CN114480445B - 一种人源超氧化物歧化酶hSOD1突变体的制备及其应用 - Google Patents
一种人源超氧化物歧化酶hSOD1突变体的制备及其应用 Download PDFInfo
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Abstract
本发明提供一种人源超氧化物岐化酶hSOD1突变体制备方法及其应用，涉及生物工程技术领域。提供一种人源超氧化物歧化酶hSOD1突变体的编码蛋白，其具有SEQIDNO.Mt1SOD1aa所示氨基酸序列；提供一种编码上述蛋白的Mt1SOD1基因，具有SEQIDNO.Mt1SOD1所示核苷酸序列。本发明以人源hSOD1为模板，通过基因突变获得了hSOD1突变体Mt1SOD1(E25G、P29T、E101V、C112S)基因，重组人源超氧化物歧化酶hSOD1突变体Mt1SOD1蛋白；在应用上解决了异源性问题。且具备高活性高稳定性，对新型功能型抗衰妆品及系列产品的开发、其他抗衰产品如功能食品、抗衰药品的开发提供了新的途径。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种人源超氧化物歧化酶hSOD1突变体的制备及其应用。
背景技术
超氧化物歧化酶(Superoxide Dismutase,SOD)是一种普遍存在于各类生物体中的活性物质，是生物体内最核心的自由基清除酶，能够专一地清除机体内细胞代谢过程中超氧阴离子，被称为生物体抗氧化系统的第一道防线。SOD可减少由光照黑色素耗氧过多而产生的氧自由基，防止过多的黑色素形成，同时也可不断地清除由脂褐素和蜡样质产生的氧自由基，避免过多的脂质发生过氧化，减少脂褐素和蜡样质的形成。此外机体中SOD的活性升高可减轻组织细胞过氧化性损伤，降低脂类过氧化物的形成，加强分解体内过多的脂质过氧化物。SOD不仅具有延缓皮肤衰老的功能，还在机体氧化应激、抗炎修复、抗皱等方面起重要作用，现已被广泛运用于医疗、食品以及化妆品行业。
目前重组SOD1已成功在大肠杆菌、毕赤酵母、乳酸菌、聚球藻、山羊乳腺细胞等表达系统中表达，然而，到目前为止，这些系统还没有能够大规模生产出产量和活性都令人满意的重组Cu/Zn-SOD(SOD1)。牦牛属于牛属牦牛亚属牦牛种，青藏高原高寒缺氧的恶劣环境使其体内的SOD活性随含氧量的降低而升高，从牦牛血中提取的牦牛SOD1活性高达13000U。例如发明专利201010169323.5号重组表达牦牛SOD1的文献，但仍然是传统的动物提取出来的SOD，存在稳定性较差，表达水平不稳定，活性难以保持等缺陷。基于目前现有技术中重组Cu/Zn-SOD(SOD1)存在的缺陷，本研究以hSOD1为模板，利用生物信息学结合分子生物学方法对比牦牛SOD1基因序列，得到hSOD1突变体，重组出来的突变体也是人源的，在应用上解决了异源性问题。
本发明通过对人源hSOD1进行氨基酸位点优化，利用大肠杆菌表达系统，优化发酵及纯化条件，获得高活性高稳定性的重组人源hSOD1突变体。此重组人源hSOD1突变体可应用于新型功能型抗衰妆品，且可用于系列产品的应用开发以及其他抗衰品(如功能食品、抗衰药品)的开发应用。
发明内容
本发明提供一种人源hSOD1突变体的制备方法及其应用。公开了人源超氧化物歧化酶hSOD1突变体的编码蛋白，其具有SEQ ID NO.Mt1SOD1 aa所示的氨基酸序列；
本发明以hSOD1为模板，利用生物信息学结合分子生物学方法对比牦牛SOD1基因序列，得到人源超氧化物歧化酶hSOD1突变体Mt1SOD1(E25G、P29T、E101V、C112S)基因，命名为Mt1SOD1。
本发明公开了一种编码上述蛋白的Mt1SOD1基因，具有SEQ ID NO.Mt1SOD1所示的核苷酸序列。
本发明还公开了一种含有上述基因的pET-28a(+)-Mt1SOD1表达载体以及含有所述载体的BL21(DE3)-pET28a(+)-Mt1SOD1工程菌。
一种重组人源超氧化物歧化酶hSOD1突变体Mt1SOD1的制备方法，步骤如下：
(1)NCBI获取序列，进行密码子优化，5‘端添加6×His-tag标签，化学合成5‘端具有6×His-tag编码序列的hSOD1、Mt1SOD1基因，化学合成由擎科公司代理完成；
(2)将hSOD1、Mt1SOD1基因片段克隆至pET-28a(+)质粒,构建pET-28a(+)-hSOD1、pET-28a(+)-Mt1SOD1表达质粒，此步骤由擎科公司代理完成；
