柔嫩艾美耳球虫RON2L2ecto蛋白的原核表达及其免疫效果评价

doi:10.11843/j.issn.0366-6964.2025.11.024

摘要/Abstract

摘要：

旨在探究重组柔嫩艾美耳球虫RON2_L2ecto蛋白对雏鸡的免疫保护作用。本试验通过柔嫩艾美耳球虫cDNA扩增编码EtRON2_L2蛋白的全长序列，并原核重组表达EtRON2_L2的胞外域部分；选取体重相近的7日龄健康罗曼灰蛋公雏120只(前期饲喂无抗球虫药物和无抗生素的雏鸡饲料，饲养于干净无球虫环境)，随机分为5组，分别为不免疫不攻虫组、不免疫攻虫组、50 μg重组蛋白免疫攻虫组、100 μg重组蛋白免疫攻虫组、200 μg重组蛋白免疫攻虫组，将重组蛋白与弗氏佐剂混合作为亚单位疫苗免疫三组重组蛋免疫攻虫组雏鸡，通过评估鸡只的体增重、盲肠病变评分、克粪便卵囊数(OPG)，计算抗球虫指数，观察盲肠病理组织学变化以及测定血清细胞因子(IL-2、IL-10、IFN-γ)、血清IgG和盲肠黏膜分泌物sIgA的水平，对重组蛋白的免疫效果进行评价。结果显示，成功表达了EtRON2_L2的胞外域部分，大小为107 ku。免疫保护试验表明，与不免疫攻虫组相比，100 μg蛋白免疫组和200 μg蛋白免疫组的平均体增重显著增加(P＜0.05)，OPG显著下降(P＜0.05)，3个剂量免疫组的盲肠病变评分均出现了显著下降(P＜0.05)，病理组织学观察发现盲肠病变程度有一定缓解，其中100 μg蛋白免疫组和200 μg蛋白免疫组的抗球虫指数大于160，具有中等抗球虫作用。3个剂量蛋白免疫组的脾脏指数均显著高于不免疫攻虫组(P＜0.05)。28日龄(攻虫后第7天)时，蛋白免疫组的血清细胞因子IL-2、IFN-γ、IL-10均极显著(P＜0.01)高于2个不免疫组，血清IgG水平显著高于不免疫攻虫组(P＜0.05)，50 μg蛋白免疫组sIgA分泌水平显著高于(P＜0.05)其他免疫组和2个不免疫组。以上结果表明，RON2_L2胞外域对雏鸡感染柔嫩艾美耳球虫有免疫保护作用。

关键词: 柔嫩艾美耳球虫, 移动连接, 棒状体颈部蛋白2_L2, 免疫保护效果

Abstract:

The aim of this study is to investigate the immune protection effect of recombinant Eimeria tenella RON2_L2ecto protein on chicks. The full-length sequence encoding EtRON2_L2 protein from Eimeria tenella cDNA was amplified and the extracellular domain of EtRON2_L2 was expressed through prokaryutic recombination in this study. A hundred and twenty 7-day-old healthy Roman Grey male chicks with similar body weight (raised in clean and coccidia-free environment, fed with diet without coccidiostats or artibiotic) were randomly allotted into 5 groups, including negative control group (without immune or coccidia infection), positive control group (without immune but infected with coccida), and 50 μg, 100 μg, 200 μg recombinant protein immune groups. Recombinant protein was mixed with Freund's adjuvant as a subunit vaccine to immunize chicks in three immune groups. The immune protection effect of recombinant protein was evaluated by measuring the body weight gain, cecal lesion score, number of oocysts per gram of feces (OPG), calculating the anticoccidial index, observing the histopathological changes in the cecum, detecting serum cytokines (IL-2, IL-10, IFN-γ) and serum IgG, and the secreting levels of cecal mucosal sIgA. The results showed that the extracellular domain of EtRON2_L2 was successfully expressed, with a size of 107 ku. The immune protection experiment indicated that compared with the non-immunized group, the average body weight gain of the 100 μg protein immunization group and the 200 μg protein immunization group significantly increased (P < 0.05), while the OPG significantly decreased (P < 0.05), the cecal lesion scores of each dose of immune group showed a significant decrease (P < 0.05), pathological histology observation shows that the degree of cecal lesions has been alleviated to some extent. The anti-coccidiosis index of the 100 μg protein immunization group and the 200 μg protein immunization group is greater than 160, indicating moderate anti-coccidiosis activity. The spleen index of three protein immune groups were also significantly increased (P < 0.05). At 28 days of age (the 7th day after infection), the serum cytokines IL-2, IFN-γ, and IL-10 in the protein immune groups were significantly higher (P < 0.01) than those in the two control groups, and the serum IgG levels were significantly higher than those in the negative control group (P < 0.05). The sIgA secretion levels in the 50 μg protein immune group were significantly higher (P < 0.05) than those in the other immune groups and the two control groups. The above results indicate that the extracellular domain of RON2_L2 has an immunoprotective effect on chicks infected with Eimeria tenella.

