斯氏艾美耳球虫3-磷酸甘油醛脱氢酶重组蛋白对兔的免疫保护效果评价

doi:10.11843/j.issn.0366-6964.2023.06.035

摘要/Abstract

摘要： 旨在评价斯氏艾美耳球虫重组蛋白Es-GAPDH对兔的免疫保护效果,为兔斯氏艾美耳球虫重组亚单位疫苗的研制奠定基础。利用相对荧光定量PCR分析Es-GAPDH在斯氏艾美耳球虫不同发育时期的转录水平,并对Es-GAPDH进行原核表达与蛋白纯化;然后将42只45日龄无球虫幼兔随机分为5组(空白对照组、不免疫攻虫组、Trx-His-S tag攻虫对照组、Quil-A saponin攻虫对照组和rEs-GAPDH免疫组),分别经颈部皮下注射1 mL PBS、1 mL PBS、1 mL PBS含100 μg pET-32a空载蛋白含1 mg Quil-A、1 mL PBS含1 mg Quil-A、1 mL 100 μg rEs-GAPDH含1 mg Quil-A,首免14 d后同等剂量加强免疫。二免14 d后除空白对照组外,其余各组实验兔经口感染1×10⁴个斯氏艾美耳球虫孢子化卵囊,攻虫后观察各组临床表现,每周定时采血、称重,感染21 d后剖检观察肝组织病理变化,并测定和统计每组的相对增重、料肉比、肝指数、卵囊排出量、生化指标、特异性IgG抗体以及细胞因子等。结果发现,Es-GAPDH在斯氏艾美耳球虫各个发育阶段均有转录,且转录水平存在差异,在孢子化卵囊阶段转录水平最高。免疫保护试验表明:感染后不免疫攻虫组出现兔肝球虫病典型症状,而rEs-GAPDH免疫组症状不明显。rEs-GAPDH免疫组的卵囊减少率达87.09%,相对增重率显著大于三个攻虫对照组(P＜0.05),特异性IgG抗体水平、细胞因子(IFN-γ、IL-2、IL-4、IL-10、TGF-β)水平均与不免疫攻虫组存在显著差异(P＜0.05),组织病理切片也显示,免疫组相较不免疫攻虫组肝组织被破坏程度低,虫体数量较少。综上,斯氏艾美耳球虫重组蛋白rEs-GAPDH可减少增重损失和卵囊排出,能引发宿主体内的细胞免疫和体液免疫应答,具有一定免疫保护作用,可作为斯氏艾美耳球虫重组亚单位疫苗的候选抗原。

关键词: 兔, 斯氏艾美耳球虫, 三磷酸甘油醛脱氢酶, 重组蛋白, 免疫保护

Abstract: The aim of this study was to evaluate the immunoprotective effect of Es-GAPDH, a recombinant protein of Eimeria stiedae, on rabbits and to lay the foundation for the development of a recombinant subunit immunization vaccine for rabbits.The transcript levels of Es-GAPDH at different developmental stages of E.stiedae were analyzed by relative fluorescence quantitative PCR, and Es-GAPDH protein was prokaryotically expressed and purified. Then 42 45-day-old coccidia-free rabbits were randomly divided into 5 groups (unimmunized and unchallenged group, unimmunized and challenged group, Trx-His-S tag-challenged group, Quil-A saponin-challenged group and rEs-GAPDH immunization group). 1 mL PBS, 1 mL PBS, 1 mL PBS containing 100 μg pET-32a and 1 mg Quil-A, 1 mL PBS containing 1 mg Quil-A, 1 mL 100 μg rEs-GAPDH and 1 mg Quil-A were respectively injected subcutaneously via the neck to the rabbits, and were booster immunized with the same dose 14 d after the first immunization. After the secondary immunization, except for the unimmunized and unchallenged group, each rabbit in the other groups was orally infected with 1×10⁴ sporulated oocysts of E.stiedae. The clinical manifestations of each group were observed after the attack. Blood samples were collected and the animals were weighed at regular intervals every week. Twenty-one days after infection, the liver pathological changes were observed by autopsy, and the relative weight gain, feed to gain ratio, liver index, oocyst excretion, biochemical indexes, specific IgG antibodies and cytokines of each experimental group were measured and counted. The results showed that Es-GAPDH was transcribed at all developmental stages of E.stiedae, and there were differences in transcript levels, with the highest transcript level in the sporulating oocyst stage. The immunoprotection test showed that typical symptoms of rabbit hepatic coccidiosis appeared in the unimmunized and challenged group after infection, while rEs-GAPDH immunized group showed no significant symptoms. rEs-GAPDH immunized group showed 87.09% oocyst reduction, and significantly greater relative weight gain than the other three challenged groups (P<0.05), specific IgG antibody levels, cytokine (IFN-γ, IL-2, IL-4, IL-10, TGF-β) levels were significantly different from those of the unimmunized and challenged group (P<0.05), and histopathological sections also showed that the liver tissue was less destroyed and the number of parasites was lower in the immunized group compared with the unimmunized and challenged group. It can be concluded that the recombinant protein rEs-GAPDH of E.stiedae can reduce weight loss and oocyst excretion, trigger cellular and humoral immune responses in the host, have certain immunoprotective effects, and can be used as a candidate antigen for E.stiedae recombinant subunit vaccine.

Key words: rabbit, Eimeria stiedae, 3-phosphoglyceraldehyde dehydrogenase, recombinant protein, immunoprotection

中图分类号:

郑若愚, 任永军, 肖洁, 白鑫, 蒲家艳, 陈浩, 杨光友. 斯氏艾美耳球虫3-磷酸甘油醛脱氢酶重组蛋白对兔的免疫保护效果评价[J]. 畜牧兽医学报, 2023, 54(6): 2581-2595.

ZHENG Ruoyu, REN Yongjun, XIAO Jie, BAI Xin, PU Jiayan, CHEN Hao, YANG Guangyou. Evaluation of the Immunoprotective Effect of Recombinant Protein of Eimeria stiedae 3-Phosphoglyceraldehyde Dehydrogenase on Rabbits[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2581-2595.

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