【作者】 余榕捷；

【导师】 周天鸿；

【作者基本信息】 暨南大学 ， 生物医学工程， 2005， 博士

【摘要】 垂体腺苷酸环化酶激活肽（pituitary adenylate cyclase activating polypeptide，PACAP）是具有重要生物学功能和极具应用价值的神经多肽。为利用基因工程技术获得非融合的重组人垂体腺苷酸环化酶激活肽（recombinant human PACAP，rhPACAP），采用rhPACAP与纤维素结合域（cellulose binding domain，CBD）融合表达，并在两者之间引入Xa因子的识别位点（Ile-Glu-Gly-Arg↓）。融合蛋白CBD-PACAP经纤维素亲和层析纯化后，Xa因子酶切释放非融合重组PACAP。HPLC进一步纯化的重组PACAP多肽经Western blot与激光飞行质谱鉴定。制备的rhPACAP具有促进胰腺癌细胞株SW1990胞内cAMP合成的活性，表明其具有生物学活性。在两个融合蛋白（CBD-PACAP和CBD-IGF）中Xa因子识别位点（Ile-Glu-Gly-Arg↓）前引入7个氨基酸组成的富含甘氨酸柔性短肽（Gly-Thr-Gly-Gly-Gly-Ser-Gly），经比较Xa因子对引入短肽前、后融合蛋白的酶切效率后发现，短肽的引入不同程度地提高了融合蛋白CBD-IGF和CBD-PACAP对Xa因子的敏感性，此研究结果提供了一种提高Xa因子酶切效率的策略。为利用蛋白内含肽介导的一步纯化实现重组PACAP（RMPACAP）的高效制备，将目的多肽融合表达在具可诱导剪切功能的亲和纯化标签的N端，实现融合蛋白RMPACAP-intein-CBD的高效表达；融合蛋白经几丁质柱纯化后，硫醇诱导蛋白内含肽自动切割释放目的多肽RMPACAP；所制备的RMPACAP与天然PACAP38相比，在其N端多了一个甲硫氨酸（Met），但其促进SW1990细胞胞内cAMP生成的活性与PACAP38标准品相同。利用此工艺，1L的菌体培养物可获得22mg的纯度可高达98％的RMPACAP多肽。此研究建立了一种高效制备重组PACAP的方法。模仿已有的VPAC2受体特异激动剂序列，结合已建立的蛋白内含肽介导的多肽制备工艺，设计并实现4条重组多肽（RMROM、RROM、RMBAY和RBAY）的制备；四条重组多肽（50ng／kg）与高浓度葡萄糖（1.8mmol／kg）共同腹腔注射NIH小鼠时，均有效协助降低血糖；其中RMBAY的降糖作用较其它重组多肽显著，并可有效促进葡萄糖依赖的胰岛素分泌。RMBAY的筛选及其制备方法的建立为开展重组多肽RMBAY治疗Ⅱ型糖尿病的研究奠定基础。为从分子和细胞水平研究所制备的重组多肽RMPACAP及RMBAY对PACAP的三个受体的选择性（Selectivity）和激动性（Potency），从分子水平诠释重组多肽的作用机制，构建分别高效表达PACAP三个受体：PAC1、VAPC1和VPAC2的中国仓鼠卵巢（Chinese hamster ovary，CHO）细胞株。RT-PCR鉴定受体基因在mRNA水平的表达；Western blot和荧光免疫法鉴定VPAC1和VPAC2受体的表达。¹²⁵I-PACAP38配体与受体结合实验检测PAC1受体的表达。重组多肽与¹²⁵I-PACAP38对受体的竞争结合实验结果显示RMPACAP对三个受体均具有选择性；而RMBAY对VPAC2受体的IC₅₀为70±5nmol／L，对VAPC1受体和PAC1受体的IC₅₀均大于10μmol／L。重组多肽RMBAY促进VPAC2-CHO细胞株胞内cAMP产生的EC₅₀为1.8±0.2nM，RMPACAP的EC₅₀为5.3±0.3nM。RMBAY对VPAC2受体具有特异的选择性和激动性，是VPAC2受体的特异激动剂。RMBAY的筛选和鉴定为具有自主知识产权的VPAC2受体特异激动剂的研究和开发铺垫道路。为提高多肽的活性和稳定性，尝试利用蛋白内含肽可诱导剪切功能和多肽硫酯环合技术实现RCBAY环肽的制备。激光飞行质谱检测制备产物中含有成分的分子量为4128.10，与环肽的理论值相符。活性检测表明含有重组环肽RCBAY的制备产物与直链肽RMBAY相比，降血糖的作用更显著和持久。本研究提示RCBAY可能成为治疗Ⅱ型糖尿病的更有效的药物。在实验室水平确定工程菌RMBAY-ER2566的生长曲线，碳源，IPTG诱导时OD₆₀₀，IPTG用量和诱导时间等参数。初步的探索结果表明：培养菌体至OD₆₀₀达到0.5-1.0时加入IPTG至工作浓度0.4mmol／L，30℃诱导表达3-4h，或37℃诱导表达2-3h，均能有保证目的蛋白的高效表达。葡萄糖作为碳源抑制目的蛋白的表达。以实验室研究为参考，利用5L发酵罐发酵，3.5L发酵液收获湿重为113g的菌体，其中目的蛋白表达量达到30-35％，可溶性成分占可溶性蛋白的28-30％。工程菌RMBAY-ER2566的发酵为开展RMBAY作为VPAC2型受体特异激动剂的生物学研究及其医学应用奠定基础。总体上，本文主要利用基因工程原理和技术，结合蛋白内含肽可诱导剪切功能实现重组PACAP及其衍生多肽的高效制备；筛选并鉴定一条可高效协助机体降低血糖的新型VPAC2受体特异激动剂——RMBAY；尝试通过环化提高其活性和稳定性；开展工程菌中试发酵的研究；为具有自主知识产权的VPAC2受体特异激动剂的研究和开发奠定基础。更多还原

