CN114805593A - 一种具有穿膜功能的Penetratin-hSOD1及其制备方法与应用 - Google Patents
一种具有穿膜功能的Penetratin-hSOD1及其制备方法与应用 Download PDFInfo
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- CN114805593A CN114805593A CN202110065861.8A CN202110065861A CN114805593A CN 114805593 A CN114805593 A CN 114805593A CN 202110065861 A CN202110065861 A CN 202110065861A CN 114805593 A CN114805593 A CN 114805593A
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Abstract
本发明提供了一种具有穿膜功能的Penetratin‑hSOD1及其制备方法与应用,属于分子与细胞生物学领域,制备方法具体为以细胞cDNA为模板,PCR扩增得到Penetratin‑hSOD1片段,将该片段同源重组到pET15b质粒,再将该质粒转化到大肠杆菌BL21中进行诱导表达,破碎菌体后,上清总蛋白通过Ni‑NTA柱纯化,得到高效可溶的Penetratin‑hSOD1。本发明的制备方法得到的Penetratin‑hSOD1可应用于制备抗氧化药物,其加上穿膜肽,可高效穿过细胞膜,与不能穿过细胞膜的SOD1相比,具有更强的细胞抗氧化能力。本发明首次采用穿膜肽Penetratin与SOD1蛋白进行重组,该蛋白与其它类似产品相比,具有更强的细胞穿膜能力和更高的稳定性。
Description
技术领域
本发明属于分子生物学与细胞生物学领域,特别涉及一种具有穿膜功能的重组蛋白Penetratin-hSOD1,还涉及到重组蛋白Penetratin-hSOD1的制备方法,重组蛋白Penetratin-hSOD1在制备抗氧化药物中的应用。
背景技术
超氧化物歧化酶(SODs)是生物体内一类重要的抗氧化酶,可以清除细胞中由于氧化压力产生的多余的活性氧。根据SODs在细胞中的定位可以分为三种(SOD1,SOD2,SOD3),其中SOD1在细胞中含量最多,分布在细胞质和线粒体中,占总SOD的90%(Zelko,I.N.,T.J.Mariani,and R.J.Folz,Superoxide dismutase multigene family:A comparisonof the CuZn-SOD(SOD1),Mn-SOD(SOD2),and EC-SOD(SOD3)gene structures,evolution,and expression.Free Radical Biology and Medicine,2002.33(3):p.337-349.)。但是SOD1为大分子蛋白,无法有效穿过细胞膜,只能在细胞外发挥作用,进而限制了其药用价值。
以往采用显微注射法,电穿孔法,脂质体法,病毒载体等方法将生物大分子导入细胞内,对细胞毒性大且效率低。穿膜肽(Cell-penetrating peptide,CPP)是由5-30个氨基酸短肽组成的家族,可以将外源性的蛋白质、DNA、siRNA、小分子药物带到细胞中。常见的穿膜肽有HIV-1转录激活蛋白TAT,多聚精氨酸Rn,黑腹果蝇触足肽Penetratin等(Raucher,D.and J.S.Ryu,Cell-penetrating peptides:strategies for anticancertreatment.Trends in Molecular Medicine,2015.21(9):p.560-570.)。
目前报道穿膜性最好的重组SOD1蛋白是TAT-SOD1,但是TAT来源于HIV-1,具有潜在的免疫反应和细胞毒性,且进入细胞内稳定性较差。Penetratin含有丰富的精氨酸和赖氨酸等正电荷氨基酸,能够迅速和带负电的细胞膜结合,可将多种低渗透性货物运送到细胞中,比如胰岛素。(El-Sayed Khafagy et al,Structural requirements of penetratinabsorption enhancement efficiency for insulin delivery,Journal of ControlledRelease,2010,143:302–310)。Penetratin-SOD1与TAT-SOD1相比,具有更强的的穿膜性和稳定性。
