CN107904251A - TAT‑hEGF融合蛋白的制备及其在隐形面膜的应用 - Google Patents
TAT‑hEGF融合蛋白的制备及其在隐形面膜的应用 Download PDFInfo
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- CN107904251A CN107904251A CN201711470311.4A CN201711470311A CN107904251A CN 107904251 A CN107904251 A CN 107904251A CN 201711470311 A CN201711470311 A CN 201711470311A CN 107904251 A CN107904251 A CN 107904251A
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Abstract
本发明涉及一种TAT‑hEGF融合蛋白的制备及其在隐形面膜的应用,属于基因工程领域。设计获得毕赤酵母高效表达TAT‑hEGF融合蛋白的核酸序列,并利用毕赤酵母高效分泌表达TAT‑hEGF融合蛋白,及在制备温敏无基布生物活性隐形面膜中的应用。有益效果是该面膜在常温状态下为液体,应用到面部后相变为凝胶状态,克服现有基布面膜与面部贴合不佳和生物活性成分利用度低的问题,同时克服了目前市售的面膜膏的观感不佳问题。
Description
技术领域
本发明涉及基因工程和化妆品领域,尤其涉及利用毕赤酵母表达系统高效表达TAT-人表皮生长因子(TAT-hEGF)融合蛋白及利用该活性蛋白制备温敏无基布生物活性隐形面膜的方法。
背景技术
表皮生长因子(Epidermal Growth Factor,EGF)是一类普遍存在于人类多种腺体及体液中且具备生物活性的多肽,参与调节机体多种细胞的增殖、迁移、分化等重要的生物学过程。1962年,Cohen等分离纯化小鼠颌下腺中神经生长因子时,意外获得的一种多肽可以促进初龄小鼠门齿的萌出、眼睑睁开程度及抑制小鼠毛发生长,将其命名为“齿睑因子(Tooth-Lid Factor)”,后体内外研究显示,此种多肽具有促进多种表皮细胞增殖及组织再生的作用,1972年Savage等提纯并发表了此肽完整53个氨基酸序列组成,改称表皮生长因子(Epidermal Growth Factor,EGF)。Gregory等首次从人尿中分离出人表皮细胞生长因子(human Epidermal Growth Factor,hEGF),具有抑制胃酸分泌作用,故早期称为尿胃抑素(urogastrone,UG)。后继研究发现尿胃抑素与人表皮细胞生长因子是同一种物质,因此统一命名为人表皮生长因子并沿用至今。
伴随对EGF的深入研究,目前已广泛开发应用于临床疾病的医治,如眼角膜损伤、皮肤烧伤和创伤、手术切口及外伤愈合等。另外EGF作为生物工程产品应用于美容护肤产品中,成为医学生物技术渗透美容业的典型开端,在细胞及分子水平上解决美容护肤问题,加快皮肤新陈代谢的速度,延缓衰老,为广大爱美女性带来科技的福音。
天然的EGF来源有限,提取工艺复杂且产量甚微,远远不能满足临床需求。随着基因工程技术的发展与日渐完善,应用基因工程技术生产出高产量、高活性的EGF,备受关注。
自然界中细胞跨膜肽的发现源于1988年,Green等研究发现HIV-1肽段上编码的反式激活蛋白TAT(Trans-activating transcriptional activator,TAT),自身可以跨膜进入细胞,反式激活HIV-1启动子,对病毒基因表达至关重要。其碱性氨基酸富集区含有11个氨基酸(YGRKKRRQRRR),包括6个精氨酸(R)和2个赖氨酸(K),Vives等的研究揭示这11个氨基酸是跨膜转导的核心区。长期研究发现,TAT可介导包括多肽、蛋白质及核酸等外源生物大分子安全有效地进入细胞,甚至可以通过血脑屏障到达脑组织发挥作用,与此同时保持其生物活性。
