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

Abstract

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

CLC Number:

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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Evaluation of the Immunoprotective Effect of Recombinant Protein of Eimeria stiedae 3-Phosphoglyceraldehyde Dehydrogenase on Rabbits

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