日本鳗鲡绿色荧光蛋白基因的克隆与表达

Cloning and expression of green fluorescent protein gene from eel muscle

[目的]克隆日本鳗鲡绿色荧光蛋白UnaG基因,表达并纯化UnaG蛋白。[方法]从日本鳗鲡中克隆了绿色荧光UnaG基因,并克隆至p ET28Aa、pc DNA-Flag质粒中。将重组质粒pc DNA-Flag用脂质体转染到293T细胞中,用荧光显微镜观察绿色荧光,同时用原核表达系统诱导His-UnaG融合蛋白的表达,用亲和层析和凝胶过滤层析纯化HisUnaGg融合蛋白。[结果]转染人293T细胞后不到24 h,荧光显微镜观察到日本鳗鲡UnaG能被激发出较强的绿色荧光;原核表达并纯化的His-UnaG融合蛋白纯度很高,Western Blot检测为单一条带,蛋白量达3μg/μl。[结论]成功克隆并纯化了UnaG,为进一步研究UnaG的应用奠定基础。

[ Objective ] To clone UnaG gene from eel muscle and express in eukaryotic cells. [ Methods ] UnaG nag gene of green fluorescent protein was cloned from Japanese eel muscle. After constructed to pET28a and pcDNA - Flag vectors, transfected vectors of pcDNA-flag-UnaG to 293T cell line with Lipo 2000. Green fluorescence was observed with fluorescent microscope. His-UnaG fusion protein was expressed using prokaryotic expression system, and purified with affinity chromatography by His beads and gel filtration chromatography. [ Results ] After transfected pcDNA-flag-UnaG for 24h, stronger green fluorescence was excitated from 293T of expression UnaG. Meanwhile His-UnaG fusion protein of prokaryotic expression was very well by Western Blot detected, and purified mono protein band. Protein content of His-Unag reach to 3 μg/μl.[ Conclusion ] UnaG gene was cloned and UnaG protein was purified , which provides a foundation for further investigation of UnaG applications.