CN117247441A - 重组贻贝粘蛋白及其表达体系和应用 - Google Patents
重组贻贝粘蛋白及其表达体系和应用 Download PDFInfo
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- CN117247441A CN117247441A CN202311523119.2A CN202311523119A CN117247441A CN 117247441 A CN117247441 A CN 117247441A CN 202311523119 A CN202311523119 A CN 202311523119A CN 117247441 A CN117247441 A CN 117247441A
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- mussel mucin
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Abstract
本发明提供了重组贻贝粘蛋白及其表达体系和应用,属于重组蛋白技术领域。本发明提供的重组贻贝粘蛋白具备美白、抗氧化、促进细胞修复以及促进创面修复的多项更优能力。体内酪氨酸酶活性及黑色素合成相关蛋白的定量数据分析表明该重组贻贝粘蛋白具备美白的功效;体外的DPPH实验表明该重组贻贝粘蛋白有一定的抗氧化能力;细胞活力实验及对皮肤屏障相关蛋白的定量实验数据分析,证实了该重组贻贝粘蛋白具备促进细胞修复,特别是可促进人体表皮皮肤屏障系统中的各种细胞的发育、更新;细胞划痕实验中对细胞迁移率统计分析发现,本发明提供的重组贻贝粘蛋白可促进成纤维细胞迁移,可作为创面敷料使用,达到创面修复的结果。
Description
技术领域
本发明涉及重组蛋白技术领域,尤其涉及重组贻贝粘蛋白及其表达体系和应用。
背景技术
贻贝是一种广泛分布于沿海和近海的甲壳类海洋生物,一种海洋软体动物,属于双壳贝类,俗称“青口”和“淡菜”,遍布世界各沿海国家。贻贝种类繁多,经济价值较高,其中紫贻贝、翡翠贻贝和后壳贻贝是主要的养殖品种。贻贝富含蛋白质、脂肪、碳水化合物、钙、磷、铁、核黄素、尼克酸等营养元素,而且具有很高的药用价值。
贻贝中的足丝腺能分泌足丝,其足丝的主要成分为贻贝足丝蛋白(mytilusedulis foot protein,mfp),也称为贻贝粘蛋白(mussel adhesive protein,MAP),该蛋白具有“海洋软黄金”的美誉。贻贝粘蛋白(MAP)因其含有赖氨酸和多巴,其可形成隔水粘附带正电荷的微观生物支架,吸引带负电荷的细胞,例如表皮细胞、成纤维细胞、血管内皮细胞、神经细胞等快速贴壁、爬行替代和生长,促进创面愈合,可以用于烧伤、激光术后等皮肤损伤的修复,并起止痒的效果。在化妆品中也可以添加该蛋白,主要是减少粉尘对于皮肤的伤害,贻贝粘蛋白形成的微观纳米级保护膜,在物理性阻隔、抗氧化、局部抗炎的同时作用下修复受损的创面。
中国专利CN115925858A提供了一种异源表达的贻贝蛋白前体及其应用,制备方法简单、成本低,大规模应用更经济,且整个工艺过程中所添加的酶制剂及加工助剂安全无害。
但是现有技术中的贻贝粘蛋白性能仍有待提升。
发明内容
本发明的目的在于提供重组贻贝粘蛋白及其表达体系和应用,具备美白、抗氧化、促进细胞修复以及促进创面修复的多项能力。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种重组贻贝粘蛋白,所述重组贻贝粘蛋白的氨基酸序列如SEQ IDNO.1所示。
