人血管内皮生长因子165克隆表达及其生物学功能的研究

发布时间：2018-06-02 01:43

本文选题：血管内皮生长因子 + 克隆　；参考：《大连医科大学》2005年硕士论文

【摘要】：目的:构建人血管内皮生长因子 165(Vascular endothelial growthfactor 165,VEGF165)原核表达载体并在大肠杆菌中诱导表达。构建VEGF165 真核表达载体并将其转入人慢性髓系白血病急变细胞株K562 中,研究其对 K562 增殖和凋亡的影响,进一步验证 VEGF165通过自分泌途径对肿瘤生长发挥作用,从而为临床上恶性血液病抗血管新生治疗的部分机理提供实验依据。方法:用 RT-PCR 法从人白血病细胞株 TF1 扩增 VEGF165 完整编码序列(不包含前面 81bp 的信号肽序列及终止密码子),将其插入质粒pUCmT中并测序鉴定。将测序正确的pUCmT-VEGF165与PET20b(+)均用 EcoRI 和 SalI 双酶切,回收纯化目的基因片段与表达载体,构建VEGF165 的原核表达载体 PET20b-VEGF165, 转化大肠杆菌BL21(DE3)pLysS,IPTG 诱导表达,表达产物用 Ni-NTA Resin 纯化,SDS-PAGE 及 Western Blot 鉴定重组蛋白。用 RT-PCR 法从人白血病细胞株 HL60 扩增 VEGF165 完整编码序列, 将其插入质粒 pUCmT 中并测序鉴定。将测序正确的 pUCmT-VEGF165 与 pcDNA3.1(-)均用 EcoR I和 HindⅢ双酶切,回收纯化目的基因片段与表达载体,构建 VEGF165的真核表达载体 pcDNA3.1-VEGF165。用脂质体介导的方法转染人慢性髓系白血病急变细胞株 K562,G418 筛选转染阳性的细胞株。RT-PCR和Western Blot检测VEGF165在RNA水平和蛋白水平的表达。用MTT
[Abstract]:Objective: to construct the prokaryotic expression vector of human vascular endothelial growth factor 165 (Vascular endothelial growthfactor 165, VEGF165) and to induce expression in Escherichia coli. The VEGF165 eukaryotic expression vector was constructed and transferred into the human chronic myeloid leukemia acute cell strain K562 to study the effect of the eukaryotic expression vector on the proliferation and apoptosis of K562 and further verify VE. GF165 can play a role in tumor growth through autocrine pathway, thus providing experimental evidence for the clinical mechanism of anti angiogenesis therapy for hematological malignancies.
Methods: RT-PCR method was used to amplify the complete coding sequence of VEGF165 from human leukemia cell line TF1 (not including the sequence of signal peptide and terminating codon of the preceding 81bp) and insert it into plasmid pUCmT and sequenced and identified. The correct pUCmT-VEGF165 and PET20b (+) were sequenced with EcoRI and SalI double enzyme, and the purified target gene fragment and expression were recovered. Vector, the prokaryotic expression vector PET20b-VEGF165 of VEGF165 was constructed, the expression of Escherichia coli BL21 (DE3) pLysS, IPTG was induced, the expression products were purified with Ni-NTA Resin, SDS-PAGE and Western Blot were used to identify the recombinant protein. The whole coding sequence was amplified by RT-PCR method and inserted into the plasmid. PUCmT and sequencing identification. The correct pUCmT-VEGF165 and pcDNA3.1 (-) were sequenced with EcoR I and Hind III double enzyme, the purified target gene fragment and expression vector were purified, and the eukaryotic expression vector of VEGF165 was constructed by liposome mediated transfection of K562 in human chronic myeloid leukemia rapid cell line, and G418 screening turned. .RT-PCR and Western Blot were used to detect the expression of VEGF165 at RNA level and protein level. MTT was used.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341

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