鸡氨肽酶N的高效可溶性表达及生物学功能分析

Expression and biological function analysis of chicken aminopeptidase N

摘要:

本研究旨为克隆鸡氨肽酶N(chAPN) 基因，高效表达可溶性目的蛋白，并测定其生物学功能。应用RT-PCR方法从鸡胚肾细胞中克隆chAPN的基因片段，经测序鉴定后再克隆至原核表达载体pCOLD-TF，构建重组原核表达质粒pCOLD-TF-chAPN，在大肠杆菌BL21(DE3) 中经不同条件诱导表达目的蛋白；利用镍柱亲和层析法纯化可溶性蛋白，并进行SDS-PAGE、Western blotting鉴定；Leu-PNA酶促反应和ELISA等方法检测目的蛋白生物学功能。结果显示，重组质粒pCOLD-TF-chAPN在大肠杆菌中以可溶形式高效表达；酶促反应及ELISA结果显示该蛋白具有酶活性，可结合传染性支气管炎病毒(IBV)，并表现为剂量依赖性。这为今后研究chAPN的酶活性、作为IBV受体及抗病毒功能奠定了实验基础。

Abstract:

To clone and express the gene encoding chicken aminopeptidase N (chAPN), and analysis the biological function of chAPN expressed in Escherichia coli (E. coli). The chAPN gene was amplified by RT-PCR from the kidney cells of chicken embryo and then cloned into the prokaryotic expression vector pCOLD-TF. Recombinant expression plasmid of pCOLD-TF-chAPN was constructed and then transformed into the competent E. coli BL21(DE3) cells for expression under different conditions such as induction time and inductor concentrations. Purified soluble recombinant chAPN was obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blotting assay. Its biological function was detected by its reaction with Leu-PNA and Enzyme-Linked Immunosorbent Assay (ELISA). The results showed that the expression product of chAPN gene in E. coli was soluble. It was able to bind infectious bronchitis virus (IBV) dose-dependently. In conclusion, chAPN gene has been successfully cloned and expressed in E. coli, which will establish a basis for further research the enzymatic activity and antiviral function.