CN116284437B - 一种超氧化物歧化酶融合多肽及其制备方法和在制备抗衰老产品中的应用 - Google Patents
一种超氧化物歧化酶融合多肽及其制备方法和在制备抗衰老产品中的应用 Download PDFInfo
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Abstract
一种超氧化物歧化酶融合多肽及其制备方法和在制备抗衰老产品中的应用，涉及基因工程、合成生物技术领域。重组SOD酶融合多肽的氨基酸序列如SEQ ID NO:3所示，是将耐热性强的mPeSOD2‑1，与高功能但不稳定的人源化细胞修复多肽进行融合表达。利用mPeSOD2‑1作为伴体蛋白，保护修复多肽，抵抗高温和降解，且保留两者的生物活性。高温孵育后，融合多肽既具有强抗氧化性，有利于清除过多有害的氧自由基，保护和修复细胞，也具有养护细胞，促细胞增殖，修复机体损伤的作用。融合多肽稳定性强，活性高，工业实用性强，在制备延缓机体衰老，促进机体和皮肤新生的抗衰老化妆品、保健食品等领域中具有广阔的应用前景。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种超氧化物歧化酶融合多肽及其制备方法和在制备抗衰老产品中的应用。
背景技术
超氧化物歧化酶(superoxide dismutase，SOD)可用于催化超氧阴离子与氢反应，产生氧气和过氧化氢，是一种重要抗氧化剂，在保护细胞免受氧自由基的毒性方面发挥着关键作用。SOD常以铜和锌、或锰、铁、或镍作为辅助因子的形式存在；几乎所有真核细胞的细胞内都含有带有铜和锌的超氧化物歧化酶(Cu-Zn-SOD，SOD1)；几乎所有的线粒体和许多细菌(如大肠杆菌)均含有结合锰的超氧化物歧化酶(Mn-SOD，SOD2)，研究表明，SOD2可用来清除对皮肤造成损害的自由基，减少肌成纤维细胞的表型逆转来减少纤维化。
活性因子，包含三对二硫键(Cys6-Cys20,Cys14-Cys31,Cys33-Cys42)，二硫键形成产生三个结构环，通过与其受体结合，刺激细胞生长、增殖和分化。根据Hang-Cheol Shin等人对活性因子定点突变研究，把L8S，I38C，D46C突变增加活性因子其自身热稳定性，与野生型活性因子相比圆二色(CD)相变温度Tm值从76℃提高到约87℃，可能与其增加了一二硫键有关，该突变体60℃处理5天，80％以上的突变体分子维持结构稳定(CD分析)。但增加二硫键会增加分子合成中正确折叠，形成活性高级结构的难度，在用大肠杆菌等表达过程中可能会引入包涵体。根据Mount C D及Song Yub Shin等人研究，活性因子的N端2-6位氨基酸或C端48-53位氨基酸删去对其与受体亲和力及促细胞生长活性无显著影响，在小鼠和人的尿液中也存在2-53,1-52，1-51.1-50氨基酸等结构。
然而无论是SOD还是活性因子，其稳定性较差，普遍存在耐高温性差、易降解等问题，影响其活性，限制其进一步的发展应用。
发明内容
为了克服现有技术的不足，本发明的目的之一在于提供一种超氧化物歧化酶融合多肽，其结构稳定，耐热性强，不易降解。
本发明的目的之二在于提供一种超氧化物歧化酶融合多肽的制备方法，其易于操作性，可重复性强。
本发明的目的之三在于提供一种超氧化物歧化酶融合多肽在制备抗衰老产品中的应用。
本发明的目的之一采用如下技术方案实现：
一种超氧化物歧化酶融合多肽，包括：(a)氨基酸序列如SEQ ID NO:3所示的蛋白质；
(b)在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有超氧化物歧化酶融合多肽活性的由(a)衍生的超氧化物歧化酶融合多肽衍生物。
进一步地，所述超氧化物歧化酶融合多肽衍生物(b)包括由超氧化物歧化酶融合多肽(a)的N端修饰多肽形成的末端修饰衍生物。
