作　　者： ; ; ; ; ; ; ; ;

机构地区： 暨南大学生命科学技术学院

出　　处： 《中国公共卫生》 2007年第12期1483-1485,共3页

摘　　要： 目的研究利用基因重组方法生产人胰高血糖素样肽-1(GLP-1)衍生物的最佳表达及纯化条件。方法选用大肠埃希菌偏爱密码子,以含人GLP-1的质粒为模板,用重叠PCR方法合成全长人GLP-1衍生物基因,并定向插入到高效表达载体pMFH中,用大肠埃希菌BL21进行表达,融合蛋白经Ni-NTA柱纯化后,用C18 Sep-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。结果利用载体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0.6 mmol/L,最佳诱导表达时间为6 h;HPLC分析和制备GLP-1衍生物最佳洗脱条件为:30 min线性梯度洗脱,流动相A(100%乙腈)由10%至70%,流动相B(100%水,0.1%三氟乙酸)由90%至30%;流速1 ml/min;检测波长280 nm;质谱鉴定GLP-1衍生物的分子量为5548Da,与理论值相符合。结论在最佳表达及纯化条件下可得到高产量和高纯度(>98%)的GLP-1衍生物。 Objective To study the optimal expression and purification condition of the derivate of GLP - 1. Methods By PCR technology synthetizing the gene of the derivate of GLP - 1 with preference codon of E. coli and the plasmid containing GLP - 1. 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 and prepared with Ni - NTA column and HPLC. The target peptide was identified by mass spectrum. Results In BL21 the optimal expression condition as follow：inducing temperature was 37℃, inducing time was 6 h, and the concentration of the IPTG was 0.6 mmol/L. The optimal chromatographic condition of getting HPLC as follow： 30 min of the linear gradient elution, from 10 % to 70 % for mobile phase A （100% CNCH3）, from 90% to 30% for mobile phase B（100% H2O, 0.1% TFA）, flow rate was 1 ml/min, and the detection wave length was 280nm. The molecular mass of the derivate of GLP - 1 was 5 548 Da through mass spectrum identification. Conclusion With the optimal expression and purification condition, the high yield and high purity（ 〉 98 % ）derivate of GLP - 1 can be obtained.

关 键 词： 基因重组 胰高血糖素样肽 衍生物 高效液相色谱 质谱

领　　域： [生物学]