本发明还提供了编码上述重组贻贝粘蛋白的基因,所述基因的核苷酸序列如SEQID NO.2所示。
本发明还提供了一种重组贻贝粘蛋白,所述重组贻贝粘蛋白的氨基酸序列如SEQID NO.3所示。
本发明还提供了编码上述重组贻贝粘蛋白的基因,所述基因的核苷酸序列如SEQID NO.4所示。
本发明还提供了一种表达重组贻贝粘蛋白的重组载体,所述重组载体包括初始载体和上述编码重组贻贝粘蛋白的基因;
或,
所述重组载体包括初始载体和上述编码重组贻贝粘蛋白的基因;
所述初始载体为pGEX系列载体、pET系列载体或pMAL系列载体。
本发明还提供了一种上述重组贻贝粘蛋白的编码基因的重组菌株。
优选的,所述重组菌株中还包含如SEQ ID NO.5所示的酪氨酸酶基因。
本发明还提供了一种氧化修饰贻贝粘蛋白的制备方法,包括以下步骤:
将上述重组贻贝粘蛋白与酪氨酸酶混合,进行氧化反应,得到氧化修饰贻贝粘蛋白;
以体积百分比计,所述酪氨酸酶的用量0.5~1.5%;
所述氧化反应中,Cu2+的终浓度为80~120µM;
所述氧化反应的时间为4~8h;
所述氧化反应的温度为35~40℃。
本发明还提供了通过上述制备方法得到的氧化修饰贻贝粘蛋白。
本发明还提供了上述重组贻贝粘蛋白、重组载体、重组菌株或氧化修饰贻贝粘蛋白在制备美白产品、抗氧化产品和/或促修复产品中的应用。
本发明的有益效果:
本发明利用基因工程技术得到了重组贻贝粘蛋白,体内酪氨酸酶活性及黑色素合成相关蛋白的定量数据分析表明该重组贻贝粘蛋白具备美白的功效;体外的DPPH实验表明该重组贻贝粘蛋白有一定的抗氧化能力;细胞活力实验及对皮肤屏障相关蛋白的定量实验数据分析,证实了该重组贻贝粘蛋白具备促进细胞修复,特别是可促进人体表皮皮肤屏障系统中的各种细胞的发育、更新;细胞划痕实验中对细胞迁移率统计分析发现,本发明提供的重组贻贝粘蛋白可促进成纤维细胞迁移,可作为创面敷料使用,达到创面修复的结果。
本发明提供的重组贻贝粘蛋白可通过酪氨酸酶进行体外或体内氧化修饰,使其DOPA含量得到提高,粘性实现增强;本发明提供的重组生产贻贝粘蛋白生产方式简单,可与蛋白保护剂和抗氧化剂等组合使用或单独使用,能够最大限度地刺激表皮细胞生长、持续实现修复作用及增效作用。
附图说明
图1为蛋白分子量检测结果图,图中A为重组蛋白MAP5的检测结果,B为MAP3+5的检测结果;
图2为蛋白粘性分析结果图,图中A为用浓蛋白溶液粘附玻璃片的示意图;B为剪切力试验(力-位移)曲线图;C为贻贝粘蛋白粘附强度统计结果图;
图3为蛋白DOPA含量分析图;
图4为0h统计促进皮肤成纤维细胞迁移结果图;
图5为6h统计促进皮肤成纤维细胞迁移结果图;
图6为24h统计促进皮肤成纤维细胞迁移结果图;
图7为促进皮肤成纤维细胞迁移率统计结果图,图中A为6h统计结果,B为24h统计结果;
图8为促皮肤成纤维细胞增殖检测结果图,图中A为MAP5样品检测结果,B为MAP5+B样品检测结果,C为MAP3+5样品检测结果,D为MAP3+5+B样品检测结果;
图9为蛋白促皮肤屏障修复的定量分析结果图,图中A为丝聚蛋白(FLG)、紧密连接蛋白(TJ)、水通道蛋自3(AQP3)和兜甲蛋自(LOR)的检测结果,B为胶原蛋白COL1的检测结果,C为胶原蛋白COL17的检测结果;
图10为蛋白促美白分析结果图;
图11为蛋白粗美白的定量分析结果图,图中A为PGE2的检测结果,B为MITF的检测结果,C为TYR的检测结果,D为TRP1的检测结果,E为TRP2的检测结果。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