进一步地，所述末端修饰衍生物的氨基酸序列如SEQ ID NO:4所示。
进一步地，超氧化物歧化酶融合多肽包括氨基酸序列如SEQ ID NO:2所示的超氧化物歧化酶片段和活性肽。
进一步地，所述超氧化物歧化酶片段由氨基酸序列如SEQ ID NO:1所示的SOD酶定点突变而成，所述定点突变包括：将所述SOD酶的第9位的天冬酰胺突变成丝氨酸，第15位的天冬酰胺突变成赖氨酸，第41位的脯氨酸突变成亮氨酸。
一种所述超氧化物歧化酶融合多肽的制备方法，包括以下步骤：
(1)将超氧化物歧化酶片段和活性肽构建融合多肽的氨基酸序列，按照表达菌株的密码子优化后，人工合成cDNA；
(2)将所述cDNA序列接入质粒中，得到重组质粒；
(3)将所述重组质粒电转入感受态表达菌株中培养并诱导表达，得到超氧化物歧化酶融合多肽。
进一步地，所述表达菌株为芽孢杆菌、大肠杆菌和酵母菌中的任一种。
进一步地，所述cDNA的核苷酸序列为SEQ ID NO:5至7所示的任一种。
进一步地，步骤(1)中，所述cDNA的C端接上His tag。
一种超氧化物歧化酶融合多肽的应用，所述的超氧化物歧化酶融合多肽在制备抗衰老产品、皮肤治疗药品和医美产品中的应用。
相比现有技术，本发明的有益效果在于：
本发明的一种超氧化物歧化酶融合多肽，将耐热性强的SOD突变体(N9S，N15K，P41L)和活性因子融合表达，以SOD突变体作为伴体使活性因子具有耐高温和延缓降解的性能，提高稳定性，并各自发挥抗氧化、促愈合、抗衰老功效。
本发明的一种超氧化物歧化酶融合多肽的制备方法，其易于操作性，可重复性强，生产效率高。
本发明的一种超氧化物歧化酶融合多肽的应用，融合多肽可刺激细胞生长、增殖和分化，修复受损组织，且抗氧化、抗衰老性能优异，在制备抗衰老产品中具有广阔的应用前景。
附图说明
图1是本发明的实施例5中SE多肽的表达纯化分析图。
具体实施方式
下面，结合具体实施方式，对本发明做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
实施例1
在NCBI数据库中搜索SOD相关基因组测序，clastalX的对比结果显示：XP_011023491.1和XP_011023492.1多肽序列完全相同，来源于同一基因座(LOC105124956)；XP_011032547.1和XP_011032548.1多肽序列也完全相同，来自于同一基因座(LOC105131320)；而XP_011034389.1和XP_011021470.1两条[CuZn]SOD2-like与其他两条序列并不属于一类，差异较大。通过对比分析，挑选假定的PeSOD2转录变异体1(XP_011032547.1)的多肽序列为蓝本(命名为PeSOD2-1)，以其为模板，氨基酸如SEQ ID NO:1所示，研究SOD高级结构和保守残基及其他植物耐热[CuZn]SOD序列，对其进行定点突变(N9S,N15K,P41L)；所述定点突变具体为：将所述超氧化物歧化酶的第9位的天冬酰胺(Asn)突变成丝氨酸(Ser)，第15位的天冬酰胺(Asn)突变成赖氨酸(Lys)，第41位的脯氨酸(Pro)突变成亮氨酸(Leu)，命名为mPeSOD2-1，其氨基酸序列如SEQ ID NO:2所示。
用SOPMA分析显示mPeSOD2-1含有3.95％α螺旋(h)，32.89％的β片层(extendedstrand,e)，6.58％的β-turn(t)，56.58％随机卷曲(c)。
实施例2
将活性因子进行定点突变，得到高稳定性的活性肽(2-52，L8S，I38C，D46C)，将实施例1的胡杨[CuZn]SOD突变体mPeSOD2-1与活性肽连接，同时在杨[CuZn]SOD突变体mPeSOD2-1与活性肽之间引入Xa因子识别位点(序列IEGR)，得到包含SOD突变体和活性肽的融合多肽，命名为SE多肽，其氨基酸序列如SEQ ID NO:3所示。在SE多肽中构建Xa因子识别位点，有利于SE多肽在皮肤尤其是创面富含Xa因子处被加工成各自游离的活性多肽，发挥抗氧化、促愈合功效，具有良好的医疗和美容的应用前景。
