鸡Mx基因原核表达载体的构建及其表达分析

Prokaryotic Expression Vector Construction and Expression Analysis of Chicken Mx Gene

旨在大肠杆菌中表达鸡抗病毒Mx蛋白,为进一步研究该基因抗病活性奠定基础。本研究通过设计一对特异性引物,从已构建好的鸡Mx基因真核表达载体pcDNA3.0-MMx上扩增出Mx基因开放式阅读框(2118bp),并将其编码区重组于原核表达载体PGEX-6P-1中,经酶切和序列鉴定分析后,重组质粒转化大肠杆菌BL21(DE3)和Rossetta-gami(DE3),并比较其表达效率。结果显示,经不同浓度IPTG诱导后,该基因在Rossetta-gami(DE3)中获得表达。SDS-PAGE检测结果显示,蛋白大小与预期结果相符,说明重组质粒pGEX-Mx在大肠杆菌中成功诱导表达出鸡Mx蛋白,为下一步鸡Mx蛋白活性检测及抗体制备奠定基础。

This study expressed the Mx antiviral proteins of chicken in E.coli,in order to lay the foundation for further study of the gene for resistance activity. A pair of specific primers were designed,the open reading frame of Mx （2 118 bp） gene was amplified from eukaryotic expression vector of pcDNA3.0-MMx of Mx gene which have been constructed from chicken,then the coding region of Mx gene was restructured in the prokaryotic expression vector of pGEX-6P-1. After restriction enzyme digestion and sequence analysis,recombinant plasmid was transformed into E.coli BL21 （DE3）and Rossetta-gami （DE3）,and the efficiency of its expression was compared. The results showed that Mx gene was expressed successfully in Rossetta-gami （DE3）which was induced by different concentrations of IPTG. After the detection by SDS- PAGE,it was found that protein size was corresponded with the expected results which illustrated the chicken Mx protein was successfully induced and expressed by the recombinant plasmid pGEX-Mx in E.coli. It was useful-lo further activity of detection of chicken Mx protein and antibody＇s preparation.