胰核糖核酸酶A的原核表达及纯化复性研究

Expression and Purification of Bovine Pancreatic RNase A

生化提取牛胰核糖核酸酶A工艺复杂、得率较低,而且动物源产品不能用于基因免疫质粒的生产。该工作采用原核表达路线解决上述问题。提取牛胰腺总RNA,利用RT-PCR扩增获得牛核糖核酸酶A基因,连入pGEM-T中测序验证。目的基因克隆到表达载体pET32a中,摇瓶培养得到带有Trx标签的融合蛋白表达产物,为包涵体表达形式。裂解上清经8mol/L尿素变性后采用镍柱纯化,在柱上用肠激酶切掉标签得到纯化的目的蛋白。目的蛋白经稀释复性后检测酶活。SDS-PAGE检测得到目的蛋白为Mr16k。经检测,复性纯化的核糖核酸酶A具有良好的RNA降解活性。

The procedure to isolate and purify calf pancreatic RNase A by biochemical methods is complicated, less efficient, and not suitable for gene plasmid production. This work will utilize the bacterial expression method to solve the above difficulties. Total RNA was isolated from bovine pancreas, cDNA encoding calf RNase A was amplified by RT-PCR, subcloned into pGEM-T, and verified by sequencing. The target gene was subcloned into the expression vector pET32a. After bacterial culture, the fusion protein with Trx tag was obtained. The supernatant from the lysate was denatured with 8mol/L urea, followed by purification through Ni-column. Purified target protein was obtained after the tag was digested with Enterokinase in the column. Purified target protein was then diluted and tested for enzymatic activity. A 16KD protein was purified and verified by SDS-PAGE electrophoresis. This RNase A displays an excellent enzymatic activity to degrade RNA.