CN104211810B - 一种可诱导细胞凋亡、有荧光标记的融合蛋白及其编码基因和应用 - Google Patents
一种可诱导细胞凋亡、有荧光标记的融合蛋白及其编码基因和应用 Download PDFInfo
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Abstract
本发明涉及生物技术和基因工程领域,公开了一种可诱导细胞凋亡、有荧光标记的融合蛋白,包括从N端到C端依次连接的豆蔻酰化位点、他克莫司结合蛋白、半胱天冬酶8、2A肽、组蛋白2B和绿色荧光蛋白,其氨基酸序列如SEQ ID No.1所示。编码这种融合蛋白的基因核苷酸序列如SEQ ID No.2所示。表达了该融合蛋白的细胞在半小时内启动凋亡,并且所有表达了该融合蛋白的细胞在2小时内死亡。通过利用荧光标记,在细胞启动凋亡程序前就能预知细胞将会死亡,使细胞凋亡可控并便于观察。
Description
技术领域
本发明涉及生物技术和基因工程领域,具体为一种可诱导细胞凋亡、有荧光标记的融合蛋白及其编码基因和应用。
背景技术
科学研究中,需要对一个群体中的部分细胞进行操作,导致其死亡,从而研究健康细胞的表现。以往的研究多使用细菌毒素及受体,使表达了毒素受体的细胞因为添加的细菌毒素致敏而死亡。但细菌毒素诱导细胞死亡,需要通过较长的信号通路,中间有各类抑制蛋白可能发挥作用,从而效率不高。而且细菌毒素需要积累的一定程度才有反应,而积累的过程相对缓慢,通常需要几个小时甚至一整天才能最终观察到特定细胞的死亡。此外,过量积累的细菌毒素对细胞会产生非特异性的杀伤。
因此,需要对现有技术加以改进,使细胞凋亡程序可控。
细胞凋亡是细胞按照需要程序性死亡的过程,受着精确的调控。各类半胱天冬酶的依次激活是细胞凋亡的重要环节。如果能对细胞凋亡加以诱导和控制,加速细胞凋亡并且能对加以预测,可以更有效地进行研究。
细胞凋亡有很多种形式,如:由CD95/Fas/APO-1受体导致的凋亡,激活半被部分酶切激活的半胱天冬酶8在细胞膜表面的多聚化是细胞凋亡过程的下游,能够十分有效地导致细胞的死亡。
Membrane oligomerization and cleavage activates the caspase-8(FLICE/MACHalpha1)death signal.
Martin DA,Siegel RM,Zheng L,Lenardo MJ.
J Biol Chem.1998Feb20;273(8):4345-9.[PMID:9468483]
绿色荧光蛋白是一种标记蛋白,融合到已知蛋白后,可以实现融合蛋白具有荧光,从而标记相应的细胞器。
在构建多基因融合载体时,连接肽的选择很重要。因为融合蛋白可能会影响彼此的表达或者折叠,2A肽是口蹄疫病毒的一段多肽,它在真核细胞中表达时会发生自剪切作用,融合蛋白会分开成两个单独的蛋白,这样既可以达到两个基因融合利于转化的目的,又能自剪切分开而行使各自的功能。
发明内容
本发明旨在提供一种具有荧光标记的、可诱导细胞凋亡的融合蛋白。
本发明还提供了上述融合蛋白的编码基因序列。
本发明另一个目的是将上述融合蛋白用于诱导细胞凋亡。
技术方案为:可诱导细胞凋亡、有荧光标记的融合蛋白,包括从N端到C端依次连接的豆蔻酰化位点、FKBP(他克莫司结合蛋白)、半胱天冬酶8、2A肽、组蛋白2B和绿色荧光蛋白。优选的,其氨基酸序列如SEQ ID No.1所示。
上述编码融合蛋白的基因,是能编码上述融合蛋白的核苷酸序列,优选的,其核苷酸序列如SEQ ID No.2所示。
将本发明的融合蛋白编码基因导入细胞,使之表达。表达了该融合蛋白的细胞在被诱导后半小时内启动凋亡,并且所有表达了该融合蛋白的细胞在2小时内死亡。通过利用荧光标记,在细胞启动凋亡程序前就能预知细胞将会死亡,使细胞凋亡可控并便于观察。
附图说明
图1为实施例2中,表达融合蛋白的细胞在加药后凋亡的图片
图2为实施例2中,表达融合蛋白的细胞与普通细胞按不同比例混合后,在加药后存活率
具体实施方式
实施例1
所构建的融合蛋白为:Myr–L1–FKBP–FKBP–L2–Caspase8Δ–L3–2A–L4–H2B–L5–EGFP
其中,Myr为豆蔻酰化位点,FKBP为他克莫司结合蛋白,Caspase8Δ为半胱天冬酶8的C端片段,2A为2A肽,H2B为组蛋白,EGFP为绿色荧光蛋白;L1~L4为连接肽段,L1:S,L2:S,L3:GST,L4:RS,L5:QDPPVAT。
所构建的融合蛋白氨基酸序列如SEQ ID No.1所示
通过根据GenBank数据库中信息由基因组反转录的cDNA中PCR获得各片段序列,从购买的pEGFP-N1质粒中PCR获得EGFP片段,构建核苷酸片段,得到的基因核苷酸序列如SEQID No.2所示。
