肠艾美耳球虫重组微线蛋白2对家兔免疫保护效果评价

Evaluation of the Immune Protective Effect of Recombinant MIC2 from Eimeria intestinalis in Rabbits

本研究旨在评价肠艾美耳球虫重组微线蛋白2(rEiMIC2)对兔的免疫保护效果,为肠艾美耳球虫重组亚单位疫苗的研究提供参考。在肠艾美耳球虫转录组数据中筛选出EiMIC2基因,进行克隆、原核表达和蛋白纯化,并采用免疫印迹检测重组蛋白的免疫反应性。将35日龄无球虫新西兰幼兔32只随机分为4组(未免疫未攻虫组、未免疫攻虫组、pET-32a(+)载体组和rEiMIC2免疫组),每组8只,未免疫未攻虫组和未免疫攻虫组每只颈部皮下注射1 mL无菌PBS,pET-32a(+)载体组和rEiMIC2免疫组每只分别接种100μg pET-32a(+)载体蛋白、rEiMIC2,首免后2周进行二免。二免14 d后,除去未免疫未攻虫组,其余3组每只新西兰兔口服接种5×10^(4)个肠艾美耳球虫孢子化卵囊;攻虫14 d后安乐死所有新西兰兔。重组蛋白免疫兔后通过观察临床症状、肠道病变、测定兔的相对增重率、卵囊减少率、抗球虫指数(ACI)值和料肉比以及检测血清中特异性IgG、IL-2、IL-4、IL-10、IFN-γ综合评价rEiMIC2的免疫保护效果。结果显示,EiMIC2基因开放阅读框长度为1 605 bp,编码的蛋白分子质量约为57.68 ku。免疫印迹显示rEiMIC2能够被阳性血清所识别,表明其具有良好的免疫反应性。免疫保护试验显示:攻虫后各组幼兔陆续出现精神沉郁和腹泻的症状,未免疫攻虫组和rEiMIC2免疫组各有1只兔死亡;rEiMIC2免疫组相对增重率为73.97%,卵囊减少率为69.98%,ACI值为136.97,料肉比为3.98∶1,与未免疫攻虫组差异显著(P<0.05);rEiMIC2免疫组肠道病变与未免疫攻虫组相比较轻,血清中特异性IgG水平显著高于未免疫攻虫组(P<0.05)。综上,rEiMIC2免疫兔后,能有效减少卵囊排出和增重损失,减轻肠道损伤,具有一定的保护效果。

The aim of this study was to evaluate the immunoprotective effect of recombinant Eimeria intestinalis microneme protein 2(rEiMIC2)in rabbits and to provide a reference for the study of subunit recombinant vaccine against Eimeria intestinalis.cation of EiMIC2 gene was identified in E.intestinalis transcriptome data,then cloning,prokaryotic expression and protein purification were conducted.The immunoreactivity of the recombinant protein was also detected using immunoblotting.Thirty-two 35-day-old coccidia-free New Zealand young rabbits were randomly divided into 4 groups(unchallenged group,challenged group,pET-32a(+)vector group and rEiMIC2 immunization group),with 8 rabbits in each group.In the challenge group unchallenged and challenged groups,1 mL sterile PBS was subcutaneously injected into the neck of each rabbit.While in the pET-32a(+)vector group and rEiMIC2 immunization group,each rabbits were inoculated with 100μg pET-32a(+)protein or rEiMIC2.A second immunization was conducted after 2 weeks of the first immunization.Fourteen days after the second immunization,excluding the unimmunized and unchallenged group,each of the remaining three groups of New Zealand rabbits was orally inoculated with 5×10^(4) sporulated oocysts of Eimeria intestinalis;All New Zealand rabbits were euthanized fourteen 14 days post-infection.After immunizing rabbits with recombinant protein,the relative weight gain rate,oocyst reduction rate,After immunizing rabbits with recombinant protein,the immune protection effect of rEiMIC2 was comprehensively evaluated by observing clinical symptoms,evaluating intestinal lesions,and measuring relative weight gain rate,oocyst reduction rate,anticoccidial index(ACI),feed-to-meat ratio,serum IgG,IL-2,IL-4,IL-10,IFN-γcomprehensive evaluation of the immune protection effect of rEiMIC2.The results showed that,the open reading frame length of EiMIC2 gene,ORF was 1605 bp,encoded a protein with a molecular mass of approximately 57.68 ku.Western blot showed that rEiMIC2 could be recognized by positive sera,indicating its good immunoreactivity.The immune protection experiment showed that after the challenge,the symptoms of depression and diarrhea appeared in each group,and one rabbit died in the each challenged group and the rEiMIC2 immunization group;the relative weight gain rate of the rEiMIC2 immunization group was 73.97%,and the oocyst reduction rate was 69.98%,the ACI was 136.97,and the feed-to-meat ratio was 3.98∶1,which were significantly different from the unimmunized and challenged group(P<0.05).Compared with the intestinal lesions in the challenged control group,the intestinal lesions in the rEiMIC2 immunization group were milder,and the level of the IgG in serum was significantly higher than that in the unimmunized and challenged group(P<0.05).In summary,after immunizing rabbits with rEiMIC2,it can effectively reduce the excretion of oocysts and the loss of weight gain,and reduce intestinal damage,which providing a certain protective effect of the rabbits.