三种呈味肽串联基因的构建和表达

通过对呈味肽GD、AD、VD结构的分析,引入甲酸和胰蛋白酶的专一性位点,设计了呈味肽的串联单体。根据大肠杆菌偏嗜密码子设计并合成寡核苷酸片段,利用拼接法合成此三种肽的8拷贝片段,将其克隆至表达载体pET-30a并转化到宿主菌BL21（DE3）中。筛选的阳性克隆经测序表明,本研究已成功构建了重组呈味肽工程菌BL21-pET30-8GD、BL21-pET30-8AD、BL21-pET30-8VD。IPTG诱导的菌体总蛋白和破碎液上清的纯化物经Tricine-SDS-PAGE电泳检测,证明目的基因得以成功表达。 

Construction and expression of three taste peptide tandem genes

According to the analysis of the structure of GD, AD and VD taste peptide, the tandem monomers were designed by introducing the specific sites for formic acid and trypsin.Oligonucleotides were designed and synthesized based on the code usage of Escherichia Coli and then 8-copy genes were structured by spliced, the corresponding genes were cloned into pET-30a which were finally transformed into BL21 (DE3) .The recombinants BL21-pET30-8GD, BL21-pET30-8AD, BL21-pET30-8VD were confirmed by positive clone screening and DNA sequencing.Expression of target genes was confirmed by modified Tricine-SDS-PAGE electrophoresis of engineering bacteria total protein and purified protein from supernatant of lysate.