本发明设计并合成编码重组贻贝粘蛋白MAP3+5的基因,所述基因的核苷酸序列为:ATGAACAACATTTCTGTGGCGGTCCTGGTAGCTCTGGTTCTGATTGGCTCTTTCGCAGTGCAGTCTGACGCAGCAGACTATTACGGTCCGAAATACGGTCCACCGCGTCGTTACGGCGGTGGTAACTACAACCGCTATGGTCGCCGTTACGGCGGTTATAAGGGCTGGAACAATGGCTGGAAACGTGGCCGTTGGGGCCGCAAATACTACATGAACAACATTTCTGTGGCGGTCCTGGTAGCTCTGGTTCTGATTGGCTCTTTCGCAGTGCAGTCTGACGCAGCAGACTATTACGGTCCGAAATACGGTCCACCGCGTCGTTACGGCGGTGGTAACTACAACCGCTATGGTCGCCGTTACGGCGGTTATAAGGGCTGGAACAATGGCTGGAAACGTGGCCGTTGGGGCCGCAAATACTACTCTTCCGAAGAATATAAAGGTGGTTACTATCCAGGCAATACCTATCACTATCACTCTGGTGGTTCCTATCACGGTTCTGGTTATCACGGTGGCTACAAAGGTAAATACTACGGCAAAGCGAAGAAATACTACTACAAATACAAGAACTCTGGTAAATACAAATATCTGAAAAAAGCTCGTAAATATCACCGTAAGGGTTACAAGAAGTATTATGGTGGCGGTTCTAGC,如SEQ ID NO.2所示。
所述基因编码的重组贻贝粘蛋白MAP3+5的氨基酸序列为:MGMNNISVAVLVALVLIGSFAVQSDAADYYGPKYGPPRRYGGGNYNRYGRRYGGYKGWNNGWKRGRWGRKYYMNNISVAVLVALVLIGSFAVQSDAADYYGPKYGPPRRYGGGNYNRYGRRYGGYKGWNNGWKRGRWGRKYYSSEEYKGGYYPGNTYHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYYGGGSS,如SEQ ID NO.1所示。
本发明设计并合成编码重组贻贝粘蛋白MAP5的基因,所述基因的核苷酸序列为:TCTTCCGAAGAATATAAAGGTGGTTACTATCCAGGCAATACCTATCACTATCACTCTGGTGGTTCCTATCACGGTTCTGGTTATCACGGTGGCTACAAAGGTAAATACTACGGCAAAGCGAAGAAATACTACTACAAATACAAGAACTCTGGTAAATACAAATATCTGAAAAAAGCTCGTAAATATCACCGTAAGGGTTACAAGAAGTATTATGGTGGCGGTTCTAGC,如SEQ ID NO.4所示。
所述基因编码的重组贻贝粘蛋白MAP5的氨基酸序列为:MGSSEEYKGGYYPGNTYHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKKYYGGGSS,如SEQ ID NO.3所示。
将上述基因由南京金斯瑞生物科技有限公司进行密码子优化、连接至初始载体pET28a以获得质粒,分别得到重组表达载体pET28a-MAP5、pET28a-MAP3+5。
将pET28a-MAP5、pET28a-MAP3+5通过热激法转化感受态细胞BL21(DE3),涂布卡纳抗性平板,37℃倒置培养12-16h获得单克隆菌体。将含有目的基因的质粒送出测序,经比对序列正确,单克隆于-80℃甘油保存。取重组菌活化、扩增,得种子液;将种子液接种到LB培养基中,37℃培养,待培养液OD600为0.6-1.0时加入诱导剂至终浓度为1mM,诱导蛋白表达,所述诱导剂优选为异丙基-β-D-硫代半乳糖苷;发酵完成后,用冷冻离心机5000rpm离心10min,收集细胞,用0.9%的NaCl溶液清洗1-2次,离心收集细胞。
