CN111808186A - 一种人源性分泌型fndc5蛋白及其制备方法和用途 - Google Patents
一种人源性分泌型fndc5蛋白及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种人源性分泌型FNDC5蛋白及其制备方法和用途,涉及基因工程技术领域;其中人源性分泌型FNDC5蛋白不含跨膜结构域,可直接分泌并释放到血液中;本发明的人源性分泌型FNDC5蛋白能用于制备治疗动脉粥样硬化药物、促进骨形成药物和治疗再生障碍性贫血药物,并且能够制备内皮细胞增殖药物。本发明的人源性分泌型FNDC5蛋白,对高脂喂养的肥胖小鼠模型空腹注射后,可有效减轻肥胖小鼠的体重,表明该蛋白可明显改善肥胖患者体脂代谢,明显降低血浆胆固醇、游离脂肪酸和甘油三脂的水平,表明该蛋白可以明显改善肥胖患者脂代谢紊乱。
Description
技术领域
本发明涉及基因工程技术领域,具体说是一种人源性分泌型FNDC5蛋白及其制备方法和用途。
背景技术
鸢尾素(irisin)是2012年发现的一种含有112个氨基酸残基的蛋白,是由一种未知蛋白酶切割III型纤连蛋白域包含蛋白5(FNDC5)的产物,鸢尾素即为FNDC5的部分切割产物。目前发现的三种FNDC5亚型均包含一段跨膜结构域(exon5),跨膜结构域将此蛋白锚定在细胞膜上,鸢尾素的序列在哺乳动物中高度保守,人和鼠、兔鸢尾素氨基酸序列完全相同;但是人源和鼠、兔(动物源)的FNDC5基因的C末端碱基顺序不同,如果动物源性FNDC5蛋白如果直接注入人体内,可能由于种属问题会出现排斥反应,人源性FNDC5蛋白除了具备动物源FNDC5蛋白所有的生物学功能外,直接注入人体不会引起排斥反应。
鸢尾素可由骨骼肌、心肌、肝脏、肾脏、神经、脂肪、胰腺等多种组织细胞产生,研究认为鸢尾素在改善肥胖,心血管疾病,骨质疏松等各种疾病方面具有良好的应用前景。目前推测鸢尾素是被一种未知的蛋白酶从FNDC5上酶切下来并释放到血液中的,但至今未发现这个蛋白酶及其确切的酶切位点,因此关于鸢尾素的特性,鉴定及鸢尾素的释放途径仍存在争议。
FNDC5作为鸢尾素的前体蛋白,目前人们对他的研究很少,不含跨膜结构域并且可直接分泌的人来源的FNDC5蛋白新亚型及其在各种疾病中的应用,目前还尚未被报道过。
发明内容
为解决上述问题,本发明的目的是提供一种人源性分泌型FNDC5蛋白及其制备方法和用途。
本发明为实现上述目的,通过以下技术方案实现:
一种人源性分泌型FNDC5蛋白,人源性分泌型FNDC5蛋白不含跨膜结构域,可直接分泌并释放到血液中,所述分泌型FNDC5蛋白的氨基酸序列为SED ID NO:1。
本发明还包括人源性分泌型FNDC5蛋白的制备方法,包括以下步骤:
⑴从FNDC5基因C末端不同位点设计引物,进行PCR,跑电泳,检测出与Pubmed已经报道的三种FNDC5异构体C末端不同的亚型(即为新的FNDC5异构体);
所述引物为:
上游引物FNDC5-F:TGAGGCCGAGAAGATGGCCTCTAA;
下游引物FNDC5-R:TAGTGACAATGGCTGCTCTCTGCC;
⑵将检测到的与三种已知的FNDC5异构体C末端不同的FNDC5亚型序列设计特异性引物,进行PCR扩增,将此PCR产物转化入DH5a感受态细胞,涂板,挑取单克隆,提取质粒,酶切确认后,转入x-33酵母菌中,YPD培养基用来扩大培养,用含0.5%甲醇的BMMH溶液来蛋白表达,最后将蛋白表达上清进行纯化,得到分泌型FNDC5蛋白;
所述特异性引物为上游引物FNDC5-F和下游引物FNDC5-R;