TAT同样能跨越动物的表皮细胞,甚至能够进入真皮层。Jin等将人肝源性过氧化氢酶基因克隆到编码HIV-1TAT的原核表达载体,表达后的融合蛋白喷淋动物皮肤,根据免疫组织化学和特定的酶活性分析,显示融合蛋白可以有效地渗透到皮下层的表皮和真皮,并且酶活性不受影响。Wu等研究发现TAT核心肽段不仅能促进外源蛋白的表达,而且还能提高外源蛋白的产量和溶解性。本发明将TAT与hEGF进行融合表达,不仅可以提高融合蛋白的表达量,更有利于活性蛋白透过角化的表皮,从而大大提高TAT-hEGF的美容效果。
尿囊素具有促进细胞生长,加快伤口愈合,杀菌防腐、止痛、抗氧化,软化角质蛋白等生理功能,能使皮肤保持水份,滋润和柔软,是皮肤创伤的良好愈合剂和抗溃疡药剂。可用作缓解和治疗皮肤干燥症、鳞屑性肤疾患、皮肤溃疡、消化道溃疡及炎症,对骨髓炎、糖尿病、肝硬化、痤疮均有较好疗效。
透明质酸(玻尿酸)HA是人皮肤表皮及真皮的主要基质成分之一,其生理功能是能使水分进入细胞间隙,并与蛋白质结合而形成蛋白凝胶,将细胞粘在一起,发挥正常的细胞代谢作用,起到保持细胞水分,保护细胞不受病原菌的侵害,加快恢复皮肤组织,提高创口愈合再生能力,减少疤痕,增强免疫力等作用。将其用于化妆品中,能起到独特的保护皮肤作用,可保持皮肤滋润光滑,细腻柔嫩,富有弹性,具有防皱、抗皱、美容保健和恢复皮肤生理功能的作用。薄荷醇,为薄荷和欧薄荷精油中的主要成分,以游离和酯的状态存在,有清凉止痒、止痛、防腐麻醉和抗炎作用,同时还具有促进有效成分透皮吸收的功能。
泊洛沙姆407为无毒无刺激的粉末,其溶液无色透明,美国FDA批准作为分散剂,乳化剂,增溶剂,润滑剂,润湿剂等用于药物或医疗器械的辅料,在日化用品中作为黏度改善剂,用于牙膏、漱口水等产品中。将其作为基质制造温敏无基布生物活性隐形面膜并未见报道。
发明内容
本发明提供一种TAT-hEGF融合蛋白的制备及其在隐形面膜的应用,要解决的技术问题是:克服现有hEGF生产过程繁琐,(比如目前常用大肠杆菌来表达和制备hEGF,往往需要繁琐的包涵体变性和复性过程)和应用于皮肤美容时难以透过角化的表皮而美容效果不佳的问题,克服现有基布面膜与面部贴合不佳和生物活性成分利用度低的问题,同时克服了目前市售的面膜膏的观感不佳问题,本发明提供一种在组成成分的协同作用下,可使生物活性物质直达皮肤的真皮层的温敏无基布生物活性隐形面膜的制备方法。
本发明采取的技术方案是:一种用于毕赤酵母表达系统高效表达TAT-hEGF融合蛋白的核酸序列,包含:Kex2信号切割位点编码序列、TAT跨膜肽编码序列、linker序列和人表皮生长因子的编码序列,如SEQ ID NO:1所示。一种TAT-hEGF融合蛋白,其中TAT为HIV病毒跨膜转导的核心区短肽,hEGF为成熟的人表皮生长因子。一种温敏无基布生物活性隐形面膜的制备方法,以质量百分比记,由以下成分构成:泊洛沙姆407 14-25%、甘油5-15%,透明质酸0.5-5%、TAT-hEGF 0.02-0.2%、尿囊素0.2-2%、薄荷醇0.5%-2%、苯氧乙醇0.8%、碘丙炔醇丁基氨甲酸酯(IPBC)0.05%。
具体地,所述TAT短肽与hEGF之间为直接融合或通过连接序列(linker)融合。
TAT-hEGF融合蛋白毕赤酵母高表达体系的构建及其制备方法,包括以下步骤:
1)人工合成编码TAT-hEGF融合蛋白的DNA序列,所述TAT为HIV病毒跨膜转导的核心区短肽,包含11个氨基酸残基;所述hEGF为人源的成熟EGF,包含53个氨基酸残基。
