishnamurthy S, Deng B, Del Rio R, Buchholz K R, Treeck M, Urban S, Boothroyd J, Lam Y W, Ward G E. Not a simple tether:binding of toxoplasma gondii AMA1 to RON2 during invasion protects AMA1 from rhomboid-mediated cleavage and leads to dephosphorylation of its cytosolic tail[J]. mBio, 2016, 7(5):e00754-16.doi:10.1128/mBio.00754-16.
[7]	范士龙, 刘丹丹, 韦丽婷, 芦星, 李思媛, 王金明, 呼尔查, 李永畅, 巴音查汗, 张伟. 马泰勒虫顶膜抗原1基因的原核表达与鉴定[J]. 中国兽医学报, 2021, 41(9):1752-1756,1769.doi:10.16303/j.cnki.1005-4545.2021.09.13.
	Fan S L, Liu D D, Wei L T, Lu X, Li S Y, Wang J M, Hu E C, Li Y C, Ba Y, Zhang W. Prokaryotic expression and identification of apical membrane antigen 1 gene of Theileria equi[J]. Chinese Journal of Veterinary Science, 2021, 41(9):1752-1756,1769.
[8]	方旭. 新孢子虫AMA1与RON2蛋白互作的鉴定及其免疫保护性研究[D]. 长春: 吉林大学, 2020.doi:10.27162/d.cnki.gjlin.2020.006949.
	Fang X. Identification of the interaction between AMA1 and RON2 protein of Neospora neospora and its immunoprotection[D]. Changchun: Jilin University, 2020.
[9]	Johnson J, Reid W M. Anticoccidial drugs:lesion scoring techniques in battery and floor-pen experiments with chickens[J]. Experimental Parasitology, 1970, 28(1):30-36.doi:10.1016/0014-4894(70)90063-9. pmid: 5459870
[10]	韩利方, 邹均平, 谢月华, 刘易扬, 闫文朝. 一种改进型麦克马斯特虫卵计数板的应用效果研究[J]. 中国农学通报, 2021, 37(21):145-150. doi: 10.11924/j.issn.1000-6850.casb2020-0497
	Han L F, Zou J P, Xie Y H, Liu Y Y, Yan W C. Application evaluation of a modified McMaster egg counting plate[J]. Chinese Agricultural Science Bulletin, 2021, 37(21):145-150. doi: 10.11924/j.issn.1000-6850.casb2020-0497
[11]	Pastor-Fernández I, Kim S, Marugán-Hernández V, Soutter F, Tomley F M, Blake D P. Vaccination with transgenic Eimeria tenella expressing Eimeria maxima AMA1 and IMP1 confers partial protection against high-level E.maxima challenge in a broiler model of coccidiosis[J]. Parasites & Vectors, 2020, 13(1):343.doi:10.1186/s13071-020-04210-2.
[12]	Bargieri D Y, Andenmatten N, Lagal V, Thiberge S, Whitelaw J A, Tardieux I, Meissner M, Ménard R. Apical membrane antigen 1 mediates api complexan parasite attachment but is dispensable for host cell invasion[J]. Nature Communications, 2013, 4:2552.doi:10.1038/ncomms3552. pmid: 24108241
[13]	Jiang L L, Lin J J, Han H Y, Dong H, Zhao Q P, Zhu S H, Huang B. Identification and characterization of Eimeria tenella apical membrane antigen-1(AMA1)[J]. PLoS One, 2012, 7(7):e41115.doi:10.1371/journal.pone.0041115.
[14]	王晔, 赵其平, 朱顺海, 董辉, 姜连连, 薛璞, 舒凡帆, 黄兵, 韩红玉. 柔嫩艾美耳球虫AMA1基因DNA疫苗的免疫保护效果[J]. 中国动物传染病学报, 2019, 27(6):1-9.doi:10.19958/j.cnki.cn31-2031/s.2019.06.001.
	Wang Y, Zhao Q P, Zhu S H, Dong H, Jiang L L, Xue P, Shu F F, Huang B, Han H Y. Immunogenicity of EtAMA1 DNA vaccine of Eimeria tenella in chickens[J]. Chinese Journal of Animal Infectious Diseases, 2019, 27(6):1-9.
[15]	Li J, Wang F, Ma C L, Huang Y C, Wang D, Ma D X. Recombinant Lactococcus lactis expressing Eimeria tenella AMA1 protein and its immunological effects against homologous challenge[J]. Experimental Parasitology, 2018, 191:1-8.doi:10.1016/j.exppara.2018.05.003.
[16]	Li W C, Zhang X K, Du L, Pan L, Gong P T, Li J H, Yang J, Li H, Zhang X C. Eimeria maxima:efficacy of recombinant Mycobacterium bovis BCG expressing apical membrane antigen1 against homologous infection[J]. Parasitology Research, 2013, 112(11):3825-3833.doi:10.1007/s00436-013-3570-5.
[17]	付少萌, 瞿彬, 翁嘉静, 付家敏, 黄婷婷, 侯睛, 梁好琴, 王琤, 韦华. 不同品种鸡消化器官指数和免疫器官指数的比较分析[J]. 湖南饲料, 2023(1):8-10.doi:10.3969/j.issn.1673-7539.2023.01.004.
	Fu S M, Qu B, Weng J J, Fu J M, Huang T T, Hou J, Liang H Q, Wang Z, Wei H. Comparative analysis of digestive organ index and immune organ index of different breeds of chickens[J]. Hunan Feed, 2023(1):8-10.
[18]	蒲响林, 潘仰栋, 相权珈, 孙晓婷, 陆明敏, 严若峰, 徐立新, 李祥瑞, 宋小凯. 巨型艾美耳球虫Th1类细胞因子刺激性分子EmARM-β对鸡PBMC和T细胞亚群免疫功能的影响[J]. 南京农业大学学报, 2024, 47(1):9-17.doi:10.7685/jnau.202302001.
	Pu X L, Pan Y D, Xiang Q J, Sun X T, Lu M M, Yan R F, Xu L X, Li X R, Song X K. Effects of EmARM-β,Eimeria maxima Th1 cytokine stimulatory molecule on immune function of chicken PBMC and T cell subsets[J]. Journal of Nanjing Agricultural University, 2024, 47(1):9-17.
[19]	Xiao J, He W, Xiong C M, Hao G, Pu J Y, Chen H, Xu L W, Zhu Y H, Ren Y J, Yang G Y. Protective efficacy of recombinant proteins AMA1 and IMP1 in rabbits infected with Eimeria intestinalis[J]. Veterinary Parasitology, 2023, 320:109985.doi:10.1016/j.vetpar.2023.109985.
[20]	袁福斌, 何剑斌, 王建民. 鸡球虫病体液免疫研究进展[J]. 畜禽业, 2009, 20(8):32-35.doi:10.19567/j.cnki.1008-0414.2009.08.013.
	Yuan F B, He J B, Wang J M. The progress of the research of humoral immunity of chicken coccidiosis[J]. Livestock and Poultry Industry, 2009, 20(8):32-35.
[21]	陈欢. 鸡球虫重组双价疫苗的制备及其效果评价[D]. 福州: 福建农林大学, 2020.doi:10.27018/d.cnki.gfjnu.2020.000022.
	Chen H. Preparation and evaluation of recombinant bivalent vaccine against chicken coccidiosis[D]. Fuzhou: Fujian Agriculture and Forestry University, 2020.

Please choose a citation manager

Content to export