CN114507290A - 一种来源于截短型人SirT2蛋白的融合蛋白、其制备方法及应用 - Google Patents
一种来源于截短型人SirT2蛋白的融合蛋白、其制备方法及应用 Download PDFInfo
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- CN114507290A CN114507290A CN202011278356.3A CN202011278356A CN114507290A CN 114507290 A CN114507290 A CN 114507290A CN 202011278356 A CN202011278356 A CN 202011278356A CN 114507290 A CN114507290 A CN 114507290A
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Abstract
本发明公开了包含SirT2蛋白的融合蛋白的制备方法,所述融合蛋白是来源于SirT2蛋白上的一段氨基酸序列与细胞穿膜多肽TAT偶联成的融合蛋白,制备方法包括以下步骤:TAT‑SirT2t表达载体的构建,将TAT克隆至Pet28a载体中;TAT‑SirT2t蛋白的原核表达;TAT‑SirT2t蛋白的纯化。本发明还公开了TAT‑SirT2t蛋白降脂的应用,包括SirT2、SirT2t促进脂滴降解、TAT‑SirT2t促进人类脂肪细胞对脂滴的降解。本发明的重组TAT‑SirT2t多肽制备方法简便,而且该重组蛋白具有潜在的降低血脂功能,可用于制备治疗和/或预防脂肪肝、肥胖的药物或保健品。
Description
技术领域
本发明属于生物制药和生物工程技术领域,尤其涉及一种来源于截短型人SirT2蛋白的融合蛋白、其制备方法及应用。
背景技术
细胞内脂肪合成和脂肪水解的平衡是维持机体代谢平衡的关键,脂代谢异常引发脂代谢紊乱性疾病,如动脉粥样硬化性心脑血管疾病、肥胖症、脂肪肝等,同时会继发如高血糖、胰岛素抵抗等代谢综合症,严重危害人类健康。脂肪组织中甘油三酯(TG)的过度积累是起始和发展脂代谢紊乱的关键原因之一,而目前对脂代谢的研究多集中于高低密度脂蛋白、胆固醇的代谢调控,对甘油三酯的代谢调控研究并不多,因此,需要深入研究甘油三酯代谢的调控机制,深入探索脂代谢紊乱的发生及调控机制,为疾病治疗提供有效的理论依据与药物靶标。
甘油三酯(TG)作为含量最多的脂类,是细胞中最主要的能量储存形式。TG在肝脏组织和脂肪组织中合成,在脂肪组织中以脂滴(LD)的形式储存。TG合成和水解的平衡决定机体的脂代谢平衡,如果在组织中TG过度积累,就会引起脂代谢紊乱性疾病的发生。TG水解的过程涉及不同的水解酶,包括脂肪甘油三酯脂肪酶ATGL、激素敏感脂肪酶HSL和单甘酯脂肪酶MGL。ATGL将TG分解成二甘油三酯(DG)和游离脂肪酸(FFA),HSL将DG分解成单甘油三酯,最后MGL将单甘油三酯水解成甘油和脂肪酸。
组蛋白去乙酰化酶Sirtuin是与酵母Sir2同源的相关酶类。Sir2是一类高度保守的NAD依赖的去乙酰化酶类。Sir2首先在酵母细胞中被分离鉴定出来,之后在线虫和果蝇中发现Sir2可以参与调节细胞的寿命,因此被称为长寿基因。2000年从哺乳动物体内分离鉴定出与酵母Sir2同源的蛋白,称为Sirtuin。Sirtuin家族由七个成员组成,SirT1-7,它们都有一个保守的NAD依赖的催化核心区,促进NAD依赖的去乙酰化酶活性以及ADP核糖转移酶活性。Sirtuins依赖于其去乙酰化活性,参与调控细胞的代谢、衰老、增殖、抗压及炎症氧化应激反应等。近年来,Sirtuins在脂肪代谢中的作用越来越重要,作为响应能量和调节代谢的关键蛋白控制机体的能量平衡。Sirtuins在脂代谢中的重要调控作用暗示其已成为治疗脂代谢紊乱性疾病(肥胖、脂肪肝、糖尿病等)防治药物研发的重要分子靶点。然而,Sirtuins活性调控及其下游活性调控机制研究并不是很透彻,针对不同的Sirtuins,深入研究其活性调控,探索其在生理胁迫及在疾病防治中的作用和功能,对靶向Sirtuins的药物研发非常重要。