其中上游引物FNDC5-F:GAATTCATGCCCCCAGGGCCGTGCGCCTG;
下游引物FNDC5-R:TCAGGCCTTGGCAGAGCCCTCTGCTCTAGA。
优选的制备方法,步骤⑵中,将检测到的新的异构体蛋白对应的cDNA序列转化入DH5a感受态细胞之前,先将检测到的新的异构体蛋白对应的cDNA序列与PPIC-ZaA质粒连接。
本发明还包括上述人源性分泌型FNDC5蛋白在制备治疗肥胖代谢紊乱药物中的应用。
本发明还包括上述人源性分泌型FNDC5蛋白在制备治疗动脉粥样硬化药物中的应用。
本发明还包括上述人源性分泌型FNDC5蛋白在制备促进内皮细胞增殖药物中的应用。
本发明还包括上述人源性分泌型FNDC5蛋白在制备促进骨形成药物中的应用。
本发明还包括上述人源性分泌型FNDC5蛋白在制备治疗再生障碍性贫血药物中的应用。
本发明相比现有技术具有以下优点:
本发明的人源性分泌型FNDC5蛋白,不含跨膜结构域,能够直接分泌并释放到血液中,结构简单,获得步骤容易操作;本发明的人源性分泌型FNDC5蛋白解决了生物学对irisin来源的机理问题,以前很长时间内,我们认为irisin是从FNDC5上通过一种未知的蛋白酶酶切下来的产物,但是一直没有发现这种蛋白酶,限制了对irisin来源的研究,而本发明的人源性分泌型FNDC5蛋白表明,irisin可能就是存在可直接分泌的FNDC5亚型的一部分,不需要酶切过程。
本发明的人源性分泌型FNDC5蛋白能用于制备治疗动脉粥样硬化药物、治疗动脉粥样硬化药物、促进骨形成药物和治疗再生障碍性贫血药物,并且能够制备内皮细胞增殖药物。
本发明的人源性分泌型FNDC5蛋白,对高脂喂养的肥胖小鼠模型空腹注射后,可有效减轻肥胖小鼠的体重,表明该蛋白可明显改善肥胖患者体脂代谢,明显降低血浆胆固醇、游离脂肪酸和甘油三脂的水平,表明该蛋白可以明显改善肥胖患者脂代谢紊乱;
本发明的人源性分泌型FNDC5蛋白,可以抑制动脉粥样硬化斑块的形成,促进血管内皮细胞的增殖;本发明的人源性分泌型FNDC5蛋白,可以显著增加皮质骨骨量和强度;本发明的人源性分泌型FNDC5蛋白,在注射入再生障碍性小鼠模型后,能够明显升高并维持造血干细胞早期造血功能的基因表达水平;人脐带血内皮细胞经分泌型FNDC5重组蛋白处理后,人脐带血内皮细胞增殖能力增强,高糖诱导的凋亡减弱;本发明的分泌型FNDC5蛋白,可以作为新的治疗代谢性疾病的药物靶点进行开发,具有非常广阔的应用前景。
附图说明
图1为给药前后小鼠体重改变示意图;
图2为检测棕色脂肪相关基因(UCP-1、PRDM16、Cidea、PGC1α和aP2)表达水平示意图;
图3为分泌型FNDC5对颈总动脉部分结扎ApoE-/-小鼠颈总动脉新生内膜面积的比值示意图;
图4为分泌型FNDC5对颈总动脉部分结扎ApoE-/-小鼠颈总动脉新生中膜面积的比值示意图;
图5为免疫组织化学染色检测分泌型FNDC5对颈总动脉斑块中免疫细胞(CD3阳性巨噬细胞)的面积示意图;
图6为免疫组织化学染色检测分泌型FNDC5对颈总动脉斑块中免疫细胞(CD68阳性T细胞)的面积示意图;
图7为qRT-PCR检测分泌型FNDC5对颈总动脉斑块中炎症基因(IL-6,MCP-1,VCAM-1,ICAM-1)表达的影响示意图;
图8为分泌型FNDC5对人脐静脉内皮细胞的[3H]胸腺嘧啶掺入率的影响示意图;
图9为采用流式细胞术观察分泌型FNDC5与高糖处理24小时后人脐静脉内皮细胞的凋亡率的影响示意图;
图10为小鼠胫骨三点弯曲试验结果示意图;
图11为分泌型FNDC5蛋白对GATA-2基因表达水平的结果示意图。
具体实施方式
