CN111411120B - 一种锌离子诱导铁蛋白自组装包埋活性小分子的方法 - Google Patents
一种锌离子诱导铁蛋白自组装包埋活性小分子的方法 Download PDFInfo
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Abstract
本发明涉及一种锌离子诱导铁蛋白自组装包埋活性小分子的方法,利用同种电荷的排斥作用将铁蛋白改造为在近中性pH条件下呈现四聚体状态(现有的重组人重链铁蛋白(recombinant human H‑chain ferritin,rHuHF)在近中性pH条件下为二十四聚体状态),而四聚体形式的重组人铁蛋白突变体在人体必需的微量金属元素锌诱导下可进一步组装为笼形二十四聚体,从而实现对脂溶性活性分子比如姜黄素、以及水溶性活性分子比如阿霉素等生物活性成分的高效率包埋,其包埋效率为酸碱诱导包埋方法的1.9~2倍。
Description
技术领域
本发明涉及一种锌离子诱导铁蛋白自组装包埋活性小分子的方法,利用锌离子在pH近中性条件下诱导铁蛋白四聚体转变为二十四聚体笼形蛋白进而包埋活性小分子。
背景技术
自然界中广泛存在的铁蛋白分子具有中空的纳米空腔,具有很好的小分子装载特性。在中性pH水溶液条件下,天然铁蛋白分子为二十四聚体状态,若想利用铁蛋白包埋具有生物活性的小分子,需要首先通过酸变性的方法将铁蛋白解聚,再利用铁蛋白可逆自组织的性质来实现包埋,这种方法所需要的条件相对苛刻且效率低下,而且部分蛋白质在包埋过程中会发生变性,变性蛋白可达到50%左右。而组成天然蛋白质的二十种氨基酸中的组氨酸在中性pH水溶液条件下带有正电荷且可与金属离子结合,利用这一特性可制备铁蛋白低聚体并实现金属离子诱导铁蛋白的组装,从而可以实现在温和条件下对活性分子的包埋。
目前,能在pH近中性条件下利用人体必需的微量元素锌诱导铁蛋白自组装,进而实现对小分子的包埋的方法在国内外还未见报道。
发明内容
本发明的目的是提供一种锌离子诱导铁蛋白自组装包埋活性小分子的方法,利用同种电荷的排斥作用将铁蛋白改造为在近中性pH条件下呈现四聚体状态(现有的重组人重链铁蛋白(recombinant human H-chain ferritin,rHuHF)在近中性pH条件下均为二十四聚体状态),而四聚体形式的重组人铁蛋白突变体在人体必需的微量金属元素锌诱导下可进一步组装为笼形二十四聚体,从而实现对脂溶性活性分子比如姜黄素、以及水溶性活性分子比如阿霉素等生物活性成分的高效率包埋,其包埋效率为酸碱诱导包埋方法的1.9~2倍。
为了实现上述目的,本发明提供了如下技术方案:
一种锌离子诱导铁蛋白自组装包埋活性小分子的方法,包括如下步骤:
(1)制备四聚体铁蛋白突变体DE6H溶液
(1.1)以重组H型人铁蛋白rHuHF作为模板,其核酸序列如SEQ ID NO.1所示,将所述核酸序列编码的重组H型人铁蛋白四重轴周围的159~164位的氨基酸替换为6个组氨酸,将替换后的氨基酸序列对应的如SEQ ID NO.2所示的核酸序列插入到蛋白表达质粒载体,得到带有四聚体铁蛋白突变体DE6H核酸序列的蛋白表达质粒载体;
(1.2)将步骤1.1制得的带有四聚体铁蛋白突变体DE6H核酸序列的蛋白表达质粒载体导入至市售BL21(DE3)感受态表达菌株中进行蛋白表达,8000-11000rpm/min高速离心菌液收集沉淀菌体,复溶于pH 6.0~8.0,20~50mM Tris-HCl缓冲液中,超声波破碎,经镍离子纯化柱纯化,得到纯四聚体铁蛋白突变体DE6H溶液;将纯四聚体铁蛋白突变体DE6H溶于pH 6.0~8.0,20~50mM Tris-HCl缓冲液中,获得浓度为1~5μM的四聚体铁蛋白突变体DE6H溶液;
(2)锌离子诱导四聚体铁蛋白包埋活性小分子
(2.1)称取一定量的活性小分子,将其溶解在无水乙酸或无水乙醇中,制备成一定浓度的活性小分子母液,4℃避光保存待用;
(2.2)按照四聚体铁蛋白突变体DE6H与活性小分子的分子摩尔比为1:50~1:500的比例,向步骤1.2制得的四聚体铁蛋白突变体DE6H溶液中缓慢加入步骤2.1中制备的活性小分子母液,室温搅拌5min;接着按照四聚体铁蛋白与锌离子的分子摩尔比为1:50~1:200的比例,缓慢加入一定浓度的Zn2+溶液,于4℃~25℃层析柜中搅拌30~150min;