(3)通过热激法将表达质粒转化至BL21(DE3)，获得BL21(DE3)-pET28a(+)-hSOD1、BL21(DE3)-pET28a(+)-Mt1SOD1工程菌，此工程菌暂存于西南交通大学生命科学与工程学院-80℃冰箱；
(4)利用大肠杆菌BL21(DE3)进行诱导表达，利用BL21(DE3)-pET28a(+)-hSOD1、BL21(DE3)-pET28a(+)-Mt1SOD1工程菌在25℃，1mMIPTG，800μMCu2+，20μMZn2+条件下诱导16h表达野生型hSOD1及突变型Mt1SOD1。
(5)利用Ni-NTA进行蛋白纯化，通过hSOD1、Mt1SOD1与Ni离子特性异性结合洗脱杂蛋白，200mM咪唑洗脱液洗脱得到hSOD1、Mt1SOD1蛋白。
重组人源超氧化物歧化酶hSOD1突变体Mt1SOD1的应用。由于天然SOD1来源有限,且具有异体蛋白免疫原性,外源SOD不易被人体接受，易引起过敏反应等缺陷,使之在应用方面受到很大限制。SOD基因工程是广开酶源,降低成本和获得无抗原性的人源SOD的有效途径。本发明以人源SOD1为模板，通过基因突变获得了突变体Mt1SOD1，在应用上解决了异源性问题，且具备高活性高稳定性。已应用于新型功能型抗衰妆品及系列产品的开发，还可应用于其他抗衰产品如功能食品、抗衰药品的开发。
与现有技术相比，本发明包括具有如下技术效果：
(1)本发明中的超氧化物歧化酶是一种普遍存在于各类生物体中的活性物质，是生物体内重要的自由基清除剂。本发明通过突变野生型人源SOD1的氨基酸位点(E25G、P29T、E101V、C112S)，获得了高活性高稳定性的人源SOD1突变体Mt1SOD1。(2)本发明根据大肠杆菌密码子使用偏好性，分别将野生型人源SOD1核酸序列、突变野生型人源SOD1核酸序列进行密码子优化，5‘端添加6×His-tag标签，构建pET-28a(+)表达载体。
(3)本发明通过在25℃条件下，添加1mMIPTG、800μMCu2+、20μMZn2+，180rpm培养实现了目的蛋白的高可溶性表达。
(4)本发明通过Ni-NTA纯化hSOD1、Mt1SOD1，通过热稳定性及PH稳定性分析，验证突变野生型人源SOD1与hSOD1相比具有高活性高稳定性。
(5)本发明将经典的抗氧化酶SOD1应用于新型功能型抗衰妆品，且可用于系列产品的应用开发。此重组的SOD1还应用于其他抗衰产品(如功能食品、抗衰药品)的开发应用。
附图说明
图1显示了根据本发明实施例1的野生型人源SOD1纯化电泳示意图，第8道为纯化的hSOD1。
图2显示了根据本发明实施例1的人源SOD1突变体纯化电泳示意图，第8道为纯化的Mt1SOD1。
图3显示了根据本发明实施例2的热稳定性测试。
图4显示了根据本发明实施例2的PH稳定性测试。
具体实施方式
结合附图与实施例对本发明作进一步的说明。
实施例中未注明具体条件的实验方法，通常按照分子克隆实验室手册(《分子克隆实验指南》，科学出版社，2002年，第三版)中所述的实验条件，或按照试剂或仪器生产厂商所建议操作说明书条件进行。
本发明以hSOD1为模板，获得了高活性高稳定性Mt1SOD1，其突变位点为E25G、P29T、E101V、C112S。突变位点的分析过程表明，利用protparam分析显示，hSOD1、牦牛SOD1理化性质相似，牦牛SOD1与hSOD1相比不稳定性指数显著偏低(表1)。其中25、29、101位对不稳定性指数有显著影响，为影响SOD活性的关键氨基酸位点结合112位半胱氨酸的突变，构建Mt1SOD1。
化学合成密码子优化的hSOD1、Mt1SOD1序列后，将其重组克隆到pET-28a(+)中，转化大肠杆菌BL21(DE3)。经1mmol/LIPTG诱导后，在含800μmol/L Cu2+和20μmol/L Zn2+的LB培养基中，25℃、180r/min诱导培养16h，hSOD1、Mt1SOD1均以可溶性形式高效表达，镍亲和层析可有效纯化重组hSOD1、Mt1SOD1。100mlLB摇瓶培养获得的hSOD1活性为71094U/mg，产率为4.57mg。Mt1SOD1活性为128506U/mg，产率为3.13mg。
对hSOD1、Mt1SOD1的酶学性质研究表明，在25～55℃之间，两种酶活力基本保持不变，75℃保温30min，hSOD1相对酶活力保持在50％左右，Mt1SOD1保持30％酶活性。在pH3.6～10.4条件下放置30min，两种SOD1均能保持70％以上的酶活力。与重组hSOD1相比，Mt1SOD1活性更高，同时稳定性良好。