本发明的目的是提供一种人源性分泌型FNDC5蛋白及其制备方法和用途,以下结合具体实施例来对本发明作进一步的描述。
本发明中分泌型FNDC5蛋白、人源性分泌型FNDC5蛋白和FNDC5蛋白均是人源性分泌型FNDC5蛋白的不同表达形式,均是一种蛋白。
本发明所用PPIC_ZaA质粒购买于美国Invitrogen公司。
实施例1
一种人源性分泌型FNDC5蛋白,不含跨膜结构域,可直接分泌并释放到血液中,所述人源性分泌型FNDC5蛋白的氨基酸序列为SED ID NO:1:
IHPGSPSAWPPRARAALRLWLGCVCFALVQADSPSAPVNVTVRHLKANSAVVSWDVLEDEVVIGFAISQQKKDVRMLRFIQEVNTTTRSCALWGLHILPSHPGFHESYPGSSCPSLEPEWCEVTGLSLHCPFLV;
实施例2
实施例1所述的人源性分泌型FNDC5蛋白的制备方法,包括以下步骤:
1.从FNDC5基因C末端不同位点设计引物,进行PCR,跑电泳,检测出与Pubmed已经报道的三种已知的FNDC5异构体C末端不同的FNDC5亚型(即为新的FNDC5异构体),具体的:
所述引物为:
上游引物FNDC5-F:TGAGGCCGAGAAGATGGCCTCTAA;
下游引物FNDC5-R:TAGTGACAATGGCTGCTCTCTGCC;
提取Blox5细胞RNA,逆转录成cDNA,使用上述引物做PCR;
PCR反应体系:
H2O:36.75μl
Go Taq DNA Polymerase:0.25μl(Promega公司)
5x Go Taq Buffer:10μl(Promega公司)
FNDC5-F:1μl
FNDC5-R:1μl
cDNA:1μl
Total:50μl
94℃5分钟;
94℃30秒,56℃30秒,72℃1分钟;30循环;
72℃7分钟;
PCR反应产物跑电泳,发现除了原来已知大小的FNDC5基因条带,还有一条比他们短的条带;
送公司测序,测序结果为一条新的来源于FNDC5基因的序列。
2.根据检测到的新的FNDC5异构体序列设计特异性引物,进行PCR扩增,将此PCR产物转化入DH5a感受态细胞,涂板,挑取单克隆,提取质粒,酶切确认后,转入x-33酵母菌中,YPD培养基用来扩大培养,用含0.5%甲醇的BMMH溶液来蛋白表达,最后将蛋白表达上清进行纯化,得到分泌型FNDC5蛋白;具体的:
2.1PCR扩增分泌型FNDC5:
特异性引物为:
上游引物FNDC5-F:GAATTCATGCCCCCAGGGCCGTGCGCCTG;
下游引物FNDC5-R:TCAGGCCTTGGCAGAGCCCTCTGCTCTAGA;
反应体系与步骤⑴中PCR反应体系相同;
2.2酶切PPIC-ZaA质粒和步骤①中PCR产物
酶切体系:
2.3目的基因回收(美国MpBio公司试剂盒:Geneclean II Kit)
2.3.1.切下含有目的基因DNA片段的凝胶;
2.3.2.称重;
2.3.3.加入3倍体积的NaI,混匀,55℃水浴5分钟,待胶全部化掉;
2.3.4.加入10μl Glassmilk,混匀,室温下放置5min;
2.3.5.14000rpm,5s,弃上清;
2.3.6.加入500μl New wash,重悬,14000rpm,5s,弃上清;
2.3.7.加入500μl New wash,重悬,14000rpm,5s,弃上清;
2.3.8.室温放置5min,以挥发残存的酒精;
2.3.9.加入ddH2O(10μl)溶解DNA;
2.3.10.离心14000rpm,1min,取上清至新EP管中,即为目的基因的PCR回收产物,测DNA浓度即可。
上述试剂均为试剂盒内试剂。