2)构建TAT-hEGF融合蛋白的表达盒,所述的蛋白表达盒依次包括Kex2信号切割位点编码序列、TAT跨膜肽编码序列、linker序列和人表皮生长因子的编码序列,如SEQ IDNO:1所示。
3)构建毕赤酵母表达载体,所述表达载体是通过基因工程手段将TAT-hEGF融合蛋白的表达盒亚克隆到毕赤酵母的表达载体(如Invitrogen公司的pPICZα)上。
4)表达与纯化TAT-hEGF融合蛋白,将获得的毕赤酵母表达载体使用核酸内切酶如Pme I线性化后,电转化毕赤酵母,而后筛选获得高表达TAT-hEGF融合蛋白的菌株,确定最佳诱导条件和时间,取发酵上清液使用40%的饱和硫酸铵沉淀蛋白,而后用凝胶层析或阳离子交换层析,获得纯化的TAT-hEGF融合蛋白。
5)鉴定TAT-hEGF融合蛋白,并检测其生物活性,通过SDS-PAGE和免疫印迹的方法对制备的融合蛋白进行分子量和免疫学鉴定,并通过刺激细胞增殖的方法检测TAT-hEGF融合蛋白的生物学活性,通过小鼠皮肤涂抹实验,验证TAT-hEGF融合蛋白透过表皮的能力。
进一步的,所述TAT序列位于hEGF的氨基段。
进一步的,所述载体为真核表达载体。
进一步的,所述真核表达载体为毕赤酵母分泌表达载体,因为原核表达系统存在缺乏翻译后修饰、不能形成正确的二硫键、需要繁琐的变性与复性过程、不能分泌表达和内毒素污染问题,而哺乳动物细胞表达系统存在表达量低和生产成本高的问题,因此本发明优选毕赤酵母分泌表达载体,如pPICZα。
进一步的,所述真核表达载体能被宿主细胞高效表达。
进一步的,所述宿主细胞为真核细胞或原核细胞,本发明优选毕赤酵母X-33。
进一步的,以此制备的TAT-hEGF为主要生物活性成分制备所述的温敏无基布生物活性隐形面膜,其特征在于:以质量百分比记,由以下成分构成:泊洛沙姆40714-25%、甘油5-15%,透明质酸0.5-5%、TAT-hEGF 0.02-0.2%、尿囊素0.2-2%、薄荷醇0.5%-2%、苯氧乙醇0.8%、碘丙炔醇丁基氨甲酸酯(IPBC)0.05%,其余为去离子水。
本发明的一种TAT-hEGF融合蛋白毕赤酵母高表达体系的构建及其制备方法,通过使用发明构建的TAT-hEGF融合蛋白表达元件,转化毕赤酵母后,可以高效分泌表达出TAT-hEGF融合蛋白,可获得600mg/L的高表达量,生产成本低。而后,利用本发明所建立的纯化方法,可获得高纯度的TAT-hEGF融合蛋白,该融合蛋白具有较好的刺激细胞增殖的活性和穿透皮肤角质层的能力。运用此方生产制备的TAT-hEGF融合蛋白为主要生物活性物质制备温敏无基布生物活性隐形面膜,其特征在于:以质量百分比记,由以下成分构成:泊洛沙姆40714-25%、甘油5-15%,透明质酸0.5-5%、TAT-hEGF 0.02-0.2%、尿囊素0.2-2%、薄荷醇0.5%-2%、苯氧乙醇0.8%、碘丙炔醇丁基氨甲酸酯(IPBC)0.05%,去离子水定容。
本发明的有益效果是:1、克服现有hEGF生产过程繁琐(比如目前常用大肠杆菌来表达和制备hEGF,往往需要繁琐的包涵体变性和复性过程)和应用于皮肤美容时难以透过角化的表皮而美容效果不佳的问题,本发明提供一种TAT-hEGF融合蛋白毕赤酵母高表达体系的构建及其制备方法。2、该面膜在常温状态下为液体,应用到面部后相变为凝胶状态,克服现有基布面膜与面部贴合不佳和生物活性成分利用度低的问题,同时克服了目前市售的面膜膏的观感不佳问题,本发明提供一种在组成成分的协同作用下,可使生物活性物质直达皮肤的真皮层的温敏无基布生物活性隐形面膜的制备方法。
附图说明
图1是不同诱导时间的TAT-hEGF表达情况,其中M为蛋白Marker;1-8:为重组毕赤酵母X-33菌株诱导0h,24h,48h,72h,96h,120h,144h,168h的发酵上清液;