因此,可否利用SirT2对脂代谢的调控作用,以开发出降脂的有效蛋白多肽药物,是本领域有待解决的问题。
发明内容
本发明的主要目的在于提供一种人源化、安全性好、具有较好降脂效果的蛋白多肽及其制备方法。为了实现上述目的,本发明包括如下几个方面的内容:
作为本发明的第一方面,在于提供一种来源于截短型人SirT2蛋白的融合蛋白,所述融合蛋白包括氨基酸序列如SEQ ID NO:1所示的截短型人SirT2蛋白和氨基酸序列如SEQID NO:2所示的TAT蛋白。所述截短型人SirT2蛋白记为SirT2t。所述融合蛋白是通过将氨基酸序列如SEQ ID NO:1所示的截短型人SirT2蛋白和氨基酸序列如SEQ ID NO:2所示的TAT蛋白分别克隆至Pet28a载体中得到。
作为本发明的第二个方面,在于提供了所述融合蛋白的制备方法,包括如下几个步骤:
步骤(1),TAT-SirT2t表达载体的构建:
定义截短型人SirT2蛋白为SirT2t;将如SEQ ID NO:2所示TAT蛋白克隆至Pet28a载体中,得到His-TAT载体,然后将SEQ ID NO:1所示SirT2t蛋白克隆至Pet28a-TAT载体中,形成His-TAT-SirT2t原核表达质粒;
步骤(2),TAT-SirT2t蛋白多肽的原核表达:
将步骤(1)构建的His-TAT-SirT2t转化进大肠杆菌感受态细胞transetta中,使用含卡那霉素抗性的固体LB平板筛选克隆,挑取克隆,活化菌种;
待12-16小时后,挑取单克隆菌落,并接种至摇管扩大培养,加入IPTG诱导蛋白表达,6-12小时后收获细胞沉淀,直接加入2X蛋白loading,煮沸后蛋白质电泳,考马斯亮蓝染色,确认表达的蛋白分子量是否正确和比较不同单克隆菌落的蛋白表达量;
选取蛋白分子量正确和表达量最高的单克隆菌落,接种至摇瓶扩大培养,加入IPTG诱导蛋白表达,6-12小时后收获细胞沉淀,获取的细胞沉淀用于后续蛋白多肽的纯化;
步骤(3),TAT-SirT2t蛋白多肽的纯化:
用裂解液裂解细胞沉淀,同时用超声破碎细胞,离心后获取裂解液上清,加入蛋白纯化柱His-beads,4℃振荡孵育4-8小时,用平衡液清洗蛋白纯化柱,再用洗脱液将TAT-SirT2t蛋白洗脱下来;
用1XPBS溶液透析12小时,最终完成TAT-SirT2t蛋白多肽的纯化。
优选的,步骤(2)中,培养容器为15ml摇管和1L摇瓶,条件为25-30℃,摇管和摇瓶转动速率为220rpm,加入IPTG终浓度为0.1-1mM,培养时间为6-12小时;
优选的,步骤(3)中,所述裂解液成分为包含6g/L NaH2PO4,17.5g/L NaCl和0.68g/L咪唑的水溶液,用量为100ml菌液10ml裂解液;
所述平衡液成分为包含6g/L NaH2PO4,17.5g/L NaCl和1.36g/L咪唑的水溶液,用量为每次10ml;
所述洗脱液成分为包含6g/L NaH2PO4,17.5g/L NaCl和17.0g/L咪唑的水溶液,用量为每100ml菌液洗脱500μl。
作为本发明的第三个方面,在于提供了所述截短型人SirT2蛋白的cDNA序列,所述cDNA序列的核苷酸序列如SEQ ID NO:3所示。
作为本发明的第四个方面,在于还提供了包含如SEQ ID NO:1所示氨基酸序列的人SirT2蛋白的应用,所述人SirT2蛋白为以下任一项:
(a)氨基酸序列为SEQ ID NO:1所示的蛋白;
(b)SEQ ID NO:1所示氨基酸序列任意的甲基化或酰基化衍生物;
(c)包含如SEQ ID NO:1所示氨基酸序列的SirT2蛋白与TAT形成的融合蛋白。