2.4回收基因连接
10μl连接体系:
目的基因回收产物1(酶切分泌型FNDC5) 2.5μl
目的基因回收产物2(酶切PPIC_ZaA质粒) 2.5μl
T4连接酶5μl
连接产物4℃过夜。
2.5将步骤④中的连接产物转化到感受态细胞中:
2.5.1.取50μl感受态DH5a或TOP10细胞置于冰上融化;
2.5.2.将连接产物加入到融化后的DH5a或TOP10细胞中,轻微上下颠倒混匀,然后冰上放置30min;
2.5.3.将带有DH5a或TOP10细胞的离心管放在42℃加热器上,90s后再次转移到冰上放置3min;
2.5.4.向上述离心管中加入300μl无菌无抗生物的LB培养基,37℃180rpm振荡培养1h;
2.5.5.取适量已转化完毕的DH5a或TOP10细胞涂布于含氨苄的LB培养板上,37℃倒置培养12-16h;
2.6挑取单克隆,提取质粒(凯杰公司质粒提取试剂盒)
2.6.1柱平衡步骤:将500μl BL加入到CP4中,15000rpm离心1min,然后将管中废液倒除;
2.6.2.将培养的菌液倒入离心管中,15000rpm离心1min,除去上清液;
2.6.3.将500μl含RNaseA溶液PI加入带有菌体的离心管中,用涡旋振荡器悬浮细胞沉淀;
2.6.4.将500μl P2加入3中,轻轻的上下翻转10-12次;
2.6.5.将700μl P3加入4中,轻轻地上下翻转10-12次,至出现白色絮状沉淀,然后离心,将白色絮状物离至管底;
2.6.6.将5中的上清倒入Cs中,然后15000rpm离心3min,将得到的溶液加入到CP4中;
2.6.7.15000rpm离心3min,将CP4放入收集管中;
2.6.8将500μl PD加入到CP4中,15000rpm离心3min,再次将CP4放入收集管中;
2.6.9.将600μl PW加入到CP4中,15000rpm离心3min,再次将CP4放入收集管中;
2.6.10.重复9;
2.6.11.将CP4放回收集管中,15000rpm离心3min;
2.6.12.向CP4中吸附膜的中间位置加入200μlTB,放于室温3min,然后,15000rpm离心2min,所得到的溶液即PPIC-sFNDC5重组质粒溶液,然后测浓度。
文中BL、CP4、含RNaseA溶液PI、P2、P3、Cs、PD、PW等均为试剂盒中配套溶液。
2.7将PPIC-sFNDC5重组质粒转入x-33酵母菌中
2.7.1感受态x-33酵母菌的制备
2.7.1.1在50ml离心管中加入10mlYPD培养基,接种x-33酵母菌种,30℃300rpm振荡过夜;
2.7.1.2室温离心500g,5min,弃上清,用10ml新鲜YPD培养基重悬X-33酵母,30℃300rpm振荡4-6h,使OD值达到0.6-1.0;
2.7.1.3室温离心500g,5min,弃上清;
2.7.1.4用10ml EasyComp Transformation Kit中试剂1重悬细胞;
2.7.1.5室温离心500g,5min,弃上清;
2.7.1.6用1ml试剂1重悬细胞,得到感受态x-33细胞;
2.7.2将PPIC-sFNDC5质粒转入感受态x-33细胞
2.7.2.1取50μl感受态X-33细胞置于冰上融化;
2.7.2.2向感受态细胞中加入3ugPPIC-sFNDC5重组质粒;
2.7.2.3再加入1ml EasyComp Transformation Kit中试剂2,震荡混匀;
2.7.2.4将混合物置于37度水浴1小时,每15min震荡混匀一次;
2.7.2.5 42℃水浴10min;
