CN103694340A - 重组蛋白igf1-24及其应用 - Google Patents
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Abstract
本发明公开了一种重组蛋白IGF1-24,氨基酸序列如SEQ ID No.2所示,是将IGF-1序列的C端与MGF-24序列的N端通过3个GLY连接子连接而得;研究结果显示,该重组蛋白能够促进成肌细胞的增殖、分化、粘附和迁移,能够促进成骨细胞的增殖和迁移,能够促进神经细胞的损伤修复,还能够促进心肌细胞的增殖,且作用明显强于IGF-1和MGF-24,可制成药物,用于治疗肌肉、骨、神经、心肌损伤等相关疾病,为改善患者生存质量发挥积极、重要的推动作用。
Description
技术领域
本发明属于基因工程技术领域,涉及一种重组蛋白及其制药用途。
背景技术
胰岛素样生长因子-1(IGF-1)是胰岛素家族的一种单链多肽类生长因子,其结构与胰岛素有高度同源性,具有细胞分化、增殖功能和胰岛素样代谢作用。对IGF-1的研究发现其在生长障碍、糖尿病、骨质疏松症、肾脏疾病、心血管疾病、肿瘤及中枢神经系统等方面都起到重要作用。
力生长因子(mechano growth factor,MGF)是IGF-1的一种剪接异构体。肌肉在受到机械刺激后,IGF-1基因会选择性剪接产生MGF,通过激活肌肉卫星细胞和合成代谢途径来修复局部肌肉损伤。MGF之所以具有不同于IGF-1其他异构体的功能,部分是由于MGF C末端的24个氨基酸(MGF-24),又称为MGF E肽,其被证明具有激活人类肌肉卫星细胞、促进肌元细胞增殖等功能,但确切的分子作用机制目前尚不清楚。
发明内容
有鉴于此,本发明的目的之一在于对IGF-1和MGF-24进行结构改造,以期获得一种具有更强活性的重组蛋白;目的之二在于提供该重组蛋白在制药领域中的应用。
为达到上述目的,经研究,本发明提供如下技术方案:
1.重组蛋白IGF1-24,氨基酸序列如SEQ ID No.2所示。
2.编码所述重组蛋白IGF1-24的基因。
优选的,所述基因的核苷酸序列如SEQ ID No.1所示。
3.含有重组蛋白IGF1-24编码基因的重组表达载体。
4.含有IGF1-24基因重组表达载体的微生物转化体。
5.重组蛋白IGF1-24在制备促进肌肉生长和损伤修复的药物中的应用。
6.重组蛋白IGF1-24在制备促进骨生长和损伤修复的药物中的应用。
7.重组蛋白IGF1-24在制备促进神经细胞损伤修复的药物中的应用。
8.重组蛋白IGF1-24在制备促进心肌修复的药物中的应用。
本发明的有益效果:本发明将IGF-1序列的C端与MGF-24序列的N端通过3个GLY连接子连接,构建了重组蛋白IGF1-24。研究结果显示,该重组蛋白能够促进成肌细胞的增殖、分化、粘附和迁移,能够促进成骨细胞的增殖和迁移,能够促进神经细胞的损伤修复,还能够促进心肌细胞的增殖,且作用明显强于IGF-1和MGF-24,可制成药物,用于治疗肌肉、骨、神经、心肌损伤等相关疾病,为改善患者生存质量发挥积极、重要的推动作用。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为重组蛋白IGF1-24诱导表达产物和纯化产物的SDS-PAGE结果,其中1为转染pGEX-4T-1/IGF1-24大肠杆菌的诱导表达产物,2为纯化蛋白。
图2为重组蛋白IGF1-24促进成肌细胞C2C12增殖。
图3为重组蛋白IGF1-24促进成肌细胞C2C12分化标记蛋白Myogenin的表达。
图4为重组蛋白IGF1-24促进成肌细胞C2C12融合。
图5为重组蛋白IGF1-24促进成肌细胞C2C12粘附。
图6为重组蛋白IGF1-24促进成肌细胞C2C12迁移。
图7为重组蛋白IGF1-24对C2C12细胞增殖的影响依赖于IGF-1受体。
图8为重组蛋白IGF1-24对C2C12细胞粘附的影响不依赖于IGF-1受体。
图9为重组蛋白IGF1-24对C2C12细胞迁移的影响不依赖于IGF-1受体。
图10为重组蛋白IGF1-24促进大鼠成骨细胞增殖。
图11为重组蛋白IGF1-24促进大鼠成骨细胞迁移。
图12为重组蛋白IGF1-24促进人神经母细胞瘤细胞SH-SY5Y的损伤修复。
图13为重组蛋白IGF1-24促进大鼠心肌细胞H9C2增殖。
具体实施方式
下面将结合附图,对本发明的优选实施例进行详细的说明。优选实施例中未注明具体条件的实验方法,通常按照常规条件,或按照试剂制造厂商所建议的条件进行。
实施例1、重组蛋白IGF1-24的获得
采用overlap PCR方法扩增IGF1-24基因。PCR引物序列如下:
