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目的:构建一种新型表达载体(pEDCC),表达并纯化得到人源胰岛素原C肽。方法:将编码截短的门冬酰胺酶突变体(ansB-C),天然C肽,人IgG1铰链区(hinge),额外的酸敏感二肽(DP)以及富含碱性氨基酸的8肽(KRKRKKSR)的核苷酸序列依次分别插入pET28a载体中,构建表达载体pEDCC。在乳糖的诱导下,融合蛋白ansB-C-hinge-DPKRKRKKSRNGS-GR-C-peptide以包涵体形式高效表达。融合蛋白经洗涤和乙醇分级沉淀纯化后,通过酸水解将PKRKRKKSRNGSGR-C-peptide释放出来。C肽N端14肽用胰蛋白酶切割,通过DE52柱与C-peptide分离。结果:构建的表达载体pEDCC序列正确,融合蛋白经分离纯化得到了高纯度的重组人源胰岛素原C肽。结论:以截短的门冬酰胺酶作为融合伙伴,并以富含碱性氨基酸的8肽调节等电点是一种生产重组人源胰岛素原C肽的有效方法。
Objective: To construct a novel expression vector (pEDCC) expressing and purifying human proinsulin C peptide. Methods: Truncated asparaginase mutants (ansB-C), native C-peptide, human IgGl hinge, additional acid sensitive dipeptide (DP), and basic amino acid rich 8 peptide KRKRKKSR) were inserted into the pET28a vector in turn to construct the expression vector pEDCC. Under the induction of lactose, the fusion protein ansB-C-hinge-DPKRKRKKSRNGS-GR-C-peptide was highly expressed in inclusion bodies. After purification of the fusion protein by washing and ethanol fractionation, PKRKRKKSRNGSGR-C-peptide is released by acid hydrolysis. The C-peptide N-terminal 14 peptide is trypsinized and separated from the C-peptide by a DE52 column. Results: The constructed expression vector pEDCC was correct. The fusion protein was isolated and purified to obtain highly purified recombinant human proinsulin C peptide. CONCLUSION: The use of truncated asparaginase as a fusion partner and the regulation of the isoelectric point with the 8-peptide, which is rich in basic amino acids, is an efficient method for the production of recombinant human proinsulin C peptide.