CN116836296A - 一种α促黑素细胞激素的融合蛋白及其制备方法和应用 - Google Patents
一种α促黑素细胞激素的融合蛋白及其制备方法和应用 Download PDFInfo
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- CN116836296A CN116836296A CN202310254150.4A CN202310254150A CN116836296A CN 116836296 A CN116836296 A CN 116836296A CN 202310254150 A CN202310254150 A CN 202310254150A CN 116836296 A CN116836296 A CN 116836296A
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Abstract
本发明属于基因工程制药领域,具体涉及一种α促黑素细胞激素的融合蛋白及其制备方法和应用。所述融合蛋白包含1个蛋白转导结构域(Protein transduction domian,PTD),1个人血清白蛋白(Human Serum Albumin,HSA)和一个α促黑素细胞激素(α‑Melanocyte stimulating hormone,α‑MSH),并在α‑MSH与HSA之间加入特定的刚性连接肽,与使用柔性连接肽相比,本发明的融合蛋白具备更强的抗炎活性,且能有效跨越血脑屏障、半衰期长,能够用于制备治疗中枢神经炎症性疾病的药物。
Description
技术领域
本发明属于基因工程制药领域,具体涉及一种α促黑素细胞激素的融合蛋白及其制备方法和应用。
背景技术
中枢神经系统炎症在各种中枢神经系统疾病的发病过程中起着决定性作用,包括神经退行性疾病如阿尔茨海默症和精神障碍。随着神经炎症对中枢神经系统疾病的危害逐渐为人们所认,开发安全且有效的内源性抗炎药是提高人类生活质量的关键因素之一。内源性免疫神经调节肽α促黑素细胞激素(α-Melanocyte stimulating hormone,α-MSH)由于在中枢神经系统炎症中发挥重要作用,在基础研究与临床治疗中都具有重要价值,然而,由于其分子量小,半衰期短,限制了其临床应用。专利CN201510957366.2中针对α-MSH半衰期短的问题加入了人血清白蛋白(Human Serum Albumin,HSA),两者之间使用了柔性连接肽制备为融合蛋白,同时为了使融合蛋白能够有效地跨过血脑屏障治疗脑部炎症,还加入了蛋白转导结构域(Protein transduction domian,PTD),但是整体效果依然不佳。
连接肽是蛋白质结构域之间的短寡肽,作为将功能域连接在一起/分离或在体内释放自由功能域的桥梁,根据其结构分为3类:柔性、刚性和可裂解的体内连接肽。柔性连接肽有利于正确折叠和减少功能域之间的空间位阻,然而,刚性连接肽在功能域分离中有更好的效果。本领域人员知晓由于刚性连接肽缺乏融合蛋白功能域之间的相互作用,所以效果不如柔性连接肽。我们之前的研究显示,柔性连接肽的连接是一种有利于融合蛋白发挥活性的方式,研究人员也通常会采用这种方法来构建融合蛋白,尽管如此,所得的融合蛋白的活性效果依然不理想。
发明人在后续的研究中意外地发现将柔性连接肽替换成特定的刚性连接肽可以进一步改良使用柔性连接肽连接构建的α-MSH融合蛋白的活性。
发明内容
本发明的目的在于提供一种α促黑素细胞激素融合蛋白,该融合蛋白在α-MSH与HSA之间加入刚性连接肽,与使用柔性连接肽相比,本发明的融合蛋白具备更强的抗炎活性,且能有效跨越血脑屏障、半衰期长,能够用于制备治疗阿尔茨海默症、帕金森症、缺血性脑卒中、多发性硬化、创伤性脑损伤、运动神经元疾病、脑部感染、抑郁症、焦虑和精神分裂症等中枢神经炎症性疾病的药物。