图2是SDS-PAGE电泳筛选目的蛋白TAT-hEGF最佳诱导pH值结果,其中M为蛋白Marker;1-6:分别为pH值5.2,4.6,4.0,3.4,2.8,2.2条件下诱导表达的发酵液上清;
图3是离子交换层析样品SDS-PAGE检测结果,A图M为蛋白Marker;1为硫酸铵沉淀后的样品;2-4:上样穿透液;5:100mmol/L NaCl洗脱峰样品;6:200mmol/L NaCl洗脱峰样品;7-9:300mmol/L NaCl洗脱峰样品;
B图M为蛋白Marker;1为硫酸铵沉淀后的样品;2-4为400mmol/L NaCl洗脱峰样品;5-7为500mmol/L NaCl洗脱峰样品;8-9为1mol/L NaCl洗脱峰样品;
图4是300mmol/L NaCl洗脱峰离子交换层析样品Western Blot检测结果,1-3为300mmol/L NaCl洗脱峰样品;
图5是重组TAT-hEGF促进细胞增殖活性测定结果;
图6是小鼠皮肤组织Western blot测定结果,其中1-2为涂抹TAT-hEGF融合蛋白组,3-4为涂抹生理盐水组。
具体实施方式
下面结合具体实施例对本发明作进一步具体的阐述,应理解,引用实施例仅用于说明本发明,而不用于限制本发明的范围。
实施例1.人工合成编码TAT-hEGF融合蛋白的DNA序列(如SEQ ID NO:1所示)
根据文献报道的HIV病毒跨膜转导的核心区短肽TAT序列,包含11个氨基酸残基,和人源的成熟EGF序列,包含53个氨基酸残基,TAT和hEGF序列之间由3个甘氨酸序列组成的linker相连接,依据酵母偏爱的密码子对上述氨基酸序列进行人工合成。进行合成时,在基因的上游引入XhoI位点和Kex2切割位点Glu-Lys-Arg序列,在基因的下游引入终止密码子和XbaI位点。
实施例2.构建表达载体
分别用XhoⅠ、XbaⅠ双酶切合成的基因片段及载体pPICZα,37℃恒温过夜。将酶切产物使用琼脂糖凝胶电泳切下目的片段后,使用琼脂糖凝胶DNA回收试剂盒回收。将回收的酶切后目的基因与载体按照3:1的摩尔比例混匀,使用T4DNA Ligase连接酶16℃PCR仪恒温连接过夜,构建pPICZα-TAT-hEGF重组质粒。将连接产物转化大肠杆菌感受态DHα,转化的感受态细胞涂均匀于含有Zeocin(25μg/mL)的低盐LB平板培养基上,待菌液被完全吸收后,37℃倒置培养12-16h。用无菌枪头从转化平板上挑取4-8个生长状态良好的单菌落,接种于5mL含Zeocin(25μg/mL)的LB培养基中,37℃强烈振荡(225rpm)培养过夜。使用快速质粒小提试剂盒提取质粒DNA,用HindⅢ单酶双切鉴定重组质粒pPICZαA-TAT-hEGF,37℃消化3h,根据内切酶谱鉴定出正确克隆。选取酶切图谱正确的克隆,送测序公司进行测序验证。
实施例3.表达与纯化TAT-hEGF融合蛋白
1.电转化毕赤酵母X-33并筛选阳性转化子
取测序结果正确的重组质粒pPICZαA-TAT-hEGF 15μg,用限制性内切酶Pme I消化质粒,电转化D-山梨醇法制备的毕赤酵母X-33感受态细胞。取50-100μL菌液均匀涂布于含Zeocin(100μg/mL)的YPD平板上,30℃培养箱倒置培养2-3天,观察转化子的生长。选择Zeocin抗性克隆接种于5mLYPD培养基里,30℃摇床培养过夜,取1ml菌液,离心收集菌体,提取酵母的基因组DNA,使用引物P1 5’-ATACTCGAGAAGAGATACGGTA-3’(如SEQ ID NO:2所示)和引物P2 5’-CGGTCTAGATTATCTCAATTCC-3’(如SEQ ID NO:3所示),进行PCR,对扩增的产物进行0.8%琼脂糖凝胶电泳。观察是否能够得到220bp左右的基因片段,分析基因是否整合到酵母基因组。
2.重组毕赤酵母X-33的诱导表达及高表达TAT-hEGF融合蛋白菌株的筛选
