CN114644705B - 一种防护皮肤急性光损伤活性多肽及其应用 - Google Patents
一种防护皮肤急性光损伤活性多肽及其应用 Download PDFInfo
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- A61K38/00—Medicinal preparations containing peptides
Abstract
本发明公开了一种防护皮肤急性光损伤活性多肽及其应用,所述从云南臭蛙的皮肤中鉴定出一种新型防护皮肤急性光损伤活性OA‑GI13。成熟OA‑GI13的序列为“GIWAPWPPRAGLC”。实验表明,OA‑GI13能直接清除自由基(2'‑联氨‑双‑3‑乙基苯并噻唑啉‑6‑磺酸(ABTS+)和2,2‑二苯基‑1‑苦肼基(DPPH)),但无溶血活性和细胞毒性。OA‑GI13在保护细胞免受过氧化氢(H2O2)刺激引起的氧化应激中发挥了有效的作用。更重要的是,OA‑GI13保护小鼠皮肤免受UVB诱导的急性光损伤,包括减轻小鼠背部皮肤红斑和水肿,减少表皮晒伤细胞,增强体内抗氧化能力。所述OA‑GI13作为防治急性光损伤的候选药物具有强大潜力。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种防护皮肤急性光损伤活性多肽及其应用。
背景技术
皮肤作为人体与环境的外部界面,是保护内脏器官和组织免受各种病原体和环境胁迫的重要屏障。紫外线辐射是与皮肤损伤有关的最重要的环境因素,影响皮肤的结构和功能。过度暴露在紫外线辐射下会造成皮肤短期和长期的有害影响,如光损伤、光老化和皮肤癌。紫外线辐射的波长可分为UVA (315-400 nm)、UVB (280-315 nm)和UVC(100-280nm)。大多数UVC射线被臭氧层吸收,很少到达地球表面,UVA和UVB射线都能穿透皮肤的真皮层和表皮层,而UVB的生物活性远高于UVA,因此,UVB是光损伤的主要原因。UVB辐射导致活性氧(ROS)的过度积累,破坏氧化和抗氧化系统之间的平衡,引起严重的氧化应激。此外,ROS介导的氧化应激可引起多种生物大分子的氧化损伤,如蛋白质、DNA和脂质。这种损伤促进乳酸脱氢酶(LDH)的释放,降低超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽(GSH)的水平,提高脂质过氧化物(LPO)和丙二醛(MDA)的水平,并触发8-羟基脱氧鸟苷(8-OHdG)的形成。既往研究表明,p38 MAPK信号通路参与了氧化损伤过程。
单次暴露于UVB照射可引起各种皮肤改变,包括红斑、晒伤、血管高通透性、水肿、疼痛和发热,这些都是急性光损伤反应的特征,通常与氧化应激有关。因此,抗氧化在急性光损伤的修复中起着重要作用。一般的抗氧化方法包括涂抹防晒霜和使用各种抗氧化剂。然而,防晒霜的使用是有限的,因为其中一些成分是有毒的,会影响环境。而使用最广泛的抗氧化剂,如维生素C和维生素E,在高剂量下可能会增加机体对有害物质的敏感性,并造成细胞损伤,或引起恶心、头痛、肠胃不适和出血性中风的风险。因此,研发抗光损伤的新型天然抗氧化剂至关重要。近年来,随着多肽治疗学的显著发展,人们对多受体蛋白和多肽进行了广泛的研究,许多多肽药物通过调节生理和病理过程来预防和治疗疾病,其中一些取得了很大的成功,如治疗骨质疏松的阿巴洛肽,治疗2型糖尿病的索马鲁肽,以及治疗慢性特发性便秘的普卡那肽等。两栖动物是活性肽的天然宝库,它们裸露的皮肤直接暴露在各种生物和非生物的危险因素中,如病原体、捕食者、辐射和物理损伤等。为了适应环境,两栖动物的皮肤逐渐进化出一种化学防御系统来对抗这些复杂的因素,该系统包括新型的抗氧化肽(AOPs),用于清除自由基,并在长时间的辐射下迅速产生,以防止皮肤损伤。这些AOPs可以被认为是除了抗氧化酶和低分子量抗氧化剂之外的第三种抗氧化系统。