猪c-Myc蛋白多克隆抗体的制备  

Preparation of Polyclonal Antibody Against c-Myc Protein in Sus scrofa

作　　者：田淑婧 陈羽彤 陆春秀 苏春宇 吕其壮 TIAN Shujing;CHEN Yutong;LU Chunxiu;SU Chunyu;LV Qizhuang(College of Biology&Pharmacy,Yulin Normal University,Yulin,Guangxi 537000,China;Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology,Yulin,Guangxi 537000,China)

机构地区：[1]玉林师范学院生物与制药学院,广西玉林537000 [2]广西农产资源化学与生物技术重点实验室,广西玉林537000

出　　处：《福建农业学报》2023年第8期894-900,共7页Fujian Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31860708);玉林师范学院大学生创新创业训练计划项目(202210606027)。

摘　　要：【目的】克隆猪c-Myc基因CDS序列并构建其原核表达载体,同时纯化c-Myc蛋白并以此为抗原制备多克隆抗体,为进一步研究猪圆环病毒2型(Porcine circovirus type 2,PCV2)Rep蛋白与宿主c-Myc蛋白之间的交互作用奠定基础。【方法】根据猪c-Myc全基因序列(GenBank No.NM_001005154)设计引物,以反转录PCR方法扩增c-Myc基因CDS序列,经Nde I/Xho I双酶切后定向克隆至原核表达载体pET-30a(+);转化至Arctic-Express^(TM)大肠杆菌后诱导表达;表达产物经变性、复性和His-Band Ni+层析柱亲和纯化后免疫新西兰白兔。抗原亲和层析法纯化抗体后,用间接ELISA法测定其效价,用Western blot检测其特异性。【结果】经检测,克隆产生的猪c-Myc基因CDS序列长度为1359 bp,c-Myc-His重组蛋白主要以包涵体形式出现,分子质量约为63 kDa,由此蛋白制备的多抗效价可以达到1∶1093500,并能特异性识别猪c-Myc蛋白和重组蛋白c-Myc-His。【结论】本研究成功制备了猪c-Myc蛋白多克隆抗体,为研究猪c-Myc蛋白功能和其与PCV2 Rep蛋白之间的相互作用奠定了良好基础。【Objective】To facilitate studies on the functions of the porcine circovirus type 2 Rep protein and its interaction with the host c-Myc protein,a polyclonal antibody against the virus was prepared.【Method】According to the complete sequence of porcine c-Myc(GenBank No.NM_001005154),primers were designed to amplify the CDS sequence of the gene by reverse transcription PCR.The CDS was double-digested by Nde I/Xho I enzymes to construct the prokaryotic expression vector pET-30a(+)and transformed into bacterium Arctic-Express^(TM)(Escherichia coli)for induction and expression.The obtained products were denatured,re-natured,and affinity purified by His-band Ni+chromatography as the antigen to prepare polyclonal antibody by immunizing New Zealand white rabbits.The resulting antibody was purified by the antigen affinity purification chromatography,and the titer and specificity determined by indirect ELISA and western blot.【Result】The cloned CDS-sequence of porcine c-Myc was 1359 bp.The recombinant protein of c-Myc-His was mainly in the form of inclusion bodies with a molecular weight of approximately 63 kDa.The titer of the polyclonal antibody prepared from the protein was greater than 1∶1093500.It could specifically recognize the target proteins including recombinant c-Myc-His and porcine c-Myc proteins.【Conclusion】The target polyclonal antibody against the porcine c-Myc protein was successfully obtained for further studies on the functions of the protein as well as its interactions with the PCV2 Rep protein.

关 键 词：猪c-Myc蛋白 原核表达与纯化 多克隆抗体 

分 类 号：S852.659.2[农业科学—基础兽医学] Q785[农业科学—兽医学]