人β防御素3与植物巴西甜蛋白基因密码子的优化及其在毕氏酵母中的表达  

Optimization mosaic gene of human β defensin 3 and plant des-pGlu1-Brazzein and its expression in Pichia pastoris

作　　者：暴元元[1] 赵青君[1] 徐婷婷[1] 陈锐博[1] 赵卫东[1] 郑振宇[1] 李春丽[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《河南农业大学学报》2013年第1期77-82,共6页Journal of Henan Agricultural University

基　　金：教育部科学技术研究重点项目(207069);河南省自然科学基金项目(04110303000)

摘　　要：根据人β防御素3和植物甜蛋白des-pGlu1-Brazzein的氨基酸序列,按照酵母密码子的偏爱性,合成了14段末端具有粘合位点的核苷酸序列,经连接、PCR扩增,使人β防御素3和植物甜蛋白des-pGlu1-Brazzein的编码序列通过1段序列(含凝血酶切割位点)连接成为嵌合基因,将其插入到pPIC9K载体中,构建重组表达载体pPIC9K-hBD3-Bra.SalⅠ,BglⅡ单酶切后,分别电击转化到毕赤酵母GS115中,构建野生型和乙醇氧化酶缺陷型酵母工程菌株GS115-pPIC9K-hBD3-Bra.筛选鉴定后,以体积分数为0.5%的甲醇进行诱导,缺陷型工程菌株表达的目的蛋白约占上清蛋白的90%,纯度较高,野生型工程菌株表达的目的蛋白约占上清蛋白的57.8%.According to the sequences of human β defensin 3 and plant sweet-tasting protein despGlul-Brazzein, fourteen pairs of oligoneucleotide with cosmid site were synthesized using yeasty biased codons. After linkage and PCR, the DNA sequence of human β defensin 3 and plant des-pGlu1- Brazzein were incorporated together with the sequences coding thrombin cleavage site between them. This recombinant gene sequence was inserted into pPIC9K which resulted in recombinant expression vector pPIC9K-hBD3-Bra. Linearized by Sal Ⅰ or Bgl Ⅱ ,the vectors were transformed into Pichia pastoris strain GS115 by electro shock to produce wild type and alcohol oxidase-defective expression strain respectively. Identified by G418 and PCR, the strain was induced by 0.5% methanol. The results showed that the target protein accounted for 57.8% and 90% of total protein in the supernatant for wild type and alcohol oxidase-defective strain respectively.

关 键 词：防御素 甜蛋白 基因合成 克隆表达 活性检测 

分 类 号：Q81[生物学—生物工程]