具体的,作为本发明的第五个方面,在于提供了人SirT2蛋白促进细胞脂滴降解的应用;在实施例1中,将Flag-SirT2转染到HepG2细胞中,24小时以后将0.4mM的oleate acid(OA)加入细胞中,12小时后用4%多聚甲醛固定细胞,打孔、封闭、加Flag一抗,荧光二抗,同时用油红(oil red)或者BODIPY显示细胞中的脂滴。在荧光显微镜下观察脂滴降解情况。接下来,将上述处理的细胞收集并裂解,用甘油三酯(TG)检测试剂盒和游离脂肪酸(FFA)检测试剂盒检测每组细胞中的TG和FFA水平。结果发现,过表达SirT2,能有效减少细胞中的脂滴聚集,从而减少细胞中的脂质堆积,增加游离脂肪酸的生成,证明SirT2能促进脂滴降解。
本发明还提供了所述截短型人SirT2蛋白,即SirT2t蛋白促进细胞脂滴降解的应用;在实施例2中,将SirT2按照功能域构建截断形式的SirT2t(65-340aa),同时构建一个缺失功能域的突变体(Del 65-340aa),分别将两段构建到pcDNA3.0的Flag表达载体上,将两个质粒分别转染到HepG2细胞中,24小时以后用0.4mM的oleate acid(OA)处理细胞,12小时后用4%多聚甲醛固定细胞,打孔、封闭、加Flag一抗,荧光二抗(绿色),同时用油红(oilred)显示细胞中的脂滴。在荧光显微镜下观察脂滴降解情况。结果显示,SirT2t(65-340aa)能促进脂滴降解。
本发明还提供了所述来源于截短型人SirT2蛋白的融合蛋白的促进细胞脂滴降解的应用,在实施例3和实施例4中,构建原核表达质粒His-TAT-SirT2t,并结合体外蛋白纯化技术,诱导表达出His-TAT-SirT2t融合蛋白,破碎裂解细胞,裂解液与His-beads孵育,纯化出TAT-SirT2t融合蛋白;将纯化的融合蛋白His-TAT-SirT2t与HepG2细胞孵育12小时,并同时处理OA(0.4mM),收集并裂解细胞,用western blot方法检测细胞中TAT-SirT2t穿膜的情况,利用TG检测试剂盒检测细胞中甘油三酯的水平,检测发现,TAT-SirT2t有效减少细胞中脂质堆积。
作为本发明的第六个方面,在于提供了所述人SirT2蛋白的融合蛋白在制备治疗和/或预防脂肪肝和肥胖药物或保健品中的应用。
优选的,所述治疗和/或预防脂肪肝和肥胖药物或保健品包括所述人SirT2蛋白的融合蛋白以及药学上可接受的载体。
与现有技术相比,本发明的有益效果是:
(1),本发明提供了一种人源化、安全性好、具有较好降脂效果的蛋白多肽及其制备方法,具体地,是将人SirT2蛋白的一段氨基酸序列与细胞穿膜肽TAT偶联,构建到原核表达载体Pet28a上,利用大肠杆菌诱导并纯化出来,该制备方法可操作性强;
(2)本发明揭示了SirT2氨基酸片段(SirT2t:SirT2-65-340aa)是降脂的有效多肽,具体地,本发明研究表明SirT2t可以促进脂滴降解,TAT-SirT2t可以有效促进脂滴降解,有效降低脂肪堆积;
(3)本发明的重组TAT-SirT2t多肽制备方法简便且产量高,一般为1-3mg/ml,而且该重组蛋白具有潜在的降低血脂功能,可用于制备治疗和/或预防脂肪肝、肥胖的药物或保健品。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为实施例1的SirT2促进脂滴降解试验,其中,图A为过表达SirT2对脂滴降解情况;图B为瞬时过表达SirT2对细胞中甘油三酯积累的影响;图C为过表达SirT2对细胞中游离脂肪酸水平的影响;
图2为实施例2的SirT2截断形式(SirT2t:SirT2-65-340aa)对脂滴降解的影响;
图3为实施例3中融合蛋白His-TAT-SirT2t的表达诱导与纯化试验,其中,图A为His-TAT-SirT2t诱导情况;图B为His-TAT-SirT2t融合蛋白的纯化情况;
图4为实施例4中融合蛋白His-TAT-SirT2t对细胞的降脂作用,其中,图A为TAT-SirT2t进细胞的情况;图B为TAT-SirT2t对细胞中脂质堆积的影响;
图5为实施例5中SirT2降低脂肪堆积与脂肪水解酶ATGL关系试验;
图6为实施例3中PCR反应条件。
具体实施方式
本发明的研究主要包括四个方面的内容:SirT2促进脂滴水解,SirT2t促进脂滴水解,His-TAT-SirT2t融合蛋白构建、表达及纯化,His-TAT-SirT2t能进入细胞,有效减少脂质聚集。