为了实现上述目的,本发明一方面提供了一种α促黑素细胞激素的融合蛋白,包含1个α促黑素细胞激素(α-Melanocyte stimulating hormone,α-MSH),1个蛋白转导结构域(Protein transduction domian,PTD),1个人血清白蛋白(Human Serum Albumin,HSA)和1个连接肽L6,所述融合蛋白自N端到C端依次为PTD、HSA、L6、α-MSH,所述连接肽L6的氨基酸序列如SEQ ID NO:1所示。
优选的,所述融合蛋白的氨基酸序列如SEQ ID NO:2所示。
本发明的另一个目的在于提供α促黑素细胞激素融合蛋白在制备治疗中枢神经炎症药物中的应用。
优选的,所述中枢神经炎症为阿尔茨海默症、帕金森症、缺血性脑卒中、多发性硬化、创伤性脑损伤、运动神经元疾病、脑部感染、抑郁症、焦虑和精神分裂症。
本发明的另一个目的在于提供上述α促黑素细胞激素融合蛋白的制备方法,其特征在于,所述方法包含以下步骤:
①合成所述融合蛋白的DNA序列,如SEQ ID NO:3所示;
②通过基因工程技术连接目的片段与酵母表达载体,获得含编码所述融合蛋白DNA序列的重组酵母表达载体;
③将步骤②所述的重组酵母表达载体转化到感受态大肠杆菌细胞中,从转入质粒的大肠杆菌细胞中提取质粒,再将所述质粒转化入酵母细胞中进行表达,即得所述融合蛋白。
优选的,所述的酵母为嗜甲醇毕赤酵母(Pichia pastoris)。
优选的,所述的嗜甲醇毕赤酵母为嗜甲醇毕赤酵母PichiaPinkTM。
本发明的另一个目的在于提供上述α促黑素细胞激素融合蛋白的制备方法在制备治疗中枢神经炎症药物中的应用。
优选的,所述中枢神经炎症为阿尔茨海默症、帕金森症、缺血性脑卒中、多发性硬化、创伤性脑损伤、运动神经元疾病、脑部感染、抑郁症、焦虑和精神分裂症。
本发明的另一个目的在于提供上述α促黑素细胞激素融合蛋白加入药学上可接受的辅料制备成注射剂。
本发明的有益效果:
本发明的融合蛋白使用特定的刚性连接肽连接α-MSH与HSA,与使用柔性连接肽连接相比,抗炎活性明显提高,能够用于治疗阿尔茨海默症、帕金森症、缺血性脑卒中、多发性硬化、创伤性脑损伤、运动神经元疾病、脑部感染、抑郁症、焦虑和精神分裂症等中枢神经炎症性疾病。
附图说明
图1为PTD-HSA-L-α-MSH融合蛋白细胞毒性和抗炎活性分析
图2为PTD-HSA-L-α-MSH融合蛋白体内抗炎活性分析
具体实施方式
主要实验仪器:
移液枪、超净工作台(安泰)、磁力搅拌器、微波炉、高温蒸汽灭菌锅、-80℃低温冰箱(Forma)、超纯水仪(Millipore)、制冰机、离心机(Hitachi)、HDB-PLUS型恒温金属浴、HZQ-F16OA型恒温振荡培养箱(上海一恒)、PCR仪(Applied Biosystems)、台式冷冻离心机(Thermo)、DYY-8B型电泳仪(伯乐)、Image Quant 300型凝胶成像仪(GE)等。
主要实验材料:
1.限制性核酸内切酶Stu I、Kpn I、Xho I、Afl II(NEB公司产品,美国)
2.小提质粒试剂盒、PCR纯化试剂盒、DNA胶回收试剂盒(生工生物,中国)
3.PrimerSTAR(Takara公司产品,中国大连)
4.载体pPinkα-HC、载体pUC-57-HSA、毕赤酵母菌株、Infusion试剂盒(Invitrogen公司产品,美国)
5.大肠杆菌TOP10(天根生化科技(北京)有限公司)
6.酵母提取物、蛋白胨(Oxford公司产品,美国)
7.LB培养基
酵母提取物5g,蛋白胨10g,NaCl 10g,溶于1000ml去离子水中,并用1mol/L的NaOH调节pH值至7.0,高压蒸气灭菌。
8.YPD培养基
酵母提取物10g,胰蛋白胨20g,Agar 20g,溶于900ml去离子水中,高压灭菌,冷却后加入100ml经滤器除菌后的20%的葡萄糖。
