C19orf18蛋白的表达与纯化及多克隆抗体制备

doi:10.13560/j.cnki.biotech.bull.1985.2016-1031

生物技术通报 ›› 2017, Vol. 33 ›› Issue (5): 176-182.doi: 10.13560/j.cnki.biotech.bull.1985.2016-1031

C19orf18蛋白的表达与纯化及多克隆抗体制备

童霄霞¹, 杨远勤¹, 陈勉², 王怀远², 胡海军², 章康健^{2, 3}

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

收稿日期:2016-11-15 出版日期:2017-05-25 发布日期:2017-05-19
作者简介:童霄霞,女,硕士,研究方向：癌症的靶向基因-病毒治疗;E-mail：xiaoxia_tong@163.com
基金资助:
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）

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

TONG Xiao-Xia¹, YANG Yuan-Qin¹, CHEN Mian², WANG Huai-Yuan², HU Hai-Jun², ZHANG Kang-Jian^{2, 3}

1. Xinyuan Institute of Medicine and Biotechnology,Zhejiang Sci-Tech University,Hangzhou 310018; 2. State Key Laboratory of Cell Biology,Shanghai Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200031; 3. Sichuan Huiyang Life Science and Technology Corp,Chengdu 610021

Received:2016-11-15 Published:2017-05-25 Online:2017-05-19

摘要/Abstract

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pET28a（+）与pET32a（+）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pET28a-C19orf18 蛋白表达量高于pET32a-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。

关键词: C19orf18蛋白, 原核表达, 抗原亲和纯化

Abstract: 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 C19orf18 protein. More importantly,C19orf18 protein with truncated amino acids was expressed in prokaryotic cells,and its corresponding rabbit polyclonal antibody with high specificity was prepared successfully.

Key words: C19orf18 protein, prokaryotic expression, antigen affinity purification

童霄霞, 杨远勤, 陈勉, 王怀远, 胡海军, 章康健. C19orf18蛋白的表达与纯化及多克隆抗体制备[J]. 生物技术通报, 2017, 33(5): 176-182.

TONG Xiao-Xia, YANG Yuan-Qin, CHEN Mian, WANG Huai-Yuan, HU Hai-Jun, ZHANG Kang-Jian. Prokaryotic Expression,Purification and Preparation of Polyclonal Antibody of C19orf18 Protein[J]. Biotechnology Bulletin, 2017, 33(5): 176-182.

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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

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