重组巴氏杆菌透明质酸合成酶的克隆、表达及活性鉴定

Molecular cloning，expression and identification of recombinant hyaluronan synthase from Pasteurella multocida

摘要: 目的：构建重组巴氏杆菌透明质酸合成酶(rPmHAS)原核表达系统，获取高纯度、有活性的rPmHAS。方法：利用大肠杆菌BL-21 pET32a(+)表达重组蛋白，镍亲和柱纯化，夹心竞争ELISA法检测酶活性。结果： Western blotting 检测显示特异性条带，重组蛋白正确表达，镍亲和柱纯化后得到重组蛋白的纯度可达90%。结论：成功构建了pET32a(+)/rPmHAS重组质粒并实现了目的蛋白高效可溶表达，为PmHAS性质相关研究及单一相对分子质量寡聚透明质酸的合成奠定了基础。

Abstract: Aim ：To set a suitable route for efficient expression and purification of recombinant hyaluronan synthase from Pasteurella multocida(rPmHAS). Methods ：Coding sequences were cloned and expressed in Escherichia coli strain of BL-21(DE3).Then rPmHAS was purified through a simple Ni-affinity chromatography and identified by hyaluronic acid binding protein (HABP) based ELISA assay. Results ：High yield expression of functional rPmHAS exceeding the highest production reported hitherto was achieved，and the purity of rPmHAS was as high as 90%. Conclusion ：In this study，an optimized system of the expression，purification and identification route for large scale preparation of rPmHAS was established，which will be helpful for accelerating further study of PmHAS and facilitating fine researches on peculiar hyaluronic acid with special properties.