将得到的融合蛋白编码基因可通过反转录病毒(基于Clontech公司pMSCV质粒改造)包装导入MDCK细胞,显示绿色荧光的细胞为表达该融合蛋白的细胞。此外,该融合蛋白还能够通过瞬时转染(基于Clontech公司pEGFP-N1质粒改造)、慢病毒(基于pLentiLox3.7质粒改造)包装等方法导入细胞。该融合蛋白在除MDCK细胞之外的293FT,NIH3T3,Hela,Caco2中都能十分有效地诱导凋亡。
对相关质粒进行测序确认SEQ IDNo.2,翻译得到蛋白质的氨基酸序列吻合SEQIDNo.1。
其中,MGSSKSKGQL是豆蔻酰化位点,使融合蛋白因为脂的修饰而定位细胞膜上,这是半胱天冬酶8激活下游细胞凋亡通路的所需的一个条件。
其下游连接首尾相连的两个拷贝的他克莫司结合蛋白:
RGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL KLET,能够在纳巴霉素(rapamycin)的作用下实现多聚化,这是半胱天冬酶8激活下游细胞凋亡通路的所需的另一个条件。实例中采用的AP缩写的药物是美国Ariad公司的产品AP20187,它是纳巴霉素的类似物,具有和FKBP更高的亲和力,以及减弱的诱导免疫反应的活性。
然后连接半胱天冬酶8的C端,即被激活的半胱天冬酶8的片段,将其在细胞膜表面多聚化是细胞凋亡信号通路的下游步骤,十分有效地导致细胞死亡:SDSPREQDSESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIYDILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEPPIYELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEADFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDKKNMGKQMPQPTFTLRKKL VFPSD。
下游连接的序列为:GSGATNFSLLKQAGDVEENPGP,这是2A肽,实现一段核酸序列在同一启动子的作用下,最终在体内表达为两个蛋白。
其后为组蛋白2B,与绿色荧光蛋白融合后定位于细胞核内;细胞进入细胞凋亡,会导致核凝结,导致绿色荧光强度增加;细胞死亡后,绿色荧光或者散入培养液,或者被周围细胞吞噬:
MPDPAKSAPAPKKGSKKAVTKVQKKDGKKRKRSRKESYSVYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGE LAKHAVSEGTKAVTKYTSSK。
采用核定位的荧光蛋白,不仅仅在加药前标志了将会凋亡的细胞,而且会标记了凋亡的过程。
组蛋白2B后连接绿色荧光蛋白。
实施例2
用实施例1所获得的融合蛋白编码基因通过pMSCV载体在MDCK细胞中表达(细节见上)。构建多细胞结构,并向其中加入药物。
多细胞结构中,表达绿色荧光蛋白的细胞在加药(AP,纳巴霉素类似物)后30分钟内进入细胞凋亡,所有细胞在2小时内完成死亡。zVad是细胞凋亡的抑制剂,作用于半胱天冬酶8的下游。zVad药物阻止了被诱导的表达绿色荧光蛋白的细胞死亡,可以证明融合蛋白诱导的细胞死亡是特异性的,而且是通过细胞凋亡途径实现的。如图1所示。
将表达了融合蛋白的细胞与普通细胞按不同比例混合培养(表达融合蛋白的细胞含量为20%~100%,对照为普通细胞),而后加药,观察细胞的死亡。可以看到融合蛋白诱导的细胞死亡是通过细胞凋亡导致的,因为通过zVad药物抑制细胞凋亡,所有的细胞都存活了。此外,表达了融合蛋白的细胞的死亡不受周围环境的影响,即使只有20%的细胞表达了融合蛋白,周围80%的细胞分泌了大量的促进存活的因子,这20%表达了融合蛋白的细胞按照既定的程序,进入凋亡后死去。如图2所示(时间单位为分钟)。
Claims (5)
1.一种可诱导细胞凋亡、有荧光标记的融合蛋白,其特征在于,包括从N端到C端依次连接的豆蔻酰化位点、两个他克莫司结合蛋白、半胱天冬酶8的C端片段、2A肽、组蛋白2B和绿色荧光蛋白,其氨基酸序列如SEQ ID No.1所示。
2.一种具有荧光标记的、可诱导细胞凋亡的融合蛋白基因,其特征在于,是编码权利要求1所述融合蛋白的核苷酸序列。
3.权利要求2所述的基因,其特征在于,其核苷酸序列如SEQ ID No.2所示。
4.权利要求1所述的融合蛋白在AP20187诱导细胞凋亡及对凋亡过程的观察中的应用。
5.权利要求2或3所述的基因在AP20187诱导细胞凋亡中的应用。
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CN101709084B (zh) * | 2009-11-26 | 2012-07-04 | 中国科学院广州生物医药与健康研究院 | 优化的串连细胞因子及其在诱导多能性干细胞中的应用 |
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