摘要：

为探究柔嫩艾美耳球虫顶膜抗原-1(AMA1)对感染柔嫩艾美耳球虫雏鸡的免疫保护效果,将AMA1基因扩增产物连接到表达载体pET-32a(+)上构建重组质粒pET-32a(+)-AMA1,并转入大肠杆菌BL21(DE3)来表达重组蛋白。将108只雏鸡随机分为6组,每组3个重复,每个重复6羽,包括不免疫不攻虫组(阴性组)、不免疫攻虫组(阳性组)、弗氏佐剂对照组及12.5,25.0,50.0 μg rEtAMA1免疫组,分别于14,21日龄肌肉注射不同浓度的重组蛋白,28日龄口服感染5×10⁴个柔嫩艾美耳球虫孢子化卵囊,35日龄处死试验鸡,以体增质量、盲肠病变评分、卵囊产量、免疫器官指数、细胞因子及抗体水平来评价重组AMA1蛋白的免疫保护效果。结果显示,各组间的平均增质量差异不显著;25.0 μg及50.0 μg rEtAMA1组的盲肠病变计分较阳性组分别显著降低了53%和41%;12.5,25.0,50.0 μg rEtAMA1组的克粪便卵囊数量(OPG)分别较阳性组显著减少16%,27%,54%;50.0 μg rEtAMA1组的法氏囊指数较阳性组显著提高27%;25.0,50.0 μg rEtAMA1组的胸腺指数均显著高于阳性组,分别增加了25%,22%;各组的脾脏指数无显著差异;在二免后7 d,12.5 μg和25.0 μg rEtAMA1组的细胞因子IL-2和IFN-γ含量均显著高于阴性组,50.0 μg rEtAMA1组血清总IgG含量较阴性组显著增加47%,且二免后含量明显高于首免后。综上,重组AMA1蛋白能够减轻盲肠病变,减少球虫卵囊产量和促进免疫器官发育,提高血清细胞因子和抗体水平,对感染柔嫩艾美耳球虫雏鸡具有一定的免疫保护效果。

关键词: 柔嫩艾美耳球虫, AMA1, 原核表达, 免疫效果

Abstract:

In order to investigate the immune protective effect of apical membrane antigen 1(AMA1)of Eimeria tenella on chickens infected with E.tenella, the AMA1 gene amplified was connected to expression vector pET-32a(+).The recombinant plasmid was transformed into E.coli BL21(DE3)to express recombinant protein.108 chickens were randomly divided into 6 groups with 3 replicates in each group and 6 chicks in each replicate,including non-immune and non-attacking group(negative control group),non-immune attack group(positive control group),Freund's adjuvant group and 12.5,25.0,50.0 μg rEtAMA1 immunized groups.All of the chickens were injected with different concentration of recombinant protein at the age of 14,21 days,and infected orally with 5×10⁴ sporulated oocysts of E.tenella at the age of 28 days and killed at the age of 35 days.The immune protection effect of rEtAMA1 was evaluated by body weight gain,lesion score,oocyst production,immune organ index and levels of cytokines and antibodies.There was no significant difference in average weight gain among the groups.Compared with the positive group,the scores of cecal lesions in 25.0 μg and 50.0 μg rEtAMA1 groups were significantly reduced by 53%,41%,the OPG in 12.5,25.0,50.0 μg rEtAMA1 groups were significantly reduced by 16%,27%,54% respectively,the immune organ index of bursa of Fabricius in 50 μg rEtAMA1 groups significantly increased by 27%,the immune organ index of thymus in 25.0 μg and 50.0 μg rEtAMA1 groups significantly increased by 25%,22% respectively.There was no significant difference in the immune organ index of spleen.Seven days after the second immunization,the levels of IL-2 and IFN-γ in 12.5 μg and 25.0 μg rEtAMA1 groups were significantly higher than the negative control group.The level of serum total IgG concentration in the 50.0 μg rEtAMA1 group significantly increased by 47% than that in the negative control group,and the concentration after the second immunization was obviously higher than that after the first immunization.The results showed that the rEtAMA1 could alleviate cecal lesions,decrease the oocyst production,promote the development of immune organs and increase the levels of cytokines and antibodies in serum,which had a certain immune protective effect on chickens infected with E.tenella.

Key words: Eimeria tenella, AMA1, Prokaryotic expression, Immune effect