实施例1
重组人源超氧化物歧化酶hSOD1及其突变体Mt1SOD1的构建方法，步骤如下：
1、重组人SOD1及其突变体的构建
从NCBI获取序列，根据大肠杆菌密码子使用偏好性进行密码子优化，5‘端添加6×His-tag标签，化学合成5‘端具有6×His-tag编码序列的hSOD1、Mt1SOD1基因；经测序证实。序列两端添加BamHI、XhoI酶切位点，并克隆到pET-28a(+)载体，此步骤由擎科公司代理完成。热激法转化大肠杆菌BL21(DE3)，挑取单菌落培养后利用pET-28a(+)载体通用引物T7、T7-ter进行菌落PCR鉴定，并将鉴定正确的阳性克隆暂存于西南交通大学生命科学与工程学院-80℃冰箱。PCR反应体系为12.5μl2×TSINGKE Master Mix、0.5μlT7(10μm)、0.5μlT7-ter(10μm)、2μl菌液，补加ddH2O至25μl。反应条件依次为94℃预变性10min，94℃变性30s、55℃退火30s，72℃延伸10min，反应35个循环。
正向引物T7：TAATACGACTCACTATAGGG
反向引物T7-ter：TGCTAGTTATTGCTCAGCGG
具体步骤简述如下：
(1)NCBI获取序列，进行密码子优化，5‘端添加6×His-tag标签，化学合成5‘端具有6×His-tag编码序列的hSOD1、Mt1SOD1基因，此步骤由擎科公司代理完成；
(2)测序验证合成的核氨酸序列经编码后分别为hSOD1、Mt1SOD1氨基酸序列；(3)序列两端添加BamHI、XhoI酶切位点，将hSOD1、Mt1SOD1基因片段克隆至pET-28a(+)质粒，构建pET-28a(+)-hSOD1、pET-28a(+)-Mt1SOD1表达质粒，此步骤由擎科公司代理完成；
(4)热激法转化大肠杆菌BL21(DE3)，挑取单菌落培养后利用pET-28a(+)载体通用引物T7、T7-ter进行菌落PCR鉴定，并将鉴定正确的阳性克隆暂存于西南交通大学生命科学与工程学院-80℃冰箱。
(5)获得BL21(DE3)-pET28a(+)-hSOD1、BL21(DE3)-pET28a(+)-Mt1SOD1工程菌；
(6)利用大肠杆菌BL21(DE3)进行诱导表达，利用BL21(DE3)-pET28a(+)-hSOD1、BL21(DE3)-pET28a(+)-Mt1SOD1工程菌在25℃，1mMIPTG，800μMCu2+，20μMZn2+条件下诱导16h表达野生型hSOD1及突变型Mt1SOD1。
2、hSOD1、Mt1SOD1在大肠杆菌中的表达
将保藏的BL21(DE3)-pET28a(+)-hSOD1、BL21(DE3)-pET28a(+)-Mt1SOD1工程菌在添加卡那霉素(50μg/mL)的LB固体培养基中活化，挑取单菌落制备饱和过夜培养物。吸取1ml至100ml新鲜LB于37℃培养至OD600＝0.6-0.8时，分别添加1mMIPTG、800μMCu2+、20μMZn2 +于25℃，180rpm诱导4h。通过离心(5000rpm，4℃，20分钟)收集菌体，重悬于5ml结合液(10mM Tris-HCl pH 7.9、500mM NaCL、20mM Imidazole)。利用超声破碎仪(30％功率，超声5s停5s)冰上破碎20min，通过在4℃11000rpm离心20min，分离上清和沉淀物。
3、hSOD1、Mt1SOD1的纯化。利用Ni-NTA柱进行纯化，得到所述人源hSOD1突变体：
将上清液加载到Ni-NTA亲和柱(上海生工生物工程有限公司)上4℃结合4h。纯化条件遵循制造商的说明。用20mM和60mM咪唑洗涤色谱柱后，用200mM咪唑洗脱目的蛋白。通过BCA蛋白质测定试剂盒(天根生化科技有限公司)检测蛋白质浓度。最终100ml菌液hSOD1产量为4.57mg，纯化倍数为12.81，hSOD1回收率97.02％。100ml菌液Mt1SOD1产量为3.13mg，纯化倍数为13.42，Mt1SOD1回收率99.45％。
最终得到的重组人源超氧化物歧化酶hSOD1经12％的SDS-PAGE电泳分析，其分子量约为20kDa，纯度达95％以上。突变体Mt1SOD1融合蛋白经12％的SDS-PAGE电泳分析，其分子量约为19kDa，纯度达95％以上。
4、BCA法测定蛋白浓度
