CN101125881A - 重组pac1受体结抗剂d(25-44)rmmax及其表达方法与应用 - Google Patents
重组pac1受体结抗剂d(25-44)rmmax及其表达方法与应用 Download PDFInfo
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- CN101125881A CN101125881A CNA2007100292673A CN200710029267A CN101125881A CN 101125881 A CN101125881 A CN 101125881A CN A2007100292673 A CNA2007100292673 A CN A2007100292673A CN 200710029267 A CN200710029267 A CN 200710029267A CN 101125881 A CN101125881 A CN 101125881A
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Abstract
本发明公开了一种重组PAC1受体结抗剂D(25－44)RMMAX及其表达方法与应用。其C端和N端均具有特殊的氨基酸序列，并且在C端通过硫代羧酸修饰，有效提高多肽的稳定性。本发明的结抗剂D(25－44)RMMAX可应用于与PAC1受体密切相关的疾病的诊断和治疗。本发明发现结抗剂D(25－44)RMMAX可协助机体降糖，显示结抗剂D(25－44)RMMAX可用于能量代谢相关疾病的研究、诊断及治疗。同时，利用本发明的方法合成的结抗剂D(25－44)RMMAX多肽硫脂的水解产物比化学合成的多肽的稳定性高10倍以上。本发明的表达方法效率高、成本低，应用前景广阔。
Description
技术领域
本发明涉及基因工程领域，具体的说，涉及重组PAC1受体结抗剂D(25-44)RMMAX及其表达方法与应用。
背景技术
PAC1受体是垂体腺苷酸环化酶激活多肽(pituitary adenylatecyclase activating polypeptide，PACAP)的特定受体，参与介导PACAP的多种生物学功能。PACAP是1989年发现的，由脑垂体分泌的或目的组织自分泌和旁分泌的具有重要生物学功能的神经多肽，属于是促胰液素/高血糖素/VIP家族中的新成员。PACAP的具有3个受体：PAC1是PACAP的特异受体；VPAC1和VPAC2是PACAP和血管活性肠肽(VIP)的共同受体；PACAP对3个受体具有相同的激动功能。其中PAC1受体主要分布于中枢神经系统、周围神经系统以及睾丸、卵巢、呼吸道、肺、胰腺等非神经组织内等。目前已证实由PAC1介导的PACAP生物学功能有：神经递质/调质、保护神经细胞；神经损伤修复(眼角膜敏感度恢复，眼部手术后神经细胞突出形成)；调节血管；促进激素分泌，调节内分泌平衡；调节性腺功能和生殖细胞的产生；参与消化活动，调节能量代谢平衡；参与调节免疫系统；调节细胞增殖分化；与摄食睡眠有关；与学习和记忆有关；与某些心理感受有；与动物的生物钟有关；等等。
鉴于PAC1介导了PACAP的多种生物学功能，因此开发PAC1的特异激动剂和特异结抗剂具有巨大的药用开发的价值。例如我们发现PAC1受体特异激动剂具有显著的升糖的作用，而本专利公开的重组PAC1受体特异拮抗剂则具有协助机体降糖的作用，因此显示其在调节能量代谢方面具有应用价值。
目前已发现的PAC1特异拮抗剂是其激动剂maxadilan缺失24-42个氨基酸构成的d(24-42)max，d(24-42)max由42个氨基酸组成的多肽，本专利公开的D(25-44)RMMAX，由54个氨基酸组成，与d(24-42)max有80％同源性，但是是通过基因工程方法制备获得的重组多肽；而且D(25-44)RMMAX与d(24-42)max相比具有不同的N端和C端序列及修饰，赋予D(25-44)RMMAX更高的稳定性和活性。研究证明此重组多肽能有效结合PAC1受体，但不能激活受体，成为PAC1的特异拮抗剂。
本专利所制备的重组多肽，由于在制备过程中C端通过硫酯修饰，成为多肽硫酯，多肽硫酯定向诱导水解成为多肽硫代羧酸，使多肽的C端受到保护，有效地提高了重组多肽的稳定性。
发明内容
本发明的目的在于克服现有技术存在的不足，提供一种具有高稳定性和高活性的PAC1受体特异拮抗剂D(25-44)RMMAX。
本发明的另一个目的是提供上述重组PAC1受体特异拮抗剂D(25-44)RMMAX的表达方法。
本发明的进一步目的是提供上述重组PAC1受体特异拮抗剂D(25-44)RMMAX的应用。
根据D(25-44)RMMAX的氨基酸序列，设计在原核表达的基因，采用基因工程的原理和技术，结合蛋白内含肽(intein)的可诱导剪切功能实现两条目的多肽的高效制备。具体包括如下步骤：(1)设计并合成D(25-44)RMMAX基因，如SEQ ID NO：1所示。