下面结合说明书附图和具体实施例进一步详细说明本发明。除非特别说明,本发明采用的试剂、设备为本技术领域常规试剂和设备。当然实施例中仪器和材料的采用并不局限于本实例的列举,而是以能够解决本发明的技术问题,并实现相应的技术效果为依据。另外,实施例中未详细说明的分子生物学方法均为本领域常规的方法,具体操作可参看分子生物指南或产品说明书。
实施例1:SirT2促进脂滴降解
将人肝癌细胞系HepG2细胞铺到24孔板中,待细胞密度达到50%以后,用Lipo2000转染试剂转染Flag-SirT2质粒到细胞里,24小时后,用0.4mM油酸(OA)处理细胞12个小时,然后将细胞培养基弃掉,PBS洗一次,用4%多聚甲醛固定细胞10分钟,用0.3%TritonX-100打孔10分钟,用2%BSA封闭30分钟,孵育Flag一抗2个小时,孵育荧光二抗2个小时,用DAPI染色5分钟,然后用油红(Oil red)染色5分钟,或者用BODIPY(10ug/ml)染色15分钟,封片,最后在荧光显微镜下观察SirT2表达及脂滴降解情况。
接下来,将人肝癌细胞系HepG2细胞铺到24孔板中,待细胞密度达到50%以后,用Lipo2000转染试剂转染Flag-SirT2质粒到细胞里,24小时后,用0.4mM油酸(OA)处理细胞12个小时,然后将细胞培养基弃掉,PBS洗一次,收集细胞,裂解,裂解液分别用甘油三酯(TG)检测试剂盒(购买于南京建成生物科技有限公司)检测细胞中的甘油三脂含量,以及用游离脂肪酸(FFA)检测试剂盒(购买于南京建成生物科技有限公司)检测细胞中的游离脂肪酸水平。
结果如图1显示,过表达SirT2能减少脂滴形成,减少甘油三酯生成,促进游离脂肪酸水平,证明SirT2有效促进脂滴降解。
实施例2:SirT2t(SirT2-65-340aa)有效降解脂滴
将SirT2按照功能域构建截断形式的SirT2t(SirT2-65-340aa),将这段氨基酸序列构建到pcDNA3.0表达载体上,同时构建功能域缺失的SirT2(SirT2-Del65-340aa)表达质粒,将两个质粒分别转染到HepG2细胞中,24小时后,用0.4mM油酸(OA)处理细胞12个小时,然后将细胞培养基弃掉,PBS洗一次,用4%多聚甲醛固定细胞10分钟,用0.3%TritonX-100打孔10分钟,用2%BSA封闭30分钟,孵育Flag一抗2个小时,孵育荧光二抗2个小时,用DAPI染色5分钟,然后用油红(Oil red)染色5分钟,封片,最后在荧光显微镜下观察SirT2表达及脂滴降解情况。
结果如图2显示,SirT2t(SirT2-65-340aa)能有效促进脂肪水解。
实施例3:TAT-SirT2t融合蛋白的诱导与纯化
首先构建His-TAT-SirT2t原核表达质粒:将TAT序列构建到原核表达载体Pet28a上,构建成His-TAT表达载体,然后将SirT2t(65-340aa)构建到His-TAT表达载体上。
质粒构建具体操作如下:
(1)SirT2t引物设计
通过浏览NCBI网站,下载SirT2基因的序列,使用相关软件查找出SirT2-65-340aa基因序列中所包含的酶切位点,研究所用载体的图谱,选择合适的限制性内切酶,通过primer-5等软件辅助设计所需的上、下游引物。
(2)PCR扩增目的基因
PCR体系如下:
将配好的PCR反应液,混匀,瞬离,放入PCR仪中反应,反应条件如图6所示。
(3)扩增的目的片段的割胶回收、酶切
PCR结束后,取适量产物进行琼脂糖凝胶电泳检测,观察胶上是否有预计分子量的主要产物带,并将目的条带进行割胶回收。
a.根据回收DNA片段的大小配制合适浓度的琼脂糖凝胶(加适量的EB)。
b.向PCR产物,加入6×DNA loading稀释到1×,开始点样。
c.加样后立即通电进行电泳,电压为120V,20分钟以后停止电泳。
d.取出凝胶,在紫外灯下观察条带所在位置,然后用凝胶成像系统拍照保存,切下目的条带,用天根琼脂胶回收试剂盒回收目的基因。