收集菌体加入裂解缓冲液(6.558g Na3PO4,0.485 g Tris,480g尿素,0.174gPMSF(蛋白酶抑制剂)补足超纯水950mL,完全溶解后,HCl调PH至7.0,定容至1L,室温保存)充分混匀;
将上述溶液进行超声破碎,设置温度25℃,输出功率及破壁条件根据其实验室破壁仪器进行调节,破壁直至溶液澄清透明;破壁后溶液进行离心12000rpm,10min,取沉淀重悬与适量溶解缓冲液(5.998 g NaH2PO4,17.532g NaCl,1.211g Tris,480g尿素,补足超纯水950mL,完全溶解后,HCl调PH至7.0,定容至1L,室温保存)中,待其充分溶解后,进行离心12000rpm,10min,收集上清;
取出4℃冰箱保存Ni柱填料,恢复室温后,颠倒混匀,吸取适量体积的Ni柱填料加入大小合适的层析柱中,静置10min,待其保护液在重力作用下缓慢流出;向层析柱中加入5倍柱体积的平衡液(5.998 g NaH2PO4,17.532 g NaCl,1.211 g Tris,补超纯水至950 mL,完全溶解后,HCI调pH至7.0,最终定容到1L,室温保存)平衡柱子,缓慢流出。将细胞破碎液上柱,使其自然流出,收集流穿液,重复上柱3-5次。
用10倍柱体积的洗涤液(5.998 g NaH2PO4,17.532 g NaCl,1.211 g Tris,2.042g咪唑,补超纯水至950 mL,完全溶解后,HCI调pH至7.0,最终定容到1L,室温保存)洗涤柱子,洗去杂蛋白,收集流出液。
用5倍柱体积的酸性洗脱液(5.998 g NaH2PO4,17.532 g NaCl,1.211 g Tris,20.423 g咪唑,480 g尿素,补超纯水至950 mL,完全溶解后,HCl调pH值到4.5,最终定容到1L,室温保存)洗脱柱子,收集流出液,即为纯化的目的蛋白溶液。
通过组氨酸标记凝胶亲和层析以及离子交换层析凝胶联用的方法获得内毒素水平<0.1Eμ/mL的无内毒素的蛋白。将纯化后的蛋白通过低温真空干燥的方法进行冻干,得到重组蛋白冻干粉并保存,上述所有实验的具体操作方法参考Sambrook et al,MolecularCloning:A LaboratoryManμal,Cold Spring Harbor Laboratory Press,ColdSpringHarbor,NY,1989。
将含有重组表达载体pET28a-MAP5、pET28a-MAP3+5的重组菌表达、纯化后所得的重组蛋白分别记为MAP5和MAP3+5。
采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)对获得的重组蛋白MAP5和MAP3+5进行分子量检测,结果如图1所示,图中,诱前代表重组蛋白未进行IPTG诱导,诱后代表重组蛋白进行了IPTG诱导。重组蛋白MAP5和MAP3+5的分子量约为10.1和19.9千道尔顿(kDa)。
实施例2
利用基因工程方法构建酪氨酸酶基因(WP_007202863.1),利用原位PCR技术将其克隆到大肠杆菌表达系统pET28a中,得酪氨酸酶表达的重组大肠杆菌pET28a-Tyrosinase;将重组质粒pET28a-Tyrosinase 通过热激法转化感受态细胞BL21(DE3),涂布卡纳抗性平板,37℃倒置培养12-16h获得单克隆菌体。将含有目的基因的质粒送出测序,经比对序列正确,单克隆于-80℃甘油保存。