采用4条引物两步法合成D(25-44)RMMAX基因：引物F2和F43行链延伸反应；然后以其产物为模板，以F1和F4为引物，进行PCR，获得D(25-44)RMMAX基因；所述引物F1-4的核苷酸序列如下所示：
F1：GGTGGTGTCAT ATG GGC AGC ATT CTG TGC GAT GCG ACC TGC CAGTTT CGTAAA GCGATT
F2：TGC CAG TTT CGT AAA GCG ATT GAT GAT TGC CAG AAA CAG GCGCATACCAGC CAG CTG C
F3：TTT CTG TTT CAT GCA TTC TTTAAA CAC GCT GTT GCC CGG CAG CTGGCTGGT ATG CGC CT
F4：GGTGGTT CTC GAG TTT GCC CGC TTT AAA TTC TTT TTT TTT CTGTTT CAT GCATTC TTT
其中，N代表保护碱基，CAT ATG为NdeI酶切位点，CTCGAG为XhoI酶切位点。
(2)用NdeI和XhoI进行酶切，将D(25-44)RMMAX基因隆到质粒pKYB的NdeI和XhoI位点间，构建重组质粒pKY-D(25-44)RMMAX；
(3)两个重组质粒转化表达宿主菌E.coli Strain ER2566，构建表达工程菌；
(4)诱导剂IPTG诱导由目的多肽、蛋白内含肽和几丁质结合域(Chitin binding domain，CBD)组成的“三元”融合蛋白的表达；
(5)融合蛋白经几丁质柱纯化，硫醇诱导蛋白内含肽的N端切割，释放目的多肽C端硫酯；
(6)透析多肽硫酯，使其在适当条件下水解产生稳定的C端硫代羧酸水解产物。
本发明的有益效果：本发明所制备的PAC1受体特异拮抗剂D(25-44)RMMAX(54AA)与现有的PAC1特异激动剂和结抗剂相比，其C端和N端均具有特殊的氨基酸序列，并且在C端通过硫代羧酸修饰，有效提高多肽的稳定性。本发明的结抗剂D(25-44)RMMAX可应用于与PAC1受体密切相关的疾病的诊断和治疗。本发明发现结抗剂D(25-44)RMMAX可协助机体降糖，显示结抗剂D(25-44)RMMAX可用于能量代谢相关疾病的研究、诊断及治疗。同时，利用本发明的方法合成的结抗剂D(25-44)RMMAX多肽硫脂的水解产物比化学合成的多肽的稳定性高10倍以上。本发明的表达方法效率高、成本低，应用前景广阔。
附图说明
图1为D(25-44)RMMAX的制备示意图；
图2为D(25-44)RMMAX基因的合成示意图；
图3为pKY-D(25-44)RMMAX质粒的构建示意图；
图4为pKY-D(25-44)RMMAX质粒的测序鉴定图；
图5为D(25-44)RMMAX的表达和几丁质柱纯化；
图6为D(25-44)RMMAX的飞行质谱检测结果
图7为D(25-44)RMMAX调节血糖活性测定。
具体实施方式
实施例1PAC1受体特异拮抗剂D(25-44)RMMAXR的制备
1.D(25-44)RMMAX基因的制备
具体见图1，按大肠杆菌的密码偏好性设计编码D(25-44)RMMAX的基因；设计4条引物，采用两步法获得D(25-44)RMMAX的基因(如图2)：①链延伸反应：引物F1(0.1A/μL)5μL1，F2(0.1A/μL)5μL，10×TaKaRa LA Buffer(含有4mMMgCl2)，10μL，dNTP 16μL，H2O 63μL，94℃，10min；降至55℃，加TaKaRa LA Taq酶1μL，保温5min；72℃，5min；所得为延伸反应液。②PCR反应：引物F3(0.01A/μL)1μL，F4(0.01A/μ1)1μL，①所得延伸反应液1μL，dNTP 10μL，10×TaKaRa ExBuffer 10μL，TaKaRa Ex Taq酶1μL，H2O 76μL，94℃，4min；94℃，45sec、55℃，45sec、72℃，60sec，35个循环；72℃，10min。
F1：TGC CAG TTT CGT AAA GCG ATT GAT GAT TGC CAG AAA CAG GCGCATACCAGC CAG CTG C
F2：TTT CTG TTT CAT GCA TTC TTT AAA CAC GCT GTT GCC CGG CAG CTGGCT GGT ATG CGC CT
F3：NNNNNNNCAT ATGGGC AGC ATT CTG TGC GAT GCG ACC TGC CAGTTT CGT AAA GCG ATT
F4：NNNNNN CTC GAGTTT GCC CGC TTT AAA TTC TTT TTT TTT CTGTTT CAT GCA TTC TTT
其中，N代表保护碱基，CAT ATG为NdeI酶切位点，CTC GAG为XhoI酶切位点。