SOD1的来源主要有以下几种:一是直接从动物和植物体提取,来源丰富,但是提取和纯化工艺较为复杂。二是利用基因工程重组生产,此方法获得的SOD重组蛋白来源稳定、产量高、周期短、工艺简单、易于纯化、可规模化生产,得到越来越多关注。
发明内容
基于现有技术存在上述问题,本发明提供一种具有穿膜功能的重组蛋白Penetratin-hSOD1(人源铜锌超氧化物歧化酶)的制备方法,首先以人细胞系的SOD1对应的cDNA为模板,通过PCR扩增得到Penetratin-hSOD1核酸片段,将该核酸片段同源重组到pET15b质粒,再将该质粒转化到大肠杆菌中进行诱导表达,破碎大肠杆菌后提取总蛋白,总蛋白经过纯化得到Penetratin-hSOD1。
其中,所述的人细胞系包括LO2、HK-2或者HaCaT;所述的大肠杆菌包括BL21(DE3)、Rosetta-gami(DE3)或者Transetta(DE3),优选为BL21(DE3),大肠杆菌经诱导处于感受态。
其中,所述的大肠杆菌的诱导表达温度为15-28℃,优选为16℃;诱导时间为4-20h,优选为16h;诱导表达时,菌液OD600值为0.5-1.0,优选为0.8,IPTG终浓度为0.2-1mM;所述的纯化是通过Ni-NTA柱进行Penetratin-hSOD1重组蛋白纯化,纯化步骤为:目的蛋白结合,杂蛋白清洗,目的蛋白洗脱;目的蛋白结合条件优选为4℃结合1h,带有his标签的目的蛋白结合到Ni-NTA柱上;杂蛋白清洗缓冲液优选为50mM NaH2PO4,300mM NaCl,20mM咪唑,pH8.0;目的蛋白洗脱缓冲液优选为50mM NaH2PO4,300mM NaCl,300mM咪唑,pH8.0。
所述的纯化蛋白Penetratin-hSOD1还需要超滤除盐;超滤除盐条件优选为将纯化得到的Penetratin-hSOD1置于超滤管中,4℃、4000rpm离心,用蛋白储存液(50mM NaH2PO4,300mM NaCl,pH8.0)置换3次。
其中,所述的PCR扩增条件为:
其中,所述的破碎大肠杆菌的条件为冰水浴超声波处理,超声波功率优选为200W,超声波处理按处理3s后停止7s的规律循环至超声波处理结束,超声波处理的时间是每10ml菌液处理10min,超声波处理后的菌液经离心得到上清液总蛋白;所述的离心优选为在12000rpm离心15min。
一种可穿膜重组蛋白Penetratin-hSOD1,其根据上述的制备方法得到,其氨基酸序列如下:
一种穿膜肽Penetratin在制备可穿膜重组蛋白Penetratin-hSOD1中的应用。
一种如上所述的可穿膜重组蛋白Penetratin-hSOD1在制备抗氧化药物中的应用,所述的抗氧化药物含有如上所述的重组蛋白Penetratin-hSOD1,包括其溶剂蛋白中的至少一种,Penetratin-hSOD1加上穿膜肽,大大增加了药物进入细胞的能力,改善原有药物的抗氧化活性。
其中,所述的抗氧化药物含有一种或多种药学上可接受的载体或赋形剂,所述的赋形剂包括稀释剂、湿润剂、润滑剂、填充剂和防腐剂中的一种或多种;所述的抗氧化药物采用本领域的常规方法制备成各种剂型,剂型包括胶囊、丸剂、片剂、口服液、颗粒剂、酊剂的剂型,及注射液。
下面补充Penetratin氨基酸序列和Penetratin-hSOD1核苷酸序列:
Penetratin氨基酸序列:
Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys
Penetratin-hSOD1核苷酸序列:
本发明相对于现有技术,具有如下的优点及有益效果:
1、本发明采用新型穿膜肽Penetratin与SOD1蛋白融合,可实现SOD1蛋白的高效穿膜,相比其它SOD1产品,大大提高了SOD1进入细胞的效率。
2、本发明提供的Penetratin-hSOD1具有其它可穿膜SOD产品不具有的穿膜性更强、稳定性更好的功能。
3、本发明方法提高SOD1的穿膜能力和稳定性后,大大提高了其在细胞中的抗氧化能力,具有良好的药用前景。
4、本发明方法采用基因重组SOD表达方法,克服了传统方法从动植物中提取SOD原料的限制问题,提供一种高活性,低成本,生产周期短,产量大的制备方法。
附图说明
图1为Penetratin-hSOD1基因PCR扩增结果图。
图2为Penetratin-hSOD1基因测序结果图。