2.7.2.6将细胞平均分至两个离心管中,并将1ml YPD培养基加入其中;
2.7.2.7 30℃孵育1h以恢复zeocin活性;
2.7.2.8 3000g室温离心5min,弃上清;
2.7.2.9每管用500μl EasyComp Transformation Kit中试剂重悬细胞,并将两管细胞合至一管中;
2.7.2.10 3000g室温离心5min,弃上清;
2.7.2.11用100~150μl EasyComp Transformation Kit中试剂重悬细胞;
2.7.2.12将细胞抹匀在含100mg/ml zeocin的YPD板上,倒置,30℃培养3~5天;
2.7.3菌落PCR验证PPIC-sFNDC5质粒转入
2.7.3.1 10%SDS配置:称取1gSDS(十二烷基硫酸钠)加入10ml ddH2O中,混匀,使其完全溶解;
2.7.3.2 0.2%SDS:取20μl 10%SDS溶液加980μl水,配成1ml0.2%的SDS液。
挑取几个菌落,分别至装有100μl 0.2%SDS的EP管中,混匀,100℃煮沸5min,然后置于冰上,涡旋震匀。
反应条件:94℃4分钟,94℃30秒,54℃30秒,72℃1分钟,72℃5分钟,30循环。
其中引物包括上游引物FNDC5-F和下游引物FNDC5-R:
上游引物FNDC5-F:ATGCCCCCAGGGCCGTGCGCCTG;
下游引物FNDC5-R:TCAGGCCTTGGCAGAGCCCTCTGC;
2.8FNDC5的表达与确认
2.8.1挑取单克隆,在10mlYPD培养基中摇18h,然后换至500mlYPD培养基中摇48h扩大培养;
2.8.2 3000g室温离心15min,用200ml含0.5%甲醇的BMMH培养基(有组氨酸的缓冲性基础甲醇培养基)重悬细胞,促进蛋白的表达;
2.8.3每24h加入0.5%甲醇,每24h取样,摇菌6d结束。
2.9重组分泌型FNDC5蛋白纯化
2.9.1重组分泌型FNDC5用60%饱和硫酸铵沉淀;
2.9.2沉淀的蛋白用溶液A(25mmol/L HEPES,pH 7.9,10%甘油,0.1mol/L KCl,0.2mmol/L EDTA,and 0.5mmol/L DTT)复溶;
2.9.3在溶液A中透析48h;
2.9.4透析后的蛋白液上至concannavalin-A凝胶柱;
2.9.5洗脱:凝胶柱用含1.5mol/L KCl的溶液A洗脱;
2.9.6洗脱得到的蛋白低温冻干后,放于-80℃,备用。
实施例3
人源性分泌型FNDC5蛋白在制备治疗肥胖相关代谢紊乱药物中的应用
肥胖模型的建立:将6周龄C57BL/6雄性小鼠随机分为3组,每组6只:
第一组:正常饮食小鼠-生理盐水组:为阴性对照组,始终给予正常饮食,12周后腹腔注射生理盐水2周;
第二组:肥胖小鼠-生理盐水组:为阳性对照组,给予高脂饲料饮食制造肥胖小鼠模型,12周后腹腔注射生理盐水2周;
第三组:肥胖小鼠-分泌型FNDC5组:为实验组,给予高脂饲料制造肥胖小鼠模型,12周后腹腔注射分泌型FDNC5蛋白2周。
给药方式及给药浓度:将分泌型FNDC5蛋白按0.5ug/g/天浓度,采用腹腔注射的方式给药,每天给药,连续给药2周。
小鼠体重检测:给药处理2周后测量小鼠体重,如图1所示,比起阴性对照组,阳性对照组的体重明显增加,而给予分泌型FNDC5蛋白处理后,小鼠体重有所降低。
血脂检测:小鼠处死前使用异氟烷麻醉小鼠,采用眼球取血的方式进行采血,室温静置1小时,300g离心15分钟,取上层血清送公司进行血脂检测。结果表1所示,阳性对照组血清中总胆固醇、甘油三酯和游离脂肪酸明显高于阴性对照组,实验组血清中三个反应血脂水平的指标与阳性对照组相比明显降低。
