CN116874576A - 一种重组人源化丝聚蛋白及其制备方法和应用 - Google Patents
一种重组人源化丝聚蛋白及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域,具体涉及一种重组人源化丝聚蛋白及其制备方法和应用,该重组人源化丝聚蛋白的核苷酸序列如SEQ ID NO.1所示,该重组人源化丝聚蛋白的核苷酸序列所对应的氨基酸序列如SEQ ID NO.2所示。本发明的重组人源化丝聚蛋白由人丝聚蛋白完整单体结构域、人丝聚蛋白天然保守linker和丝素蛋白轻链融合而成,不仅能够高效表达,而且生物活性高,能够明显提高角质层含水量,降低经皮水分散失量,对维持和增强皮肤屏障有促进作用,广泛应用于保湿化妆品的制备中。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种重组人源化丝聚蛋白及其制备方法和应用。
背景技术
丝聚蛋白(Filaggrin,FLG)及其分解产物对维持屏障功能和皮肤水合度有重要作用,其分子量为37kDa。人类FLG(Filaggrin)基因位于染色体1q21,产物为表达在表皮颗粒层细胞内的FLG的前体——profilaggrin(proFLG)蛋白,分子量大于400kDa,proFLG高度磷酸化且不溶于水,其结构如图1所示,从N端到C端分别为:保守的S100钙结合区,NLS(核定位信号)、不完整FLG结构域、10-12个完整FLG单体结构域、不完整FLG结构域以及C末端结构域。在颗粒层向角质层转化过程中,proFLG在脱磷酸后,经SASPase、KLK5和matriptase等酶切割重复单体FLG结构域间的linker(序列为FLYQVST),最终形成单体FLG。单体FLG与角蛋白丝结合形成角蛋白束,维持了角质层完整性和机械强度,防止水分丢失并减少应变原和微生物的入侵,是保持皮肤屏障作用的关键因素。在上层角质层中,FLG与角蛋白丝解离并释放,在caspase14,calpain1和博来霉素水解酶等作用下降解为如谷氨酰胺、组氨酸等游离氨基酸,游离氨基酸最终被加工为如吡咯烷酮羧酸(PCA)、反式尿刊酸(trans-UCA)等产物,这些产物与乳酸、金属离子等共同构成天然保湿因子(NMF),保持和调节角质层含水量;根据研究报道,天然保湿因子(NMF)和吸湿性混合物中超50%的成分来自于FLG的降解产物。降解产物中的trans-UCA是角质层中主要的紫外吸收物质,降低皮肤的紫外损伤;研究显示,在吸收紫外线后,trans-UCA异构化为顺式尿刊酸(cis-UCA),cis-UCA能降低郎格罕细胞的抗原呈递功能,影响细胞因子的分泌,抑制皮肤接触超敏反应和迟发性超敏反应。
鉴于proFLG串联结构和其因高度磷酸化而不溶于水、无功能活性的特点,重组丝聚蛋白多选择FLG单体结构域构建融合蛋白,但目前重组丝聚蛋白结构存在缺陷:1.融合蛋白间linker不是天然序列,导致linker不能被颗粒层和角质层内的天然蛋白酶识别并切割,融合蛋白内的FLG结构域不能被释放并行使功能;2.未包含完整的FLG单体结构域序列,导致表达的重组蛋白不能发挥FLG单体结构域的功能;3.未包含完整的FLG单体结构域序列,导致其分解产物不包含全部天然FLG分解产物,不利于天然保湿因子的形成;4.参与组成融合蛋白的非FLG部分不具有明确的护肤作用。
另外,蚕丝蛋白的实际应用已较为广泛,但其主要来源依然为天然蚕丝,其成分复杂,除蚕丝本身外还含有金属离子和可能携带致病菌或其成分等,存在致敏风险。天然蚕丝包含多种蛋白成分,首先需脱去具有致敏性的丝胶,在碱性蛋白酶、木瓜蛋白酶、胰蛋白酶等水解得到丝素肽,但其水解度不高,且后期纯化等步骤繁复,限制了丝素蛋白功能的发挥。