图3为Penetratin-hSOD1表达的SDS-PAGE凝胶电泳图。
图4为Penetratin-hSOD1纯化的SDS-PAGE凝胶电泳图。
图5为Penetratin-hSOD1的Western Blotting鉴定结果图。
图6为Penetratin-hSOD1的酶活力检测结果图。
图7为Penetratin-hSOD1的抗氧化能力检测结果图。
图8为Penetratin-hSOD1在Vero细胞中的穿膜效果结果图。
图9为Penetratin-hSOD1在Vero细胞中的稳定性结果图。
图10为Penetratin-hSOD1对过氧化氢引起的Vero细胞损伤保护作用结果图。
图11为Penetratin-hSOD1对顺铂引起的Vero细胞损伤保护作用结果图。
附图均为实施例中的实验检测结果,是为了证明和突出本发明提供的重组蛋白Penetratin-hSOD1和现有技术的区别之处,其中的颜色是展示区别点必须存在的要素且不会对本领域的技术人员理解本发明公开的技术方案造成影响。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此,下列实施例中使用试剂均可从商业渠道获得。
实施例1,具有穿膜功能的Penetratin-hSOD1的制备。
以LO2的SOD1对应的cDNA为模板进行PCR扩增得到Penetratin-hSOD1基因,PCR扩增条件:
结果如图1所示(M:DNA marker,5K;1:Penetratin-hSOD1基因),Penetratin-hSOD1基因片段为507bp。
将得到的Penetratin-hSOD1基因片段用重组酶Exnase连接到表达载体pET15b质粒上,转化克隆感受态细胞DH5α,在含有氨苄青霉素的平板上培养,挑取单克隆送测序,测序结果如图2所示。
将测序正确的质粒转化到表达感受态BL21大肠杆菌中,在含有氨苄青霉素的平板上培养:挑取单克隆于5ml含有100μg/ml氨苄青霉素的LB培养基中,37℃、200rpm培养12h,取500μL菌液至100ml含有100μg/ml氨苄青霉素的LB培养基中,37℃、200rpm培养至OD600为0.8,加入IPTG(异丙基-β-D-硫代半乳糖苷)至终浓度为0.5mM,18℃、180rpm继续诱导培养16h。
菌液在4℃、6000rpm的条件下离心10min,弃上清;向菌体中加入5ml PBS,用移液枪吹散菌体,冰上超声10min(200W,5s开,7s关),至菌液基本保持澄清;再在4℃、10000rpm的条件下离心20min,留取上清,即为粗酶样品;取5ml粗酶样品至5ml镍柱中,流走样品,带有标签的目的蛋白会结合在镍柱上,用清洗液(50mM NaH2PO4,20mM咪唑,300mM NaCl,pH8.0)清洗3次;用洗脱液(50mM NaH2PO4,300mM咪唑,300mM NaCl,pH 8.0)洗脱。
将洗脱液转移至超滤管(Merck,Germany),4℃4000rpm 30min,用蛋白储存液(50mM NaH2PO4,300mM NaCl,pH8.0)置换3次,得到纯化蛋白浓缩液。
分别取诱导表达前后的菌体,超声破碎后进行SDS-PAGE电泳,结果如图3所示(1:诱导后,2:诱导前,M:Marker),结果表明Penetratin-hSOD1的分子量约为23kDa,诱导后Penetratin-hSOD1的表达明显增加。将洗脱液进行SDS-PAGE电泳,结果如图4所示(M:蛋白Marker;1:纯化前蛋白;2:纯化后蛋白),结果表明,粗酶经过纯化步骤,得到纯度较高的Penetratin-hSOD1蛋白。
实施例2,具有穿膜功能的Penetratin-hSOD1的制备,本实施例没详细描述部分与实施例1一致。
以HK-2cDNA为模板进行PCR扩增得到Penetratin-hSOD1基因,将其用重组酶连接到表达载体pET15b上,转化克隆感受态细胞DH5α,挑取测序正确的质粒转化表达感受态Rosetta-gami,挑取单克隆在LB中37℃、200rpm培养至OD600为0.5,加入IPTG至终浓度为0.5mM,18℃、180rpm继续诱导培养16h。收集菌液进行超声破碎,用Ni-NTA柱进行蛋白纯化,用超滤管进行蛋白除盐,得到纯度较高的Penetratin-hSOD1蛋白。
实施例3,具有穿膜功能的Penetratin-hSOD1的制备,本实施例没详细描述部分与实施例1一致。