Key words: Eimeria tenella, moving junction, RON2_L2, immune protection effect

中图分类号:

S852.723

单乙戈, 刘永宁, 周爽, 刘倩琳, 李翌琳, 段思彰, 安健, 张建军. 柔嫩艾美耳球虫RON2_L2ecto蛋白的原核表达及其免疫效果评价[J]. 畜牧兽医学报, 2025, 56(11): 5649-5659.

SHAN Yige, LIU Yongning, ZHOU Shuang, LIU Qianlin, LI Yilin, DUAN Sizhang, AN Jian, ZHANG Jianjun. Prokaryotic Expression and Immune Efficacy Evaluation of RON2_L2ecto Protein from Eimeria tenella[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5649-5659.

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参考文献 25

1	SHIRLEY M W , SMITH A L , TOMLEY F M . The biology of avian Eimeria with an emphasis on their control by vaccination[J]. Adv Parasitol, 2005, 60, 285- 330.
2	SHIRLEY M W , SMITH A L , BLAKE D P . Challenges in the successful control of the avian coccidia[J]. Vaccine, 2007, 25 (30): 5540- 5547.
3	YUN C H , LILLEHOJ H S , CHOI K D . Eimeria tenella infection induces local gamma interferon production and intestinal lymphocyte subpopulation changes[J]. Infect Immun, 2000, 68 (3): 1282- 1288.
4	FERNANDO M A , LAWN A M , ROSE M E , et al. Invasion of chicken caecal and intestinal lamina propria by crypt epithelial cells infected with coccidia[J]. Parasitology, 1983, 86 (Pt 3): 391- 398.
5	AIKAWA M , MILLER L H , JOHNSON J , et al. Erythrocyte entry by malarial parasites. A moving junction between erythrocyte and parasite[J]. J Cell Biol, 1978, 77 (1): 72- 82.
6	SUSS-TOBY E , ZIMMERBERG J , WARD G E . Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore[J]. Proc Natl Acad Sci USA, 1996, 93 (16): 8413- 8418.
7	LEBRUN M , MICHELIN A , EL HAJJ H , et al. The rhoptry neck protein RON4 re-localizes at the moving junction during Toxoplasma gondii invasion[J]. Cell Microbiol, 2005, 7 (12): 1823- 1833.
8	ALEXANDER D L , MITAL J , WARD G E , et al. Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles[J]. PLoS Pathog, 2005, 1 (2): e17.
9	BESTEIRO S , MICHELIN A , PONCET J , et al. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion[J]. PLoS Pathog, 2009, 5 (2): e1000309.
10	STRAUB K W , CHENG S J , SOHN C S , et al. Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements[J]. Cell Microbiol, 2009, 11 (4): 590- 603.
11	LAMARQUE M , BESTEIRO S , PAPOIN J , et al. The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites[J]. PLoS Pathog, 2011, 7 (2): e1001276.
12	SRINIVASAN P , BEATTY W L , DIOUF A , et al. Binding of Plasmodium merozoite proteins RON2 and AMA1 triggers commitment to invasion[J]. Proc Natl Acad Sci USA, 2011, 108 (32): 13275- 13280.
13	TYLER J S , BOOTHROYD J C . The C-terminus of Toxoplasma RON2 provides the crucial link between AMA1 and the host-associated invasion complex[J]. PLoS Pathog, 2011, 7 (2): e1001282.
14	JOHNSON J , REID W M . Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens[J]. Exp Parasitol, 1970, 28 (1): 30- 36.
15	郭致君, 刘澜, 布仁阿古达木. 克迪球预防人工感染鸡球虫病效果的观察[J]. 畜禽业, 2008 (2): 63- 64.
	GUO Z J , LIU L , BUREN A G D M . Studies on the preventive effects of Kediqiu on coccidiosis in chickens[J]. Livestock and Poultry Industry, 2008 (2): 63- 64.
16	潘严, 姜子睿, 周超, 等. 肠道寄生虫粪便的病原学检查方法[J]. 中国医药科学, 2016, 6 (20): 213- 217.
	PAN Y , JIANG Z R , ZHOU C , et al. Pathogenic examination methods of Intestinal parasite stool[J]. China Medicine and Pharmacy, 2016, 6 (20): 213- 217.
17	索勋, 李国清. 鸡球虫病学[M]. 北京: 中国农业出版社, 1998.
	SUO X , LI G Q . Coccidia and coccidiosis of domestic fowl[M]. Beijing: China Agriculture Press, 1998.
18	SHIRLEY M W , CHAPMAN H D , KUCERA J , et al. Enzyme variation and pathogenicity of recent field isolates of Eimeria tenella[J]. Res Vet Sci, 1989, 46 (1): 79- 83.
19	YAN M , CUI X , ZHAO Q , et al. Molecular characterization and protective efficacy of the microneme 2 protein from Eimeria tenella[J]. Parasite, 2018, 25, 60.
20	刘贤勇, 索勋. 鸡球虫病及其控制策略[J]. 中国农业科技导报, 2006 (5): 31- 37.
	LIU X Y , SUO X . Chicken coccidiosis and the control strategies[J]. Journal of Agricultural Science and Technology, 2006 (5): 31- 37.
21	JEURISSEN S H . Structure and function of the chicken spleen[J]. Res Immunol, 1991, 142 (4): 352- 355.
22	马锡慧, 肖漓. 淋巴细胞亚群成员研究进展[J]. 中华细胞与干细胞杂志(电子版), 2017, 7 (3): 168- 172. doi: 10.3877/cma.j.issn.2095-1221.2017.03.009
	MA X H , XIAO L . Research progress in members of lymphocyte subsets[J]. Chinese Journal of Cells and Stem Cells (Electronic Edition), 2017, 7 (3): 168- 171. doi: 10.3877/cma.j.issn.2095-1221.2017.03.009
23	王莉. Th1/Th2极化: 多因素的参与和调控[J]. 上海免疫学杂志, 2001 (6): 376-378, 384.
	WANG L . Th1/Th2 polarization: involvement and regulation of multiple factors[J]. Shanghai Journal of Immunology, 2001 (6): 376-378, 384.
24	LILLEHOJ H S , TROUT J M . Avian gut-associated lymphoid tissues and intestinal immune responses to Eimeria parasites[J]. Clin Microbiol Rev, 1996, 9 (3): 349- 360.
25	LILLEHOJ H S , MIN W , DALLOUL R A . Recent progress on the cytokine regulation of intestinal immune responses to Eimeria[J]. Poult Sci, 2004, 83 (4): 611- 623.

