肠艾美耳球虫重组微线蛋白2对家兔免疫保护效果评价

doi:10.11843/j.issn.0366-6964.2024.06.030

摘要/Abstract

摘要：

本研究旨在评价肠艾美耳球虫重组微线蛋白2(rEiMIC2)对兔的免疫保护效果，为肠艾美耳球虫重组亚单位疫苗的研究提供参考。在肠艾美耳球虫转录组数据中筛选出EiMIC2基因，进行克隆、原核表达和蛋白纯化，并采用免疫印迹检测重组蛋白的免疫反应性。将35日龄无球虫新西兰幼兔32只随机分为4组(未免疫未攻虫组、未免疫攻虫组、pET-32a(+)载体组和rEiMIC2免疫组)，每组8只，未免疫未攻虫组和未免疫攻虫组每只颈部皮下注射1 mL无菌PBS，pET-32a(+)载体组和rEiMIC2免疫组每只分别接种100 μg pET-32a(+)载体蛋白、rEiMIC2，首免后2周进行二免。二免14 d后，除去未免疫未攻虫组，其余3组每只新西兰兔口服接种5×10⁴个肠艾美耳球虫孢子化卵囊；攻虫14 d后安乐死所有新西兰兔。重组蛋白免疫兔后通过观察临床症状、肠道病变、测定兔的相对增重率、卵囊减少率、抗球虫指数(ACI)值和料肉比以及检测血清中特异性IgG、IL-2、IL-4、IL-10、IFN-γ综合评价rEiMIC2的免疫保护效果。结果显示，EiMIC2基因开放阅读框长度为1 605 bp，编码的蛋白分子质量约为57.68 ku。免疫印迹显示rEiMIC2能够被阳性血清所识别，表明其具有良好的免疫反应性。免疫保护试验显示：攻虫后各组幼兔陆续出现精神沉郁和腹泻的症状，未免疫攻虫组和rEiMIC2免疫组各有1只兔死亡；rEiMIC2免疫组相对增重率为73.97%，卵囊减少率为69.98%，ACI值为136.97，料肉比为3.98∶1，与未免疫攻虫组差异显著(P < 0.05)；rEiMIC2免疫组肠道病变与未免疫攻虫组相比较轻，血清中特异性IgG水平显著高于未免疫攻虫组(P < 0.05)。综上，rEiMIC2免疫兔后，能有效减少卵囊排出和增重损失，减轻肠道损伤，具有一定的保护效果。

关键词: 肠艾美耳球虫, 微线蛋白2, 兔, 免疫保护

Abstract:

The aim of this study was to evaluate the immunoprotective effect of recombinant Eimeria intestinalis microneme protein 2 (rEiMIC2) in rabbits and to provide a reference for the study of subunit recombinant vaccine against Eimeria intestinalis. EiMIC2 gene was identified in E. intestinalis transcriptome data, then cloning, prokaryotic expression and protein purification were conducted. The immunoreactivity of the recombinant protein was also detected using immunoblotting. Thirty-two 35-day-old coccidia-free New Zealand young rabbits were randomly divided into 4 groups (unchallenged group, challenged group, pET-32a(+) vector group and rEiMIC2 immunization group), with 8 rabbits in each group. In the unchallenged and challenged groups, 1 mL sterile PBS was subcutaneously injected into the neck of each rabbit. While in the pET-32a(+) vector group and rEiMIC2 immunization group, each rabbits were inoculated with 100 μg pET-32a(+) protein or rEiMIC2. A second immunization was conducted after 2 weeks of the first immunization. Fourteen days after the second immunization, excluding the unchallenged group, each of the remaining three groups of New Zealand rabbits was orally inoculated with 5×10⁴ sporulated oocysts of Eimeria intestinalis; All New Zealand rabbits were euthanized 14 days post-infection. After immunizing rabbits with recombinant protein, the immune protection effect of rEiMIC2 was comprehensively evaluated by observing clinical symptoms, evaluating intestinal lesions, and measuring relative weight gain rate, oocyst reduction rate, anticoccidial index (ACI), feed-to-meat ratio, serum IgG, IL-2, IL-4, IL-10, IFN-γ. The results showed that, the open reading frame length of EiMIC2 gene was 1 605 bp, encoded a protein with a molecular mass of approximately 57.68 ku. Western blot showed that rEiMIC2 could be recognized by positive sera, indicating its good immunoreactivity. The immune protection experiment showed that after the challenge, the symptoms of depression and diarrhea appeared in each group, and one rabbit died in each challenged group and the rEiMIC2 immunization group; the relative weight gain rate of the rEiMIC2 immunization group was 73.97%, and the oocyst reduction rate was 69.98%, the ACI was 136.97, and the feed-to-meat ratio was 3.98∶1, which were significantly different from the challenged group (P < 0.05). Compared with the intestinal lesions in the challenged group, the intestinal lesions in the rEiMIC2 immunization group were milder, and the level of the IgG in serum was significantly higher than that in the challenged group (P < 0.05). In summary, after immunizing rabbits with rEiMIC2, it can effectively reduce the excretion of oocysts and the loss of weight gain, and reduce intestinal damage, which providing a certain protective effect of the rabbits.