挑选转化成功的酪氨酸酶表达工程菌进行摇瓶发酵,取重组菌活化、扩增,得种子液;将种子液接种到LB培养基中,37℃培养,待培养液OD600为0.6-1.0时加入诱导剂至终浓度为1mM,诱导蛋白表达,所述诱导剂优选为异丙基-β-D-硫代半乳糖苷;发酵完成后,用冷冻离心机5000rpm离心10min,收集细胞,用0.9%的NaCl溶液清洗1-2次,离心收集细胞。菌体重悬5ml提取缓冲液(20mMTris-HCL,PH8.5)。超声破碎仪破胞,工作条件:功率200W,工作时间3s,间隔时间7s,60次,超声结束离心12000rpm,5min,收集上清过0.45μm滤膜过滤。将上清液滤液加载到Ni-NTA柱上,用平衡液(50mMNaH2PO4,300mMNaCl,2mM咪唑,用NaOH调pH至8.0,最终定容到1L,室温保存)洗涤,最后用酸洗脱液(50mMNaH2PO4,300mM NaCl,50mM咪唑,补超纯水至950 mL,完全溶解后,用NaOH调pH至8.0,最终定容到1L,室温保存)洗脱蛋白质。获取酪氨酸酶粗提液和纯蛋白。有所不同的是:在加入IPTG诱导蛋白表达的同时,加入终浓度为100 μM 的 Cu2+(CAS号: 7758-98-7),使酪氨酸酶能够发挥活性。
取实施例1得到的贻贝粘蛋白菌体加入到裂解缓冲液中,进行超声破碎收集上清得到粗蛋白提取液,粗蛋白提取液浓度为20mg/mL,用酪氨酸酶进行体外酶促氧化,得到氧化修饰后的蛋白。
氧化修饰后的蛋白,记为MAP5-酪氨酸酶(MAP5-T)和MAP3+5-酪氨酸酶(MAP3+5-T)。
氧化后MAP5-T和MAP3+5-T的平均分子量约为10.1和20.0千道尔顿(kDa)。
设置酪氨酸酶的用量为1%(v/v),Cu2+(硫酸铜,CAS号: 7758-98-7)终浓度为100µM,反应时间为6h,反应温度为37℃,过程中每50min取样检测,结果如下表1所示:
表1 氧化结果
实验例1 粘性功能测试
将实施例1中获得的重组蛋白MAP5和MAP3+5进行粘胶性能测试。同时,用pET的空载体菌株做阴性对照。
使用万能试验机测试玻璃片粘合后能承受的最大剪切力,执行标准为:金属材料室温拉伸试验(GB/T228-2002);拉伸速度为:2.0 mm/min,制样过程如下:选用标准级显微镜载玻片:大小为25 mm×75 mm,厚度为1-1.2mm,使用前用超纯水洗净,用95%的酒精浸泡,取出后自然晾干。
称取5 mg样品粉末到载玻片上,用10μL的5%的乙酸溶液溶解。盖上另一个玻璃片,粘合面积为18mm×18mm,即324mm2,在自然条件下风干7-10天后测试其承受的最大剪切力。
用万能试验机测试得到的阴性对照以及不同的贻贝粘蛋白样品所能承受的最大剪切力(N),数据结果如表2所示。
表2 粘性测试结果
粘合强度(/Pa)由最大剪切力(N)除以粘接重叠面积(/m2)得到,该方法依据ASTMD1002标准方法。
根据表1中的最大拉力和粘合面积324mm2可以计算出各样品的粘合强度,如图2所示。
由图2可知,与阴性对照相比,本发明提供的重组贻贝粘蛋白具有较好的粘附性能,且重组蛋白MAP3+5的综合粘附性能更好。
实验例2 多巴含量测定
使用多巴脱羧酶DOPA elisa试剂盒(ZS16356S)进行多巴含量检测。结果如图3所示。
由图3可知,氧化修饰后贻贝粘蛋白MAP3+5-T的DOPA含量最高,由此可推测,本发明提供的氧化修饰方法对提高贻贝粘蛋白中DOPA含量具有良好成效。
实验例3 蛋白促皮肤成纤维细胞迁移功能分析
