甜味蛋白Brazzein基因酵母表达系统的建立  

Construction of Yeast Expression System of Sweet Protein Brazzein

作　　者：史勇[1] 张明军[1] 张英[1] 李莉[1] 汤丹[1] 刘松财[1] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062

出　　处：《吉林农业大学学报》2010年第1期43-46,共4页Journal of Jilin Agricultural University

基　　金：吉林省科技发展计划项目(20070574)

摘　　要：根据甜味蛋白Brazzein基因成熟区的氨基酸序列,结合毕赤酵母的密码子偏爱性以及Brazzein蛋白质结构的相关研究,对甜味蛋白Brazzein基因进行改造,使表达的目的蛋白中包含3个突变氨基酸(29Asp→Lys,31His→Ala,41Glu→Lys)。采用重叠PCR(SOE-PCR)合成目的基因并克隆到pMD18-T载体,经测序鉴定,序列正确的质粒用EcoRⅠ和XbaⅠ双酶切。将回收得到的目的基因连接到经同样双酶切处理的pGAPZαA载体上,经PCR、酶切和测序鉴定,证明已成功构建pGAPZαA-Bra真核表达载体。将构建好的重组载体pGAPZαA-Bra用AvrⅡ线性化,电转进入巴斯德毕赤酵母中。筛选ZeocinTM阳性克隆菌,进行蛋白表达,SDS-PAGE结果表明蛋白表达成功。According to amino acid sequences of the sweet protein Brazzein, the biased codons of Pichia pastoris yeast and the latest research advances about the protein structure and mechanism, the gene of Brazzein, which includes 3 sites of mutation（29Asp→Lys,31His→Ala,41Glu→Lys） , was reformed. The Brazzein geneamplified by SOE - PCR was inserted into the plasmid pMD18-T, then sequenced. The recombinant plasmid pMD18-T-Bra with the right sequence was digested by EcoRⅠ and Xba Ⅰ , the retained gene was connected to pGAPZaA which was also digested by Eco R Ⅰ and Xba Ⅰ . The recombinant plasmid pGAPZaA-Bra was obtained. The pGAPZaA-Bra was linearized by Avr Ⅱ , then introduced into Pichia pastoris yeast with the Gene Pulser System. The ZeocinTM positive strains were screened, then cultured in YPD medium. After 4 days, the protein was analyzed with SDS - PAGE.The results proved the expectation.

关 键 词：甜味蛋白 BRAZZEIN基因 pGAPZαA-Bra 巴斯德毕赤酵母 

分 类 号：Q786[生物学—分子生物学]