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目的 :构建p185 HER -2 蛋白胞外区的原核表达载体 ,实现p185 HER -2 蛋白胞外区的表达。以纯化p185 HER -2 蛋白胞外区为靶 ,从噬菌体肽库中筛选其结合肽 ,用于肿瘤导向治疗。方法 :从含有HER 2全长cDNA的质粒psv2 -cerbB -2 中克隆了人p185 HER -2 蛋白胞外区cDNA ,将此cDNA克隆到pET 30a +表达载体中 ,构建p185蛋白胞外区的表达载体。表达的p185 HER -2 蛋白胞外区通过Zn2 + 螯合层析柱进行纯化。以纯化的p185 HER -2 蛋白胞外区为靶 ,通过淘洗方法进行噬菌体肽库筛选。结果与结论 :构建了p185 HER -2 蛋白胞外区的原核表达载体 ,实现了p185 HER -2蛋白胞外区的高表达。通过Zn2 + 螯合层析柱实现一步纯化 ,p185 HER -2 蛋白胞外区的纯度达到 95 %。从噬菌体随机环七肽库中找到了一组具有核心序列p185 HER -2 蛋白胞外区结合肽。这些p185 HER -2 蛋白胞外区结合肽有可能成为肿瘤治疗的新的候选分子。
OBJECTIVE: To construct a prokaryotic expression vector for the extracellular domain of p185 HER-2 protein and to express the extracellular domain of p185 HER-2 protein. Purified p185 HER-2 protein extracellular domain as a target phage peptide library screening of its binding peptide for tumor-oriented treatment. METHODS: The extracellular domain of human p185 HER-2 protein was cloned from the psv2 -cerbB-2 plasmid containing HER2 full-length cDNA. The cDNA was cloned into pET30a + expression vector to construct the extracellular region of p185 protein Vector. The expressed extracellular region of p185 HER-2 protein was purified by Zn2 + chelation chromatography. The purified p185 HER-2 protein extracellular domain was used as a target and the phage peptide library was screened by panning method. RESULTS AND CONCLUSION: The prokaryotic expression vector p185 HER-2 was constructed and the overexpression of p185 HER-2 protein was achieved. One-step purification by Zn2 + chelation chromatography resulted in a purity of 95% in the extracellular region of the p185 HER-2 protein. A set of extracellular domain binding peptides with the core sequence p185 HER-2 protein was found from the phage randomized heptapeptide library. These p185 HER-2 extracellular domain binding peptides are likely to become new candidates for cancer therapy.