IGF1-24a F:5'CGCGGATCCGGACCGGAGACGCTCTGC3'(SEQ ID No.3);
IGF1-24b R:5'CTGATAACCGCCACCAGCTGACTTGGCAGGCT3'(SEQ ID No.4);
IGF1-24c F:5'GGTGGCGGTTATCAGTATCAGCCCCCATCTA3'(SEQ ID No.5);
IGF1-24d R:5'CCGCTCGAGCTACTTGTGTTCTTCAAATGT3'(SEQ ID No.6)。
以来自于人IGF-1Ec的cDNA(GenBank Ax147742)为模板,用引物IGF1-24a F和IGF1-24bR扩增IGF1基因,用引物IGF1-24c F和IGF1-24d R扩增MGF-24基因;再以所得IGF1基因和MGF-24基因为共同模板,用引物IGF1-24a F和IGF1-24d R进行PCR扩增,获得两端带有BamHI和XhoI酶切位点的IGF1-24基因。将该DNA片段用BamHI和XhoI双酶切后,与同样经BamHI和XhoI双酶切的质粒pGEX-4T-1连接,获得重组质粒pGEX-4T-1/IGF1-24。
将空质粒pGEX-4T-1及重组质粒pGEX-4T-1/IGF1-24分别转化大肠杆菌,用0.2mM IPTG于20℃诱导表达3-5小时,离心收集菌体,超声波破碎,离心收集沉淀,SDS-PAGE检测蛋白表达情况,再用glutathione-Sepharose4B柱对蛋白进行纯化,SDS-PAGE检测蛋白纯化情况。结果见图1,转染pGEX-4T-1/IGF1-24大肠杆菌的诱导表达产物在37Kd处显示明显的蛋白条带,与重组蛋白IGF1-24的分子量一致,说明成功诱导表达了重组蛋白IGF1-24;纯化蛋白除了37Kd处的主带以外,几乎不存在其它杂带,表明重组蛋白IGF1-24纯化成功,可用于后续实验。
实施例2、重组蛋白IGF1-24促进成肌细胞的增殖、分化、粘附和迁移
将小鼠成肌细胞C2C12用含10%胎牛血清的DMEM培养基,在温度37℃、饱和湿度、CO2气体体积分数为0.05的条件下孵育。
将C2C12细胞接种96孔板,培养过夜,再用含有2%胎牛血清与50ng/mL IGF1-24的DMEM高糖培养基培养细胞,每天换液,48小时后CCK-8检测细胞增殖情况,同时分别设置空白对照、IGF-1对照和MGF-24对照。结果如图2所示,重组蛋白IGF1-24能够促进C2C12细胞的增殖,作用与IGF-1相当,而MGF-24几乎不促进C2C12细胞的增殖。
将C2C12细胞接种96孔板,培养过夜,再用含有2%胎牛血清与50ng/mL IGF1-24的DMEM高糖分化诱导培养基培养细胞,每天换液,5天后收集细胞,裂解,离心收集蛋白,western blot检测肌分化标记蛋白Myogenin的表达,同时分别设置空白对照、IGF-1对照和MGF-24对照。结果如图3所示,经IGF1-24处理后,肌分化标记蛋白Myogenin的表达上调,且上调量明显高于IGF-1对照组和MGF-24对照组。
将C2C12细胞接种96孔板,培养过夜,再用含有2%胎牛血清与50ng/mL IGF1-24的DMEM高糖分化诱导培养基培养细胞,每天换液,5天后固定细胞,与荧光标记的肌球蛋白重链(MyHC)抗体共孵育,荧光下观察MyHC的表达及细胞的融合状态,计算细胞融合率,同时分别设置空白对照、IGF-1对照和MGF-24对照。结果如图4所示,经IGF1-24处理后,肌细胞特异标志物MyHC的表达量上调,细胞融合率约为空白对照组的4倍,且高于IGF-1对照组和MGF-24对照组。
将96孔板用10μg/mL Fn于4℃包被过夜,再用1%BSA室温封闭1小时;将密度为5×104/mL的C2C12细胞悬浮液添加50ng/mL的IGF1-24预处理30分钟,再接种至封闭处理后的96孔板,37℃孵育10分钟,PBS洗去未粘附的细胞,CCK-8检测细胞粘附能力,同时分别设置空白对照、IGF-1对照和MGF-24对照。结果如图5所示,重组蛋白IGF1-24能够促进C2C12细胞的粘附,且作用明显强于IGF-1和MGF-24。
取24-well Transwell(8.0mm pore size)板,在含100μL无血清培养基的上室中接种5×104个C2C12细胞,在含500μL2%血清培养基的下室中添加50ng/mL IGF1-24,进行细胞迁移实验,6小时后用棉签擦去上室中未迁移的细胞,迁移的细胞用结晶紫染色并照相计数,同时分别设置空白对照、IGF-1对照和MGF-24对照。结果如图6所示,重组蛋白IGF1-24能促进C2C12细胞的迁移,其作用与MGF-24相当,而强于IGF-1。
