猪ELF4基因的克隆、蛋白表达及其多克隆抗体制备  

Cloning and Expression of Porcine ELF4 Gene and Preparation of Its Polyclonal Antibody

作　　者：王瑾[1] 唐青海[1,2] 谷雅静 李晓楠[1] 李羽[1] 王雪雨 姚伦广[1] 阚云超[1] 

机构地区：[1]南阳师范学院河南省伏牛山昆虫生物学重点实验室/昆虫生物反应器河南省工程实验室,南阳473061 [2]衡阳师范学院生命科学与环境学院生物药物研究所,衡阳421008

出　　处：《畜牧兽医学报》2017年第3期425-435,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金青年基金(31101837);河南省重点科技攻关项目(142102110101);衡阳师范学院引进人才专项项目;河南省高等学校重点科研项目(16A230023)

摘　　要：本研究旨在克隆猪转录因子ELF4基因,体外表达ELF4蛋白并制备抗该蛋白的多克隆抗体。采用RTPCR扩增猪ELF4基因,运用生物信息学软件分析其核苷酸和编码氨基酸序列特征,将其克隆至原核表达载体pET28a,构建重组表达载体pET28a-pELF4,转化Rosetta(DE3)菌株,在不同温度、IPTG浓度、诱导时间等条件下进行诱导表达,采用SDS-PAGE和Western blot分析蛋白表达,重组pELF4蛋白(rpELF4)经纯化免疫鸡制备抗pELF4抗体。结果表明,pELF4开放阅读框为1 989bp(GenBank No.:KU097322),编码662个氨基酸,该基因与已经测定的人、牛、小鼠的ELF4的核苷酸相似性依次为89.4%、90.7%和81.1%,与牛等大动物的亲缘关系相对较近,与人和小鼠亲缘关系相对较远;结果显示,成功表达了重组蛋白pELF4,rpELF4分子量为95ku,该蛋白以可溶性和包涵体两种形式存在,在37℃条件下,以终浓度为0.8mmol·L-1IPTG诱导8h包涵体表达量达到最高。第4次免疫后第13天,鸡抗rpELF4抗体滴度达到1︰256 000倍以上。本试验利用原核表达系统成功制备了重组pELF4蛋白,并制备了免疫活性和特异性良好的鸡抗pELF4多克隆抗体,为该蛋白生物学功能及相关疾病研究提供了基础材料。This research aimed to clone and express the porcine transcription factor E74-like factor4（pELF4）gene in vitro and to prepare its polyclonal antibody.The pELF4 gene was cloned by RT-PCR（Reverse Transcription-Polymerase Chain Reaction）,and the characteristics of its nucleotides and coding amino acids were analyzed by the bioinformatics softwares.The recombinant expression vector pET28a-pELF4 was constructed by inserting the pELF4 gene into the prokaryotic expression vector pET28 a.Then pET28a-pELF4 was transformed into E.coli Rosetta（DE3）and to investigate its expression under different temperatures,IPTG concentrations and induction time.The expressed proteins were analyzed by SDS-PAGE and Western blot.The anti-pELF4immuno-globulin egg yolk antibody（IgY）was prepared by immunizing hens after purification of the recombination pELF4protein（rpELF4）.The results showed that the ORF（Open reading frame）of pELF4gene（GenBank Accession number：KU097322）was 1 989 bp,coding 662 amino acids.The similarity between pELF4 gene and that of human,cattle and mouse were 89.4%,90.7% and 81.1%,respectively,therefore it was closer to the animals such as cattle while it was further to human and mouse.The relative molecular weight of rpELF4 was 95ku,and it existed in the form of protein and inclusion body.The amount of inclusion body reached highest when the expression combination was 37℃,0.8mmol·L-1 of IPTG and 8h.The chicken anti-rpELF4 antibody titer was above 1 ︰ 256 000 fold at the 13 rd days post the 4th vaccination.The recombinant protein pELF4 was successfully expressed by prokaryotic expression system,the chicken anti-pELF4 polyclonal antibodies with the wonderful activity and specifity was prepared,which provided basic material for the research of the biofunction of this protein and the related diseases.

关 键 词：猪ELF4蛋白 原核表达 免疫 

分 类 号：S828[农业科学—畜牧学] S813.1[农业科学—畜牧兽医]