CAJ | 학술논문

对原核表达载体pET3c进行改建，消去载体上非必需的EcoRⅠ、HindⅢ限制酶位点，然后在克隆位点BamHⅠ处引入LacZ片段，经此二步改建后的载体命名为pJN982。该载体保留了pET3c高效表达外源基因的能力，同时，其融合克隆位点由1个增至7个，并获得以目视法筛选重组子的能力。应用该载体在E.coliBL21(DE3)pLysS中融合表达牛碱性成纤维细胞生长因子，表达量占菌体总蛋白30.04%。破碎菌体上清经阳离子交换和肝素亲和两步层析，得纯度为95%的重组蛋白，活性检测显示，重组蛋白具有与参照品一致的促有丝分裂活性。
대원핵표체재체pET3c진행개건，소거재체상비필수적EcoRⅠ、HindⅢ한제매위점，연후재극륭위점BamHⅠ처인입LacZ편단，경차이보개건후적재체명명위pJN982。해재체보류료pET3c고효표체외원기인적능력，동시，기융합극륭위점유1개증지7개，병획득이목시법사선중조자적능력。응용해재체재E.coliBL21(DE3)pLysS중융합표체우감성성섬유세포생장인자，표체량점균체총단백30.04%。파쇄균체상청경양리자교환화간소친화량보층석，득순도위95%적중조단백，활성검측현시，중조단백구유여삼조품일치적촉유사분렬활성。
The prokaryote expression vector pET3c was modified by destroyingthe EcoRⅠ,HindⅢ restriction enzyme sites and inserting LacZ fragment in the BamH Ⅰ site .The derivative, named as pJN982,not only retained the ability of pET3 c to express exogenous gene efficiently but also had an increased number of fus ion cloning sites from 1 to 7,as well as gained the ability to identity the rec ombinant by visual screening of E.coli colonies .Using pJN982,fused bovine basic fibroblast growth factor(B-bFGF) was expressed in E.coli BL21(DE3)pLysS to a level of 30.04% of total celluar protein . The target protein was purified by cation exchange and heparin affinity chromatography from the supernant of bacteria lysate .It was found that the recombinant B-bFGF had an identical bio-activit y with the standard bFGF.