组别 Group	平均体增重/g Average weight gain	平均OPG/(×10⁵·g^-1) Average OPG
不免疫不攻虫组 Negative control group	65.29±1.32^a	0^a
不免疫攻虫组 Positive control group	42.58±1.11^b	7.04±0.26^b
50 μg蛋白免疫组 50 μg rEtRON2_L2ecto immune group	41.87±0.75^b	4.01±0.27^c
100 μg蛋白免疫组 100 μg rEtRON2_L2ecto immune group	47.02±0.86^c	0.40±0.19^d
200 μg蛋白免疫组 200 μg rEtRON2_L2ecto immune group	46.83±1.26^c	0.24±0.06^d

组别 Group	盲肠病变评分 Score of cecal lesions
不免疫不攻虫组 Negative control group	0^a
不免疫攻虫组 Positive control group	1.5±0.53^b
50 μg蛋白免疫组 50 μg rEtRON2_L2ecto immune group	0.62±0.74^ac
100 μg蛋白免疫组 100 μg rEtRON2_L2ecto immune group	0.25±0.46^ac
200 μg蛋白免疫组 200 μg rEtRON2_L2ecto immune group	0.25±0.46^ac

组别 Group	存活率/% Survival rate	相对增重率/% Relative weight gain rate	病变值 Lesion value	卵囊值 Oocyst value	抗球虫指数 ACI
不免疫不攻虫组 Negative control group	100	100.0	0	0	200
不免疫攻虫组 Positive control group	100	65.2	15	40	110.2
50 μg蛋白免疫组 50 μg rEtRON2_L2ecto immune group	100	64.1	6.2	20	137.9
100 μg蛋白免疫组 100 μg rEtRON2_L2ecto immune group	100	72.0	2.5	5	164.5
200 μg蛋白免疫组 200 μg rEtRON2_L2ecto immune group	100	71.7	2.5	5	164.2

组别 Group	脾脏指数 Spleen index	法氏囊指数 Bursa of Fabricius index
不免疫不攻虫组 Negative control group	21.8±1.0^a	52.6±2.2^a
不免疫攻虫组 Positive control group	24.6±2.6^a	44.5±1.1^b
50 μg蛋白免疫组 50 μg rEtRON2_L2ecto immune group	29.7±1.5^b	31.0±1.9^c
100 μg蛋白免疫组 100 μg rEtRON2_L2ecto immune group	31.3±4.5^b	24.7±4.0^d
200 μg蛋白免疫组 200 μg rEtRON2_L2ecto immune group	33.8.±2.2^b	30.5±1.3^c

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