CN116836264B - 一种重组人源化纤连蛋白rhFEB及应用 - Google Patents
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Abstract
本发明属于基因工程技术领域,公开了一种重组人源化纤连蛋白rhFEB及应用。本发明通过对不同的多肽类物质进行分析、筛选、组合,利用基因工程手段,在人源化纤连蛋白EDB片段N端顺序插入人III型胶原促细胞增殖的功能基序,获得一种多肽类融合蛋白rhFEB,其氨基序列如SEQ ID NO:1所示。通过基因工程的方法在体外进行表达纯化,得到具抗衰性能的重组人源化纤连蛋白rhFEB,所述rhFEB具有较高的促细胞恢复能力,可溶性、高吸收性。本发明的重组人源化纤连蛋白rhFEB具有抗衰,并且有影响细胞粘附、促进微小血管形成、修复受损皮肤细胞的功能,可作为原料应用于化妆品、护肤品及细胞培养基质等领域中。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一种重组人源化纤连蛋白rhFEB及应用。
背景技术
抗衰老指抑制、延缓机体的衰老过程。皮肤和其他器官一样,随着年龄不断增加,都逐渐趋向衰老,而抗衰老的意义就是减缓肌肤衰老的脚步,让人的皮肤更光滑细腻,减少皱纹。
真皮细胞外基质(ECM)在皮肤老化中起着至关重要的作用,它们的损伤与皮肤老化密切有关,胶原蛋白流失会直接导致皮肤下垂、老化、弹性下降。ECM蛋白质主要包括胶原蛋白、纤连蛋白、弹性蛋白和层粘连蛋白。纤连蛋白(FN)有多个功能性结构域,在细胞黏附,迁移发挥着重要作用,同时还可与肝素,整合素受体,胶原等结合可启动各种细胞内信号通路,此外,还可维持ECM的稳定性。FN第12至14个重复序列形成混杂的结合结构域,称为II型肝素结合结构域,能与大多数血小板衍生生长因子相互作用。II型肝素结合结构域通过与细胞表面硫酸乙酰肝素蛋白聚糖的结合,在细胞粘附中也起着关键作用。纤连蛋白的“细胞结合域”FNIII10(后文简称FN10)包含有RGD序列(Arg-Gly-Asp),具有促进细胞附着的位点,可与细胞表面上多种整合素(如αⅤβ3、αⅤβ5、αⅤβ6等)相结合。整合素来自于血液的干细胞,如造血干细胞,骨髓干细胞表面都表达整合素β3。干细胞位于由邻近细胞、ECM、自分泌和旁分泌可溶性生长因子等组成的环境中。因此,研发一种抗衰老作用优异的相关肽,是抗衰老产品研究的重点方向。
发明内容
本发明的目的在于克服现有技术的不足之处而提供一种重组人源化纤连蛋白rhFEB及应用。
为实现上述目的,本发明采取的技术方案如下:
第一方面,本发明提供了一种重组人源化纤连蛋白rhFEB,其氨基序列如SEQ IDNO:1所示。
本发明利用基因工程手段,在人源化纤连蛋白EDB片段N端顺序插入人III型胶原促细胞增殖的功能基序,形成新的一种多肽融合蛋白,通过基因工程的方法在体外进行表达纯化,得到具抗衰性能的重组人源化纤连蛋白rhFEB,其具有高渗透性、高修复性、高安全性等特点。本发明的重组人源化纤连蛋白rhFEB具有抗衰,并且有影响细胞粘附、促进微小血管形成、修复受损皮肤细胞的功能,在护肤品和化妆品中具有广阔的应用前景。
第二方面,本发明提供了一种编码所述的重组人源化纤连蛋白rhFEB的基因。
作为本发明所述的基因的优选实施方式,其核苷酸序列如SEQ ID NO:2所示。
第三方面,本发明提供了一种融合多肽,包含所述的重组人源化纤连蛋白rhFEB。
作为本发明所述的融合多肽的优选实施方式,还包含在所述重组人源化纤连蛋白rhFEB的N端和/或C端的至少一个His标签。
作为本发明所述的融合多肽进一步的优选实施方式,其氨基序列如SEQ ID NO:3所示。
作为本发明所述的融合多肽进一步的优选实施方式,其核苷酸编码序列如SEQ IDNO:4所示。
第四方面,本发明提供一种重组表达载体,包含上述基因。优选的,所述表达载体为pET-20b。
第五方面,本发明提供一种重组菌,包含上述重组表达载体。优选的,所述重组菌的宿主细胞为BL21。
第六方面,本发明将所述的重组人源化纤连蛋白rhFEB、所述的基因、所述的融合多肽在制备a~d任一种产品中应用:
a.用于细胞抗光老化的化妆品或护肤品;
b.促细胞修复的化妆品或护肤品;
c.促细胞修复的细胞培养基质;