近年来,对两栖类AOPs的研究显著增加,例如,从热带蛙皮肤中提取的多肽antioxidin-I (TWYFITPYIPDK)具有潜在的抗氧化活性,从蝾螈皮肤分泌物中提取的抗氧化肽salamandrin-I(FAVWGCADYRGY-NH2)具有清除自由基的能力。然而,应用两栖类AOPs来保护皮肤免受UVB引起的光损伤仍然很少见。目前已知的用于治疗光损伤的两栖类AOPs有:来自云南臭蛙的OA-VI12(VIPFLACRPLGL)和AOP-P1(FLPGLECVW),来自大绿臭蛙的antioxidin-RL(AMRLTYNRPCIYAT),来自花臭蛙的OS-LL11 (LLPPWLCPRNK)和来自绿臭蛙的OM-GL15(GLLSGHYGRASPVAC)。有趣的是,OA-VI12、AOP-P1、antioxidin-RL和OS-LL11都被用于治疗慢性光损伤,只有OM-GL15用于治疗急性光损伤。因此,需要进一步探索两栖类AOPs在急性光损伤中的应用。
发明内容
本发明的第一目的在于提供一种抗皮肤光损伤活性多肽,本发明的第二目的是提供所述抗皮肤光损伤活性多肽的应用,本发明的第三目的是提供编码所述抗皮肤光损伤活性多肽前体的基因。
本发明的第一目的是这样实现的,所述多肽的氨基酸序列如SEQ ID NO.1所示,分子量为1423.68Da。
本发明的第二目的是这样实现的,所述多肽的应用为在制备抗氧化制剂中的应用以及在抗皮肤光老化或抗衰老功效的护肤品中的应用和在制备用于预防或修复由UVB引起的皮肤光损伤的制剂中的应用。
本发明的第三目的是这样实现的,所述多肽前体基因的核苷酸序列如SEQ IDNO.2所示。
本发明的有益效果为:
本发明从云南臭蛙的皮肤中鉴定出一种新型肽(OA-GI13)。成熟OA-GI13的序列为“GIWAPWPPRAGLC”。实验表明,OA-GI13能直接清除自由基(2'-联氨-双-3-乙基苯并噻唑啉-6-磺酸(ABTS+)和2,2-二苯基-1-苦肼基(DPPH)),但无溶血活性和细胞毒性。OA-GI13在保护细胞免受过氧化氢(H2O2)刺激引起的氧化应激中发挥了有效的作用。更重要的是,OA-GI13保护小鼠皮肤免受UVB诱导的急性光损伤,包括减轻小鼠背部皮肤红斑和水肿,减少表皮晒伤细胞,增强体内抗氧化能力。本发明提供的OA-GI13作为防治急性光损伤的候选药物具有强大潜力。
附图说明
图1为本发明编码抗损伤多肽OA-GI13的cDNA序列图;
图2为本发明抗损伤多肽OA-GI13对体外的ABTS+和DPPH自由基清除率图;
图3 为本发明抗损伤多肽OA-GI13对HaCaT细胞的细胞毒性图;
图4为本发明抗损伤多肽OA-GI13对HaCaT细胞活力影响图;
图5为本发明抗损伤多肽OA-GI13对H2O2刺激下HaCaT细胞氧化应激水平的影响;
图6为本发明抗损伤多肽OA-GI13对UVB诱导的小鼠背部红斑的减轻效果;
图7为本发明抗损伤多肽OA-GI13对UVB诱导的小鼠背部皮肤弹性和水分的影响;
图8为本发明抗损伤多肽OA-GI13对UVB诱导的小鼠皮肤的表皮与真皮的保护作用;
图9为本发明抗损伤多肽OA-GI13对UVB诱导的小鼠皮肤氧化应激水平的影响。
具体实施方式
下面结合附图和实施例对本发明作进一步的详细说明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变换或改进,均落入本发明的保护范围。
实施例1 OA-GI13的制备
1、 云南臭蛙动物样本捕获
从云南省野外捕捉了成年云南臭蛙(Odorrana andersonii),将其饲养于一个体积为50 cm ×65 cm ×65 cm的特定容器中,用面包虫作为饲料喂养云南臭蛙,定期换水,适应环境7天。
2、cDNA 合成,Illumina MiSeq 测序及数据处理