实施例3
将实施例2的SE多肽的N端去掉3个氨基酸后连接多肽，得到修饰的SE多肽，并在其C端接上His tag标签以便于后续纯化和鉴定，氨基酸序列如SEQ ID NO:4所示。本实施例SE多肽加入多肽，其渗透能力增强，容易被吸收。
实施例4
取实施例3的SE多肽，按照枯草芽孢杆菌优化密码子及mRNA高级结构后人工合成cDNA序列，获得的cDNA序列如SEQ ID NO:5所示，然后BamHI和XbaI双酶切连入pHT1469中，构建pHT1469-SE重组质粒。
实施例5
制备电转感受态的枯草芽孢杆菌WB800N(MoBiTec Gmbh,PBS022)：挑选新鲜LB平板单菌落接种于3ml LB培养基中，30℃培养过夜。取过夜培养后的菌种接种至50ml SLB培养基(LB培养液+0.5M山梨醇，pH值为7.2)中，控制接种量为OD＝0.19-0.2之间，30℃，250rpm下培养至OD600＝0.8-1.0(约4h)。将全部菌液冰水浴10min，然后下5000rpm，8min，4℃下离心收集菌体。用40ml预冷超纯水或1mM Hepes液(pH 7.0)，4℃，5000rpm，8min下洗涤菌体3次。再将菌体用5ml PM洗涤，加1ml PM重悬，分装100μl/管，-80冻存。
取一支WB800N感受态加3μl重组载体(～200ng)/100μl感受态，混均移入预冷1mm电转杯中，冰浴5min；2000V,25uF,200Ω电击一次，立即加入1ml RM复苏培养基(LB培养液+0.5M山梨醇+0.38M甘露醇，pH值为7.2)混合，30℃静置复苏3h，5000rpm离心3min，吸弃上清剩余约50μl，吹打均匀全涂在含氯霉素(5-10μg/ml)LB平板上，倒扣30℃培养过夜，得到重组芽孢杆菌。
挑取单克隆，接入1ml含20μg/ml氯霉素LB培养基中，放30℃摇床250rpm摇至OD600＝0.8-1.0，按1:100接入15ml 2YT培养基中(含20μg/ml氯霉素)，30℃，250rpm摇至OD600＝0.8-1.0，加入1mM IPTG诱导表达，分别在24h、48h、3天、4天、5天和第6天各收上清。发酵菌液在室温12000rpm离心2min，取上清40μl作为原上清液样本。
发酵液离心去除菌体后上清，加入4倍体积的甲醇，混均，加1倍体积氯仿，混均，再加3倍体积纯水，涡旋20秒，14000g×4℃×1min，吸弃上层水相保留蛋白沉淀和氯仿，直接再加入4倍体积甲醇，轻混匀，吸弃溶液，14000g×2min，去上清，沉淀室温干燥后加适当水重悬，作为发酵上清浓缩样本。
原上清液样本和浓缩样本各加入相应体积的5×SDS-PAGE上样缓冲液，混均，100℃加热5min，置冰上，10000rpm离心2min，各取20μl上清上样用SDS-PAGE凝胶电泳分析表达情况，结果如图1所示。
参照图1，与未诱导样本相比较，诱导的菌体中在理论分子量约24kDa处有明显的目的带。诱导48h后有较明显的目的蛋白表达，72h时基本到达表达最高点，随着时间的推移，杂蛋白表达量也增多(M(beyotime,P0075)，泳道9：未诱导上清；泳道7：24h原液；泳道8：24h原液浓缩50x；泳道5：48h原液，泳道5：48h原液浓缩15x；泳道3：3d原液，泳道4：3d原液浓缩25x；泳道1：5d原液，泳道2：5d原液浓缩25x。)。
实施例6
取实施例3的SE多肽，按照大肠杆菌(E.coli)优化密码子及mRNA高级结构后人工合成cDNA序列，获得的cDNA序列如SEQ ID NO:6所示，然后NdeI和XhoI双酶切连入pET28a中，构建pET28a-SE重组质粒。其表达方法与实施例5的表达方法近似，在此不再重复论述。
实施例7
