鸡毒害艾美耳球虫ROP30蛋白的原核表达及其对鸡免疫保护效果的观察

doi:10.11843/j.issn.0366-6964.2025.03.040

摘要/Abstract

摘要：

旨在克隆表达鸡毒害艾美耳球虫棒状体EnROP30基因，并用重组蛋白rEnROP30免疫雏鸡，评价其免疫保护效果。以毒害艾美耳球虫第二代裂殖子(MZ-2)的总RNA为模板，RT-PCR扩增EnROP30基因，连接至pGEM^Ⓡ-T-Easy载体，测序后构建pET28a(+)-EnROP30表达载体，转化至大肠杆菌BL21(DE3)，重组菌经测序鉴定后诱导表达，并纯化重组蛋白。用重组蛋白免疫BALB/c小鼠，制备抗rEnROP30的多克隆抗体。利用多克隆抗体，分别用Western blot和间接免疫荧光试验检测子孢子(SZ)和MZ-2中的天然EnROP30蛋白及其定位。最后，应用荧光定量PCR分析EnROP30基因在SZ和MZ-2中的转录水平。用重组蛋白按高剂量(200 μg·羽^-1)、中剂量(100 μg·羽^-1)和低剂量(50 μg·羽^-1)免疫雏鸡，同时设置未免疫攻虫组和未免疫未攻虫组为对照，以成活率、平均增重、相对增重率、卵囊减少率、病变记分和抗球虫指数(ACI)为指标，评价重组蛋白的免疫保护力。结果显示：该基因全长1 605 bp，编码534个氨基酸，推导相对分子质量为55.55 ku；重组蛋白大小约为65 ku，以可溶形式存在，能被小鼠抗His标签单抗和鸡抗毒害艾美耳球虫阳性血清特异性识别；在SZ和MZ-2中均检测到天然EnROP30蛋白，其分子质量大约为62 ku；EnROP30蛋白定位在SZ和MZ-2的顶端；MZ-2中EnROP30转录水平显著高于SZ(P < 0.05)。各试验组的成活率均为100%；免疫组的平均增重增加，病变记分显著降低(P＜0.05)；中剂量组的相对增重率(92.96%)、卵囊减少率(66.87%)和ACI值(158.96)均为最高。综上：成功克隆表达了EnROP30基因，重组蛋白rEnROP30对毒害艾美耳球虫具有一定的免疫保护力。

关键词: 毒害艾美耳球虫, EnROP30, 克隆, 表达, 免疫保护力

Abstract:

This study aimed to clone and express EnROP30 gene of Eimeria necatrix, and to evaluate the immune protection of recombinant protein EnROP30 in chicken. EnROP30 gene was cloned from total RNA of the second generation merozoites (MZ-2) of E. necatrix by RT-PCR, and inserted into the pGEM^Ⓡ-T-Easy vector by TA cloning. After sequencing analysis, EnROP30 cDNA was subcloned to the pET-28a(+) vector to obtain a recombinant prokaryotic plasmid. After transformed into E. coli BL21, the recombinant plasmid pET28a(+)-EnROP30 was induced to express by IPTG, and the recombinant protein rEnROP30 was identified and purified. BALB/c mice were immunized with the purified recombinant protein to prepare the anti-rEnROP30 polyclonal antibodies, which was used to detect the native protein EnROP30 and its localization in sporozoites (SZ) and MZ-2 of E. necatrix by Western blot and indirect immunofluorescence assay, respectively. Finally, the transcriptional level of EnROP30 in SZ and MZ-2 was analyzed by qRT-PCR. Three groups of chickens were immunized with rEnROP30 at three different doses (200 μg, 100 μg and 50 μg per chicken), respectively. At the same time, two groups of chickens, namely the unimmunized and challenged (UC) group, and the unimmunized and unchallenged (UU) group, were used as the controls. The immune protective effects were evaluated based on the survival rate, weight gain, relative weight gain, oocyst reduction, lesion score and anticoccidial index (ACI) of each group. The results showed that the target gene was 1 605 bp, coding 534 amino acids with a predicted molecular weight of 55.55 ku. The recombinant protein was about 65 ku and predominantly expressed as soluble form. Western blot analysis showed that the recombinant protein could be specifically recognized by mouse anti-His monoclonal antibodies and the convalescent serum of the chickens infected with E. necatrix. Native protein EnROP30 was detected in SZ and MZ-2 and had a molecular weight of 62 ku. EnROP30 protein was located in the top of SZ and MZ-2. The transcription level of EnROP30 in MZ-2 was significantly higher than that in SZ (P < 0.05). The survival rate of all the groups was 100%. The weight gain of the immunized groups was higher than that of the UC group, but their lesion scores were significantly lower than that of the UC group (P < 0.05). In the group immunized with 100 μg rEnROP30 per chicken, the relative weight gain (92.96%), the oocyst reduction (66.87%) and ACI (158.96) were the highest. It was showed that EnROP30 gene was cloned and expressed successfully; rEnROP30 had a good protective efficacy against E. necatrix.