(2.3)将步骤2.2所得溶液置于透析袋中,于4℃~25℃层析柜中透析至含50~200mM NaCl的pH 6.0~8.0,20~50mM Tris-HCl缓冲液中,获得包埋有活性小分子的铁蛋白溶液。
所述步骤1.2中的纯化条件为:平衡缓冲液:20mM Tris,10mM咪唑,500mM NaCl,pH=8.0;洗脱缓冲液:20mM Tris,500mM咪唑,500mM NaCl,pH=8.0。
所述步骤2.2中Zn2+溶液为ZnSO4溶液。
所述活性小分子包括姜黄素、虾青素、β-胡萝卜素、阿霉素。
所述方法的包埋效率为酸碱诱导的包埋效率的1.9~2倍且包埋反应在近中性pH溶液条件下进行,比酸碱诱导更为温和。
与现有技术相比,本发明的有益效果在于:
本发明方法使生物活性小分子更高效地被包裹在铁蛋白空腔内部。该方法与传统的酸碱诱导的铁蛋白包埋方法相比,首先,锌离子诱导的包埋使用的条件很温和(pH 6.0~8.0),即接近中性条件就可以;其次,锌离子诱导的包埋在包埋过程中蛋白质不会发生变性,而传统的酸碱诱导的铁蛋白包埋方法在包埋过程中,蛋白质变性可达到50%,这样锌离子诱导的包埋可以大大节约蛋白质样品;第三,锌离子诱导的铁蛋白包埋效率为酸碱诱导的包埋效率的1.9~2倍。因此,扩大了其应用范围,以满足不同的生产需要。
具体实施方式
下面结合实施例对本发明进行进一步说明。
一种锌离子诱导铁蛋白自组装包埋活性小分子的方法,包括如下步骤:
(1)制备四聚体铁蛋白突变体DE6H溶液
(1.1)为了使得铁蛋白包埋产物具有可食性,本发明以重组H型人铁蛋白(rHuHF)作为模板,其核酸序列如SEQ ID NO.1所示,考虑到组氨酸在中性pH条件下带有正电荷且易与金属离子结合的性质,将所述核酸序列编码的重组H型人铁蛋白四重轴周围的159~164位(从启动子后一位作为序列首位)的氨基酸,替换为6个组氨酸,将得到的核酸序列(SEQID NO.2)插入到蛋白表达质粒载体,得到带有四聚体铁蛋白突变体DE6H核酸序列的蛋白表达质粒载体。优选地,该步骤由基因合成公司完成。
(1.2)将步骤1.1制得的带有突变体的四聚体铁蛋白突变体DE6H核酸序列的蛋白表达质粒载体导入至市售BL21(DE3)感受态表达菌株中进行蛋白表达,8000-11000rpm/min高速离心菌液收集沉淀菌体,复溶于pH 6.0~8.0,20~50mM Tris-HCl缓冲液中,超声波破碎,经镍离子纯化柱纯化,纯化条件:平衡缓冲液:20mM Tris,10mM咪唑,500mM NaCl,pH=8.0;洗脱缓冲液:20mM Tris,500mM咪唑,500mM NaCl,pH=8.0,得到纯四聚体铁蛋白突变体DE6H溶液;将纯四聚体铁蛋白DE6H溶于pH 6.0~8.0,20~50mM Tris-HCl缓冲液中,获得浓度为1~5μM的四聚体铁蛋白突变体DE6H溶液。
该四聚体铁蛋白突变体DE6H由于大量同种电荷富集到铁蛋白四重轴处时,同种电荷产生排斥作用,在溶液pH 6.0~8.0条件下,铁蛋白突变体会由二十四聚体的状态裂解为四聚体的状态,得到四聚体铁蛋白突变体DE6H。
(2)锌离子诱导四聚体铁蛋白包埋活性小分子
(2.1)称取一定量的活性小分子,将其溶解在无水乙酸或无水乙醇中,制备成一定浓度的活性小分子母液,4℃避光保存待用;
(2.2)按照四聚体铁蛋白突变体DE6H与活性小分子的分子摩尔比为1:50~1:500的比例,向步骤1.2制得的四聚体铁蛋白突变体DE6H溶液中缓慢加入步骤2.1中制备的活性小分子母液,室温搅拌5min;接着按照四聚体铁蛋白突变体DE6H与锌离子的分子摩尔比为1:50~1:200的比例,缓慢加入一定浓度的Zn2+溶液,于4℃~25℃层析柜中搅拌30~150min;所述Zn2+溶液为ZnSO4溶液。
(2.3)将步骤2.2所得溶液置于透析袋中,于4℃~25℃层析柜中透析至含50~200mM NaCl的Tris-HCl缓冲液(pH 6.0~8.0,20~50mM)中,获得包埋有活性小分子的铁蛋白溶液。
所述活性小分子包括姜黄素、虾青素、β-胡萝卜素、阿霉素等。
实施例-锌离子诱导铁蛋白自组装包埋姜黄素
具体过程如下:
(1)制备四聚体铁蛋白突变体DE6H溶液