此外,重组蛋白表达系统有多种选择,主要分为原核表达系统和真核表达系统。原核表达系统常选用大肠杆菌为工程菌,容易形成包涵体和污染内毒素(细菌内毒素是强致热源,1-5ng/kg体重即可引体温上升),导致蛋白分离纯化繁琐,分离得到的包涵体蛋白需复性以恢复构象和活性,但复性操作繁琐且复性比例低,导致蛋白活性不能满足要求。真核表达系统常选用酿酒酵母为工程菌,由于酿酒酵母发酵密度低、难以高密度培养,缺少强力且严格的启动子,蛋白产量通常很低;酿酒酵母表达分泌蛋白通常会发生过度糖基化问题,使分泌蛋白构象与天然蛋白差异较大,且核心寡糖有α1,3-半乳糖残基,具有超抗原性,不利于安全使用;酿酒酵母表达蛋白的分泌效率低,如蛋白分子量较大则无法分泌,这都造成了蛋白的分离纯化的困难,限制了产量。
因此,找到一种既能够克服目前重组丝聚蛋白结构存在的缺陷,又能够在生产工艺上较大规模生产的产品是当务之急。
发明内容
本发明意在提供一种重组人源化丝聚蛋白,以期解决现有重组丝聚蛋白在蛋白结构、功能和生产工艺上的弊端。
为了达到上述目的,本发明采用以下技术方案实现:
本发明提供了一种重组人源化丝聚蛋白,所述重组人源化丝聚蛋白的核苷酸序列如SEQ ID NO.1所示。
进一步地,所述重组人源化丝聚蛋白的氨基酸序列如SEQ ID NO.2所示。
进一步地,将所述重组人源化丝聚蛋白核苷酸序列SEQ ID NO.1中的部分密码子替换为酵母偏爱型,并在序列前后加入限制性内切酶位点,得到如SEQ ID NO.3所示的含有酶切位点的酵母偏爱型重组人源化丝聚蛋白核苷酸序列。
本发明还提供一种毕赤酵母分泌表达重组载体,所述毕赤酵母分泌表达重组载体含有如SEQ ID NO.3所示的核苷酸序列。
进一步地,所述毕赤酵母分泌表达重组载体的构建包括以下步骤:
人工合成SEQ ID No.3所示的核苷酸序列,将毕赤酵母分泌表达载体pPICZαA与SEQ IDNo.3所示的核苷酸序列分别用相同的限制性内切酶酶切,将酶切产物经纯化后用连接酶环化,经转化工程菌并经抗生素筛选、双酶切鉴定筛选阳性克隆,并将阳性克隆测序,得到毕赤酵母分泌表达重组载体。
本发明还提供一种重组人源化丝聚蛋白毕赤酵母表达菌株,所述重组人源化丝聚蛋白毕赤酵母表达菌株表达含有如SEQ ID NO.2所示序列的蛋白。
本发明还提供一种重组人源化丝聚蛋白毕赤酵母表达菌株的构建方法,包括以下步骤:
将上述的毕赤酵母分泌表达重组载体经限制性内切酶酶切线性化,以电转化的方法转化入毕赤酵母GS115中,将转化后酵母菌液均匀涂布于抗性平板,培养后挑取单克隆菌落,经PCR鉴定后得到重组人源化丝聚蛋白毕赤酵母表达菌株。
本发明还提供上述重组人源化丝聚蛋白在制备保湿化妆品中的应用。
本发明的有益效果在于:
1、本发明利用毕赤酵母表达系统表达的重组人源化丝聚蛋白由人丝聚蛋白完整单体结构域、人丝聚蛋白天然保守linker和丝素蛋白轻链融合而成,命名为hFLG-Fib-l。该重组蛋白内的天然保守linker能被颗粒层和角质层中的蛋白酶识别并切割,释放完整37kDa大小的丝聚蛋白单体结构域,此结构域协助角蛋白丝聚集成致密的角蛋白纤维束,加强皮肤屏障作用,减少应变原和微生物的入侵,维持角质层水分。释放出的丝聚蛋白单体结构域在与角蛋白解离后,被降解为游离氨基酸,经酶加工后参与形成天然保湿因子,阻挡紫外线的trans-UCA,trans-UCA在吸收紫外线后转变为cis-UCA,降低皮肤超敏反应和迟发超敏反应。
2、重组人源化丝聚蛋白(hFLG-Fib-l)的linker被蛋白酶切割后,释放融合蛋白中的丝素蛋白轻链结构域,其免疫原性非常低,且具有良好的吸湿性,可以增加角质层含水量,减少水分丢失,保持和提高皮肤屏障作用。另外,皮肤表面的皮脂膜pH值在4.5-6.5之间,弱酸环境能抑制致病微生物生长,皮脂膜pH值过低或过高,均会造成皮肤屏障作用受损,而丝素蛋白轻链为弱酸性蛋白,其pI=5.2,有利于将皮肤pH值保持在正常范围内,维持皮肤正常的屏障作用。分析显示,丝素蛋白轻链的半衰期较长,这一特性有利于丝素蛋白轻链长时间发挥其保持角质层水分等功能,同时是良好的融合蛋白载体,延长融合蛋白的半衰期。丝素蛋白轻链最终会降解为短肽和氨基酸,部分可参与天然保湿因子的形成,维持和增加角质层含水量。