根据试剂盒要求，定量取溶液A：溶液B＝50:1，混匀制成BCA工作液，称取10mg试剂C，用250μl溶液D和250μl蒸馏水溶解，涡旋振荡1min，得到溶液F。配制2mg/mL BSA蛋白母液，分别稀释成终浓度为0.0625mg/mL、0.125mg/mL、0.25mg/mL、0.5mg/mL、1.0mg/mL、2mg/mL。各孔加入5μl溶液F，37℃水浴中保温30min，再往每孔中加入200μl BCA工作液，迅速混匀,在37℃水浴中保温30min。冷却至室温后，测定各孔的562nm处的吸光度值。分别以蛋白浓度和吸光度为横纵坐标绘制标准曲线。将待检测的样品蛋白用ddH2O稀释4倍，使其浓度在0.0625mg/mL-2mg/mL，吸光度测定方法同上，根据样品稀释液A562的平均值，在标准曲线上计算出该样品经过稀释后的蛋白质浓度，再乘以稀释倍数计算出原样品的蛋白浓度。
实施例2
1、重组人源超氧化物歧化酶hSOD1及其突变体Mt1SOD1的酶学性质
(1)测定原理及方法介绍:
采用羟胺法总超氧化物歧化酶(T-SOD)测定试剂盒检测重组hSOD1、Mt1SOD1的催化活性。通过黄嘌呤及黄嘌呤氧化酶反应系统产生超氧阴离子自由基，后者氧化羟胺形成亚硝酸盐，在显色剂作用下呈现紫红色。根据生产厂家的要求，预先配制了试剂四应用液。初步实验确定了hSOD1的最佳加入量，使SOD的抑制率在15％～55％之间。将一定量的SOD样品、各试剂混匀在37℃孵育40min，室温放置30min在450nm处检测吸光度。根据厂家说明书中的公式计算hSOD1、Mt1SOD1的活性。
(2)酶活性及其稳定性的测定：
将hSOD1、Mt1SOD1以及从牛血中提取的标准品SOD1分别置于pH 3.6、5.8、7.4、8.6、10.4的缓冲液中，放置30min，测定酶在不同pH条件下残余的酶活力。将hSOD1、Mt1SOD1以及从牛血中提取的标准品SOD1分别置于25、37、55、75、90℃下保温30min，测定残余酶活力。结果清晰显示出在pH为3.6-10.4之间时，SOD1相对活力较高，两种SOD1均能在酸碱环境下保持70％以上的酶活力。在25-55℃之间，两种SOD1活力基本保持不变，在温度达到75℃时hSOD1相对酶活力保持在50％左右，Mt1SOD1保持30％酶活性，当温度升高至90℃时，hSOD1基本失活，Mt1SOD1仅残余16％的酶活力。说明hSOD1与Mt1SOD1均具有较好的热稳定性。与hSOD1相比，Mt1SOD1活性更高且稳定性良好。
实施例3
重组hSOD1、Mt1SOD1电泳检测
分别收集上述表达及纯化过程中的菌液和洗脱液，加入5×蛋白上样缓冲液，95℃金属浴8min。10000rpm，离心10min，吸取8μl上清液进行常规SDS-PAGE检测(5％浓缩胶，12％分离胶)。电泳仪(北京六一仪器厂)工作模式为80V进行30min随后调至120V进行1h30min。电泳结束后采用考马斯亮蓝染色液染色2h，后更换脱色液直到条带清晰。图1为hSOD1表达纯化过程，其中M为蛋白预染Maker，1为未诱导的菌液可溶性片段，2为添加IPTG诱导的全菌体裂解液，3为诱导后超声的上清液，4为诱导后超声的不溶性沉淀，5为流穿液，6为20mmol/L咪唑洗脱液产物，7为60mmol/L咪唑洗脱液产物，8为200mmol/L咪唑洗脱液产物。图2为Mt1SOD1表达纯化过程，其条带注释与图1相同。
上述实施例为本发明示例性的实施方式，但本发明的实施方式并不受实施例的限制，其它任何未背离本发明的精神实质与原理下所做的改变、修饰、组合、替代、简化均应为等效替换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 西南交通大学
<120> 一种人源超氧化物歧化酶hSOD1突变体的制备及其应用
<130> 专利申请
<160> 2
<170> PatentIn version 3.5
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<213> 人工序列 (Artificial Sequence)
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Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val
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His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His