以LO2 cDNA为模板进行PCR扩增得到Penetratin-hSOD1基因,将其用重组酶连接到表达载体pET15b上,转化克隆感受态细胞DH5α,挑取测序正确的质粒转化表达感受态Transetta,挑取单克隆在LB中37℃、200rpm培养至OD600为1.0,加入IPTG至终浓度为0.5mM,18℃、180rpm继续诱导培养16h。收集菌液进行超声破碎,用Ni-NTA柱进行蛋白纯化,用超滤管进行蛋白除盐,得到纯度较高的Penetratin-hSOD1蛋白。
实施例4,具有穿膜功能的Penetratin-hSOD1的制备,本实施例没详细描述部分与实施例1一致。
以LO2 cDNA为模板进行PCR扩增得到Penetratin-hSOD1基因,将其用重组酶连接到表达载体pET15b上,转化克隆感受态细胞DH5α,挑取测序正确的质粒转化表达感受态BL21,挑取单克隆在LB中37℃、200rpm培养至OD600为0.5,加入IPTG至终浓度为0.5mM,25℃、180rpm继续诱导培养4h。收集菌液进行超声破碎,用Ni-NTA柱进行蛋白纯化,用超滤管进行蛋白除盐,得到纯度较高的Penetratin-hSOD1蛋白。
实施例5,具有穿膜功能的Penetratin-hSOD1的制备,本实施例没详细描述部分与实施例1一致。
以LO2 cDNA为模板进行PCR扩增得到Penetratin-hSOD1基因,将其用重组酶连接到表达载体pET15b上,转化克隆感受态细胞DH5α,挑取测序正确的质粒转化表达感受态BL21,挑取单克隆在LB中37℃、200rpm培养至OD600为1.0,加入IPTG至终浓度为0.3mM,20℃、200rpm继续诱导培养10h。收集菌液进行超声破碎,用Ni-NTA柱进行蛋白纯化,用超滤管进行蛋白除盐,得到纯度较高的Penetratin-hSOD1蛋白。
实施例6:Penetratin-hSOD1蛋白检测。
用Western Bloting方法检测Penetratin-hSOD1蛋白:用SOD1抗体与Penetratin-hSOD1蛋白结合,再用HRP标记的二抗与一抗结合,最后用ECL发光液显示条带,结果如图5所示(1:低浓度,2:高浓度),结果表明纯化得到的蛋白为SOD蛋白。
实施例7:Penetratin-hSOD1蛋白酶活检测。
用WST-1法对Penetratin-hSOD1蛋白进行酶活检测:酶活为1776U/mg,不加穿膜肽的SOD1酶活为1232U/mg,结果如图6所示。
实施例8:Penetratin-hSOD1蛋总抗氧化能力检测。
用FRAP法对Penetratin-hSOD1蛋白进行体外总抗氧化能力(T-AOC)检测:1mgPenetratin-hSOD1具有相当于10mM Vitamin E的抗氧化能力,1mg SOD1具有相当于8mMVitamin E的抗氧化能力,结果如图7所示。
实施例9:Penetratin-hSOD1蛋白穿膜能力检测。
用免疫荧光方法对Penetratin-hSOD1蛋白进行穿膜能力检测:分别用SOD1和Penetratin-hSOD1处理Vero细胞1h,将细胞固定、透化后,用his一抗孵育过夜,再用带有荧光标记的二抗室温孵育2h,荧光显微镜观察,结果如图8所示,结果表明Penetratin-hSOD1可以高效的穿过细胞膜到达Vero细胞内,不加穿膜肽的SOD1无法穿过细胞膜。
实施例10:Penetratin-hSOD1蛋白稳定性检测。
用Western Blot方法检测Penetratin-hSOD1蛋白在Vero细胞中的稳定性:分别用Penetratin-hSOD1和TAT-hSOD1处理Vero细胞1h,撤去培养基,在不同时间点收取蛋白,进行Western Blot检测,结果如图9所示,结果表明Penetratin-hSOD1相比于已知穿膜性较好的TAT-hSOD1蛋白,具有更强的穿膜性和更高的稳定性。
实施例11:Penetratin-hSOD1蛋白细胞内抗氧化能力检测。
(1)Penetratin-hSOD1可有效缓解过氧化氢引起的Vero细胞损伤:用Penetratin-hSOD1处理Vero细胞1h,加入2mM过氧化氢培养3h,加入10μL 5mg/ml MTT,继续培养4h,吸走培养基,加入100μL DMSO,490nm处测量吸光度,计算细胞存活率,结果如图10所示,结果表明用过氧化氢处理细胞后,细胞存活率降为50%左右,Penetratin-hSOD1预处理再加过氧化氢,细胞存活率为80%左右,并且呈剂量依赖性,表明Penetratin-hSOD1可以有效缓解过氧化氢引起的Vero细胞损伤。