9.YPDS培养基
酵母提取物10g,蛋白胨20g,山梨糖醇182.2g,溶于900ml去离子水中,高压灭菌,冷却后加入100ml经滤器除菌后的20%的葡萄糖。
10.BMGY液体培养基
酵母提取物10g,蛋白胨20g,无氨基酸酵母氮源13.4g,甘油10g,磷酸钾26.631g,溶于1000ml双蒸水中高压灭菌,冷至室温,调节pH至6.0,4℃保存备用。
11.1%琼脂糖凝胶的配置
根据用量,每100ml的TAE缓冲液,加入1g琼脂糖,使用微波炉加热煮沸,使琼脂糖完全融解,室温冷却至不烫手时滴加少量溴化乙锭(EB),混匀后将其倒入事先摆放好梳子的胶槽中,待到室温冷却至完全凝固后拔去梳子即可使用。
实施例1pPinkα-HC/PTD-HSA-L-α-MSH
一、pPinkα-HC/PTD-HSA-L-α-MSH载体的构建
1.设计PCR引物:
表1PTD-HSA-Linker1-7-α-MSH引物设计
引物,进行PCR扩增。反应条件如下:①变性:94℃,5min;②变性:94℃,1min;③复性:55℃,30S;④延伸:72℃,2min;⑤返回步骤“②”,进行35循环;⑥延伸:72℃,5min,总循环次数为30次。将PCR产物进行1%琼脂糖凝胶电泳,结果显示扩增出约1.8kb大小的部分PTD-HSA-L-α-MSH条带。
3.第二轮PCR扩增:以第一轮PCR扩增的产物为模板,采用对应上、下游引物,进行PCR扩增。反应条件如下:①变性:94℃,5min;②变性:94℃,1min;③复性:55℃,30S;④延伸:72℃,2min;⑤返回步骤“②”,进行35循环;⑥延伸:72℃,5min,总循环次数为30次。将PCR产物进行1%琼脂糖凝胶电泳,结果显示扩增出约1.8kb大小的完整PTD-HSA-L-α-MSHDNA条带,将以上PCR产物进行胶回收。
4.Kpn I和Stu I双酶切pPinkα-HC(Invitrogen公司产品)质粒DNA,胶回收获得pPinkα-HC(Kpn I/Stu I)载体片段,利用Infusion试剂盒将上述胶回收的pPinkα-HC(KpnI/Stu I)载体片段和PTD-HSA-L-α-MSH DNA目的基因片段进行重组反应,反应产物转化大肠杆菌感受态TOP10,涂于氨苄抗性LB板37℃培养过夜,筛选阳性克隆。所得克隆送Invitrogen公司测序,序列正确的克隆命名为pPinkα-HC/PTD-HSA-L-α-MSH。
另外,我们还可以委托公司进行全序列合成。
二、融合蛋白在酵母中的表达
将测序正确的带有融合蛋白争取序列的质粒pPinkα-HC用Afl II酶切回收后得到线性化片段,分别转化嗜甲醇毕赤酵母,然后将转化菌液接种于PAD平板,30℃培养3-4天,挑取阳性克隆。将得到阳性克隆分别接种BMGY液体培养基,30℃培养48小时,然后转接至BMMY培养基中诱导表达,持续96小时后,1500rpm低温离心15分钟,取上清,SDS-PAGE电泳检测蛋白表达情况。融合蛋白分子量约为70kDa。
实施例2双荧光素酶检测PTD-HSA-L-α-MSH融合蛋白抑制NF-kB功能比较一、实验材料
pNF-κB-luc购自于碧云天生物技术研究所。A172细胞购于中国科学院上海生科院细胞资源中心。
二、实验仪器
高纯度无热源质粒大量提取试剂盒及Lipofectamine 3000脂质体转染试剂盒购于Thermo Fisher公司。DMEM基础培养基、胎牛血清均购于Gbico公司。LPS,炎症因子TNF-α及α-MSH标准品购于Sigma公司。。Dual-Luciferase Reporter Assay System试剂盒和细胞被动裂解液购于Promega公司。
三、实验方法
1.A172细胞铺板及转染