d.抗皮肤衰老和修复的制剂;优选的,所述制剂为化妆品或护肤品。
与现有技术相比,本发明的有益效果为:
本发明通过对不同的多肽类物质进行分析、筛选、组合,利用基因工程手段,在人源化纤连蛋白EDB片段N端顺序插入人III型胶原促细胞增殖的功能基序,获得一种多肽类融合蛋白rhFEB。通过基因工程的方法在体外进行表达纯化,得到具抗衰性能的重组人源化纤连蛋白rhFEB,所述rhFEB具有较高的促细胞恢复能力,可溶性、高吸收性。本发明的重组人源化纤连蛋白rhFEB具有抗衰,并且有影响细胞粘附、促进微小血管形成、修复受损皮肤细胞的功能,可作为原料应用于化妆品、护肤品及细胞培养基质等领域中。
附图说明
图1为本发明实施例中所用的大肠杆菌重组表达质粒pET20b-rhFEB的构建图谱。
图2为本发明的纤连蛋白rhFEB的核酸DNA序列的扩增片段的琼脂糖凝胶电泳图。图中,“M”指的是琼脂糖凝胶电泳marker,“-”指的是阴性对照,“+”指的是目的片段的扩增,约1860bp。
图3为本发明的rhFEB的蛋白菌种诱导聚丙烯酰胺凝胶电泳图。图中,“M”指的是聚丙烯酰胺凝胶电泳marker,“1”指的是未诱导前的菌种的样品,“2”指的是诱导后的菌种生产蛋白的样品。
图4为本发明的rhFEB的蛋白进行蛋白免疫印迹实验的结果。图中,“M”指的是聚丙烯酰胺凝胶电泳marker,“1”指的是目的蛋白的样品。
图5及图6为本发明的rhFEB对Hacat细胞粘附检测的结果。本发明的rhFEB对细胞促粘附能力有明显作用,且与EGF的促粘附作用功效相似。
图7及图8为本发明的rhFEB的细胞划痕修复活性检测结果。图7显示细胞划痕实验给药24h和48h后。结果显示,本发明的rhFEB的细胞划痕修复活性对于对照组有明显修复效果,且与EGF功效相近。
图9及图10为本发明的rhFEB对于HSF细胞紧致、抗皱功效实验结果。结果显示,本发明的rhFEB对细胞具有紧致、抗皱功效的作用。
图11为本发明的rhFEB对弹性蛋白酶的抑制效果。
图12为本发明的rhFEB对巨噬细胞表型向M1转换的qpcr结果。
图13为本发明的rhFEB诱导ECV304细胞管腔形成。
图14为db/db小鼠创面给药后不同时间的创面恢复情况。
图15为db/db小鼠创面愈合率。
图16为db/db小鼠给药29d后创面组织HE染色。结果显示,给药组rhFEB毛囊形成情况最好,表皮厚度没有显著性差异。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。本领域技术人员应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。除非另外指出,所用术语具有本领域技术人员理解的一般技术含义。
实施例中所用的试验方法如无特殊说明,均为常规方法;所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:重组人源化纤连蛋白rhFEB的表达和纯化
(1)pET20b-rhFEB重组质粒构建与鉴定
本发明所述的重组人源化纤连蛋白rhFEB,其氨基酸序列如SEQ ID NO:1所示,其编码核苷酸序列如SEQ ID NO:2所示。
本发明采用人工方法构建pET20b-rhFEB重组质粒。在编码核苷酸序列的5’端和3’端分别引入Nde I和Xho I酶切位点(参见SEQ ID NO:4)。为方便后续纯化,在5’端和3’端分别加上6个组氨酸标签(参见SEQ ID NO:3),通过Nde I/Xho I双酶切将其连入pET20b载体(购自金唯智公司),得到pET20b-rhFEB重组质粒(如图1所示)。
(2)rhFEB蛋白的表达
将基因表达载体pET20b-rhFEB进行转化大肠杆菌BL21,在LB固体培养基中(含100μg/mL的氨苄青霉素)挑选阳性克隆,并将阳性克隆接种于LB液体培养基中(含100μg/mL的氨苄青霉素),37度培养3~5小时至OD600为0.5,加入IPTG至终浓度为1mM,37摄氏度诱导培养4小时,离心收集菌体。
(3)rhFEB蛋白的纯化