去离子水冲洗云南臭蛙的皮肤,洗干净后将臭蛙处死,将其皮肤迅速剥离切成片状后,为了防止RNA降解在液氮中迅速研磨。使用 RNAiso (TaKaRa,Dalian,China) 提取皮肤总 RNA,按照规范步骤使用 Absolutely mRNA 纯化试剂盒 (Stratagene,Canada) 纯化mRNA, 基于实验室前期研究经验,利用 SMART 技术合成第一和第二链 cDNA,利用从基因库中筛选出来的编码臭蛙皮肤生物活性前体的 cDNA,用特异性的 5’PCR 引物 (5’-CCAAA(G/C)ATGTTCACC(T/A)TGAAGAAA-3’) 编码信号肽区和 3’PCR 引 物 (5’-ATTCTAGAGGCCGAGGCGGCCGACATG-3’)。PCR 产物经 DNA 凝胶提取试剂盒回收,最后进行Illumina MiSeq 测序。主要步骤如下:50-100 ng PCR 产物用 KAPA HiFi 热启动预混料再次扩增3个 周 期,使用 注 入 引 物 F(AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT- 特 异 性 5’PCR 引 物 ) 和 注 入 引 物 R (CAAGCAGAAGACGGCATACGAGATCTCAGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-特异性 3’PCR引物),扩增子使用 Agencourt AMPure XP beads (Beckman Coulter Diagnostics, Inc.,USA) 按照说明进行纯化,并使用 Qubit 2.0 荧光仪 (Life Invitrogen, Inc.,Carlsbad, CA, USA) 进行定量。纯化的扩增子汇集在等分子量的扩增子中,并在 Illumina MiSeq 平台上按照通用生物技术 (Shanghai, China) 的标准协议进行配对测序 (2×300)。使用 Trim Galore软件从原始文件中删除适配器序列。配对端读取采用 FLASH-1.2.11 软件进行装配,符合以下条件:重叠在 10 bp 到 300 bp 之间,最大失配率为 0.1。然后得到一个 fastq 格式的文件,将该文件转换为 fasta 格式,使用 Fastx-toolkit 软件去除重复序列。
测序结果:cDNA的核苷酸序列如SEQ ID NO.2所示,cDNA序列全长325bd(含有325个碱基),编码59个氨基酸残基,成熟肽序列为“GIWAPWPPRAGLC”(图1中碱基序列下划线部分)。通过在NCBI数据库的BLAST搜索,我们发现不存在与该前体相同的两栖类生物活性肽,因此我们认为该肽是一种新肽,并将其命名为OA-GI13(OA:物种的缩写,GI:两个初始残基,13:肽的长度)。
3、OA-GI13多肽的制备
根据编码OA-GI13多肽的cDNA,推导的氨基酸序列,由武汉百意欣生物技术有限公司人工合成OA-GI13的成熟肽,成熟肽的纯度高于95%,分子量为1423.68 Da,氨基酸序列为“GIWAPWPPRAGLC”。
实施例2 OA-GI13多肽抗氧化活性测定
1、ABTS+自由基清除率检测
检测方法:首先制备ABTS+ 自由基的原液:用ddH2O溶解2.8 mM的 过 硫 酸 钾(Sigma-Aldrich, St Louis,MO,USA)后与7 mM ABTS(Sigma-Aldrich,St Louis,MO,USA)混合,将混合液放置暗室中反应 6 h 使之生成ABTS+ 自由基,然后在室温下避光备用。在检测样品的ABTS+自由基清除活性前,先将ABTS+ 自由基的原液用ddH2O进行稀释。将各样品与稀释后的原液按1:1的比例混合,然后分别加入96孔板中,用锡箔纸包裹96孔板10分钟后在415 nm处测量吸光度。用Vc作为阳性对照,样品溶剂即ddH2O作为阴性对照。根据吸光度值的降低来评估样品的自由基清除活性,ABTS+自由基清除率(%)=(A空白 – A样品)× 100/A空白。
2、DPPH自由基清除活性检测
