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  

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

作　　者：李慧中 张弛 严丹丽 宋鹏慧 汪飞燕 冯茜茜 刘丹丹[1,2] 许金俊 陶建平[1,2] LI Huizhong;ZHANG Chi;YAN Danli;SONG Penghui;WANG Feiyan;FENG Qianqian;LIU Dandan;XU Jinjun;TAO Jianping(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,扬州225009 [2]江苏动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《畜牧兽医学报》2025年第3期1419-1430,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31972698);江苏省高校优势学科建设四期工程项目(2021);高等学校学科创新引智计划资助(D18007)。

摘　　要：旨在克隆表达鸡毒害艾美耳球虫棒状体En ROP 30基因,并用重组蛋白rEnROP30免疫雏鸡,评价其免疫保护效果。以毒害艾美耳球虫第二代裂殖子(MZ-2)的总RNA为模板,RT-PCR扩增En ROP 30基因,连接至pGEM-T-Easy载体,测序后构建pET28a(+)-EnROP30表达载体,转化至大肠杆菌BL21(DE3),重组菌经测序鉴定后诱导表达,并纯化重组蛋白。用重组蛋白免疫BALB/c小鼠,制备抗rEnROP30的多克隆抗体。利用多克隆抗体,分别用Western blot和间接免疫荧光试验检测子孢子(S Z)和MZ-2中的天然EnROP30蛋白及其定位。最后,应用荧光定量PCR分析En ROP 30基因在SZ和MZ-2中的转录水平。用重组蛋白按高剂量(200μg·羽-1)、中剂量(100μg·羽-1)和低剂量(50μg·羽-1)免疫雏鸡,同时设置未免疫攻虫组和未免疫未攻虫组为对照,以成活率、平均增重、相对增重率、卵囊减少率、病变记分和抗球虫指数(ACI)为指标,评价重组蛋白的免疫保护力。结果显示:该基因全长1605 bp,编码534个氨基酸,推导相对分子质量为55.55 ku;重组蛋白大小约为65 ku,以可溶形式存在,能被小鼠抗His标签单抗和鸡抗毒害艾美耳球虫阳性血清特异性识别;在S Z和MZ-2中均检测到天然EnROP30蛋白,其分子质量大约为62 ku;EnROP30蛋白定位在S Z和MZ-2的顶端;MZ-2中En ROP 30转录水平显著高于S Z(P<0.05)。各试验组的成活率均为100%;免疫组的平均增重增加,病变记分显著降低(P<0.05);中剂量组的相对增重率(92.96%)、卵囊减少率(66.87%)和ACI值(158.96)均为最高。综上:成功克隆表达了En ROP 30基因,重组蛋白rEnROP30对毒害艾美耳球虫具有一定的免疫保护力。This study aimed to clone and express En ROP 30 gene of Eimeria necatrix,and to evaluate the immune protection of recombinant protein EnROP30 in chicken.En ROP 30 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,En ROP 30 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 En ROP 30 in S Z and MZ-2 was analyzed by qRT-PCR.Three groups of chickens were immunized with rEnROP30 at three different doses(200μg,100μg and 50μg 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 1605 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 S Z and MZ-2.The transcription level of En ROP 30 in MZ-

关 键 词：毒害艾美耳球虫 En ROP 30 克隆 表达 免疫保护力 

分 类 号：S852.723[农业科学—基础兽医学] S855.9[农业科学—兽医学]