取实施例3的SE多肽，按照毕赤酵母(Pichia pastoris)优化密码子及mRNA高级结构后人工合成cDNA序列，获得的cDNA序列如SEQ ID NO:7所示，然后XhoI和NotI双酶切连入pPICZaA中，构建pPICZaA-SE重组质粒。其表达方法与实施例5的表达方法近似，在此不再重复论述。
性能测试
1、抗氧化生物活性测试
取实施例5的表达SE多肽的枯草芽孢杆菌，按1:100接种2xYT培养基中(含50μg/ml新霉素)，置于30℃×250rpm摇床中培养表达，分别在12h、24h、36h、48h、60h、3天、4天、5天和第6天各收菌500μl(60h时补加2％葡萄糖)离心取上清稀释后测活性，活性分析按照国标GB/T 5009.171-2003中所述第一法(邻苯三酚法)进行，具体参照国标。
结果测得上清中SOD活性很低，比如第5天的发酵上清中SE多肽约11U/ml。当取第5天的发酵上清液，加入不同浓度的CuSO4和ZnSO4溶液，60℃孵育30min置冰上，分别测活性，其中1.8mM的CuSO4和0.4mM的ZnSO4有较好的活性，用1.8mMCuSO4和1.2mM的ZnSO4共同处理SE多肽后，其活性增加约37倍。
2、耐热性测试
取Cu2+和Zn2+离子重构后的SE多肽母液，在60℃、70℃、80℃、90℃、100℃处理1h后放冰上，12000rpm离心2min，检测各离心上清溶液中SOD和修复多肽活性。SOD的活性分析按照国标GB/T 5009.171-2003中所述第一法(邻苯三酚法)进行。
修复多肽促生长增殖活性用NIH3T3细胞分析，NIH3T3细胞用含10％新生牛血清的DMEM培养至密度80％时接入96孔板中，每孔4000个细胞，37℃，5％CO2培养过夜，换入200ul新鲜5％新生牛血清培养基，分别加60℃、70℃、80℃、90℃、100℃处理1h的SE各2ul，每个温度处理样本做4个平行孔，处理24h后，各孔培养基换成100ul 1×PBS，每孔加10ul MTT溶液(5mg/ml用PBS配)，继续孵育4h，小心吸弃孔内培养上清液。每孔加150ul DMSO，振荡10min，使结晶物充分融解。在酶联免疫监测仪上测定各孔OD490(参考波长570nm)值，结果如表1所示。
表1温度对SE抗氧化和细胞增殖活性的影响
如表1所示，90℃以内孵育1小时对SE多肽自身的抗氧化和促细胞增殖活性无显著影响。但90℃和100℃处理1h后对抗氧化和细胞生长增殖作用具有显著影响，尤其是100℃处理1h，其几乎失去抗氧化和促NIH3T3细胞增值效果，与室温对照无显著差异。
上述实施方式仅为本发明的优选实施方式，不能以此来限定本发明保护的范围，本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (6)
1. 一种超氧化物歧化酶融合多肽，其特征在于，是：（a）氨基酸序列如SEQ ID NO:3所示的蛋白质。
2.一种权利要求1所述超氧化物歧化酶融合多肽的制备方法，其特征在于，包括以下步骤：
（1）将超氧化物歧化酶片段和活性肽构建融合多肽的氨基酸序列，按照表达菌株的密码子优化后，人工合成cDNA；
（2）将所述cDNA序列接入质粒中，得到重组质粒；
（3）将所述重组质粒电转入感受态表达菌株中培养并诱导表达，得到超氧化物歧化酶融合多肽。
3.如权利要求2所述的超氧化物歧化酶融合多肽的制备方法，其特征在于：所述表达菌株为芽孢杆菌、大肠杆菌和酵母菌中的任一种。
4.如权利要求3所述的超氧化物歧化酶融合多肽的制备方法，其特征在于：所述cDNA的核苷酸序列为SEQ ID NO:5至7所示的任一种。
5.如权利要求2所述的超氧化物歧化酶融合多肽的制备方法，其特征在于：步骤（1）中，所述 cDNA的C端接上His tag。
