脑型一氧化氮合酶钙调蛋白结合区的克隆表达与活性鉴定

Recombinant Expression and Activity Assay of nNOS/CAM

应用PCR法扩增了脑型一氧化氮合酶 (nNOS)的钙调蛋白CaM结合区基因 ,将扩增出的 0 6kbDNA片段经NdeI/EcoRI酶切 ,重组于表达载体pET/2 8a( + ) 质粒中 ,用电穿孔法导入E Coli,筛选获得基因工程菌。在IPTG诱导下 ,目的蛋白在工程菌的表达量为菌体蛋白的 1 5 %～ 2 0 % ,应用His、Tagsepharose金属螯合亲和层析和SDS PAGE纯化 ,获得电泳纯的目的蛋白、相对分子质量为 2 2 0 0 0 ,以它为抗原 ,制备了免疫克隆抗血清 ,经纯化获得高度专一性的nNOSCaM结合区蛋白的抗体 ,实验证实所得抗体与nNOS抗原特异结合 ,与诱导型一氧化氮合酶 (iNOS)

Using the PCR technique, we obtained the DNA sequenc e of CaM binding domain from the nNOS, then insert 06 kb DNA fragment obtained f rom PCR into the PET system vectors PET/28 a+ plasmid, digested by NdeI/E coRI. Cloned and high revel expressed in Ecoli, after being induced by IPTG, the recombinant bacteria expressed an additional protein(binding with CaM in nNOS) which expression quantities is about 15%～20 % of total p rotein in bacteria, the recombinant protein with molecular weight 22 kD was pre p ared by the affinity chromatography with His-Tag sepherose and SDS-PAGE.The immuniged rabbits by pure recombinant protein could yield t he pol ycloned antibody which is high selective for the nNOS/CaM recombinant protein. The bioactive assay comformed that anti-nNOS/CaM antibody could well immuno-reacted with nNOS protein mark, without cross reaction with iNOS protein