人鼻病毒3C蛋白酶基因的克隆、表达、纯化及活性

Gene Cloning,Expression,Purification and Activity of Human Rhinovirus 3C Protease

目的获得重组3C蛋白酶,开发一种新型的基因工程工具酶。方法通过RT-PCR方法获得人鼻病毒3C蛋白酶基因,克隆入表达载体pBV220,构建非融合表达载体pBV220-3C,转化大肠杆菌BL21(Gold)进行表达。表达产物经变性阳离子交换层析纯化后复性,再经Superdex-75凝胶过滤进一步纯化,获得的3C蛋白酶在4℃条件下,酶切融合蛋白IL-11,进行活性测定。结果3C蛋白酶在大肠杆菌中获得了稳定、高效表达,表达量约占菌体总蛋白的25%,以包涵体形式存在。经过纯化、复性,纯度达90%以上,获得的3C蛋白酶能够有效切割含3C酶切位点的融合蛋白。结论已成功开发了一种新型的基因工程工具酶。

Objective To obtain human rhinovirus 3C protease as a novel tool enzyme for gene engineering. Methods Amplify the gene encoding human rhinovirus 3C protease and clone into expression vector pBV220. Transform the constructed recombinant plasmid pBV220-3C to E. coll BI21 （Gold） for expression. The expressed product was primarily purified by CM-Sepharose FF cationexchange chromatography,then refolded and further purified by Superdex-75 gel filtration. The purified 3C protease was determined for activity by digestion of fusion protein IL-11 at 4℃. Results Human rhinovirus 3C protease was stably and highly expressed in E. coli. The expressed product,in a form of inclusion body, contained about 25 % of total somatic protein and reached a purity of more than 90% after purification and refolding. The obtained 3C protease was effective in digestion of fusion protein IL-11. Conclusion A novel tool enzyme for gene engineering was successfully developed.