Hepcidin的基因克隆及其在毕赤酵母中的分泌表达

Cloning and Secretion Expression of Hepcidin in Pichia pastoris

摘要:

根据已知hepcidin氨基酸序列，参照毕赤氏巴斯德酵母( Pichia pastoris) 密码子偏好性，设计合成了hepcidin目的基因。所合成的hepcidin基因全长96bp，其5′ 端引入KEX2基因产物（Kex2）的特异性识别位点序列，以保证表达产物具有天然N端。通过基因重组的方法将hepcidin基因克隆到pPicZαA 载体中，构建了分泌型重组酵母表达载体pPICZαA_Hepc，经电转至毕赤酵母GS115中表达。使用浓度高达1500 μg/mL的Zeocin 筛选得到高拷贝插入GS115菌株，经摇瓶发酵和甲醇诱导，上清液有明显的hepcidin表达，表达量达到100 mg/L。初步抗菌特性研究表明,该表达产物对枯草芽孢杆菌有明显的抑菌作用，而对大肠杆菌抑菌效果不明显。

Abstract:

Hepcidin is a liver_expressed, small cysteine rich peptide that acts as a regulator of systemic iron homeostasis. In this work, according to the partiality codon of Pichia pastoris, a DNA fragment containing the coding sequence of hepcidin was designed and synthesized, especially a Kex2 signal cleavage site was fused in 5′end of the antibacterial peptide genes. Then the modified hepcidin gene was inserted into the Pichia pastoris expression vector plasmid pPICZα_A. After electroporation of the resulting vector, pPICZα_A_Hepc, into the yeast host strain GS115, transformants with high copy inserts were selected by 1500mg/L Zeocin selection. Under the control of the promoter AOX1 (alcohol oxidase 1), recombinant hepcidin secreted from P. pastoris had a molecular weight of 2.7kD. After optimization of the flask_shaking culture fermentation, the yield of hepcidin reached 100mg/L in the clarified broth. Through antibacterial assay, the recombinant hepcidin displayed obvious antibacterial activity against Bacillus subtilis. But it could not distinctly inhibit the growth of E. coli BL21(DE3).