进行细胞瞬时转染前,先将生长状态良好汇合度达到80%的细胞用胰酶消化并计数,以1×105的密度接种于24孔板中,在CO2培养箱中培养过夜。本文使用脂质体转染的方法将外源质粒DNA导入A172细胞,每孔使用质粒DNA总量(μg):脂质体3000(μL)为0.5μg:0.75μL,将质粒和脂质体3000按照上述比例混匀制备成转染复合物。转染前将24板中的培养基置换成opti-DMEM减血清培养基,将制备好的转染复合物加入24孔板中轻轻混匀,置于CO2培养箱中培养。转染6h后更换为含有10% FBS的DMEM完全培养基。
2.双荧光素酶法检测融合蛋白对NF-кB转录因子的抑制
细胞转染24h后,进行短暂的饥饿处理,加入ɑ-MSH/PTD-HSA-L-α-MSH或TNF-α进行诱导或者同时加入TNF-α与ɑ-MSH/PTD-HSA-L-α-MSH进行处理,药物处理6h后用Dual-Luciferase Reporter Assay System进行萤光素酶活力的分析。弃掉24孔板中的细胞培养上清,用无菌PBS清洗贴壁细胞,加入300μL新鲜配置的细胞被动裂解液,反复吹打裂解细胞。然后在37℃,100rpm振荡15min,将35μL细胞裂解液加入不透光96孔板中,首先加入35μLDual-Glo Luciferase Reagent,室温孵育10min后,使用荧光酶标仪检测萤火虫荧光素酶催化底物产生的荧光强度。然后每孔加入35μL Dual-Glo Stop&Glo Reagent,孵育10min后,检测海洋腔肠荧光素酶催化底物产生的荧光强度。两种荧光素酶底物荧光强度的比率即代表NF-кB转录因子的活力。每组实验重复3次,取平均值进行分析。
3.实验数据处理
数据用平均值±标准差表示,使用GraphPad Prism 8进行各组均数之间单因素方差分析。
四、实验结果
本领域人员知晓由于刚性连接肽缺乏融合蛋白功能域之间的相互作用,所以效果不如柔性连接肽。我们之前的研究也显示,柔性连接肽的连接是一种有利于融合蛋白发挥活性的方式(专利CN201510957366.2),研究人员也通常采用这种方法来构建融合蛋白,但是我们意外地发现特定的刚性连接肽能够达到更好的效果。
实验结果如图1所示,本实验将L0-L4柔性连接肽和L5-L7刚性连接肽用于融合蛋白中α-MSH和HSA蛋白结构域的连接,其中L0为专利CN201510957366.2中所使用的融合蛋白连接肽。根据图2的C图和D图的MTT实验结果选择合适的融合蛋白浓度范围,所有融合蛋白的细胞毒性均低于30%(0.05-8μM)。
双荧光素酶法测定中所使用的融合蛋白的浓度范围为0.1-0.8μM,用10ng/mL诱导TNF-α,根据上述MTT细胞活性试验结果,在该浓度范围里细胞活力均在90%左右。在0.1-0.8μM浓度范围内选取NF-κB抑制率最好的融合蛋白浓度绘制成图2的E图和F图,其中,E图融合蛋白对A172正常细胞的NF-κB抑制率,F图为融合蛋白对TNF-α诱导A172细胞的NF-κB抑制率,可以看出,本发明特定刚性连接肽连接的PTD-HSA-L6-α-MSH融合蛋白能够在最低浓度0.1μM下达到最好的NF-κB抑制效果,抗炎作用达到最佳。
实施例3PTD-HSA-L-α-MSH融合蛋白抑制中枢神经炎症实验
1.实验仪器
注射器、移液枪、离心机(Hitachi)、超纯水仪(Millipore)、组织匀浆机、恒温培养箱(上海一恒)、酶标仪(Thermo)等。HSA Elisa试剂盒(Cygnus Technologies)
2.实验动物
50只标准体重昆明小鼠,购于兰州大学实验动物中心。
3.实验方法
小鼠的分组及给药方式:
脂多糖LPS(Lipopolysaccharides)即革兰氏阴性菌内毒素是革兰氏阴性细菌的细胞壁组成成分,小鼠尾静脉注射LPS能够引起大脑中的神经退行性病变及炎症反应,本实验以小鼠脑部海马组织中的IL-6为指标,检测本发明所述的α-MSH的融合蛋白在小鼠由LPS引起的脑部炎症模型中的药效。