6.一种超氧化物歧化酶融合多肽的应用，其特征在于：权利要求1所述的超氧化物歧化酶融合多肽在制备抗衰老产品中的应用。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002080395A (ja) * | 2000-08-31 | 2002-03-19 | Ltt Institute Co Ltd | 心筋傷害処置剤 |
| CN104140457A (zh) * | 2013-05-08 | 2014-11-12 | 浙江日升昌药业有限公司 | 一种肿瘤细胞靶向性穿膜肽 |
| KR20170031068A (ko) * | 2016-09-09 | 2017-03-20 | 한양대학교 산학협력단 | 피부 투과성 펩티드 및 그 이용방법 |
| CN113332448A (zh) * | 2017-03-17 | 2021-09-03 | 浙江孚诺医药股份有限公司 | 肿瘤靶向多肽、制备方法及其应用 |
| CN114480445A (zh) * | 2022-01-26 | 2022-05-13 | 西南交通大学 | 一种人源超氧化物歧化酶hSOD1突变体的制备及其应用 |
-
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- 2022-12-14 CN CN202211609986.3A patent/CN116284437B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002080395A (ja) * | 2000-08-31 | 2002-03-19 | Ltt Institute Co Ltd | 心筋傷害処置剤 |
| CN104140457A (zh) * | 2013-05-08 | 2014-11-12 | 浙江日升昌药业有限公司 | 一种肿瘤细胞靶向性穿膜肽 |
| KR20170031068A (ko) * | 2016-09-09 | 2017-03-20 | 한양대학교 산학협력단 | 피부 투과성 펩티드 및 그 이용방법 |
| CN113332448A (zh) * | 2017-03-17 | 2021-09-03 | 浙江孚诺医药股份有限公司 | 肿瘤靶向多肽、制备方法及其应用 |
| CN114480445A (zh) * | 2022-01-26 | 2022-05-13 | 西南交通大学 | 一种人源超氧化物歧化酶hSOD1突变体的制备及其应用 |
Non-Patent Citations (4)
| Title |
|---|
| ‘洛阳红’花期花瓣抗氧化酶SOD、POD和CAT活性的研究;刘银萍;;河南林业科技(03);全文 * |
| Impact of Acinetobacter baumannii Superoxide Dismutase on Motility, Virulence, Oxidative Stress Resistance and Susceptibility to Antibiotics;Magdalena Heindorf等;《plos one》;e101033 * |
| Protective Effect of Penetratin Analogue-Tagged SOD1 on Cisplatin-Induced Nephrotoxicity through Inhibiting Oxidative Stress and JNK/p38 MAPK Signaling Pathway;Xiao-lu Wang等;《Oxidative Medicine and Cellular Longevity》;5526053 * |
| 王晓勋 等.高效hCu,Zn-SOD-PTD融合蛋白的表达及其穿膜功能的研究.《分子诊断与治疗杂志》.2016,第88-93页. * |
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