3、毕赤酵母表达系统能高水平地分泌表达重组人源化丝聚蛋白,且酵母自身分泌的蛋白很少,因此培养基中蛋白成分单纯,非常利于重组蛋白的纯化回收。毕赤酵母对分泌蛋白的糖基化修饰很少,蛋白结构更接近天然结构,且不含有超抗原性的结构,重组蛋白不产生免疫反应。
附图说明
图1为人proFLG蛋白结构示意图;
图2为重组载体pPICZαA-hFLG-Fib-l双酶切鉴定琼脂糖凝胶电泳图;
图3为重组载体pPICZαA-hFLG-Fib-l插入序列测序部分结果图;
图4为酵母转化结果PCR鉴定琼脂糖凝胶电泳图;
图5为毕赤酵母培养基上清液经SDS-PAGE电泳和Western Blot鉴定结果图;
图6为纯化回收的重组人源化丝聚蛋白经SDS-PAGE电泳和Western Blot鉴定结果图;
图7为使用重组人源化丝聚蛋白后前臂试验区和对照区的角质层含水量测定结果图;
图8为使用重组人源化丝聚蛋白后前臂试验区和对照区的经表皮水散失率测定结果图。
具体实施方式
为了使本发明的内容更加便于理解,下面结合附图和具体实施方式对本发明所述的技术方案做进一步说明,但是本发明不仅限于此。
实施例1重组载体的构建
在本实施例中,结合表达载体pPICZαA所提供可使用的限制性内切酶位点和重组人源化丝聚蛋白核苷酸序列SEQ ID NO.1,最终选择限制性内切酶EcoR I和Sal I酶切位点,在不改变核苷酸序列SEQ ID NO.1所对应的氨基酸序列(如SEQ ID NO.2所示)前提下,将重组人源化丝聚蛋白核苷酸序列SEQ ID NO.1中的部分密码子替换为酵母偏爱型,并在序列前后分别插入EcoR I限制性内切酶位点和Sal I限制性内切酶位点,得到如SEQ IDNO.3所示的含有酶切位点的酵母偏爱型重组人源化丝聚蛋白核苷酸序列。
人工合成如SEQ ID NO.3所示的含有酶切位点酵母偏爱型重组人源化丝聚蛋白基因片段,并命名为hFLG-Fib-l片段。将hFLG-Fib-l片段和空载体pPICZαA分别用EcoR I和Sal I限制性内切酶双酶切,反应体系如下:
37℃金属浴酶切15分钟后,65℃金属浴20分钟失活限制性内切酶。失活处理后,经Cycle Pure Kit纯化回收酶切产物,用T4 DNA连接酶进行连接,反应体系如下:
16℃金属浴孵育5小时后,将连接产物经热激法转化入大肠杆菌DH5α感受态,热激后向大肠杆菌DH5α的EP管中加入1mL空白LB培养基,于37℃、100RPM条件下振荡培养1小时,再将大肠杆菌DH5α浓缩后均匀涂布Zeocin抗性的固体LB平板。将平板倒置放于37℃的孵箱中,培养14小时后,挑取5个阳性菌落分别接种于Zeocin抗性的液体LB培养基中,在37℃、250RPM的条件下过夜振荡培养。
各吸取1mL过夜培养的菌液行质粒小量抽提,将提取得到的5个重组载体行双酶切鉴定,反应条件如下:
将5个双酶切产物行琼脂糖凝胶电泳,观察是否含有重组基因片段。结果如图2所示,M表示该泳道为DNA Marker,1-5表示该泳道内分别为1-5号重组载体样品双酶切产物,电泳结果提示重组载体均含有重组基因片段。将1号泳道对应的pPICZαA-hFLG-Fib-l重组载体样品进行测序,测序的部分结果如图3所示。将测序结果与序列SEQ ID NO.3比对,显示重组基因序列正确。至此,得到pPICZαA-hFLG-Fib-l重组载体。
实施例2酵母转化、蛋白表达及鉴定
本实施例中,将实施例1中得到的pPICZαA-hFLG-Fib-l重组载体和不含有重组片段的pPICZαA空载体以限制性内切酶Sac I酶切得到线性化载体,经Cycle Pure Kit纯化后,通过电转化方法将线性化载体转化进毕赤酵母GS115感受态细胞内,酶切反应条件如下:
电转操作如下:
冰浴电转杯,分别将10μL线性化pPICZαA-hFLG-Fib-l和pPICZαA与80μL毕赤酵母GS115感受态细胞混合,混匀后加入0.2cm电转杯中。采用Bio-Rad电转仪,电击条件为:电压1700V,25uF,200Ω。
