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重组PACAP衍生物RBAY-2的高效制备及二肽基肽酶对其酶解研究 被引量:2

Study on Highly Efficient Preparation of Recombinant PACAP Derivate RBAY-2 and Its Enzymolysis by Dipeptidyl Deptidase
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摘要 为了利用基因工程技术高效制备具有治疗Ⅱ型糖尿病功能的垂体腺苷酸环化酶激活肽(PACAP)衍生物RBAY-2,研究二肽基肽酶Ⅳ(DPPⅣ)对其酶解效应,采用基因重组技术表达重组肽RBAY-2,纯化并鉴定目的肽RBAY-2,检测其生物活性;通过LC-MASS技术检测并绘制PACAP衍生物RBAY-2在体外模拟环境中的浓度-时间曲线,测定其体外模拟条件下的半衰期。实验结果表明:利用重组肽技术制备的RBAY-2的Mr为3.756k,纯度大于96%,其产率为18.3毫克/(每升发酵产物);制备的RBAY-2可与VPAC2受体特异结合并显著促进VPAC2-CHO细胞cAMP的产生,其受体半激活浓度(EC50)为0.93 nmol/L,目的肽RBAY-在体外可被DPPⅣ酶解,其半衰期为45.1 min。由此表明,利用本研究确立的表达、纯化策略可实现高活性RBAY-2的高效制备,应用LC-MASS技术研究药物肽体外酶解效应是一种方便、准确的检测方法,可用于药物肽稳定性的初步检测和高稳定性药物肽的体外筛选。 In order to efficiently prepare the pituitary adenylate cyclase activating polypeptide (PACAP) derivate RBAY-2 against typelldiabetes by genetic engineering technology and studying the enzymolysis of RBAY-2 by dipeptidyl peptidase Ⅳ (DPP Ⅳ) in vitro, genetic recombination technology was used to express RBAY-2. After purifing and identifing recombinant peptide RBAY-2, the amount of RBAY-2 at different times in simulated environment in vitro was assayed by LC-MASS method and the concentration-time curve for RBAY-2 in vitro was drawed. The experimental results show that the molecular weight of RBAY-2 is 3. 756k. , its purity is greater than 96% and its yield was 18. 3mg/L fermentation product. Prepared RBAY-2 could specifically active VPAC2 with a half-maximal stimulatory concentration (ECA0) of 0. 91nmol/L and significantly enhanced cAMP accumulation in CHO cells, . RBAY-2 can be hydrolyzed by DPP IV and its half-life in vitro is 45. 1 min. This shows that the strategy established for expression and purification by this study can achieve the efficient preparation of recombinant peptide with high activity. LC-MASS technology is a convenient and accurate detection method for studying the enzymolysis of drug peptides and LC-MASS could be used for preliminary detection of stability for drug peptides and screening drug peptides with high stability in vitro.
出处 《药物生物技术》 CAS CSCD 2009年第5期409-413,420,共6页 Pharmaceutical Biotechnology
基金 国家高技术发展计划("863"计划)(2006AA02Z125) 广东省重大专项(2007A032100006)
关键词 液质联用质谱 基因重组 PACAP衍生物 二肽基肽酶Ⅳ LC-MASS, Genetic recombination, PACAP derivate, Dipeptidyl peptidase Ⅳ (DPP Ⅳ)
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