将50只昆明小鼠分为五组,体重18~22g左右:1-对照组,2-LPS组,3-α-MSH+LPS组,4-PTD-HSA-L6-α-MSH+LPS组。对照组分两次注射300μL生理盐水,每次注射量为150μL;LPS组先注射150μL LPS(5mg/kg),后注射150μL生理盐水;3-α-MSH+LPS组先注射150μL LPS(5mg/kg),后注射150μLα-MSH;4-PTD-HSA-L6-α-MSH+LPS组先注射150μL LPS(5mg/kg),后注射150μL PTD-HSA-L-α-MSH(1μM/kg);6小时后,处死小鼠,眼球取血后迅速取出海马组织及全脑组织,预冻存后进行组织匀浆。本实验所述注射方式均为尾静脉注射。ELISA法检测组织匀浆及血清中的IL-6水平。
4.实验结果
实验结果如图2所示。与对照组相比,LPS组小鼠海马组织中IL-6水平明显升高,说明模型建立成功;与α-MSH+LPS组相比,PTD-HSA-L6-α-MSH组中小鼠海马组织中IL-6显著降低,说明本发明所述PTD-HSA-L6-α-MSH能够显著地降低中枢神经系统的炎症,能够用于治疗脑部炎症及相关疾病。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提。还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
Claims (10)
1.一种α促黑素细胞激素的融合蛋白,其特征在于,所述融合蛋白包含1个α促黑素细胞激素(α-Melanocyte stimulating hormone,α-MSH),1个蛋白转导结构域(Proteintransduction domian,PTD),1个人血清白蛋白(Human Serum Albumin,HSA)和1个连接肽L6,所述融合蛋白自N端到C端依次为PTD、HSA、L6、α-MSH,所述连接肽L6的氨基酸序列如SEQID NO:1所示。
2.如权利要求1所述的融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如SEQ IDNO:2所示。
3.如权利要求1或2任一所述的融合蛋白在制备治疗中枢神经炎症药物中的应用。
4.如权利要求3所述的融合蛋白在制备治疗阿尔茨海默症、帕金森症、缺血性脑卒中、多发性硬化、创伤性脑损伤、运动神经元疾病、脑部感染、抑郁症、焦虑和精神分裂症药物中的应用。
5.一种如权利要求1或2任一所述的融合蛋白的制备方法,其特征在于,所述方法包含以下步骤:
①合成所述融合蛋白的DNA序列,如SEQ ID NO:3所示;
②通过基因工程技术连接目的片段与酵母表达载体,获得含编码所述融合蛋白DNA序列的重组酵母表达载体;
③将步骤②所述的重组酵母表达载体转化到感受态大肠杆菌细胞中,从转入质粒的大肠杆菌细胞中提取质粒,再将所述质粒转化入酵母细胞中进行表达,即得所述融合蛋白。
6.如权利要求5所述的一种融合蛋白的制备方法,其特征在于,所述的酵母细胞为嗜甲醇毕赤酵母(Pichia pastoris)。
7.如权利要求6所述的一种融合蛋白的制备方法,其特征在于,所述的嗜甲醇毕赤酵母为嗜甲醇毕赤酵母PichiaPinkTM。
8.如权利要求5所述的融合蛋白的制备方法在制备治疗中枢神经炎症药物中的应用。
9.如权利要求8所述的融合蛋白的制备方法在制备治疗阿尔茨海默症、帕金森症、缺血性脑卒中、多发性硬化、创伤性脑损伤、运动神经元疾病、脑部感染、抑郁症、焦虑和精神分裂症药物中的应用。
10.如权利要求1-2任一所述融合蛋白加入药学上可接受的辅料制备成注射剂。
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