以重组H型人铁蛋白为模板,以重组H型人铁蛋白rHuHF作为模板,其核酸序列如SEQ ID NO.1所示,由基因合成公司,将核酸序列编码的重组H型人铁蛋白四重轴周围的159~164位(从启动子后一位作为序列首位)的氨基酸替换为6个组氨酸,所得氨基酸对应的核酸序列如SEQ ID NO.2所示,将得到的核酸序列插入蛋白表达质粒载体,得到带有四聚体铁蛋白突变体DE6H核酸序列的蛋白表达质粒载体。
将上述制得的带有突变体的四聚体铁蛋白突变体DE6H核酸序列的蛋白表达质粒载体导入至市售BL21(DE3)感受态表达菌株中进行蛋白表达,8000-11000rpm/min高速离心菌液收集沉淀菌体,复溶于pH8.0,20mM Tris-HCl缓冲液中,超声波破碎,经镍离子纯化柱纯化后得到纯四聚体铁蛋白突变体DE6H溶液,将纯四聚体铁蛋白突变体DE6H溶于pH 8.0,20mM Tris-HCl缓冲液中,最终获得浓度为2μM的四聚体铁蛋白突变体DE6H溶液。
(2)锌离子诱导四聚体铁蛋白包埋姜黄素的包
(2.1)称取一定量的姜黄素,将其溶解在无水乙酸中,制备成终浓度5mM的姜黄素母液,4℃避光保存待用。
(2.2)将5mL 2μM四聚体铁蛋白突变体DE6H溶液(pH 8.0,20mM Tris-HCl缓冲液)置于50mL离心管中,内置小转子缓慢搅拌。
缓慢加入200μL姜黄素母液,使分子摩尔比四聚体铁蛋白突变体DE6H:姜黄素为1:100,室温搅拌5min。
接着缓慢加入200μL 5mM ZnSO4溶液使分子摩尔比四聚体铁蛋白突变体DE6H:Zn2+为1:100,于4℃层析柜中搅拌2h。
(2.3)将所得溶液置于孔径为12,000~14,000Da透析袋中,用150mM NaCl的Tris-HCl缓冲液(pH 8.0,20mM)透析三次,每6小时换一次透析缓冲液,除去游离的姜黄素,此步骤在4℃层析柜中避光操作;最后将包埋有姜黄素的四聚体铁蛋白(DE6H)溶液,置于4℃避光保存备用。
将包埋有姜黄素的四聚体铁蛋白突变体DE6H溶液用1M HCl调节pH值到2.0左右,解离四聚体铁蛋白突变体DE6H亚基,释放包埋的姜黄素分子并通过HPLC检测。通过标曲计算姜黄素浓度。通过最后计算得出,平均一个四聚体铁蛋白突变体DE6H分子装载了约28.2个姜黄素分子。
这一结果相比传统酸变性包埋法(平均一个铁蛋白分子装载了约14.7个姜黄素分子)包埋效率提高了近2倍。
序列表
<110> 中国农业大学
<120> 一种锌离子诱导铁蛋白自组装包埋活性小分子的方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 552
<212> DNA
<213> Homo sapiens
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tactttgacc gcgatgatgt ggctttgaag aactttgcca aatactttct tcaccaatct 180
catgaggaga gggaacatgc tgagaaactg atgaagctgc agaaccaacg aggtggccga 240
atcttccttc aggatatcca gaaaccagac tgtgatgact gggagagcgg gctgaatgca 300
atggagtgtg cattacattt ggaaaaaaat gtgaatcagt cactactgga actgcacaaa 360
ctggccactg acaaaaatga cccccatttg tgtgacttca ttgagacaca ttacctgaat 420
gagcaggtga aagccatcaa agaattgggt gaccacgtga ccaacttgcg caagatggga 480
gcgcccgaat ctggcttggc ggaatatctc tttgacaagc acaccctggg agacagtgat 540
aatgaaagct aa 552
<210> 2
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<212> DNA
<213> 人工序列(Artificial Sequence)
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atgacgaccg cgtccacctc gcaggtgcgc cagaactacc accaggactc agaggccgcc 60
