犬ELF4基因原核表达及其卵黄抗体的制备  

Prokaryotic expression and egg yolk antibody of canine ELF4 gene

作　　者：喻娇 曹馨 唐青海 侯鑫军 高翠翠 韩涛涛 黎露 刘会敬 唐斯萍 YU Jiao;CAO Xin;TANG Qing-hai;HOU Xin-jun;GAO Cui-cui;HAN Tao-tao;LI Lu;LIU Hui-jing;TANG Si-ping(College of Life Sciences and Environment/Hunan Key Laboratory for Conservation and Utilization of Biological Resources in the Nanyue Mountainous Region,Hengyang Key Laboratory of New Detection Technology and Biological Agents of Animal Microorganism,Hengyang Normal University,Hengyang,Hunan 421008,China)

机构地区：[1]衡阳师范学院生命科学与环境学院/南岳山区生物资源保护与利用湖南省重点实验室/动物微生物新型检测技术及生物制剂衡阳市重点实验室,湖南衡阳421008

出　　处：《南方农业学报》2022年第8期2359-2372,共14页Journal of Southern Agriculture

基　　金：国家级大学生创新创业训练计划项目(202110546001);湖南省自然科学基金面上项目(2021JJ30060);湖南省教育厅科学研究项目重点项目(21A0442);湖南省重点研发计划项目(2021NK2026)。

摘　　要：【目的】克隆犬转录因子ELF4基因(cELF4),体外表达重组c ELF4蛋白(rcELF4)并制备抗该蛋白的卵黄抗体(IgY),为深入探究犬ELF4蛋白功能提供基础材料。【方法】采用RT-PCR扩增cELF4基因,将目的基因克隆至pMD19-T载体构建重组质粒并测序,运用生物信息学软件分析其核苷酸序列及编码的氨基酸序列特征。将c ELF4基因克隆至原核表达载体pET-28a,构建重组表达质粒pET28a-cELF4,分别转化大肠杆菌BL21(DE3)和Rosetta(DE3)感受态细胞,诱导表达后对表达产物进行SDS-PAGE检测和Western blotting鉴定。将rcELF4蛋白分别与3种免疫佐剂(ISA 15AVG、ISA 71VG、ISA 201VG)乳化制备免疫原,通过免疫蛋鸡检测抗体滴度和IgY含量。构建真核重组表达载体pEGFP-cELF4,转染细胞后,利用免疫过氧化物酶细胞单层试验(IPMA)分析c ELF4特异性IgY的免疫活性。【结果】cELF4基因(GenBank登录号MZ198105)的开放阅读框(ORF)为1992 bp,编码663个氨基酸残基。cELF4基因与狐ELF4基因的核苷酸相似性最高(99.3%),而与小鼠的相似性最低(81.5%)。结合系统进化结果发现,c ELF4基因与狐、大熊猫、欧洲雪貂、北美水獭等动物ELF4基因的亲缘关系较近,而与人和猴等灵长类动物及啮齿类鼠ELF4基因的亲缘关系相对较远。rcELF4蛋白分子量大小约100 kD,可刺激鸡产生相应抗体,ISA 15AVG、ISA 71VG和ISA201VG组血清抗体滴度均在首免后38 d达峰值,其中ISA 201VG组效价最高(>128000倍),且3组rcELF4特异性IgY抗体均在首免后14 d转阳,首免后35 d达峰值,其中ISA 201VG组血清抗体滴度最高,为256000倍。3组鸡蛋卵黄中所提取的IgY含量最高值为4.09 mg/10 mL卵黄液,与rcELF4蛋白呈特异性反应、且与人ELF4蛋白存在交叉反应。rcELF4蛋白定位在细胞核。【结论】rcELF4蛋白在大肠杆菌中成功表达,且存在翻译后修饰现象,不同类型的免疫佐剂对rcELF4蛋白的免疫原性产生不同的影响,所制备rcELF4抗体免疫【Objective】To clone canine transcription factor ELF4 gene(cELF4),recombinant c ELF4 protein(rcELF4)expressed in vitro and prepare egg yolk antibody(IgY)against this protein,so as to provide fundamental material for exploring canine ELF4 protein function.【Method】The c ELF4 gene was amplified by RT-PCR,and the target gene was cloned into p MD19-T vector to construct recombinant plasmid and sequenced.Then nucleotide sequence and amino acid sequence characteristics were analyzed by bioinformatics software.The cELF4 gene was cloned into the prokaryotic expression vector pET-28a,and the recombinant expression plasmid pET28a-cELF4 was constructed and transformed into Escherichia coli BL21(DE3)and Rosetta(DE3)competent cells,respectively.After expression induction,expression product was identified by SDS-PAGE and western blotting.The rcELF4 protein was emulsified with 3 adjuvants(ISA15AVG,ISA 71VG and ISA 201VG),immunized with egg chickens,antibody titers and IgY content were detected.The eukaryotic recombinant expression vector p EGFP-c ELF4 was constructed,cells were transfected,and the immune activity of cELF4-specific IgY was analyzed by the immunoperoxidase cell monolayer assay(IPMA).【Result】The c ELF4 gene(GenBank accession number MZ198105)had an open reading frame of 1992 bp and encoded 663 amino acid residues.The cELF4 gene had the highest similarity with the predicted fox ELF4 gene(99.3%),while it had the lowest similarity with small mouse(81.5%).The phylogenetic analysis showed that canine cELF4 gene were most closely related to the gene in animals such as foxes,pandas,European ferrets,Lontra canadensis and were relatively distantly related to the gene in primates such as humans and monkeys and rodent rats.Molecular weight of rcELF4 protein was about 100 kD and the protein can stimulate chicken to produce antibodies.Titers of serum antibodies in ISA 15AVG,ISA 71VG and ISA201VG groups peaked at 38 d after the first immunization,and the titer of ISA 201VG group was the highest(>128000times).The rcELF4

关 键 词：犬ELF4蛋白 原核表达 真核表达 佐剂 卵黄抗体 

分 类 号：S858.292[农业科学—临床兽医学]