(1)选择阳性重组菌接种于10mL BMGY培养基中,30℃振荡培养24h,至OD600达2.0-6.0时收集细胞;
(2)等体积(10mL)BMMY重悬细胞沉淀,30℃振荡培养,诱导表达。诱导过程中,每24h补充一次甲醇直至其终浓度0.5%,同时补充灭菌蒸馏水,使发酵液保持总体积不变;
(3)连续诱导培养7天,每24h取1mL发酵液,离心菌体,分离上清与沉淀,上清用于SDS-PAGE蛋白分析,筛选高表达目的蛋白的菌株,并确定最佳发酵时间长度,结果表明诱导第1天即有目的蛋白的表达,随着诱导时间的延长,表达量升高,第6天时表达量达到峰值(如图1所示)。
3.毕赤酵母X-33诱导表达TAT-hEGF最佳pH值的确定及分析
(1)选取TAT-hEGF高表达量的毕赤酵母工程菌,在10mLYPD培养基中30℃、225rpm振荡培养24h;
(2)将扩增的毕赤酵母工程菌接种于10mL BMGY中,pH值为6.0、28℃、220rpm振荡培养24h左右,使其OD600达2.0-6.0。此方法使酵母扩增的起始条件相同,为以后确定诱导最佳pH值奠定基础;
(3)室温离心(4000rpm)5min,弃去上清,加入前述未加缓冲液的BMMY 9mL,按下表的量加入1mol/L Na2HPO4和0.5mol/L柠檬酸配制成不同pH值的BMMY,如表1,30℃、225rpm振荡培养,诱导过程中每24h补充一次甲醇至终浓度为0.5%,同时补充灭菌蒸馏水,使发酵液总体积保持不变;
表1
(4)取第7d各个pH值样品上清进行SDS-PAGE分析,确定最佳诱导的pH值,结果显示最佳诱导的pH值为pH4.6(如图2所示)。
4.融合蛋白TAT-hEGF的纯化
1)硫酸铵盐析
(1)在25℃条件下,配置饱和度100%的硫酸铵溶液,称取767g的硫酸铵固体,边搅拌边加入到1L的蒸馏水中,完全溶解后,用氨水或者硫酸调节pH至7.0。取发酵液上清60mL,调节pH值至目的蛋白等电点8.5附近。
(2)将发酵液上清分成6份,每份分别边搅拌边缓慢加入饱和硫酸铵溶液,至浓度分别为20%、30%、40%、50%、60%,70%。4℃过夜,使蛋白质充分沉淀。
(3)4℃离心,11000rpm,10min,保留沉淀与上清。分析沉淀中是否含有目的蛋白,以确定沉淀目的蛋白所需要的最佳饱和硫酸铵溶液的浓度,结果确定使用40%饱和度的硫酸铵可以完全沉淀目的蛋白,并可以去除部分杂蛋白。
(4)沉淀用10mL 30mmol/L醋酸-醋酸钠(pH5.2)缓冲液溶解,进行后续的凝胶层析纯化。
2)凝胶过滤层析
用3-5倍柱床体积的30mmol/L醋酸-醋酸钠(pH5.2)缓冲液冲洗Superdex 75prepgrade凝胶柱至基线稳定。将上述硫酸铵沉淀并溶解在30mmol/L醋酸-醋酸钠(pH5.2)缓冲液的蛋白上样到Superdex 75prep grade凝胶柱,使用30mmol/L醋酸-醋酸钠(pH5.2)缓冲液洗脱,并分部收集蛋白峰,SDS-PAGE确定目的蛋白所在的峰位置。
3)离子交换层析
使用3-5倍柱床体积的30mmol/L醋酸-醋酸钠(pH5.2)缓冲液平衡CM SepharoseF.F层析柱,而后上样,使用3-5倍柱床体积的30mmol/L醋酸-醋酸钠(pH5.2)缓冲液冲至基线,依次使用0.1、0.2、0.3、0.4、0.5、1mol/L NaCl-30mmol/L醋酸-醋酸钠(pH5.2)洗脱液,进行梯度洗脱。
分部收集各个蛋白峰,SDS-PAGE确定目的蛋白所在的峰位置,,结果发现0.3mol/L的NaCl可以较好地洗脱TAT-hEGF融合蛋白,获得纯度大于95%的目的蛋白(如图3所示)。将含有目的蛋白的组分使用超滤离心浓缩管(截留分子量3kDa)进行脱盐和浓缩处理,定量后用于生物活性检测。
实施例4.鉴定TAT-hEGF融合蛋白