电击结束后,向电转杯中加入1mL预冷的山梨醇,吹打混匀后将液体全部转移到5mL EP管中,30℃静置2小时后,分别吸取100μL、200μL和500μL菌液涂布与含有Zeocin抗生素(100μg/mL)的YPDS平板,于30℃培养至平板上长出单克隆菌落后,挑取8个转化pPICZαA-hFLG-Fib-l重组载体的单菌落和5个转化pPICZαA空载体的单菌落,分别接种到含有Zeocin浓度为100μg/mL的YPD液体培养基中,30℃、250RPM振荡过夜培养。
提取过夜培养的重组毕赤酵母基因组,使用α-factor引物(α-factor引物的核苷酸序列如SEQ ID NO.4所示)和3'AOX1引物(3'AOX1引物的核苷酸序列如SEQ ID NO.5所示),进行PCR鉴定插入基因序列,引物参考Invitrogen pPICZαA操作手册设计如下:
PCR鉴定结果如图4所示,根据扩增产物大小判断,8个转化pPICZαA-hFLG-Fib-l载体的重组毕赤酵母中都含有重组hFLG-Fib-l基因片段,5个转化pPICZαA空载体的重组毕赤酵母中只含有载体序列片段,不含有重组hFLG-Fib-l基因片段。
鉴定完毕后,先将含有hFLG-Fib-l基因片段的重组毕赤酵母单克隆菌落接种到BMGY培养基中,30℃、250RPM振荡培养至OD600=5.0~6.0,然后离心收集菌体转入BMMY培养基中,30℃、250RPM条件下振荡培养5天,加入甲醇诱导表达重组人源化丝聚蛋白,期间每24小时补充甲醇,是甲醇浓度维持1.0~1.5%。通过SDS-PAGE电泳和Western Blot检测培养基中重组人源化丝聚蛋白,一抗为小鼠抗人FLG抗体,结果如图5所示。图5中共3个泳道,M表示该泳道为蛋白Maker,1泳道为pPICZαA空载体对照组,2泳道为pPICZαA-hFLG-Fib-l重组载体实验组。结果显示转化pPICZαA-hFLG-Fib-l载体的重组毕赤酵母液体培养基中含有重组人源化丝聚蛋白,转化pPICZαA空载体的重组毕赤酵母液体培养基中不含重组蛋白。
将成功表达重组人源化丝聚蛋白的重组毕赤酵母菌株放大培养,以甲醇诱导表达,并经镍柱纯化回收重组人源化丝聚蛋白,操作过程如下:
先使用5倍柱体积的去离子水冲洗出Ni-NTA预装柱,再用5倍体积的裂解缓冲液平衡预装柱,通过蠕动泵上样,上样速度为15mL/h。上样完毕后,用20倍柱体积清洗缓冲液冲洗预装柱,直到OD280吸收峰稳定。清洗预装柱后,用5倍柱体积的含有高浓度咪唑的洗脱缓冲液洗脱结合的重组人源化丝聚蛋白,并以SDS-PAGE电泳和Western Blot检测,一抗为小鼠抗人FLG抗体,结果如图6所示。图6中共2个泳道,M表示该泳道为蛋白Marker,1泳道为纯化回收的重组人源化丝聚蛋白。结果显示在约68kDa大小左右可见清晰条带,说明本发明提供的重组酵母菌株,可以表达重组人源化丝聚蛋白。
实施例3重组人源化丝聚蛋白的应用
本实施例提供了一种重组人源化丝聚蛋白在保湿化妆品中的应用。为了验证实施例2中得到的重组人源化丝聚蛋白的保湿作用,本实施例操作流程如下:
1.受试者入选标准:(1)20-40岁健康中国女性40人;(2)非孕期,非哺乳期,无重大基础疾病和免疫系统疾病,无过敏史;(3)近3个月未使用过激素类药物和免疫抑制药物;(4)试验开始前3天停用任何化妆品;(5)愿意参与本试验,能严格遵守试验方案并完成随访。
2.试验仪器:(1)皮肤水分测试仪Corneometer CM 825(德国Courage+Khazaka公司);(2)经皮水分散失测试仪Tewameter TM 300(德国Courage+Khazaka公司)。