表1血清脂质代谢结果表
检测指标 | 阴性对照组 | 阳性对照组 | 实验组 |
胆固醇 | 2.10±0.03 | 4.60±0.23<sup>###</sup> | 3.63±0.16** |
甘油三酯 | 1.82±0.28 | 2.52±0.17 | 1.18±0.24** |
游离脂肪酸 | 0.37±0.07 | 0.87±0.04<sup>###</sup> | 0.52±0.06* |
数据均以均值±标准误(mean±SE)表示,每组样本有9个,*P<0.05有统计学差异,#与正常饮食-生理盐水处理组(阴性对照组)相比;*与高脂饮食-生理盐水处理组(阳性对照组)相比。
分泌型FNDC5对白色脂肪棕色化的影响:上述三组不同处理组小鼠处死后取腹股沟皮下白色脂肪组织,采用RT-qPCR(实时定量PCR)方法,检测棕色脂肪相关基因(UCP-1、PRDM16、Cidea、PGC1α和aP2)表达水平。
如图2所示,阳性对照组皮下白色脂肪表达棕色脂肪相关基因水平明显低于阴性对照组,实验组小鼠棕色脂肪相关基因表达水比阳性对照组明显升高。
实施例4
人源性分泌型FNDC5蛋白在制备治疗动脉粥样硬化药物中的应用
将10只8周龄的ApoE-/-小鼠行左侧颈总动脉部分结扎,即用6-0的缝合线结扎左侧颈内动脉、颈外动脉及枕动脉,仅保留甲状腺上动脉。结扎24小时后,将小鼠随机分成2组,每组5只,实验组分别给予0.5ug/g/天的分泌型FNDC5,对照组给予等量的生理盐水腹腔注射,术后继续高脂喂养4周。
实验结束后,用过量的戊巴比妥钠腹腔注射麻醉使其安乐死。然后将小鼠仰卧位固定于操作台上,先用手术剪剪开胸腔,暴露出心脏,用1ml注射器于左心室穿刺取血。取血后将输液器的针头插入左心室并固定,然后剪开右心耳,用生理盐水持续灌洗,当暗红色的肝脏逐渐变为黄白色时,即认为已将心脏及血管内血液冲洗干净,然后用4%多聚甲醛溶液灌注内固定约20分钟。灌注后,在解剖镜下仔细分离颈总动脉,然后钝性分离主动脉,去除周围组织。在主动脉弓处将颈总动脉和主动脉分离;在升主动脉处用显微剪将心脏和主动脉分离。主动脉及颈总动脉放于4%多聚甲醛溶液中持续固定超过24小时,进行切片染色。
结果如图3所示,在颈总动脉部分结扎的ApoE-/-小鼠中,与对照组相比,腹腔注射分泌型FNDC5 4周能显著减少新生内膜的形成,降低新生内膜面积。
结果如图4所示,在颈总动脉部分结扎的ApoE-/-小鼠中,与对照组相比,腹腔注射分泌型FNDC5 4周能显著减少新生内膜的形成,降低中膜面积的比值。
将分离出的颈总动脉进行免疫组化染色,检测小鼠颈总动脉斑块中CD3阳性巨噬细胞和CD68阳性T细胞的面积,结果如图5所示,分泌型FNDC5组的小鼠颈总动脉斑块中CD3阳性巨噬细胞较对照组小鼠明显减少,如图6所示,分泌型FNDC5组的小鼠颈总动脉斑块中CD68阳性T细胞的面积较对照组小鼠明显减少。
将分离出的颈总动脉采用RT-qPCR方法进行基因表达水平检测,结果如图7所示,野生型即为非颈总动脉部分结扎的ApoE-/-小鼠,由图7可以看出,分泌型FNDC5可以明显降低ApoE-/-小鼠颈总动脉斑块中IL-6、MCP-1、ICAM-1、VCAM-1的mRNA水平。
实施例5
人源性分泌型FNDC5蛋白在制备促进内皮细胞增殖药物中的应用。
