【摘要】 目的 :构建蛋白质转导结构域 8个精氨酸聚合体 (8R)与 MUC1核心肽融合蛋白的原核表达载体 ,并表达、纯化出具有生物学活性的 8R- MUC1核心肽融合蛋白。方法 :以 PCR法从 HSP6 5 - MUC1- p ET2 8a质粒中钓取 MUC1核心肽 2个重复序列片段 ,连入 p MD18- T载体 ,然后用酶切、连接的方法从 T载体上获得 MU C1核心肽 6重复序列片段 (MU CPT) ,将其克隆入含 8R的 p ET2 6 b+ 原核表达载体中构建 8R与 MU CPT融合蛋白(8R- MU CPT)表达载体。用 IPTG诱导转化 8R- MU CPT表达载体的大肠杆菌 BL2 1(DE3) ,并以镍螯合层析法纯化 8R- MU CPT融合蛋白。结果 :构建了 8R与 MU C1核心肽 6个重复序列片段的融合蛋白原核表达载体8R- MUCPT- p ET2 6 b+ ,并在 BL2 1(DE3)中表达了 8R- MU CPT融合蛋白 ,经镍螯合层析获得纯度为 95 .5 %的8R- MUC1核心肽融合蛋白。结论 :构建了 8R- MU C1核心肽融合蛋白原核表达载体并成功表达与纯化出具有生物学活性的融合蛋白。更多还原

【Abstract】 Objective To construct the prokaryotic expression vector of the fusion protein composed of protein transduction domain-eight arginine (8R) and MUC1 core peptides, and to express and purify the 8R-MUC1 core peptide fusion protein which should possess biological activity. Methods Two tandem repeat sequence of MUC1 core peptides were gotten from HSP65-MUC1-pET28a plasmid by PCR and cloned into pMD18-T vector. The T vector was digested by endonuclease for releasing the fragment of two repeats of MUC1 core peptides which was used for constructing six tandem repeat sequence of MUC1 core peptides by ligation. The fragment of six repeats of MUC1 core peptides was cloned into pET26b + plasmid, which contained 8R, for constructing expression vector of the fusion protein composed of 8R and MUCPT (8R-MUCPT). The E.coli BL21(DE3) transformed with 8R-MUCPT-pET26b + were induced by IPTG for expression of 8R-MUCPT fusion protein. The protein was purified by Ni 2+ affinity chromatography. Results The prokaryotic expression vector, 8R-MUCPT-pET26b +, was constructed with 8R and six tandem repeat sequence of MUC1 core peptides. The fusion protein of 8R-MUCPT was expressed in BL21(DE3). The purity of the fusion protein was 95.5% after purified by Ni 2+ affinity chromatography. Conclusion The prokaryotic expression vector of 8R-MUC1 core peptide fusion protein has been constructed, and the fusion protein with biological activity has been successfully expressed and purified.更多还原