2.用NdeI和XhoI进行酶切，将D(25-44)RMMAX基因克隆到质粒pKYB的NdeI和XhoI位点间，构建重组质粒pKY-D(25-44)RMMAX，构建图如图3，测序鉴定图如图4。
3.重组质粒pKY-D(25-44)RMMAX转化表达宿主菌E.coli StrainER2566，构建表达工程菌pKY-DRMMAX-ER；挑取单克隆于5mL含50μg/mL卡那霉素的LB培养基，摇菌培养过夜，以1∶20接于1L含50mg/L卡那霉素的LB培养基，37℃摇菌至OD600为0.5-0.8，加入IPTG至终浓度0.1mmol/L，30℃诱导4h。离心收集菌体，重悬于60mL的溶液A(20mM Tris.HCl，500mM NaCl，1mM EDTA，pH8.0)，然后超声破碎，20,000g离心30min，收集上清。
取10mL几丁质珠装填2.5×10cm层析柱，不少于10倍体积的溶液A(20mM Tris-HCl，0.5M NaCl，1mM EDTA，pH 8.0)洗柱；以0.5mL/min的流速上破碎上清；大于10倍体积的溶液A以2mL/min洗去杂蛋白，30mL溶液B(20mM Tris-HCl，0.5M NaCl，1mM EDTA，50mM β-巯基乙醇，pH 8.0)快速过柱，使β-巯基乙醇均匀分布并浸泡柱内填料，16℃诱导蛋白肽切割16h。溶液A洗脱目的多肽，分管收集，每2mL为一管，目的多肽大多存在于前10管中。Tricine-SDS-PAGE鉴定目的多肽；4℃透析过夜除去β-巯基乙醇。采用滤过分子量为10kD的超滤管滤除大分子蛋白杂蛋白。RMMAX制备过程的SDS-PAGE检测如图5。通过飞行质谱检测，重组多肽分子量为5851.67(图6)，与理论值相符。
实施例2重组多肽D(25-44)RMMAX协助机体降低血糖的功能
清洁级(SPF级)KW雄性小鼠(使用许可证号：粤检证字2002-2009；合格证号：粤检证字2003A076)，由第一军医大学实验动物中心提供，体重25±5g，按体重随机分组，每组8只。称重，编号，禁食18h，5nmol/kg的重组D(25-44)RMMAX与高浓度葡萄糖(1.8mmol/kg)同时腹腔注射KW小鼠，注射溶媒作为空白对照。用OneTouch Ultra血糖自动分析仪(美国JOHNSON公司)测定注射后一小时内血糖的变化。
实验结果(表1，表2，图7)显示：与注射溶酶的对照组相比，D(25-44)RMMAX组小鼠血糖在注射20min后开始下降，而对照组注射20min后仍在上升。按公式AUC(the glucose area under the curve)＝(PG0+PG60)/2+PG10+PG20+PG30计算D(25-44)RMMAX组及溶媒对照组的AUC，结果组的AUG (Control)＝69.1±10.9；AUG(D(25-44)RMMAX)＝56.7±8.7；两组间经t检验，P＜0.05。而注射PAC 1受体激动剂的RMMAX组小鼠血糖一直在升高，没有降低的趋势，并且在注射后90min，其各个个体血糖均超过仪器测量上限，显示RMMAX具有强烈的，持续的升血糖的作用。因此重组多肽D(25-44)RMMAX具有协助机体降糖的功能，估计是通过与天然PACAP竞争结合并封闭PAC1受体，阻断PAC1受体介导的升糖作用，从而发挥协助机体降糖的功能。
表1.D(25-44)RMMAX组
表2.对照组
| KM小鼠时间 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | AVG |
| 0min | 9.8 | 10.6 | 10.1 | 9.3 | 10.9 | 7.4 | 9.7 | 9.2 | 9.6±1.1 |
| 10min | 24.3 | 20.3 | 20.2 | 18.3 | 20.5 | 15.2 | 16.3 | 19.1 | 19.3±2.8 |
| 20min | 24.3 | 22.9 | 19.9 | 19.1 | 23.3 | 15.3 | 15.2 | 18.9 | 19.9±3.5 |
| 30min | 20.1 | 21.4 | 13.9 | 13.0 | 24.4 | 12.8 | 12.7 | 16.6 | 16.9±4.6 |
| 60min | 11.9 | 16.6 | 11.1 | 10.5 | 18.1 | 10.1 | 10.1 | 10.1 | 12.3±3.2 |
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F2：TGC CAG TTT CGT AAA GCG ATT GAT GAT TGC CAG AAA CAG GCG CAT ACC AGC CAG CTG C
F3：TTT CTG TTT CAT GCA TTC TTT AAA CAC GCT GTT GCC CGG CAG CTG GCT GGT ATG CGC CT
