诱生型一氧化氮合酶FAD结合区的克隆、表达和活性鉴定

Cloning and Expression of Inducible Nitric Oxide Synthase FAD-Binding Domain and Its Binding Activity to FAD

诱生型的一氧化氮合酶 (iNOS)的表达可以被多种细胞因子、IPS、多种试剂及创伤等诱导 ,很多疾病的产生发展都与iNOS的过度表达有关。为进一步研究iNOSFAD结合区的功能和特异性抑制肽的筛选 ,用PCR方法克隆出iNOSFAD结合区编码基因片段 (iNOS2 0 31 2 6 6 0bp) ,并在大肠杆菌中得到高效表达 ,得到表达率 >30 %的包涵体 ;经过Ni金属螯合亲和柱层析和电泳纯化 ,得到纯度 >95 %的重组蛋白 ,相对分子质量为2 3 0 0 0。运用波长扫描 ,证实纯化蛋白具有与FAD的结合活性 。

To study the function of iNOS FAD binding domain and to screen iNOS specific inhibiting peptides from phage peptide library, We have cloned the coding gene of iNOS FAD binding domain(iNOS2031 2660 bp) and expressed it effectively in E.coli. The recombinant protein in size 23 000u was purified (over 95% in purity) by His Tag sepharose column chromatography. Its FAD binding activity was detected with spectrum scan within 200nm and 700nm . Bovine serum albumin was used as a nagtive control. Since the specific sequence and effective binding activity.The protein could be used as an ideal target for screening iNOS specific peptides from the peptide library.