Key words: Eimeria intestinalis, microneme proteins 2, rabbit, immune protective

中图分类号:

S852.723

陈浩, 郝哥, 蒲家艳, 肖洁, 熊常明, 何维, 朱煜华, 许力文, 姜庆, 杨光友. 肠艾美耳球虫重组微线蛋白2对家兔免疫保护效果评价[J]. 畜牧兽医学报, 2024, 55(6): 2588-2598.

Hao CHEN, Ge HAO, Jiayan PU, Jie XIAO, Changming XIONG, Wei HE, Yuhua ZHU, Liwen XU, Qing JIANG, Guangyou YANG. Evaluation of the Immune Protective Effect of Recombinant MIC2 from Eimeria intestinalis in Rabbits[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2588-2598.

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组别 Group	动物数量 Rabbits number	免疫原及剂量 Immunogen and dosage	免疫方式 Immunization route	免疫次数 Immunization times	攻虫 Challenge dosage
未免疫未攻虫组 Unchallenged group	8	PBS 1 mL	颈部皮下注射	2次，同等剂量，间隔14 d	未感染
未免疫攻虫组 Challenged group	8	PBS 1 mL	颈部皮下注射	2次，同等剂量，间隔14 d	口服5×10⁴孢子化卵囊
pET-32a(+)组 pET-32a(+) vector group	8	100 μg载体蛋白+1 mg皂素溶于无菌PBS 1 mL	颈部皮下注射	2次，同等剂量，间隔14 d	口服5×10⁴孢子化卵囊
rEiMIC2免疫组 rEiMIC2 immunized group	8	100 μg rEiMIC2+ 1mg皂素溶于无菌PBS 1 mL	颈部皮下注射	2次，同等剂量，间隔14 d	口服5×10⁴孢子化卵囊

评分 Score	小肠病变记分的判定标准 Criteria for scoring intestinal lesions
0	空肠、回肠未见明显病理改变，表现正常
1	肠壁可见少量白色结节或少量出血点，未见增厚
2	出血点或白色结节较多，肠壁稍增厚
3	可见大量出血点或白色结节，肠壁增厚
4	动物死亡；肠壁广泛出血，白色结节连成斑块；肠壁极度扩张增厚

试验组卵囊数占感染非免疫组比例 The proportion of oocyst in the experimental group to unimmunized and challenged group	卵囊值 Oocyst value
0~1%	0
2%~25%	1
26%~50%	10
51%~75%	20
76%~100%	40

范围 Range	抗球虫效果 Anti coccidiosis effect
ACI＜120	无
120≤ACI＜160	中等
160≤ACI≤180	良好
ACI＞180	优秀

组别 Group	攻虫前平均增重/g Average body weight gain before challenge	攻虫后平均增重/g Average body weight gain after challenge	相对增重率/% Relative weight gain	OPG/10⁵	卵囊减少率/% Oocyst decrease ratio	料肉比 Feed conversion ratio	平均病变评分 Mean lesion scores	ACI
未免疫未攻虫组	510.00±91.92^a	716.50±98.48^a	—	—	—	3.00∶1	—	200
Unchallenged group
未免疫攻虫组	481.62±66.25^a	438.28±81.60^b	61.17	9.45±8.79^a	—	4.71∶1	2.03±1.39	88.37
Challenged group
pET-32a(+)组	511.18±90.82^a	419.37±80.64^b	58.53	9.32±10.01^a	-1.4	4.95∶1	2.67±0.75	91.83
pET-32a(+) vector group
rEiMIC2免疫组	471.87±82.97^a	530.00±90.18^c	73.97	2.83±3.22^b	69.98	3.98∶1	1.45±1.19	136.97
rEiMIC2 immunization group