F4：GGTGGTT CTC GAG TTT GCC CGC TTT AAA TTC TTT TTT TTT CTG TTT CAT GCA TTC TTT
Claims (7)
1.重组PAC1受体拮抗剂D(25-44)RMMAX，其氨基酸序列如SEQID NO：2所示。
2.编码权利要求1所示重组PAC1受体拮抗剂D(25-44)RMMAX的核苷酸序列，如SEQ ID NO：1所示。
3.一种权利要求1所述重组PAC1受体特异拮抗剂D(25-44)RMMAX的表达方法，其特征在于包括如下步骤：
(1)设计并合成D(25-44)RMMAX的基因：
(2)将D(25-44)RMMAX基因克隆到质粒pKYB的NdeI和XhoI位点间，构建重组质粒pKY-D(25-44)RMMAX；
(3)重组质粒转化表达宿主菌E.coli Strain ER2566，构建表达工程菌；
(4)用诱导剂IPTG诱导由目的多肽、蛋白内含肽和几丁质结合域组成的“三元”融合蛋白的表达；
(5)融合蛋白经几丁质柱纯化，硫醇诱导蛋白内含肽的N端切割，释放目的多肽C端硫酯，多肽C端硫酯水解得到重组多肽D(25-44)RMMAX。
4.如权利要求3所述的表达方法，其特征在于步骤(1)所述的D(25-44)RMMAX基因序列如SEQ ID NO：1所示。
5.权利要求1所述重组PAC1受体特异拮抗剂D(25-44)RMMAX在制备协助机体降糖或PAC1受体相关疾病药物中的应用。
6.如权利要求5所述的应用，其特征在于所述重组PAC1受体特异拮抗剂D(25-44)RMMAX在制备治疗肥胖、高血糖或高血脂药物中的应用。
7.如权利要求5所述的应用，其特征在于所述PAC1受体相关疾病为脑损伤、脑缺血、防止神经细胞死亡、抗炎、角膜敏感性恢复、眼部神经损伤修复、男性阳痿、性冷淡、不孕不育、神经痛、神经病、神经混乱、焦虑症、记忆损伤、痴呆、认知混乱、中枢神经系统疾病、偏头痛、神经畏缩、心肌缺血、心肌梗死/纤维化、与生物钟睡眠及痛觉相关的疾病、动脉硬化、糖尿病、胃肠功能紊乱、肌萎缩症、胃溃疡、高血压、内毒性休克、血栓症、视网膜病变、心血管疾病、肾衰竭、心衰竭或脱发。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009033489A3 (en) * | 2007-09-11 | 2009-05-07 | Glostrup Hospital | Selective pacl inhibitors for use in the treatment of migraine and headaches |
| CN101985464A (zh) * | 2010-10-22 | 2011-03-16 | 暨南大学 | 高稳定性pac1型受体特异激动剂mpapo及其制备方法与应用 |
| WO2018222991A3 (en) * | 2017-06-02 | 2019-01-31 | Amgen Inc. | PAC1 ANTAGONIST PEPTIDES |
-
2007
- 2007-07-20 CN CNA2007100292673A patent/CN101125881A/zh active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009033489A3 (en) * | 2007-09-11 | 2009-05-07 | Glostrup Hospital | Selective pacl inhibitors for use in the treatment of migraine and headaches |
| CN101985464A (zh) * | 2010-10-22 | 2011-03-16 | 暨南大学 | 高稳定性pac1型受体特异激动剂mpapo及其制备方法与应用 |
| CN101985464B (zh) * | 2010-10-22 | 2013-01-16 | 暨南大学 | 高稳定性pac1型受体特异激动剂mpapo及其制备方法与应用 |
| WO2018222991A3 (en) * | 2017-06-02 | 2019-01-31 | Amgen Inc. | PAC1 ANTAGONIST PEPTIDES |
| US11643440B2 (en) | 2017-06-02 | 2023-05-09 | Amgen Inc. | Peptide PAC1 antagonists |
| AU2018275270B2 (en) * | 2017-06-02 | 2024-06-06 | Amgen Inc. | Peptide PAC1 antagonists |
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