检测方法:首先制备DPPH自由基的储备液:用甲醇溶解DPPH自由基(Sigma-Aldrich,StLouis,MO,USA)配成浓度为 5×10-5 M 的储备液,避光保存。拿出96孔板,每孔加入10 μL样品与190 μLDPPH储备液,重复3次。加样完毕后充分混匀,用锡箔纸包裹96孔板并静置30min。最后在517 nm处测量吸光度,用Vc作为阳性对照,样品溶剂即ddH2O作为阴性对照,DPPH自由基清除率(%)=(A空白 – A样品)× 100/A空白。
检测结果:如图2A和图2B所示,OA-GI13在1nM和10nM浓度下表现出清除ABTS+和DPPH自由基的能力,其效果与维生素C(Vc)的效果一致;但在浓度为100pM时,OA-GI13与溶剂组无差异,且对ABTS+或DPPH无清除活性。
实施例3 OA-GI13溶血活性检测
检测方法:用小鼠红细胞检测OA-GI13的溶血活性,具体方法参考已报道的文献并稍作修改。具体方法为:用0.9%的生理盐水轻洗小鼠红细胞3次,然后2000-3000×g离心5min,去上清,如此重复3次后得到红细胞沉淀。然后用生理盐水将红细胞沉淀稀释成2%的红细胞溶液。接着在1.5 mL的离心管中按照1:1的比例加入样品与2%的红细胞溶液各200-300μL,混合后放入37°C水浴锅孵育30min。实验中,用0.9%的生理盐水作为阴性对照,用0.1%Triton X-100作为阳性对照。孵育完毕后,室温下2000-3000×g离心5 min取上清,在540nm处测量吸光度。溶血活性(%)的计算公式为:(A样品)× 100 / Atriton× 100。
表1:OA-GI13对小鼠红细胞的溶血活性
检测结果:从表1可知,在不同浓度的OA-GI13中未观察到溶血活性,表明OA-GI13对小鼠红细胞无溶血活性。
实施例4 OA-GI13对HaCaT细胞的毒性测定
检测方法:HaCaT细胞分为4组,即正常组、OA-GI13 100pM组、OA-GI13 1nM组和OA-GI13 10nM组。将细胞以每孔3×105细胞/毫升的密度接种到12孔板中。待细胞贴壁后,正常组加入1mL载体(无血清培养基),OA-GI13组分别加入1mL不同浓度的OA-GI13(100pM,1nM,10nM),然后将细胞放入37°C培养箱继续培养12 h。在此之后,用磷酸盐缓冲液(PBS)轻柔地洗涤细胞2次,并按照Calcein/PI细胞活力/细胞毒性测定试剂盒(碧云天,上海,中国)提供的步骤进行实验。最后,通过共焦激光扫描荧光显微镜(Axio Observer Z1,ZEISS,Germany)对活细胞和死细胞进行荧光成像和分析。
检测结果:如图3所示,几乎所有细胞都被钙黄绿素染色为绿色,而PI染色的死亡细胞很少被观察到。这表明OA-GI13对HaCaT细胞无细胞毒性。
实施例5 OA-GI13对过氧化氢(H2O2)处理的HaCaT细胞活力的影响
检测方法:HaCaT细胞分7组,即正常组、H2O2组、溶剂对照组(Vehicle)、阳性对照组(Vc,10μM)和OA-GI13组(100pM,1nM,10nM)。将细胞以每孔3000个的密度接种到含有 90 μLDMEM/F12(无血清)的96孔板中。待细胞贴壁后,正常组和H2O2组用DMEM/F12(无血清)预处理,溶剂对照组用ddH2O预处理,阳性对照组用Vc(10μM)预处理,OA-GI13组用各个浓度的OA-GI13(100pM,1nM,10nM)预处理,然后在37°C下培养2 h。2 h后,除了正常组,其他各组的细胞用200 μM H2O2(Sigma,St. Louis,MO,USA)刺激2 h。在此之后,正常组和H2O2组用DMEM/F12(无血清)处理,溶剂对照组用ddH2O处理,阳性对照组用Vc(10μM)处理,OA-GI13组用各个浓度的OA-GI13(100pM,1nM,10nM)处理,然后在37°C培养箱中继续培养24 h。最后,向每个孔添加10 μL MTS试剂(Promega,Madison,USA)并放入培养箱继续培养2-4 h,在490nm处测量其吸光度值。