12%的SDS-PAGE分离目的蛋白,而后湿法转膜。将PVDF膜在使用前在甲醇中活化30秒,在转膜液中按从下到上海绵、滤纸、胶、PVDF膜、滤纸、海绵的顺序夹好放入转膜槽,避免产生气泡,200mA恒流冰浴转膜30min。转膜结束后取出PVDF膜,置于TBST中漂洗,以免PVDF干燥影响实验结果。常温下1%BSA摇床封闭1h,除去封闭液,根据抗体说明比例使用1%BSA稀释抗hEGF特异性抗体,4℃摇洗孵育过夜。一抗回收后,TBST洗3次,10min/次。根据二抗说明书按比例使用TBST稀释二抗。室温孵育1h。TBST洗3次,10min/次。吸弃TBST洗液,加入ECL显影液孵育片刻,成像仪器下显影,分析结果。结果在预期分子量位置出现条带,表明重组蛋白可以与hEGF特异性抗体结合(如图4所示)。
实施例5.检测TAT-hEGF融合蛋白的生物活性
1)TAT-hEGF融合蛋白促进细胞增殖活性检测
(1)NIH/3T3细胞培养:培养基选用含10%(V/V)FBS的DMEM,在37℃、5%二氧化碳条件下培养,细胞浓度控制在每1mL含1.0×105-5.0×105个细胞;
(2)细胞接种:传代24-36小时后,细胞消化离心,用含10%(V/V)FBS的DMEM培养基重悬,稀释到每100μL含5.0×103个细胞的浓度,接种于96孔板,每孔加入100μl上述稀释好的细胞悬液,37℃、5%二氧化碳条件下培养24小时;
(3)血清饥饿:培养基更换为含4%(V/V)FBS的DMEM维持培养基,37℃、5%二氧化碳条件下培养12小时;
(4)添加生长因子:用维持培养基将参考品和重组TAT-hEGF融合蛋白稀释成不同的浓度,替换原来的维持培养基,对照组加入不含参考品及供试品的维持培养基,37℃、5%二氧化碳条件下培养60-72小时;
(5)检测细胞活性:弃去含参考品及TAT-hEGF融合蛋白的培养基,PBS洗两遍,然后用细胞活性试剂盒检测细胞的增殖情况。每孔加入含20μL细胞活性检测试剂的100μL维持培养基,37℃、5%二氧化碳条件下孵育1-2小时,用酶标仪在490nm波长下检测细胞增殖情况,并记录和计算(如图5所示)。
2)TAT-hEGF融合蛋白穿透小鼠皮肤的活性检测
取BALB/c小鼠4只,去除其背部皮肤,分为两组每组2只,一组使用100μL生理盐水涂抹,另一组使用100μL含50μgTAT-hEGF融合蛋白涂抹,每30分钟涂抹一次,共涂抹2次,第二次涂抹后1小时,使用蒸馏水冲洗涂抹部位皮肤,擦干后取涂抹部位皮肤,匀浆后取蛋白上清50μg,使用Western blot来检测TAT-hEGF融合蛋白穿透小鼠皮肤的活性。结果表明TAT-hEGF融合蛋白具有较好的穿透小鼠皮肤的活性(如图6所示)。
实施例6.温敏无基布生物活性隐形面膜的制备
1)基质中泊洛沙姆含量的确定
我们通过对胶凝温度、相转变温度、凝胶强度以及粘度进行研究考察,确定了温敏面膜中泊洛沙姆407的最佳含量为18%。
表2.泊洛沙姆温度敏感隐形面膜的胶凝温度
泊洛沙姆浓度 | 胶凝温度 |
14% | 53.6±0.1 |
16% | 45.3±0.2 |
18% | 36.7±0.3 |
20% | 27.5±0.2 |
22% | 25.6±0.2 |
24% | 22.9±0.3 |
2)温敏无基布生物活性隐形面膜的制备
称取180g泊洛沙姆407加入800ml去离子水中,搅拌溶解后,加入甘油70ml,透明质酸20g,搅拌溶解,而后逐次加入尿囊素5g、薄荷醇5g、TAT-hEGF 0.5g、苯氧乙醇8ml、碘丙炔醇丁基氨甲酸酯(IPBC)0.5g,充分搅拌溶解后,加入去离子水定容至1000ml。