e.选用pcDNA3.0 Flag载体,将载体和PCR产物用同样的酶分别同时进行酶切,以产生相同的酶切位点。酶切条件为37℃下3-4小时。用琼脂糖凝胶电泳分离载体和PCR产物的酶切产物,然后用胶回收试剂盒(试剂盒购自天根生化科技有限公司)回收需要的产物。具体步骤见试剂盒说明书。
(4)连接
实验原理:在一定的条件下,T4 DNA连接酶可以催化两个双链DNA片段相邻的5’端磷酸和3’端羟基之间形成磷酸二酯键,从而将两个片段连接起来。向体系中加入1μl载体,1μl T4连接酶,1μl buffer,7μl目的基因片段,混合均匀后,放到16℃水浴锅中8-12小时。
(5)转化
利用热激法原理进行转化,将50微升刚融化的感受态加入到连接产物中,冰上放置30分钟,然后放入42℃水浴锅中热激60秒,然后放到冰上5分钟,再加入700微升的无抗性的LB,37度摇床复苏45分钟,低速离心后,涂板到相应抗性的平板上,倒置放到37度培养箱培养12小时。
(6)小摇并提取质粒
在超净工作台中,挑取转化所得的克隆,接种到相应抗性的LB培养基中,37℃,200rpm/min振荡培养12-16小时,然后保菌,将剩余的菌液使用天根质粒小提试剂盒提取质粒,详细步骤可查阅说明书。
(7)酶切鉴定、测序分析及验证表达
取1ug质粒,选择对应的酶和Buffer进行酶切鉴定。37℃酶切处理3h,然后用琼脂糖凝胶电泳检测。将酶切验证正确的质粒挑取一个克隆送去测序公司测序,将反馈的结果进行分析,检测是否构建成功。
然后将His-TAT-SirT2t转化到大肠杆菌感受态细胞transetta(DE3)中,然后用卡那霉素抗性的LB平板筛选克隆,挑取克隆,活化菌种。待12-16小时后,挑取单克隆菌落,并接种至15ml摇管扩大培养,加入IPTG诱导蛋白表达,6-12小时后收获细胞沉淀,直接加入2X蛋白loading,煮沸后蛋白质电泳,考马斯亮蓝染色,确认表达的蛋白分子量是否正确和比较不同单克隆菌落的蛋白表达量;选取蛋白分子量正确和表达量最高的单克隆菌落,接种至1L摇瓶扩大培养,加入IPTG诱导蛋白表达,6-12小时后收获细胞沉淀,获取的细胞沉淀用于后续蛋白多肽的纯化。
用裂解液裂解细胞沉淀,同时用超声破碎细胞,离心后获取裂解液上清,加入蛋白纯化柱His-beads,4℃振荡孵育4-8小时,用平衡液清洗蛋白纯化柱,再用洗脱液将TAT-SirT2t蛋白洗脱下来;用1XPBS溶液透析12小时,最终完成TAT-SirT2t蛋白多肽的纯化,所述裂解液成分为包含6g/L NaH2PO4,17.5g/L NaCl和0.68g/L咪唑的水溶液,用量为100ml菌液10ml裂解液;所述平衡液成分为包含6g/L NaH2PO4,17.5g/L NaCl和1.36g/L咪唑的水溶液,用量为每次10ml;所述洗脱液成分为包含6g/L NaH2PO4,17.5g/L NaCl和17.0g/L咪唑的水溶液,用量为每100ml菌液洗脱500μl。结果如图3所示。
实施例4:TAT-SirT2t融合蛋白有效降低细胞内的脂质堆积
将纯化得到的TAT-SirT2t以及同时得到的His-TAT(TAT-Control,即为空载体构建的蛋白,作为TAT-SirT2t的有效对照,纯化方法同TAT-SirT2t)分别与HepG2细胞孵育12小时,同时对细胞处理0.4mM的OA,12小时以后,收集细胞,分成两份裂解,一份裂解液做western blot检测细胞中进入的TAT-SirT2t,另外一份裂解液用TG检测试剂盒检测细胞中TG含量。
根据图4中图A所示,TAT-SirT2t能有效进入细胞,图4中图B显示,采用TAT-SirT2t+OA处理后细胞中甘油三酯含量明显低于其他两组,可见,TAT-SirT2t的引入可以有效降低细胞中脂质的堆积。
实施例5:SirT2降解脂肪的能力依赖于脂肪水解酶ATGL
1、首先构建ATGL-Si的质粒(同时Plko.1为空载体,作为实验的对照),构建方法如下:
a.根据实验需求,选择具有嘌呤霉素抗性的慢病毒Plko.1载体;