将实施例1中获得的重组蛋白MAP5和MAP3+5进行促皮肤成纤维细胞迁移功能检测。
将实施例1中获得的重组蛋白MAP5和MAP3+5分别与蛋白保护剂(海藻糖)以2:1比例混合,记为样品MAP5+B和MAP3+5+B。
将皮肤原代成纤维细胞接种到改良杜氏伊格尔培养基(Dulbecco's ModifiedEagle Medium ,DMEM)中,37℃培养至对数生长期,再接种到6孔细胞培养板中(200000个细胞/孔,l毫升培养基/孔)。将细胞培养至铺满培养孔表面(覆盖度为100%)后,用细胞刮刀进行轻轻划痕,划痕宽度约为8mm。
用磷酸缓冲液(137mM氯化钠,2.7mM氯化钾,10mM磷酸二氢钠,2mM磷酸二氢钾,10EU凝血酶,1mM氯化钙以及0.5mM氯化镁,pH=7.2)稀释蛋白冻干粉至1mg/mL,然后按比例加入到细胞铺满培养孔表面的细胞培养板,使蛋白的终浓度分别为5μg/mL(5ppm)、10μg/mL(10ppm)、50μg/mL(50ppm)及100μg/mL(10ppm),以加入磷酸缓冲液的样品为空白对照组,每组进行三个重复。
37℃培养0、6、24小时后,分别利用倒置显微镜进行观察并记录细胞迁移的距离,结果如图4~6所示。
计算迁移率,结果如图7所示。
结果显示,与空白对照组(HACAT,CT1)相比,MAP5在5-10ppm浓度下蛋白以浓度依赖的方式促进皮肤成纤维细胞迁移,MAP5-10ppm的迁移效果最好,MAP3+5在5-10ppm浓度下蛋白不以浓度依赖的方式促进皮肤成纤维细胞迁移,MAP3+5-5ppm+保护剂的迁移效果最好,且在相同剂量下,MAP3+5该蛋白促皮肤成纤维细胞迁移的能力远强于阳性对照HACAT及MAP5蛋白。
实验例4 蛋白促皮肤成纤维细胞增殖功能的分析
样品同实验例3。
将皮肤原代成纤维细胞接种到改良杜氏伊格尔培养基(Dulbecco's ModifiedEagle Medium ,DMEM)中,37℃培养至对数生长期,再接种到96孔细胞培养板中(5000个细胞/孔,100微升培养基/孔)。
用磷酸缓冲液(137mM氯化钠,2.7mM氯化钾,10mM磷酸二氢钠,2mM磷酸二氢钾,10EU凝血酶,1mM氯化钙以及0.5mM氯化镁, pH=7.2)稀释蛋白冻干粉至1mg/mL,然后按比例加入到96孔细胞培养板,使重组贻贝粘蛋白的终浓度依次至5μg/mL、10μg/mL、50μg/mL及100μg/mL,以加入磷酸缓冲液的样品为空白对照组。每种浓度处理各3个重复孔。在37℃培养48小时后,加入终浓度为5毫克/毫升的噻唑兰溶液(MTT,10微升/孔),37℃继续培养4小时。弃去培养基,每孔加入150μL的二甲基亚砜(DMSO),然后置于摇床上缓慢振荡10分钟。
最后利用酶标仪测定各孔的吸光度(测定波长为490nm),结果如图8所示。
结果显示,与空白对照组相比(HACAT,CT1),MAP5蛋白在10ppm浓度下存活率为104.41%,MAP3+5蛋白在5ppm浓度下存活率为102.73%,MAP3+5+蛋白保护剂复合蛋白在5ppm浓度下存活率为102.88%,本发明提供的重组贻贝粘蛋白对皮肤成纤维细胞有明显的促增殖作用。
实验例5 蛋白促皮肤屏障修复的定量分析
通过对修护皮肤屏障相关信号通路的调研分析,选择丝聚蛋白(filaggrin,FLG)、紧密连接蛋白(tight junction,TJ)、水通道蛋自3(aquaporin 3,AQP3)、兜甲蛋自(loricrin,LOR)、胶原蛋白(COL1、COL17)进行皮肤屏障修护定量实验。
用样本前处理试剂盒中的样本稀释液将样品(同实验例3)溶解成4 mg/mL原液,即为待测样本。
