重组PACAP衍生物RMBYLL的高效制备及其体外生物学效应

Highly Efficient Preparation of Recombinant PACAP Derivate RMBYLL and Its Biological Effects in Vitro

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RMBYLL,并在体外研究其生物学效应,采用基因重组技术表达重组肽RMBYLL,纯化、制备并鉴定重组肽RMBYLL后,利用特定细胞系和Western-blot等技术,检测RMBYLL对VPAC1、VPAC2和PAC1受体的选择性和激动活性,以及重组肽RMBYLL对胰岛素受体介导的信号传导最终效应物—葡萄糖转运蛋白4(GLUT4)表达的影响。实验结果表明:利用重组肽技术制备的RMBYLL的M_r为3.901k,纯度大于95%,其产率为18.7 mg/(L发酵产物);制备的RMBYLL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(EC_(50))为0.90 nmol/L,目的肽RMBYLL可显著增强3T3-L1脂肪细胞GLUT4的表达,阳性实验组是对照组的1.91倍。由此表明,利用本研究确立的表达、纯化策略可实现高活性RMBYLL的高效制备,应用前期构建的含有特定受体的CHO细胞和3T3-L1脂肪细胞模型,可方便有效地进行相关药物肽的体外生物学效应研究。

In order to efficiently prepare the pituitary adenylate cyclase activating polypeptide （PACAP） derivate RMBYLL against type Ⅱ diabetes by genetic engineering technique and to study its biological effects in vitro, genetic recombination technique was used to express the recombinant RMBYLL. After purifying, preparation and identifing recombinant peptide RMBYLL, using selective cells with Western-blot and other techniques ,the selectivity and potency of RMBYLL at human VPAC1 ,VPAC2 and PAC1 receptor were assayed. And the effect of RMBYLL on the expression of glucose transporter 4 （ GLUT4 ） , the last effector protein of insulin signaling, was determined. The experimental results showed that the molecular weight of RMBYLL was 3. 901k,its purity was greater than 95% and its yield was 18.7 mg/（ L fermentation product）. Prepared RMBYLL could specifically act on VPAC2 with a half-maximal stimulatory concentration （ EC50 ） of 0.90 nmol/L and significantly enhanced cAMP accumulation in CHO cells. RMBYLL may significantly induce GLUT4 expression in differentiated 3T3-L1 adipocytes. GLUT4 protein was increased 0.91-fold in the treated cells with RMBYLL. These results showed that the strategy established for expression and purification by this study can achieve the effi- cient preparation of recombinant peptide with high activity. The biological effects in vitro could be conveniently and accurately investigated with CHO cells and 3T3-L1 adipocytes that were constructed in previous study.