作　　者： ; ; ;

机构地区： 暨南大学生命科学技术学院生物工程研究所

出　　处： 《中国生物工程杂志》 2009年第6期1-6,共6页

摘　　要： 为研究利用基因重组方法生产人胰高血糖素样肽-1(GLP-1)衍生多肽的最佳表达及纯化条件,选用大肠杆菌偏爱密码子,以含人GLP-1的质粒为模板,用PCR方法合成全长人GLP-1衍生多肽基因,并定向插入到高效表达载体pMFH中,用大肠杆菌BL21进行表达,融合蛋白经Ni-NTA柱纯化后,用C18Sep-Pak反相柱脱盐,然后融合蛋白经甲酸水解,水解产物经Ni-NTA柱和高效液相色谱(HPLC)纯化制备后,目的肽由质谱鉴定。实验结果表明:利用载体pMFH在BL21中,GLP-1衍生物的最佳诱导表达温度为37℃、诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)的最佳浓度为0.6mmol/L,最佳诱导表达时间为6h;HPLC分析和制备GLP-1衍生物最佳条件为:流动相A(10%CNCH3∶90%H2O,0.1%TFA),流动相B(100%CNCH3,0.1%TFA),流速1ml/min,30min线性梯度洗脱,B相至70%,检测波长280nm;质谱鉴定GLP-1衍生物的分子量为5.492kDa,与理论值相符合。在最佳表达及纯化条件下可得GLP-1衍生多肽的产量可达到11.6mg/L发酵产物,纯度≥98%。 In order to study the optimal expression and purification condition of the GLP-1 derived polypeptide, by PCR technology synthetizing the gene of the GLP-1 derived polypeptide with preference codon of E. coli with the plasmid containing human wild-type GLP-1 gene. Expressed fusion proteins were purified and desalted with Ni-NTA column and C18 Sep-Pak column, respectively. After chemical cleavaged by formic acid hydroformicant, the hydrolysis products were purified with Ni-NTA column and HPLC. The target peptide was identified by mass spectrum. Experiment results showed in E. coli BL21 the optimal expression condition as follow： inducing temperature is 37℃, inducing time is 6h, and the concentration of the IPTG is 0. 6mmol/L. The optimal chromatographic condition of getting HPLC as follow： mobile phase A （ 10% CNCH3： 90% H2O ,0.1% TFA）, mobile phase B （ 100% CNCH3 ,0. 1% TFA） ,flow rate is l ml/min, 30 rain of the linear gradient elution, B phase reaches 70% and the detection wave length is 280nm. The molecular mass of the GLP-1 derived polypeptide is 5.492 kDa, through mass spectrum identification. The yield of GLP-1 derived polypeptide prepared with the optimal expression and purification condition may reach 11.6mg/L fermentation product and its purity is equal or greater than 98%.

关 键 词： 基因重组 胰高血糖素样肽 衍生多肽 高效液相色谱 质谱

领　　域： [生物学]