atcaaccgcc agatcaacct ggagctctac gcctcctacg tttacctgtc catgtcttac 120
tactttgacc gcgatgatgt ggctttgaag aactttgcca aatactttct tcaccaatct 180
catgaggaga gggaacatgc tgagaaactg atgaagctgc agaaccaacg aggtggccga 240
atcttccttc aggatatcca gaaaccagac tgtgatgact gggagagcgg gctgaatgca 300
atggagtgtg cattacattt ggaaaaaaat gtgaatcagt cactactgga actgcacaaa 360
ctggccactg acaaaaatga cccccatttg tgtgacttca ttgagacaca ttacctgaat 420
gagcaggtga aagccatcaa agaattgggt gaccacgtga ccaacttgcg caagatgcat 480
catcatcatc atcatttggc ggaatatctc tttgacaagc acaccctggg agacagtgat 540
aatgaaagct aa 552
Claims (5)
1.一种锌离子诱导铁蛋白自组装包埋活性小分子的方法,其特征在于:包括如下步骤:
(1)制备四聚体铁蛋白突变体DE6H溶液
(1.1)以重组H型人铁蛋白rHuHF作为模板,其核酸序列如SEQ ID NO.1所示,将所述核酸序列编码的重组H型人铁蛋白四重轴周围的159~164位的氨基酸替换为6个组氨酸,将替换后的氨基酸序列对应的如SEQ ID NO.2所示的核酸序列插入到蛋白表达质粒载体,得到带有四聚体铁蛋白突变体DE6H核酸序列的蛋白表达质粒载体;
(1.2)将步骤1.1制得的带有四聚体铁蛋白突变体DE6H核酸序列的蛋白表达质粒载体导入至市售BL21(DE3)感受态表达菌株中进行蛋白表达,8000-11000rpm/min高速离心菌液收集沉淀菌体,复溶于pH 6.0~8.0,20~50mM Tris-HCl缓冲液中,超声波破碎,经镍离子纯化柱纯化,得到纯四聚体铁蛋白突变体DE6H溶液;将纯四聚体铁蛋白突变体DE6H溶于pH6.0~8.0,20~50mM Tris-HCl缓冲液中,获得浓度为1~5μM的四聚体铁蛋白突变体DE6H溶液;
(2)锌离子诱导四聚体铁蛋白包埋活性小分子
(2.1)称取一定量的活性小分子,将其溶解在无水乙酸或无水乙醇中,制备成一定浓度的活性小分子母液,4℃避光保存待用;
(2.2)按照四聚体铁蛋白突变体DE6H与活性小分子的分子摩尔比为1:50~1:500的比例,向步骤1.2制得的四聚体铁蛋白突变体DE6H溶液中缓慢加入步骤2.1中制备的活性小分子母液,室温搅拌5min;接着按照四聚体铁蛋白与锌离子的分子摩尔比为1:50~1:200的比例,缓慢加入一定浓度的Zn2+溶液,于4℃~25℃层析柜中搅拌30~150min;
(2.3)将步骤2.2所得溶液置于透析袋中,于4℃~25℃层析柜中透析至含50~200mMNaCl的pH 6.0~8.0,20~50mM Tris-HCl缓冲液中,获得包埋有活性小分子的铁蛋白溶液。
2.根据权利要求1所述的锌离子诱导铁蛋白自组装包埋活性小分子的方法,其特征在于:所述步骤1.2中的纯化条件为:平衡缓冲液:20mM Tris,10mM咪唑,500mM NaCl,pH=8.0;洗脱缓冲液:20mM Tris,500mM咪唑,500mM NaCl,pH=8.0。
3.根据权利要求1所述的锌离子诱导铁蛋白自组装包埋活性小分子的方法,其特征在于:所述步骤2.2中Zn2+溶液为ZnSO4溶液。
4.根据权利要求1所述的锌离子诱导铁蛋白自组装包埋活性小分子的方法,其特征在于:所述活性小分子包括姜黄素、虾青素、β-胡萝卜素、阿霉素。
5.根据权利要求1-4任一项所述的锌离子诱导铁蛋白自组装包埋活性小分子的方法,其特征在于:所述方法的包埋效率为酸碱诱导的包埋效率的1.9~2倍且包埋反应在近中性pH溶液条件下进行,比酸碱诱导更为温和。
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