检测结果:如图4A所示,OA-GI13(100 pM,1 nM,10 nM)对HaCaT细胞的活力没有明显影响。当受到H2O2刺激时,HaCaT细胞活力显著降低(图4B),但Vc或OA-GI13(1nM,10nM)预处理显著提高了细胞活力,这表明OA-GI13可以在H2O2刺激下维持HaCaT细胞活力。
实施例6 OA-GI13细胞水平氧化应激相关指标检测
检测方法:首先进行细胞建模,用胰酶将培养瓶内长满至90%的HaCaT细胞消化下来接种到6孔板中,并将细胞分为7个组,即正常组、H2O2组、溶剂对照组(Vehicle)、阳性对照组(Vc,10 μM)和OA-GI13组(100pM,1nM,10nM)。待细胞贴壁后,正常组和H2O2组用DMEM/F12(无血清)预处理,载体组用ddH2O预处理,阳性对照组用Vc(10μM)预处理,OA-GI13组用各个浓度的OA-GI13(100pM,1nM,10nM)预处理,然后在37°C下培养2 h。2 h后,除了正常组,其他各组的细胞用200 μM H2O2刺激2 h。在此之后,正常组和H2O2组用DMEM/F12(无血清)处理,载体组用ddH2O处理,阳性对照组用Vc(10μM)处理,OA-GI13组用各个浓度的OA-GI13(100pM,1nM,10nM)处理,然后在37°C培养箱中继续培养24 h。细胞建模24 h后,收集细胞培养液用以检测LDH(过氧化氢酶测定试剂盒,南京建城,南京,中国)。用预冷的PBS清洗6孔板内的细胞2~3次,每孔加入RIPA裂解液(PMSF:RIPA=1:100)100-200uL,等待10-15min。用细胞收集刮刀轻轻地刮取细胞,然后收集进离心管,全程在冰上操作。在低温离心机上离心(4℃,10000 ×g),离心10-15分钟后收集上清液,按照试剂盒的操作说明进行后续SOD(超氧化物歧化酶测定试剂盒,南京建城,南京,中国)、GSH(谷胱甘肽测定试剂盒,南京建城,南京,中国)、CAT(过氧化氢酶测定试剂盒,南京建城,南京,中国)的检测。
检测结果:如图5A和C所示,当H2O2刺激HaCaT细胞时,抗氧化酶SOD和CAT的水平显著减少。然而,OA-GI13(1nM,10nM)预处理可提高SOD和CAT水平。同样,在H2O2刺激的HaCaT细胞中,非酶抗氧化剂GSH水平显著降低,LDH水平显著升高。然而,OA-GI13预处理使GSH和LDH水平正常化(图5B,D)。
实施例7 OA-GI13对UVB辐射刺激下的急性光损伤的保护活性检测
检测方法:育养22g雌性昆明小鼠,将它们随机分为7个组即正常组、UVB组、溶剂对照组(Vehicle)、阳性对照组(Vc,10μM)和OA-GI13组(100pM,1 nM,10nM),每组3只小鼠。再构建小鼠急性光损伤动物模型:首先,使用剃须刀将小鼠的背部皮肤区域(2×3 cm2)进行去毛。然后,除正常组外均使用UVB灯(TL20w/01, Phi-lips, Netherlands)进行UVB照射,照射强度为 2J/cm。用紫外线辐射计(TM-213,Tenmars,Taiwan,China)监测照射强度。将粉末状的OA-GI13样品溶解在ddH2O中,并配制成100pM,1nM和10nM三种不同浓度。 UVB照射后,立即用1 mL以上试剂涂抹在小鼠背部的照射区域。溶剂对照组(Vehicle)涂抹ddH2O,阳性对照组(Vc)涂抹浓度为10 μMVc,OA-GI13(100pM)组涂抹100 pM的 OA-GI13,OA-GI13(1nM)组涂抹1 nM的OA-GI13,OA-GI13(10nM)组涂抹10nM的 OA-GI13。正常组和UVB 组不涂抹任何试剂。 24h后,对小鼠背部皮肤进行拍照记录红斑生成情况,使用Mior智能屏显肌肤测试仪对小鼠背部皮肤进行水分和弹性的测试,然后将所有小鼠麻醉并安乐死以进行下一步的实验:对小鼠急性光损伤的真皮及表皮的保护作用检测(H&E染色及Masson染色试验)、氧化应激水平检测。
检测结果:
1、OA-GI13 对UVB辐射后小鼠背部红斑的生成的影响