按照上述技术方案所述的方法,通过给60名30-55岁的女性每周2次来检测本发明实施例6制备得到的祛斑美白面膜的使用效果,检测结果为该面膜在常温状态下为液体,应用到面部后相变为凝胶状态,使用本发明实施例6提供的美白抗衰老面膜3周后,皮肤白皙细腻、皮肤滋润有光泽、皮肤细纹等衰老干燥黄枯现象消失,表明本发明实施例6提供的温敏无基布生物活性隐形面膜具有较好的美白抗衰老的效果。
综上所述仅是本发明的优选实施方式,应当指出对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以作成若干个变形和改进,这些也应视为本发明的保护范围之内。
序列表
<110> 吉林大学
<120> TAT-hEGF融合蛋白的制备及其在隐形面膜的应用
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attgaagctt tggataagta cgcttgtaac tgtgttgttg gttacattgg tgaaagatgt 180
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Claims (9)
1.一种利用毕赤酵母表达系统高效表达TAT-人表皮生长因子(TAT-hEGF)融合蛋白的方法,其特征在于:设计获得毕赤酵母高效表达TAT-hEGF融合蛋白的核酸序列,并利用毕赤酵母高效分泌表达TAT-hEGF融合蛋白。
2.根据权利要求1所述一种利用毕赤酵母表达系统高效表达TAT-人表皮生长因子(TAT-hEGF)融合蛋白的方法,其特征在于:所述用于毕赤酵母高效表达TAT-hEGF融合蛋白的核酸序列如SEQ ID NO:1所示,包含:Kex2信号切割位点编码序列、TAT跨膜肽编码序列、linker序列和人表皮生长因子的编码序列。
3.根据权利要求1所述一种利用毕赤酵母表达系统高效表达TAT-人表皮生长因子(TAT-hEGF)融合蛋白的制备方法,其特征在于,包括以下步骤:
1)人工合成编码TAT-hEGF融合蛋白的DNA序列;
2)构建TAT-hEGF融合蛋白的表达盒;
3)构建表达载体;
4)表达与纯化TAT-hEGF融合蛋白;
5)鉴定TAT-hEGF融合蛋白,并检测其生物活性。
4.根据权利要求3所述的利用毕赤酵母表达系统高效表达TAT-人表皮生长因子(TAT-hEGF)融合蛋白的制备方法,其特征在于:所述TAT-hEGF融合蛋白的表达盒依次包括Kex2信号切割位点编码序列、TAT跨膜肽编码序列、linker序列和人表皮生长因子的编码序列。
5.根据权利要求3利用毕赤酵母表达系统高效表达TAT-人表皮生长因子(TAT-hEGF)融合蛋白的制备方法,其特征在于:所述构建表达载体是通过分子生物学和基因工程的手段将TAT-hEGF融合蛋白的表达盒亚克隆到具有真核表达元件载体上。
6.根据权利要求5利用毕赤酵母表达系统高效表达TAT-人表皮生长因子(TAT-hEGF)融合蛋白的制备方法,其特征在于:所述载体为真核表达载体。
7.根据权利要求6利用毕赤酵母表达系统高效表达TAT-人表皮生长因子(TAT-hEGF)融合蛋白的制备方法,其特征在于:所述真核表达载体为毕赤酵母表达载体。
8.一种利用毕赤酵母表达系统高效表达TAT-人表皮生长因子(TAT-hEGF)融合蛋白在制备温敏无基布生物活性隐形面膜中的应用。
9.如权利要求8所述的温敏无基布生物活性隐形面膜的制备方法,其特征在于:以质量百分比记,由以下成分制成:泊洛沙姆407 14-25%、甘油5-15%,透明质酸0.5-5%、TAT-hEGF 0.02-0.2%、尿囊素0.2-2%、薄荷醇0.5%-2%、苯氧乙醇0.8%、碘丙炔醇丁基氨甲酸酯(IPBC)0.05%,去离子水定容,该面膜在常温状态下为液体,应用到面部后相变为凝胶状态。
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