取待测样本100μL,加至1.5 mL干净的离心管中,加入21 pg的E.coli controlDNA标准品,混匀。
用试剂盒中提供的DNA稀释缓冲液将E.coli control DNA进行梯度稀释,稀释浓度依次为10、50 pg/μL。
实时荧光定量PCR检测:
参照E.coli残留DNA检测试剂盒(PCR-荧光探针法)方法,通过Archined X系列实时荧光定量PCR仪,分别取不同浓度的DNA标准品、待测样本和加标回收样本10μL,pre-mix(2*Tagman master 15μL10×E.coli q PCR 3μL,RNase-ree H20 2μL)20μL进行q PCR扩增。扩增条件:50℃10 min,95℃10 min;95℃10 s ;58℃1 min。45个循环。每个样品做3个复孔。
定量检测结果如图9所示,图中对照是未加样品的空细胞,model是过氧化氢刺激细胞组。
由结果可知,MAP3+5、MAP5较对照组(H202,刺激HaCaT细胞)来说,出现促进丝聚蛋白(filaggrin, FLG)、水通道蛋自3(aquaporin 3,AQP3)和兜甲蛋自(loricrin,LOR)基因水平上调表达,推测其具有明显修复效果,其中MAP3+5+B在10ppm浓度下修复效果最好。
实验例6 蛋白促皮肤美白效果检测
阳性对照:用pH6.8磷酸氢二钠-柠檬酸缓冲液稀释成系列浓度梯度用以验证试验系统。
受试物处理:样品同实验例3,分别用磷酸氢二钠-柠檬酸缓冲液稀释为多级浓度样品。使用10mL试管设立样品管(T)、样品本底(T0)、酶反应管(C)和溶剂本底(C0),每一样品的每个受试浓度的样品管(T)需设立3支平行管,同时酶反应管(C)也需设立3支平行管。在样品管(T)和样品本底(T0)中各加入1mL相同浓度的样品溶液,酶反应管(C)和溶剂本底(C0)则分别加入1mL磷酸氢三钠-柠檬酸缓冲液。
在样品管(T)和酶反应管(C)中各加入0.5mL酪氨酸酶溶液,样品本底(T)与溶剂本底(C0)以0.5mL磷酸氢二钠-柠檬酸缓冲液代替,将样品和酪氨酸酶充分混匀,置37℃水浴槽孵育10分钟。
依次在各管中加入2mL的左旋多巴溶液,控制每管反应时间为5分钟,即刻将各管反应溶液移入比色皿中,在475nm处测定吸光值。计算酪氨酸酶抑制率:
(1)式中:
T——样品管吸光值,即样品与酪氨酸酶反应后溶液吸光值;
T0——样品本底吸光值;
C——酶反应管吸光值3次平均值,即未加样品时酪氨酸酶和多巴反应的吸光值;
C0——溶剂本底吸光值。
以L-酪氨酸为底物,考察贻贝粘蛋白MAP5、MAP3+5对酪氨酸酶活性抑制作用,结果如图10所示。
在实验浓度范围内,MAP5贻贝粘蛋白对L-酪氨酸的酶促氧化进程表现出明显抑制作用,高于对照熊果苷的抑制作用;且在低浓度的情况下,就能呈现较好的抑制效果;MAP3+5贻贝粘蛋白对L-酪氨酸的酶促氧化进程也表现出抑制作用,但贻贝粘蛋白MAP5对L-酪氨酸的酶促氧化进程抑制效果更好,特别是贻贝粘蛋白MAP5加了蛋白保护剂和抗氧化剂后,抑制效果翻倍。
同时参考实验例5方法,选取PGE2、MITF、TYR、TRP1、TRP2五个基因进行相关美白定量分析,结果如图11所示,实验结果与酪氨酸酶抑制结果一致。
实验例7 蛋白促抗氧化的分析
样品同实验例3。
阳性对照物用95%乙醇溶解稀释成:1μg/mL、2μg/mL、4μg/mL、4μg/mL、6μg/mL、8μg/mL、10μg/mL系列浓度梯度用以验证试验系统。使用10mL试管设立样品管(T)、样品本底(T0)、DPPH管(C)和溶剂本底(C0),每一样品的每个受试浓度的样品管(T)需设立3支平行管,同时 DPPH管(C)也需设立3支平行管。