用5μg/mL IGF-1受体抑制剂PQ401(Sigma)预处理C2C12细胞,再按照前述方法检测重组蛋白IGF1-24对细胞增殖、粘附和迁移的影响。结果如图7-9所示,抑制IGF-1受体后,IGF1-24对细胞的增殖作用消失,表明IGF1-24对C2C12细胞增殖的影响依赖于IGF-1受体;而抑制IGF-1受体后,IGF1-24仍然能够促进C2C12细胞的粘附和迁移,表明IGF1-24对C2C12细胞粘附和迁移的影响不依赖于IGF-1受体。
上述实验结果显示,重组蛋白IGF1-24能够促进成肌细胞的增殖、分化、粘附和迁移,可作为一种损伤修复因子,用于肌肉萎缩、肌营养不良和杜氏肌营养不良症等疾病的治疗。
实施例3、重组蛋白IGF1-24促进成骨细胞的增殖和迁移
通过组织块法从新生大鼠颅骨中获得成骨细胞,用含10%胎牛血清、100IU/mL青霉素和100μg/mL链霉素的DMEM/F12培养基,在温度37℃、饱和湿度、CO2气体体积分数为0.05的条件下孵育。
将成骨细胞接种96孔板,培养过夜,再用含50ng/mL IGF1-24的无血清DMEM/F12培养基培养细胞,每天换液,48小时后CCK-8检测细胞增殖情况,同时设置空白对照。结果如图10所示,重组蛋白IGF1-24能够显著促进成骨细胞的增殖。
取24-well Transwell(8.0mm pore size)板,在含100μL无血清培养基的上室中接种5×104个成骨细胞,在含500μL2%血清培养基的下室中添加50ng/mL IGF1-24,进行细胞迁移实验,6小时后用棉签擦去上室中未迁移的细胞,迁移的细胞用结晶紫染色并照相计数。结果如图11所示,重组蛋白IGF1-24能够显著促进成骨细胞的迁移。
上述实验结果显示,重组蛋白IGF1-24能够促进成骨细胞的增殖和迁移,从而可用于骨损伤以及骨相关疾病如骨质疏松等的治疗。
实施例4、重组蛋白IGF1-24促进神经细胞的损伤修复
将人神经母细胞瘤细胞SH-SY5Y用含10%胎牛血清、100IU/mL青霉素和100μg/mL链霉素的DMEM/F12培养基,在温度37℃、饱和湿度、CO2气体体积分数为0.05的条件下孵育。
将SH-SY5Y细胞接种96孔板,培养过夜,用神经毒素6-hydroxydopamine(6-OHDA)和IGF1-24共处理24小时,检查细胞存活情况,同时设置6-OHDA单独处理对照。结果如图12所示,6-OHDA单独处理可造成约55%的细胞损伤,而共孵育IGF1-24后,细胞损伤减少,存活率达到90%左右。
将神经细胞SH-SY5Y接种96孔板,培养过夜,用1-methyl-4-phenylpyridinium(MPP+)和IGF1-24共处理48小时,检查细胞存活情况,同时设置MPP+单独处理对照。结果如图12所示,MPP+单独处理可造成约60%的细胞损伤,而共孵育IGF1-24后,细胞损伤减少,存活率达到95%左右。
上述实验结果表明,重组蛋白IGF1-24可作为一种神经营养因子,参与缺血、缺氧后神经损伤的修复过程,对受累神经细胞起到保护作用,可用于神经损伤相关疾病如脑梗死、帕金森病等的治疗。
实施例5、重组蛋白IGF1-24促进心肌细胞的增殖
将大鼠心肌细胞H9C2用含10%胎牛血清、100IU/mL青霉素和100μg/mL链霉素的DMEM/F12培养基,在温度37℃、饱和湿度、CO2气体体积分数为0.05的条件下孵育。
将心肌细胞H9C2接种96孔板,培养过夜,用含50ng/mL IGF1-24的无血清DMEM/F12培养基培养细胞,每天换液,48小时后CCK-8检测细胞增殖情况,同时设置空白对照。结果如图13所示,重组蛋白IGF1-24能够显著促进心肌细胞的增殖,从而可用于心脏保护和心肌损伤相关疾病如心肌梗死等的治疗。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
Claims (9)
1.重组蛋白IGF1-24,其特征在于,氨基酸序列如SEQ ID No.2所示。
2.编码权利要求1所述重组蛋白IGF1-24的基因。
3.如权利要求2所述的基因,其特征在于,核苷酸序列如SEQ ID No.1所示。
4.含有权利要求2或3所述基因的重组表达载体。
5.含有权利要求4所述重组表达载体的微生物转化体。
6.权利要求1所述重组蛋白IGF1-24在制备促进肌肉生长和损伤修复的药物中的应用。
7.权利要求1所述重组蛋白IGF1-24在制备促进骨生长和损伤修复的药物中的应用。
8.权利要求1所述重组蛋白IGF1-24在制备促进神经细胞损伤修复的药物中的应用。
9.权利要求1所述重组蛋白IGF1-24在制备促进心肌修复的药物中的应用。
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