b.对ATGL基因设计构建shRNA,在已发表的文章中或者公司网站中找到siRNA序列,经退火延伸连接到Plko.1载体上;
c.测序鉴定正确的克隆;
d.筛选高效的shRNA,将shRNA转染到293T细胞中,通过western blot分析蛋白水平的变化,蛋白降低的越多表明基因沉默的效率越好。
2、包装病毒
a.转染:将ATGL-shRNA(10ug)、psPAX2(10ug)及pMD2G(5ug)三种质粒以磷酸钙转染法转染到预先铺好的10cm的293T细胞内。
b.换液:4小时左右,将转染后的细胞换成新鲜的培养基,继续培养。
c.收集病毒:按转染后的时间为0h计,收集24h,48h的病毒颗粒上清。
d.病毒浓缩:将收集好的病毒先低速离心:3000rpm/5min,除去残留的细胞碎片,然后将上清加入病毒浓缩柱子里,4度离心:4000g/2h,离心结束后,将柱子里的上清吸出。
e.保存:将病毒分装成小管,-80度冰箱保存。
3、将人肝癌细胞系HepG2细胞铺到24孔板中,待细胞密度达到50%以后,将上述病毒(对照病毒Plko.1及ATGL-Si)分别加入到细胞中,同时将SirT2t蛋白也加入到细胞中,48小时后,用0.4mM油酸(OA)处理细胞12个小时,然后将细胞培养基弃掉,PBS洗一次,收集细胞,裂解,裂解液分别用甘油三酯(TG)检测试剂盒(购买于南京建成生物科技有限公司)检测细胞中的甘油三脂含量,以及用游离脂肪酸(FFA)检测试剂盒(购买于南京建成生物科技有限公司)检测细胞中的游离脂肪酸水平。
结果如图5显示,SirT2t有效促进脂滴降解,然而在ATGL敲低的细胞中(ATGL-Si病毒处理组),SirT2t降解脂滴的能力丧失。证明,SirT2t通过脂肪水解酶ATGL降解脂肪。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 上海市第一人民医院
<120> 一种来源于截短型人SirT2蛋白的融合蛋白、其制备方法及应用
<130> 20201112
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gaaggggtgg cccggtacat gcagagcgaa cgctgtcgca gagtcatctg tttggtggga 60
gctggaatct ccacatccgc aggcatcccc gactttcgct ctccatccac cggcctctat 120
gacaacctag agaagtacca tcttccctac ccagaggcca tctttgagat cagctatttc 180
aagaaacatc cggaaccctt cttcgccctc gccaaggaac tctatcctgg gcagttcaag 240
ccaaccatct gtcactactt catgcgcctg ctgaaggaca aggggctact cctgcgctgc 300
tacacgcaga acatagatac cctggagcga atagccgggc tggaacagga ggacttggtg 360
gaggcgcacg gcaccttcta cacatcacac tgcgtcagcg ccagctgccg gcacgaatac 420
ccgctaagct ggatgaaaga gaagatcttc tctgaggtga cgcccaagtg tgaagactgt 480
cagagcctgg tgaagcctga tatcgtcttt tttggtgaga gcctcccagc gcgtttcttc 540
tcctgtatgc agtcagactt cctgaaggtg gacctcctcc tggtcatggg tacctccttg 600
caggtgcagc cctttgcctc cctcatcagc aaggcacccc tctccacccc tcgcctgctc 660
atcaacaagg agaaagctgg ccagtcggac cctttcctgg ggatgattat gggcctcgga 720
