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

作　　者：张卉[1] 袁其朋[1] 

机构地区：[1]北京化工大学北京生物加工过程重点实验室,北京100029

出　　处：《生物工程学报》2007年第3期381-385,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金资助项目(No.20576010)资助。~~

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1500μg/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100mg/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。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 pustoris expression vector plasmid pPICZa-A. After electroporation of the resulting vector, pPICZa-A-Hepc, into the yeast host strain GSll5, 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）.

关 键 词：HEPCIDIN 毕赤酵母 分泌表达 抗菌活性 

分 类 号：R346[医药卫生—基础医学]