C19orf18蛋白的表达与纯化及多克隆抗体制备  

Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of C19orf18 Protein

作　　者：童霄霞 杨远勤[1] 陈勉[2] 王怀远[2] 胡海军[2] 章康健[2,3] 

机构地区：[1]浙江理工大学,杭州310018 [2]中国科学院上海生命科学研究院生化细胞所,上海200031 [3]四川辉阳生命工程股份有限公司,成都610021

出　　处：《生物技术通报》2017年第5期176-182,共7页Biotechnology Bulletin

基　　金：四川科技项目(2013ZZ0004);四川辉阳生命工程股份有限公司研究项目(Y363S21763)

摘　　要：旨在得到较高浓度和纯度的C19orf18蛋白,再将得到的目的蛋白用于制备其特异性的多克隆抗体。利用PCR技术扩增出C19orf18基因的胞内段和胞外段,中间用柔性肽连接,再将目的片段克隆到p ET28a(+)与p ET32a(+)载体上,通过诱导表达少量的目的蛋白,筛选出表达量较高的载体,再用筛选得到的表达量较高的载体大量表达目的蛋白,通过镍柱纯化得到较纯的C19orf18蛋白。用得到的蛋白免疫新西兰白兔,得到的抗血清先经过Protein A纯化,再进行抗原亲和纯化得到C19orf18蛋白多克隆抗体。结果显示,载体p ET28a-C19orf18蛋白表达量高于p ET32a-C19orf18,经过镍柱纯化后得到了较高浓度与纯度的目的蛋白,利用得到的目的蛋白作为抗原成功制备了其特异性的多克隆抗体。在原核细胞中成功表达了C19orf18重组蛋白,并得到了其特异性多克隆抗体,所制备的多克隆抗体可应用于ELISA、蛋白免疫印迹实验。The purpose of this study is to clone the human C19orf18 gene, construct the prokaryotic expression system of C19orf18, and prepare the recombinant human C19orf18 protein and its corresponding rabbit polyclonal antibody. According to the sequence reported in GenBank database, we used PCR to amplify intracellular and extracellular segments, and ligated them by flexible peptide, and inserted the target segment into prokaryotic expression vectors pET28a and pET32a. Then the vectors of high expression were selected by inducing a little expression of target protein. Further the highly expressing vector was employed for expressing the great amount of target protein. The C19orf18 protein was purified by Ni-NTA affinity column, and then identified by SDS-PAGE. Following that, New Zealand white rabbits were immunized with the purified proteins four times to produce polyclonal antibody. Specificity of the product polyclonal antibody was affirmed by Western blotting. Our results showed that pET28a was a better vector than pET32a for the expression of C 19orf18 protein. More importantly, C 19orfl 8 protein with truncated amino acids was expressed in prokaryotic cells, and its corresponding rabbit polyclonal antibody with high specificity was prepared successfully.

关 键 词：C19orf18蛋白 原核表达 抗原亲和纯化 

分 类 号：Q78[生物学—分子生物学]