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血管内皮细胞生长抑制因子抑制肿瘤部位新生血管的形成 ,切断肿瘤细胞营养供应及废物排泄通道 ,抑制肿瘤细胞恶性增殖。将编码成熟的人血管内皮细胞生长抑制因子基因克隆到表达载体pPICZα中 ,电转化P .pasto risGS115菌株 ,抗生素ZeocinTM 浓度梯度筛选高抗性转化子 ,PCR筛选阳性重组菌株。经表型鉴定后 ,用甲醇进行诱导表达 ,SDS PAGE和Western印迹杂交结果证实了表达产物为重组人血管内皮细胞生长抑制因子 his6融合蛋白 ,表达量约为 5mg L。经细胞毒性试验测定 ,表达产物对人脐静脉内皮细胞HUVEC增殖具有较明显的抑制作用。
Vascular endothelial growth inhibitory factor (VEGF) inhibits the formation of neovasculature at the tumor site, cuts off the nutrient supply of tumor cells and the waste excretion channel, and inhibits the malignant proliferation of tumor cells. The mature human vascular endothelial cell growth inhibitory factor gene was cloned into the expression vector pPICZα and electroporated into P. pastosa risGS115 strain. The antibiotic ZeocinTM concentration gradient was screened for high resistant transformants, and the positive recombinant strains were screened by PCR. After phenotypic identification, the expression was induced by methanol. The result of SDS PAGE and Western blot confirmed that the expressed product was recombinant human vascular endothelial cell growth inhibitory factor his6 fusion protein with an expression level of 5 mg L -1. The cytotoxicity test showed that the expression product had a significant inhibitory effect on the proliferation of human umbilical vein endothelial cells HUVEC.