Key words: Eimeria necatrix, EnROP30, cloning, expression, protective efficacy

中图分类号:

李慧中, 张弛, 严丹丽, 宋鹏慧, 汪飞燕, 冯茜茜, 刘丹丹, 许金俊, 陶建平. 鸡毒害艾美耳球虫ROP30蛋白的原核表达及其对鸡免疫保护效果的观察[J]. 畜牧兽医学报, 2025, 56(3): 1419-1430.

LI Huizhong, ZHANG Chi, YAN Danli, SONG Penghui, WANG Feiyan, FENG Qianqian, LIU Dandan, XU Jinjun, TAO Jianping. Prokaryotic Expression of Eimeria necatrix ROP30 Protein and Its Immunoprotective Efficacy in Chicken[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1419-1430.

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组别 Group	免疫剂量/(μg·羽^-1) Immunization dose	免疫日龄/d Immunization age	免疫途径 Immunization route	攻虫日龄/d Challenge age	攻虫剂量(×10⁴) Challenge dose (×10⁴)
rEnROP30高剂量组 rEnROP30 high-dose group	200	5、12	皮下注射 Subcutaneous injection	19	2.5
rEnROP30中剂量组 rEnROP30 medium-dose group	100	5、12	皮下注射 Subcutaneous injection	19	2.5
rEnROP30低剂量组 rEnROP30 low-dose group	50	5、12	皮下注射 Subcutaneous injection	19	2.5
未免疫攻虫组 Unimmunized and challenged group	/	/	/	19	2.5
未免疫未攻虫组 Unimmunized and unchallenged group	/	/	/	/	/

组别 Group	平均增重1/g Average weight gain 1	相对增重率1/% Relative weight gain rate 1	平均增重2/g Average weight gain 2	相对增重率2/% Relative weight gain rate 2
rEnROP30高剂量组 rEnROP30 high-dose group	271.21±15.55^a	98.96	227.96±31.61^cd	79.79
rEnROP30中剂量组 rEnROP30 medium-dose group	265.67±42.90^a	96.94	265.57±17.17^ab	92.96
rEnROP30低剂量组 rEnROP30 low-dose group	261.99±28.46^a	95.59	226.57±20.61^cd	79.31
未免疫攻虫组 Unimmunized and challenged group	270.85±12.90^a	98.82	220.96±21.86^d	77.34
未免疫未攻虫组 Unimmunized and unchallenged group	274.07±13.99^a	100.00	285.69±33.80^a	100.00

组别 Group	平均病变记分($\bar x \pm s$) Average lesion score	卵囊产量(×10⁶个) Oocyst output (×10⁶)			卵囊减少率/% Oocyst reduction rate
组别 Group	平均病变记分($\bar x \pm s$) Average lesion score	粪便/羽 Fecal/bird	盲肠/羽 Cecal/bird	总计/羽 Total/bird	卵囊减少率/% Oocyst reduction rate
rEnROP30高剂量组 rEnROP30 high-dose group	2.35±0.53^bc	0.96	0.58	1.54	57.22
rEnROP30中剂量组 rEnROP30 medium-dose group	2.40±0.52^bc	0.83	0.35	1.18	66.87
rEnROP30低剂量组 rEnROP30 low-dose group	2.55±0.55^abc	1.21	0.39	1.60	55.41
未免疫攻虫组 Unimmunized and challenged group	2.90±0.46^a	2.67	0.92	3.59	0.00
未免疫未攻虫组 Unimmunized and unchallenged group	0.00±0.00^d	0.00	0.00	0.00	0.00

组别 Group	存活率/% Survival rate	相对增重/% Relative weight gain	病变值(0~40) Lesion score (0-40)	卵囊值(0~40) Oocyst score (0-40)	ACI
rEnROP30高剂量组 rEnROP30 high-dose group	100	79.79	23.5	10	146.29
rEnROP30中剂量组 rEnROP30 medium-dose group	100	92.96	24.0	10	158.96
rEnROP30低剂量组 rEnROP30 low-dose group	100	79.31	25.5	10	143.81
未免疫攻虫组 Unimmunized and challenged group	100	77.34	29.0	40	108.34
未免疫未攻虫组 Unimmunized and unchallenged group	100	100.00	0.0	0	200.00

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