目前,世界心血管疾病发病率越来越高,严重威胁人民群众健康。而大多数血管病伴随内皮细胞(ECs)的损伤和功能失调。在多种慢性代谢性疾病,如代谢综合征,II型糖尿病等,均伴随内皮细胞的损伤和功能障碍。血管内膜的完整性是血管发挥功能的保障和前提条件,而在许多慢性代谢性疾病中,血管内膜的完整性受到内皮细胞增殖与凋亡的影响。基于此,通过调节内皮细胞的增殖和凋亡修复损伤后的内皮细胞,从而修复损伤的血管内膜,使其恢复完整性对许多代谢性疾病引起的血管病的转归具有重要意义。因此,在培养人脐静脉内皮细胞过程中,用50nM剂量的分泌型FNDC5进行处理40小时。
①分泌型FNDC5对人脐静脉内皮细胞增殖的影响。为研究irisin对人脐静脉内皮细胞增殖的作用,采用了[3H]胸腺嘧啶掺入实验。结果如图8所示,与对照组相比,无血清条件下分泌型FNDC5处理,增加了人脐静脉内皮细胞的[3H]胸腺嘧啶掺入率。
②分泌型FNDC5减少了高糖诱导的人脐静脉内皮细胞的凋亡。
采用流式细胞术观察分泌型FNDC5与高糖处理24小时后人脐静脉内皮细胞的凋亡。结果如图9所示,分泌型FNDC5有效减少了高糖诱导的人脐静脉内皮细胞的凋亡。分泌型FNDC5处理24小时后,细胞凋亡率从高糖条件下的33.8±3.2%下降到22.0±2.4%。
实施例6
人源性分泌型FNDC5蛋白在制备促进骨形成(增殖)药物中的应用
体育锻炼对新陈代谢和骨骼健康有广泛的益处,并经常被用作各种疾病治疗方案的非药物干预。身体活动水平的降低,例如前运动员,会导致骨质的逐渐流失。虽然体育活动与骨骼的维持之间存在明显的联系,但肌肉是如何调节骨量的问题在很大程度上仍未解决。
将10只6周龄C57BL/6雄性小鼠随机分为2组,一组腹腔注射人源性分泌型FNDC5,另一组给予同等剂量的生理盐水进行腹腔注射。
给药方式及给药浓度:将分泌型FNDC5蛋白按0.5ug/g浓度,采用腹腔注射的方式给药,每周给药2次,连续给药4周。结果如图10所示,胫骨三点弯曲试验表明,比起生理盐水处理组,分泌型FNDC5处理组胫骨弯曲强度显著提高。
实施例7
人源性分泌型FNDC5蛋白在制备治疗再生障碍性贫血药物中的应用
再生障碍性贫血是一组由多种病因所致的骨髓造血功能衰竭性综合征,以骨髓造血细胞增生减低和外周血全血细胞减少为特征,临床以贫血、出血和感染为主要表现。研究表明,许多转录因子参与造血干细胞早期造血功能,体内外研究表明GATA-2在造血干细胞早期造血过程中是必不可少的一种转录因子。
建立再生障碍性贫血小鼠模型(AA小鼠模型构建:根据前期试验数据,6Gy TBI联合6h内父系(C57BL/6)淋巴细胞输注1×107个/只,第6天成模),10只小鼠随机分为两组,一组腹腔注射人源性分泌型FNDC5,另一组给予同等剂量的生理盐水进行腹腔注射。
给药方式及给药浓度:将分泌型FNDC5蛋白按0.5ug/g浓度,采用腹腔注射的方式给药,每天给药,连续给药10天。小鼠处死前使用异氟烷麻醉小鼠,剥离小鼠股骨和胫骨,用生理盐水冲洗小鼠骨髓腔,磁珠法分离出CD38阳性造血干细胞,实时定量PCR法检测细胞GATA-2表达水平。结果如图11所示,比起生理盐水处理组,小鼠骨髓造血干细胞GATA-2表达水平在分泌型FNDC5处理组明显升高。
序列表
<110> 山东大学第二医院
<120> 一种人源性分泌型FNDC5 蛋白及其制备方法和用途
<130> 20200720A-1
<141> 2020-07-20
<150> 2019113179559
<151> 2019-12-19
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 134
<212> PRT
<213> 智人(Homo sapiens)
<400> 1
Ile His Pro Gly Ser Pro Ser Ala Trp Pro Pro Arg Ala Arg Ala Ala
1 5 10 15
