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

Abstract

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

CLC Number:

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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References 20

1	FATOBA A J , ADELEKE M A . Diagnosis and control of chicken coccidiosis: a recent update[J]. J Parasit Dis, 2018, 42 (4): 483- 493.
2	马艺飞, 杜彩娟, 王乐乐, 等. 毒害艾美耳球虫蔗糖非酵解解旋酶2基因片段的克隆与原核表达[J]. 畜牧与兽医, 2017, 49 (8): 72- 75.
	MA Y F , DU C J , WANG L L , et al. Cloning and prokaryotic expression of partial sequence of SNF2 gene in Eimeria necatrix[J]. Animal Husbandry & Veterinary Medicine, 2017, 49 (8): 72- 75.
3	高阳. 毒害艾美耳球虫入侵宿主细胞关键分子的筛选与鉴定及其功能研究[D]. 扬州: 扬州大学, 2021.
	GAO Y. The screening, identification and functional analysis of the key molecules involved in cellular invasion of Eimeria necatrix[D]. Yangzhou: Yangzhou University, 2021. (in Chinese)
4	GAO Y , SUDING Z , WANG L L , et al. Full-length transcriptome sequence analysis of Eimeria necatrix unsporulated oocysts and sporozoites identifies genes involved in cellular invasion[J]. Vet Parasitol, 2021, 296, 109480.
5	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.
6	程振扬. 柔嫩艾美耳球虫配子体GAM22蛋白的真核表达及其免疫保护效力评价[D]. 扬州: 扬州大学, 2021.
	CHENG Z Y. Eukaryotic expression of gametophyte protein GAM22 of Eimeria tenella and its immunological protection[D]. Yangzhou: Yangzhou University, 2021. (in Chinese)
7	索勋, 李国清. 鸡球虫病学[M]. 北京: 中国农业大学出版社, 1998.
	SUO X , LI G Q . Coccidia and coccidiosis of domestic fowl[M]. Beijing: China Agricultural University Press, 1998.
8	DOGGA S K , MUKHERJEE B , JACOT D , et al. A druggable secretory protein maturase of Toxoplasma essential for invasion and egress[J]. eLife, 2017, 6, e27480.
9	HÅKANSSON S , CHARRON A J , SIBLEY L D . Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole[J]. EMBO J, 2001, 20 (12): 3132- 3144. doi: 10.1093/emboj/20.12.3132
10	KEMP L E , YAMAMOTO M , SOLDATI-FAVRE D . Subversion of host cellular functions by the apicomplexan parasites[J]. FEMS Microbiol Rev, 2013, 37 (4): 607- 631.
11	ROGER N , DUBREMETZ J F , DELPLACE P , et al. Characterization of a 225 kilodalton rhoptry protein of Plasmodium falciparum[J]. Mol Biochem Parasitol, 1988, 27 (2-3): 135- 141.
12	COLLINS W E , WALDUCK A , SULLIVAN J S , et al. Efficacy of vaccines containing rhoptry-associated proteins RAP1 and RAP2 of Plasmodium falciparum in Saimiri boliviensis monkeys[J]. Am J Trop Med Hyg, 2000, 62 (4): 466- 479.
13	PEIXOTO L , CHEN F , HARB O S , et al. Integrative genomic approaches highlight a family of parasite-specific kinases that regulate host responses[J]. Cell Host Microbe, 2010, 8 (2): 208- 218.
14	EL HAJJ H , DEMEY E , PONCET J , et al. The ROP2 family of Toxoplasma gondii rhoptry proteins: proteomic and genomic characterization and molecular modeling[J]. Proteomics, 2006, 6 (21): 5773- 5784.
15	TALEVICH E , KANNAN N . Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors[J]. BMC Evol Biol, 2013, 13, 117.
16	王炳祥. 鸡柔嫩艾美尔球虫棒状体蛋白EtROP30和EtROP35的鉴定及功能研究[D]. 泰安: 山东农业大学, 2022.
	WANG B X. Identification and functional studies of Eimeria tenella rhoptry protein EtROP30 and EtROP35[D]. Taian: Shandong Agricultural University, 2022. (in Chinese)
17	王颖. Restin核定位信号序列的鉴定及其入核分子机制的初步探讨[D]. 西安: 第四军医大学, 2006.
	WANG Y. Identification of Restin nuclear localization signal sequences and preliminary discussion of the molecular mechanism of nuclear entry[D]. Xi 'an: Fourth Military Medical University, 2006. (in Chinese)
18	JENSEN K D C , HU K , WHITMARSH R J , et al. Toxoplasma gondii rhoptry 16 kinase promotes host resistance to oral infection and intestinal inflammation only in the context of the dense granule protein GRA15[J]. Infect Immun, 2013, 81 (6): 2156- 2167.
19	SONAIMUTHU P , CHING X T , FONG M Y , et al. Induction of protective immunity against toxoplasmosis in BALB/c mice vaccinated with Toxoplasma gondii rhoptry-1[J]. Front Microbiol, 2016, 7, 808.
20	石凯. 隐孢子虫棒状体cgd8₂530蛋白sushi片段亚单位疫苗和核酸疫苗的制备及其免疫保护性研究[D]. 成都: 四川农业大学, 2014.
	SHI K. Preparation and immunoprotective study of subunit and nucleic vaccine of Cryptosporidium parvum rhoptry protein cgd8₂530 sushi domain fragment[D]. Chengdu: Sichuan Agricultural University, 2014. (in Chinese)

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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Prokaryotic Expression of Eimeria necatrix ROP30 Protein and Its Immunoprotective Efficacy in Chicken

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