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采用PCR扩增hVEGF165基因 ,经DNA序列分析后 ,插入含AOX1启动子和α分泌信号肽序列的Pichi apastoris酵母表达载体中 ,构建重组质粒 pPIC9K/hVEGF165,经转化酵母宿主菌KM 71,筛选多拷贝整合转化子 ,摇瓶培养 ,1%甲醇诱导表达。表达产物经Heparin SepharoseCL6B亲和层析纯化后 ,以人脐静脉内皮细胞(HUVEC)测定其生物学活性。SDS PAGE分析显示 ,表达产物以可溶性分子形式存在于上清中 ,诱导 4天的表达量达上清总蛋白质的 6 0 %以上。Western印迹表明 ,表达产物具有良好的抗原性和特异性。经Heparin SepharoseCL6B亲和层析纯化 ,rhVEGF165的纯度可达到 90 %以上。体内、外生物学活性研究证实其具有刺激HUVEC增殖和促进肢体缺血性动物模型侧枝循环建立的功能
The hVEGF165 gene was amplified by PCR. After DNA sequence analysis, the Pichia pastoris yeast expression vector containing the AOX1 promoter and α secretion signal peptide sequence was inserted into the recombinant plasmid pPIC9K / hVEGF165. The transformed yeast host strain KM 71 was transformed into multiple copies Transformants were integrated, shake flask culture, 1% methanol induced expression. The expressed product was purified by Heparin Sepharose CL6B affinity chromatography and its biological activity was determined by human umbilical vein endothelial cells (HUVEC). SDS PAGE analysis showed that the expressed product was soluble in the supernatant in the form of soluble molecules, and the expression level reached more than 60% of the total protein in the supernatant for 4 days. Western blot showed that the expressed product has good antigenicity and specificity. Purified by Heparin Sepharose CL6B affinity chromatography, the purity of rhVEGF165 can reach more than 90%. In vivo and in vitro biological activity studies have shown that it has the function of stimulating HUVEC proliferation and promoting collateral circulation in limb ischemic animal models