Leu Arg Leu Trp Leu Gly Cys Val Cys Phe Ala Leu Val Gln Ala Asp
20 25 30
Ser Pro Ser Ala Pro Val Asn Val Thr Val Arg His Leu Lys Ala Asn
35 40 45
Ser Ala Val Val Ser Trp Asp Val Leu Glu Asp Glu Val Val Ile Gly
50 55 60
Phe Ala Ile Ser Gln Gln Lys Lys Asp Val Arg Met Leu Arg Phe Ile
65 70 75 80
Gln Glu Val Asn Thr Thr Thr Arg Ser Cys Ala Leu Trp Gly Leu His
85 90 95
Ile Leu Pro Ser His Pro Gly Phe His Glu Ser Tyr Pro Gly Ser Ser
100 105 110
Cys Pro Ser Leu Glu Pro Glu Trp Cys Glu Val Thr Gly Leu Ser Leu
115 120 125
His Cys Pro Phe Leu Val
130
Claims (8)
1.一种人源性分泌型FNDC5蛋白,其特征在于:人源性分泌型FNDC5蛋白不含跨膜结构域,可直接分泌并释放到血液中,所述分泌型FNDC5蛋白的氨基酸序列为SED ID NO:1。
2.权利要求1所述的人源性分泌型FNDC5蛋白的制备方法,其特征在于:包括以下步骤:
⑴从FNDC5基因C末端不同位点设计引物,进行PCR,跑电泳,检测出与三种已知的FNDC5异构体C末端不同的FNDC5亚型;
所述引物为:
上游引物FNDC5-F:TGAGGCCGAGAAGATGGCCTCTAA;
下游引物FNDC5-R:TAGTGACAATGGCTGCTCTCTGCC;
⑵根据检测到的与三种已知的FNDC5异构体C末端不同的FNDC5亚型序列设计特异性引物,进行PCR扩增,将此PCR产物转化入DH5a感受态细胞,涂板,挑取单克隆,提取质粒,酶切确认后,转入x-33酵母菌中,YPD培养基用来扩大培养,用含0.5%甲醇的BMMH溶液来蛋白表达,最后将蛋白表达上清进行纯化,得到分泌型FNDC5蛋白;
所述特异性引物为上游引物FNDC5-F和下游引物FNDC5-R;
其中上游引物FNDC5-F:GAATTCATGCCCCCAGGGCCGTGCGCCTG;
下游引物FNDC5-R:TCAGGCCTTGGCAGAGCCCTCTGCTCTAGA。
3.根据权利要求2所述的人源性分泌型FNDC5蛋白的制备方法,其特征在于:步骤⑵中,将检测到的新的异构体蛋白对应的cDNA序列转化入DH5a感受态细胞之前,先将检测到的新的异构体蛋白对应的cDNA序列与PPIC-ZaA质粒连接。
4.权利要求1所述的人源性分泌型FNDC5蛋白的应用,其特征在于:在制备治疗肥胖代谢紊乱药物中的应用。
5.权利要求1所述的人源性分泌型FNDC5蛋白的应用,其特征在于:在制备治疗动脉粥样硬化药物中的应用。
6.权利要求1所述的人源性分泌型FNDC5蛋白的应用,其特征在于:在制备促进内皮细胞增殖药物中的应用。
7.权利要求1所述的人源性分泌型FNDC5蛋白的应用,其特征在于:在制备促进骨形成药物中的应用。
8.权利要求1所述的人源性分泌型FNDC5蛋白的应用,其特征在于:在制备治疗再生障碍性贫血药物中的应用。
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