在样品管(T)和样品本底(T0)中各加入1mL相同浓度的样品溶液。在所有试管中(T、T0、C、C0)补充溶剂,水溶性样品用水,油溶性样品用95%乙醇,补足3mL,混匀。
在样品管(T)和DPPH管(C)中加入DPPH乙醇溶液1mL,样品本底(T0)和溶剂本底(C0)用95%乙醇代替,轻轻摇匀,室温下静置30min。
将各支反应溶液移入96孔板中,在517nm处测定吸光值。
计算DPPH自由基清除率:
(2)式中:
T—样品管吸光值,即样品与DPPH反应后溶液吸光值;
T0—样品本底吸光值;
C—DPPH管吸光值3次平均值,即未加样品时DPPH 溶液吸光值;
C0—溶剂本底吸光值。
DPPH是一种在有机溶剂中很稳定的自由基,在517nm处有很强的吸光值。当体系中加入自由基清除剂(抗氧化剂)时,DPPH自由基上的孤对电子被配对,其紫色变浅,在517nm处的吸光值变小,通过颜色的深浅与吸光度的测定可以评价自由基清除剂的抗氧化效果。
通过本发明得到的重组贻贝粘蛋白对DPPH的清除率效果如表3所示:
表3 抗氧化效果
结果显示,本发明提供的贻贝粘蛋白MAP5对DPPH的IC50值为2.672mg/mL(P<0.05),贻贝粘蛋白MAP3+5对DPPH的IC50值为1.061mg/mL(P<0.05),Vc对DPPH的IC50值为1.975µg/mL(P<0.05)。当贻贝粘蛋白MAP5、MAP3+5浓度均达到2.4mg/mL时,对DPPH清除率分别达最大值61.20%和82.88%,具有优异的抗氧化效果。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种重组贻贝粘蛋白,其特征在于,所述重组贻贝粘蛋白的氨基酸序列如SEQ IDNO.1所示。
2.编码权利要求1所述重组贻贝粘蛋白的基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO.2所示。
3.一种重组贻贝粘蛋白,其特征在于,所述重组贻贝粘蛋白的氨基酸序列如SEQ IDNO.3所示。
4.编码权利要求3所述重组贻贝粘蛋白的基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO.4所示。
5.一种表达重组贻贝粘蛋白的重组载体,其特征在于,所述重组载体包括初始载体和权利要求2所述的编码重组贻贝粘蛋白的基因;
或,
所述重组载体包括初始载体和权利要求4所述的编码重组贻贝粘蛋白的基因;
所述初始载体为pGEX系列载体、pET系列载体或pMAL系列载体。
6.一种包含权利要求2所述的重组贻贝粘蛋白的编码基因或权利要求4所述的重组贻贝粘蛋白的编码基因的重组菌株。
7.根据权利要求6所述的重组菌株,其特征在于,所述重组菌株中还包含如SEQ IDNO.5所示的酪氨酸酶基因。
8.一种氧化修饰贻贝粘蛋白的制备方法,其特征在于,包括以下步骤:
将权利要求1或权利要求3所述的重组贻贝粘蛋白与酪氨酸酶混合,进行氧化反应,得到氧化修饰贻贝粘蛋白;
以体积百分比计,所述酪氨酸酶的用量0.5~1.5%;
所述氧化反应中,Cu2+的终浓度为80~120µM;
所述氧化反应的时间为4~8h;
所述氧化反应的温度为35~40℃。
9.通过权利要求8所述的制备方法得到的氧化修饰贻贝粘蛋白。
10.权利要求1或3所述的重组贻贝粘蛋白、权利要求5所述的重组载体、权利要求6或7任一项所述的重组菌株或权利要求9所述的氧化修饰贻贝粘蛋白在制备美白产品、抗氧化产品和/或促修复产品中的应用。
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