【Abstract】 Pituitary adenylate cyclase activating polypeptide （PACAP） is a neuropeptidewith multiple important bioactivities and worthy of medical application. In order toproduce non-fusion recombinant human pituitary adenylate cyclase activatingpolypeptide （rhPACAP） using gene engineering technology, a fusion protein with arecognized site of factor Xa （Ile-Glu-Gly-Arg↓） between CBD （cellulose bindingdomain） and rhPACAP was expressed and purified by cellulose affinitychromatography. Non-fusion rhPACAP was released by the cleavage of factor Xa,then purified by HPLC and identified by Western blot and Laser flying massspectrometry. The preliminary bioactivity assay indicated that the product had theactivity of promoting cAmp in the cell line SW1990 of human pancreas carcinoma.A short flexible peptide abundant in Gly（Gly-Thr-Gly-Gly-Gly-Ser-Gly） wasadded before the recognized site by factor Xa. The compare of the cleavage efficiencyof factor Xa on both two-group fusion proteins （CBD-IGF and CBD-PACAP） withand without short flexible peptide indicated that the short peptide helped to improvethe sensitivity of fusion protein to factor Xa in different degree. A strategy ofpromoting the cleavage efficiency of factor Xa was presented.In order to obtain the recombinant human PACAP efficiently by intein-mediatedsingle column purification, the target protein was over-expressed as a fusion to theN-terminus of a self-cleavable affinity tag. After the PACAP-intein-CBD fusionprotein was purified by chitin-affinity chromatography, the self-cleavage activity ofthe intein was induced by thiol and the RMPACAP was released from thechitin-bound intein tag. The activity assay showed that RMPACP, which has an extraMet at its N-terminus compared with the native human PACAP, had the similaractivity of stimulating cAmp accumulation with the standard PACAP38 in theSW1990 cells. Twenty-two milligrams of RMPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. A new efficientproduction procedure of the active recombinant human PACAP was established.Four recombinant PACAP derived peptides （RMROM, RROM, RMBAY andRBAY） as potential therapies for TypeⅡDiabetes were designed based on theknown agonist of VPAC2 receptor, and were prepared using intein-mediatedpurification system. All peptides （50ng/kg） decreased the level of plasma glucose afterinfused with high concentration glucose （1.8mmol/kg） to the enterocoelia of NIHmouse. RMBAY with the highest activity of reducing the level of plasma glucosestimulated the glucose-dependent insulin secretion. The screening and preparation ofRMBAY laid the foundation for development of RMBAY as potential therapy forType 2 Diabetes.The receptor-transfected Chinese hamster ovary （CHO） cell clones expressingthree receptors subclasses of PACAP respectively were constructed and selected. Theresults of RT-PCR, immunoflourescence assay and the binding assay of¹²⁵I-PACAP38 to the receptor showed that three receptors were expressed on threecells （VPAC1-CHO, VPAC2-CHO and PAC1-CHO） respectively. The competitionbinding of ¹²⁵I-PACAP38 on membranes from CHO cells expressing each of threesubtypes of human PACAP receptors was used to identify the selectivity of RMBAYand RMPACAP on three receptors. RMPACAP competitively displaced¹²⁵I-PACAP38 on all three receptor subclasses. RMBAY competitively displaced¹²⁵I-PACAP38 on VPAC2 with a half-maximal inhibitory concentration （IC50） of 70±5nmol/L, whereas the IC50 of RMBAY at human VPAC1 or PAC1 was observedup to 10μmol/L1. RMBAY stimulated the cAmp in VPAC2-CHO cells with ahalf-maximal stimulatory concentration （EC50） of 1.8±0.2nmol/L. Thus, RMBAYis a selective agonist ligand for VPAC2 receptor subclass.The intein-mediated purification and cyclization of unprotected peptidethioesters were tried to produce a recombinant cyclic peptide （RCBAY） for thepurpose of improving the stability and activity of the peptide. The mass spectrometryassay showed that the products contained the component with the molecular weight of4128.10 consistent with the theoretic value of RCBAY. The mixture containing RCBAY displayed a stronger effect on reducing the level of plasma glucose and lastedlonger time than RMBAY. It was suggested that RCBAY might be a more effectivetherapy for typeⅡdiabetes.Some parameters were studied, e.g. the growth curve of the engineered strainRMBAY-ER2566, the value of OD₆₀₀ at which the expression of the target protein wasinduced, the amount of IPTG and the time spent on inducing protein expression. Thetarget protein was effectively induced by 0.4mmol/L IPTG for 3-4hr at 30℃or for2-3hr at 37℃, when the OD₆₀₀ of the culture reached 0.5-1. Glucose inhibited theexpression of the target fusion protein. Based on the conditions mentioned above, thestrain was fermented with high density in 5L fermentor. One hundred and thirteengrams of wet bacteria was obtained from 3.5L culture. The expression of target fusionprotein reached 32-35% of total bacterial protein and 28% of the total soluble proteins.The fermentation of the engineered strain RMBAY-ER2566 ensured the furtherresearch and medical development of RMBAY.All in a word, the study focused on the efficient preparation of recombinantPACAP and its derived peptides, the screening and identification of RMBAY as anovel agonist of VPAC2 receptor with bioactivity of reducing plasma glucose,producing cyclopeptide RCBAY based on RMBAY for improving the bioactivity andstability of the peptide, and the fermentation of the engineered strain for the furtherresearch and application of the novel agonist of VPAC2 receptor of which we ownedthe property right.更多还原