摘要
本研究旨在通过克隆猫源C11orf96(Felis catus C11orf96,fC11orf96)基因,并制备其多克隆抗体分析该基因在细胞中的定位。以猫肾细胞cDNA为模板克隆fC11orf96基因,并利用无缝重组连接成功构建重组质粒pET-32a(+)-fC11orf96-Fe。随后将重组质粒转化至BL21(DE3)感受态细胞,经IPTG诱导后成功表达fC11orf96-Fe重组蛋白,并利用该重组蛋白制备多克隆抗体。最后利用Western blot及间接免疫荧光试验分析多克隆抗体的有效性及其细胞定位。结果表明,成功获得猫C11orf96基因CDS序列,全长372 bp,可编码124个氨基酸,表达的fC11orf96-Fe蛋白主要以包涵体形式存在,蛋白大小约49 ku。由该重组蛋白制备的多克隆抗体能够识别细胞内源性fC11orf96蛋白和外源真核表达蛋白,并且发现fC11orf96蛋白定位于细胞质。本研究成功克隆得到猫C11orf96基因,并且fC11orf96蛋白定位于细胞质,为后续研究C11orf96的生物学功能奠定了基础。
The aim of this study was to construct of Felis catus C11orf96(fC11orf96)gene,prepare polyclonal antibody with good reactivity to the protein and in order to analyze the localization of the C11orf96 gene in cells.Firstly,the fC11orf96 gene cloned by using Crandell Reese Feline Kidney cells(CRFK)cDNA as the template,the recombinant plasmid pET-32a(+)-fC11orf96-Fe was successfully constructed by seamless recombination.Then,the recombinant expression vector was transformed into BL21(DE3),and the recombinant protein fC11orf96-Fe was successfully expressed after IPTG induction.The fC11orf96-Fe protein was used to immunize rabbits to prepare polyclonal antibody.Finally,Western blot and indirect immunofluorescence assay(IFA)were used to analyze the validity and subcellular localization of polyclonal antibody.The results showed that the CDS sequence of the fC11orf96 gene was 372 bp,encoding 124 amino acids.The recombinant protein fC11orf96-Fe exists mainly in inclusion bodies,and the protein size was 49 ku.The polyclonal antibody prepared from the recombinant protein could identify endogenous fC11orf96 protein and eukaryotic expressed foreign protein.And we found that fC11orf96 protein was localized in the cytoplasm.In this study,the fC11orf96 gene was successfully cloned and localized in the cytoplasm,which laid a foundation for further research on the biological functions of the C11orf96 protein.
作者
杨洪早
朱杰
郭宏元
汤傲星
陈少宇
刘春草
张达
袁莉刚
刘光清
YANG Hongzao;ZHU Jie;GUO Hongyuan;TANG Aoxing;CHEN Shaoyu;LIU Chuncao;ZHANG Da;YUAN Ligang;LIU Guangqing(College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2022年第7期2390-2395,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金项目(32000109)
上海市扬帆计划项目(20YF1457700)。
作者简介
杨洪早(1992-),男,云南凤庆人,博士生,主要从事小动物病毒分子机制研究,E-mail:yhz03008@163.com;通信作者:袁莉刚,主要从事动物生殖生理学研究,E-mail:yuan2918@126.com;刘光清,主要从事病毒分子生物学研究,E-mail:liugq@shvri.ac.cn。