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Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg
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His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala
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Asp Val Ser Ile Val Asp Ser Val Ile Ser Leu Ser Gly Asp His Ser
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Ile Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly
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<213> 人工序列 (Artificial Sequence)
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atggcaacca aagcggtttg tgttctgaaa ggggacgggc cggttcaggg gattattaat 60
tttgagcaga aaggtagcaa cggtacagtt aaagtttggg ggtcgattaa aggactgacc 120
gaaggactgc atggttttca tgttcatgag tttggggaca ataccgcagg gtgtacaagc 180
gcaggtccgc attttaatcc gctgagccgt aaacatggtg gtccgaaaga tgaagaacgt 240
catgttggtg atctgggtaa tgttaccgcg gataaagatg gcgtagcgga tgttagcatt 300
gttgattcag ttatctcact gtcaggtgat catagcatta ttggccgtac actggttgtt 360
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gcaggcagtc gtctggcatg tggtgtgatt ggaattgcac agtaa 465
Claims (7)
1.一种人源超氧化物歧化酶hSOD1突变体的基因，其特征在于，命名为Mt1SOD1，其核苷酸序列如SEQ ID NO.2所示。
2.含有权利要求1所述基因的表达载体pET-28a-Mt1SOD1。
3.一种含有权利要求2所述载体pET-28a-Mt1SOD1的BL21(DE3)-pET28a-Mt1SOD1工程菌。
4.权利要求1所述基因编码得到的重组Mt1SOD1突变体蛋白，其氨基酸序列如SEQ IDNO.1所示。
5.一种重组人源超氧化物歧化酶hSOD1突变体Mt1SOD1的制备方法，其特征在于，所述突变体Mt1SOD1的氨基酸序列如SEQ ID NO.1所示，步骤如下：
(1)NCBI获取序列，进行密码子优化，5’端添加6×His-tag标签，化学合成5’端具有6×His-tag编码序列的hSOD1、Mt1SOD1基因；
(2)测序验证；
(3)将hSOD1、Mt1SOD1基因片段克隆至pET-28a+质粒；
(4)构建pET-28a-Mt1SOD1表达质粒；
(5)获得BL21(DE3)-pET28a-SOD1目的基因工程菌；
(6)利用大肠杆菌BL21(DE3)进行诱导表达，利用BL21(DE3)-pET28a-SOD1工程菌在25℃，1mMIPTG，800μMCu2+，20μMZn2+条件下诱导16h分别表达野生型hSOD1及突变型Mt1SOD1。
6.一种重组人源超氧化物歧化酶hSOD1突变体Mt1SOD1的应用，所述突变体Mt1SOD1的氨基酸序列如SEQ ID NO.1所示，其特征在于，应用于功能型抗衰化妆品的开发或应用于其他抗衰产品的开发。
7.如权利要求6所述重组人源超氧化物歧化酶hSOD1突变体Mt1SOD1的应用，其中所述其他抗衰产品是功能食品或抗衰药品。
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