8R-MUC1核心肽融合蛋白的原核表达及纯化

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HSP65-MUC1-pET28a质粒中钓取MUC1核心肽2个重复序列片段，连入pMD18-T载体，然后用酶切、连接的方法从T载体上获得MUC1核心肽6重复序列片段(MUCPT)，将其克隆入含8R的pET26b⁺原核表达载体中构建8R与MUCPT融合蛋白（8R-MUCPT）表达载体。用IPTG诱导转化8R-MUCPT表达载体的大肠杆菌BL21(DE3)，并以镍螯合层析法纯化8R-MUCPT融合蛋白。 结果：构建了8R与MUC1核心肽6个重复序列片段的融合蛋白原核表达载体8R-MUCPT-pET26b⁺，并在BL21(DE3)中表达了8R-MUCPT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-MUC1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。

Prokaryotic expression and purification of 8R-MUC1 core peptide fusion protein

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.