(2)Penetratin-hSOD1可有效缓解顺铂引起的Vero细胞损伤:用Penetratin-hSOD1处理Vero细胞1h,加入10μM顺铂培养24h,加入10μL 5mg/ml MTT,继续培养4h,吸走培养基,加入100μL DMSO,490nm处测量吸光度,计算细胞存活率,结果如图11所示,结果表明顺铂处理后,细胞存活率为50%左右,Penetratin-hSOD1预处理再加顺铂,细胞存活率为70%左右,并且呈剂量依赖性,表明Penetratin-hSOD1可以有效缓解顺铂引起的Vero细胞损伤。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种具有穿膜功能的重组蛋白Penetratin-hSOD1的制备方法,其特征在于,首先以人细胞系的SOD1对应的cDNA为模板,通过PCR扩增得到Penetratin-hSOD1核酸片段,将该核酸片段同源重组到pET15b质粒,再将该质粒转化到大肠杆菌中进行诱导表达,破碎大肠杆菌后提取总蛋白,总蛋白经过纯化得到Penetratin-hSOD1。
2.根据权利要求1所述的Penetratin-hSOD1的制备方法,其特征在于,所述的人细胞系包括LO2、HK-2或者HaCaT;所述的大肠杆菌包括BL21(DE3)、Rosetta-gami(DE3)或者Transetta(DE3),大肠杆菌经诱导处于感受态。
3.根据权利要求1所述的Penetratin-hSOD1的制备方法,其特征在于,所述的大肠杆菌的诱导表达温度为15-28℃,诱导时间为4-20h;诱导表达时,菌液OD600值为0.5-1.0,IPTG终浓度为0.2-1mM;所述的纯化是通过Ni-NTA柱进行Penetratin-hSOD1重组蛋白纯化。
5.一种可穿膜重组蛋白Penetratin-hSOD1,其特征在于,其根据权利要求1-4任一所述的制备方法得到。
6.根据权利要求5所述的重组蛋白Penetratin-hSOD1,其特征在于,其氨基酸序列如下:
1 Arg Trp Phe Lys Ile Gln Met Gln Ile Arg Arg Trp Lys Asn Lys Lys Ala ThrLys
20 Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn PheGlu Gln
39 Lys Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu ThrGlu Gly
58 Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr Ala Gly Cys ThrSer Ala
77 Gly Pro His Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp GluGlu Arg
96 His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala AspVal Ser
115 Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile Ile Gly ArgThr Leu
134 Val Val His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu SerThr Lys
153 Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly Ile Ala Gln。
7.一种穿膜肽Penetratin在制备可穿膜重组蛋白Penetratin-hSOD1中的应用。
8.一种如权利要求5或6所述的可穿膜重组蛋白Penetratin-hSOD1在制备抗氧化药物中的应用。
9.根据权利要求8所述的应用,其特征在于,所述的抗氧化药物含有如权利要求6或7所述的重组蛋白Penetratin-hSOD1。
10.根据权利要求8所述的应用,其特征在于,所述的抗氧化药物含有一种或多种药学上可接受的载体或赋形剂,所述的赋形剂包括稀释剂、湿润剂、润滑剂、填充剂和防腐剂中的一种或多种;所述的抗氧化药物的剂型包括胶囊、丸剂、片剂、口服液、颗粒剂、酊剂的剂型,及注射液。
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