3.试验操作:(1)受试者提前进入测试房间(环境温度25℃,相对湿度50~60%),保持安静状态至少30分钟;(2)首次试验时,指导受试者清洁前臂后,在受试者左右前臂屈侧、手腕上10cm处,划出3cm×3cm的测试区并编号1#(左臂)和2#(右臂)。根据随机数表,不同受试者试验区可能为1#或2#测试区,选定试验区后,另一区域则为对照区。(3)使用Corneometer CM 825和Tewameter TM 300测量两处试验区的角质层含水率(StratumCorneum Hydration,SC Hydration)和经表皮水散失率(Transepidermal Water Loss,TEWL)并记录数据;(4)指导受试者在试验区擦涂重组人源化丝聚蛋白溶液(浓度:0.2mg/mL,体积:0.5mL),对照区擦涂0.5mL去离子水;(5)受试者在此后的28天内,每天夜间分别在各自指定试验区擦涂一次重组人源化丝聚蛋白溶液和去离子水;(6)受试者分别在试验开始后第7天、14天和28天来到实验室检测试验区和对照区的角质层含水率和经表皮水散失率,并记录结果;(6)整理试验数据并进行统计学分析。
4.试验结果:
试验共进行28天,期间3人脱组,共有37人完成试验。
如图7所示,使用0.2mg/mL重组人源化丝聚蛋白的试验区SC Hydration较对照区明显上升。
如图8所示,使用0.2mg/mL重组人源化丝聚蛋白的试验区TEWL较对照区明显降低。综合以上结果,说明重组人源化丝聚蛋白能明显提升角质层含水量,降低经皮水分散失量,对维持和增强皮肤屏障有促进作用。
以上的仅是本发明的实施例,该发明不限于此实施案例涉及的领域,方案中公知的具体结构及特性等常识在此未作过多描述,所属领域普通技术人员知晓申请日或者优先权日之前发明所属技术领域所有的普通技术知识,能够获知该领域中所有的现有技术,并且具有应用该日期之前常规实验手段的能力,所属领域普通技术人员可以在本申请给出的启示下,结合自身能力完善并实施本方案,一些典型的公知结构或者公知方法不应当成为所属领域普通技术人员实施本申请的障碍。应当指出,对于本领域的技术人员来说,在不脱离本发明结构的前提下,还可以作出若干变形和改进,这些也应该视为本发明的保护范围,这些都不会影响本发明实施的效果和本发明的实用性。本申请要求的保护范围应当以其权利要求的内容为准,说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (8)
1.一种重组人源化丝聚蛋白,其特征在于:所述重组人源化丝聚蛋白的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的重组人源化丝聚蛋白,其特征在于:所述重组人源化丝聚蛋白的氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求1所述的重组人源化丝聚蛋白,其特征在于:将所述重组人源化丝聚蛋白核苷酸序列SEQ ID NO.1中的部分密码子替换为酵母偏爱型,并在序列前后加入限制性内切酶位点,得到如SEQ ID NO.3所示的含有酶切位点的酵母偏爱型重组人源化丝聚蛋白核苷酸序列。
4.一种毕赤酵母分泌表达重组载体,其特征在于:所述毕赤酵母分泌表达重组载体含有如SEQ ID NO.3所示的核苷酸序列。
5.一种重组人源化丝聚蛋白毕赤酵母表达菌株,其特征在于:所述重组人源化丝聚蛋白毕赤酵母表达菌株表达含有如SEQ ID NO.2所示序列的蛋白。
6.根据权利要求4所述的毕赤酵母分泌表达重组载体,其特征在于:所述毕赤酵母分泌表达重组载体的构建包括以下步骤:
人工合成SEQ ID No.3所示的核苷酸序列,将毕赤酵母分泌表达载体pPICZαA与SEQ IDNo.3所示的核苷酸序列分别用相同的限制性内切酶酶切,将酶切产物经纯化后用连接酶环化,经转化工程菌并经抗生素筛选、双酶切鉴定筛选阳性克隆,并将阳性克隆测序,得到毕赤酵母分泌表达重组载体。
7.一种重组人源化丝聚蛋白毕赤酵母表达菌株的构建方法,其特征在于:包括以下步骤:
将权利要求6中所述的毕赤酵母分泌表达重组载体经限制性内切酶酶切线性化,以电转化的方法转化入毕赤酵母GS115中,将转化后酵母菌液均匀涂布于抗性平板,培养后挑取单克隆菌落,经PCR鉴定后得到重组人源化丝聚蛋白毕赤酵母表达菌株。
8.如权利要求1-3中任一项所述的重组人源化丝聚蛋白在制备保湿化妆品中的应用。
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