ggaggcatgg actttgactc caagaaggcc tacagggacg tggcctggct gggtgaatgc 780
gaccagggct gcctggccct tgctgagctc cttggatgga agaaggag 828
Claims (10)
1.一种来源于截短型人SirT2蛋白的融合蛋白,其特征在于,所述融合蛋白包括氨基酸序列如SEQ ID NO:1所示的截短型人SirT2蛋白和氨基酸序列如SEQ ID NO:2所示的TAT蛋白。
2.根据权利要求1所述的一种来源于截短型人SirT2蛋白的融合蛋白,其特征在于,所述融合蛋白是通过将氨基酸序列如SEQ ID NO:1所示的截短型人SirT2蛋白和氨基酸序列如SEQ ID NO:2所示的TAT蛋白分别克隆至Pet28a载体中得到。
3.根据权利要求1或2所述的融合蛋白的制备方法,其特征在于,包括如下几个步骤:
步骤(1),TAT-SirT2t表达载体的构建:
将如SEQ ID NO:2所示TAT蛋白克隆至Pet28a载体中,得到His-TAT载体,然后将SEQ IDNO:1所示SirT2t蛋白克隆至His-TAT载体中,形成His-TAT-SirT2t原核表达质粒;
步骤(2),TAT-SirT2t蛋白多肽的原核表达:
将步骤(1)构建的His-TAT-SirT2t转化进感受态细胞transetta中,使用含卡那霉素抗性的固体LB平板筛选克隆,挑取克隆,活化菌种;
待12-16小时后,挑取单克隆菌落,并接种至摇管扩大培养,加入IPTG诱导蛋白表达,6-12小时后收获细胞沉淀,直接加入2X蛋白loading,煮沸后蛋白质电泳,考马斯亮蓝染色,确认表达的蛋白分子量是否正确和比较不同单克隆菌落的蛋白表达量;
选取蛋白分子量正确和表达量最高的单克隆菌落,接种至摇瓶扩大培养,加入IPTG诱导蛋白表达,6-12小时后收获细胞沉淀,获取的细胞沉淀用于后续蛋白多肽的纯化;
步骤(3),TAT-SirT2t蛋白多肽的纯化:
用裂解液裂解细胞沉淀,同时用超声破碎细胞,离心后获取裂解液上清,加入蛋白纯化柱His-beads,4℃振荡孵育4-8小时,用平衡液清洗蛋白纯化柱,再用洗脱液将TAT-SirT2t蛋白洗脱下来;
用1XPBS溶液透析12小时,最终完成TAT-SirT2t蛋白多肽的纯化。
4.根据权利要求3所述的制备方法,其特征在于,步骤(2)中,温度条件为25-30℃,摇管和摇瓶转动速率为220rpm,加入IPTG终浓度为0.1-1mM,培养时间为6-12小时。
5.根据权利要求3所述的制备方法,其特征在于,步骤(3)中,所述裂解液成分为包含6g/L NaH2PO4,17.5g/L NaCl和0.68g/L咪唑的水溶液,用量为100ml菌液10ml裂解液;
所述平衡液成分为包含6g/L NaH2PO4,17.5g/L NaCl和1.36g/L咪唑的水溶液,用量为每次10ml;
所述洗脱液成分为包含6g/L NaH2PO4,17.5g/L NaCl和17.0g/L咪唑的水溶液,用量为每100ml菌液洗脱500μl。
6.根据权利要求1所述的截短型人SirT2蛋白的cDNA序列,其特征在于,所述cDNA序列的核苷酸序列如SEQ ID NO:3所示。
7.包含如SEQ ID NO:1所示氨基酸序列的人SirT2蛋白的应用,其特征在于,促进细胞脂滴降解。
8.根据权利要求7所述的应用,其特征在于,所述人SirT2蛋白为以下任一项:
(a)氨基酸序列为SEQ ID NO:1所示的蛋白;
(b)SEQ ID NO:1所示氨基酸序列任意的甲基化或酰基化衍生物;
(c)包含如SEQ ID NO:1所示氨基酸序列的SirT2蛋白与TAT形成的融合蛋白。
9.根据权利要求8所述的人SirT2在制备治疗和/或预防脂肪肝和肥胖药物或保健品中的应用。
10.根据权利要求9所述的药物或保健品,其特征在于,包括所述人SirT2蛋白以及药学上可接受的载体。
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