过度暴露于UVB辐射可导致皮肤红斑、水肿、晒伤和血管疾病。因此,我们检测了OA-GI13对UVB辐射诱发红斑的影响。UVB照射后,UVB组小鼠背部皮肤出现明显红斑,溶剂组和OA-GI13(100pM)组小鼠背部仍有大量红斑。然而,经OA-GI13(1nM,10nM)或Vc(10μM)治疗后,小鼠背部皮肤上的红斑显著减少。OA-GI13在10nM剂量下的修复效果最好(图6)。
2、OA-GI13对UVB辐射后小鼠背部皮肤弹性和水分的影响
为了进一步研究OA-GI13对UVB诱导的水肿的影响,采用Mior智能屏显肌肤测试仪对小鼠背部皮肤的弹性和水分。如图7所示,与正常组相比,UVB组的弹性和水分增加。溶剂组和OA-GI13(100pM)组没有改善这种情况,但OA-GI13(1nM,10nM)组和Vc(10μM)组的弹性和水分均降低,即OA-GI13抑制了UVB辐射引起的小鼠背部皮肤弹性和水分的增多。
3、OA-GI13对小鼠皮肤急性光损伤的保护作用
晒伤细胞的形成是UVB辐射损伤的主要指标。与正常组相比,UVB组出现大量晒伤细胞,表皮完整性受损。与UVB组相比,溶剂组和OA-GI13(100pM)组之间没有显著差异,Vc组和OA-GI13(1nM)组中仍然观察到少量晒伤细胞。然而,对于OA-GI13(10nM)组,表皮完整,几乎没有晒伤细胞,与正常组相似(图8A,H&E染色)。Masson三色染色显示正常组真皮胶原纤维排列均匀。UVB辐射后,胶原纤维变得紊乱和不规则,但Vc和OA-GI13处理都抑制了这一过程,10nM OA-GI13显示出最佳效果(图8B)。
4、OA-GI13对UVB诱导的小鼠皮肤氧化应激水平的影响
UVB辐射可导致ROS的过度产生。ROS介导的氧化应激消耗内源性抗氧化剂,产生许多脂质过氧化产物,并导致DNA氧化损伤。如图9A-C所示,UVB照射后,SOD、GSH和CAT的水平明显降低,但OA-GI13(1nM,10nM)或Vc(10μM)治疗使其水平升高。ROS诱导的LPO是氧化应激的主要表现,MDA是LPO的主要副产物,8-OHdG是DNA损伤的标志物。因此,我们测量了UVB照射和治疗后LPO、MDA和8-OHdG的水平。与正常组相比,UVB组的LPO、MDA和8-OHdG水平显著升高,但OA-GI13(1nM,10nM)和Vc(10μM)有效地抑制了UVB诱导的LPO、MDA和8-OHdG的升高(图9D-F)。说明OA-GI13可降低UVB诱导的小鼠皮肤氧化应激水平。
SEQUENCE LISTING
<110> 云南民族大学
<120> 一种防护皮肤急性光损伤活性多肽及其应用
<130> 2022
<160> 2
<170> PatentIn version 3.3
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<211> 13
<212> PRT
<213> 人工合成
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<213> 人工合成
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tgatgcttct attttgttct tcttgtgctg atgatgcgaa ttgccgtctc gataaattgt 300
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Claims (5)
1.一种防护皮肤急性光损伤活性多肽,其特征在于,所述多肽的氨基酸序列如SEQ IDNO.1所示,分子量为1423.68Da。
2.权利要求1所述多肽在制备抗氧化制剂中的应用。
3.权利要求1所述多肽在制备抗皮肤光老化或抗衰老功效的护肤品中的应用。
4.权利要求1所述多肽在制备用于预防或修复由UVB引起的皮肤光损伤的制剂中的应用。
5.权利要求1所述防护皮肤急性光损伤活性多肽的前体基因,其特征在于,所述前体基因的核苷酸序列如SEQ ID NO.2所示。
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