将所收集菌体加入pH=7.4的PBS缓冲液,采用均质机600bar进行破碎,然后高速离心20分钟,将收集得到的上清经镍柱(Ni Sepharose 6Fast Flow,购自GE公司)纯化,上样流速0.7mL/min。再用pH=7.4的PBS缓冲液平衡,采用咪唑梯度洗脱,在300mM时收集流出峰,洗脱样品经G25脱盐,即得到高纯度的蛋白。通过SDS-PAGE蛋白胶以及WesternBlotting对重组蛋白的分子量大小和免疫学验证(如图2、图3和图4所示),得纯化的rhFEB蛋白。
实施例2:纤连蛋白rhFEB的Hacat细胞粘附实验。
于96孔板中加入每孔50μl不同蛋白浓度实施例1的纤连蛋白rhFEB溶液、125μmol/L EGF(EGF组)和空白水溶液对照(Ctrl组),无菌条件下吹干。调整Hacat细胞(人类永生化角质细胞,购自ATCC)浓度,按照每孔1.2×104个铺板,37℃孵育,PBS清洗,培养数小时后每孔加入MTT 10μL,37℃、5%CO2培养箱中培养4h,吸去培养液,加入终止液DMSO,在酶标仪下比色(以630nm为参比波长,在波长570nm处测定吸光度,记录测定结果)。
纤连蛋白rhFEB促细胞粘附实验如图5和图6所示:在8nM~2000nM的浓度之间,rhFEB对Hacat细胞具有促进粘附作用。在125μmol/L的浓度下,rhFEB对Hacat细胞的促进粘附作用最佳。Ctrl组细胞数量少,且细胞呈圆形,未铺展开;EGF组和rhFEB组相比于Ctrl组,细胞黏附数量显著增多,且细胞呈铺展状态。
实施例3:纤连蛋白rhFEB的Hacat细胞划痕实验。
培养板接种细胞之前先用marker笔在12孔板背面画横线标记(方便拍照时定位同一个视野)。细胞消化后接入12孔板,数量以贴壁后铺满板底为宜(数量少时可培养一段时间至铺满板底,确保细胞达到100%融合的密度)。细胞长满板底后,用100μL枪头垂直于孔板,沿板背面划线的相同位置制造细胞划痕,尽量保证各个划痕宽度一致。吸去细胞培养液,用PBS冲洗孔板三次,洗去划痕产生的细胞碎片。加入含实施例1的rhFEB蛋白(浓度125nM)的含1%血清的培养基,以及相同摩尔浓度的EGF、FN(医药中心重组表达,rhFEB蛋白中FN片段)、CC(一种胶原样蛋白,由COLA1设计,ID:79BC036531.2,2120-2164nt,由GL生物化学人工合成)做为对照组,拍照记录。依据做细胞划痕实验结果所设置的浓度,将培养板放入培养箱培养24h、48h取出拍照。根据收集图片,利用Image J软件(NationalInstitutes of Health)分析得到划痕区域面积,整理数据得到实验结果。
伤口愈合百分比(Wound Healing Percentage)的计算:根据最开始的划痕面积,跟某一时间愈合的那一部分面积(最开始面积-某时间点的面积)与最初面积的比值即可得到伤口愈合的百分比。即:
划痕试验结果由图7和图8所示:给药48h后,与空白组相比,给予rhFEB蛋白的组别均有促进划痕愈合的作用,化学合成胶原片段CC没有明显促进作用,FN蛋白起到了促进划痕愈合的作用,但效果比rhFEB蛋白差,rhFEB融合蛋白起到了协同做用,rhFEB组的愈合率比EGF组的愈合率相近。本发明的rhFEB对皮肤创面具有高效的修复能力。
实施例4:纤连蛋白rhFEB的紧致、抗皱功效测试实验
按照上海日用化学品行业协会发布团体标准T/SHrh 031-2020进行实验,通过WB检测HSF细胞给药后I型胶原的表达差异。TGFβ1购自ABclonal,货号:RP02514;I型胶原抗体购自CST,货号:72026s。
结果如图9和图10所示:浓度为2nM和20nM的rhFEB促进了I型胶原的分泌,本发明的rhFEB具有紧致、抗皱功效。
实施例5:弹性蛋白酶抑制试验
配制含有400mmol/L NaCl和10mmol/L CaCl2的Tricine缓冲液(50mmol/L、pH8.5),利用Tricine缓冲液配制20mU/mL的弹性蛋白酶溶液和200μmol/L的AAAPAN(N-琥珀酰-丙氨酸-丙氨酸-丙氨酸-p-硝基苯胺(N-succinyl-Ala-Ala-Ala-p-nitroanilide,上海源叶生物科技有限公司)溶液。将10μL不同质量浓度的rhFEB溶液与90μL 20mU/mL的弹性蛋白酶溶液,混匀后于25℃恒温孵育20min,再加入100μL 2mmol/L的AAAPAN溶液,30min后在405nm处测定吸光度值,以EGCG(表没食子儿茶素没食子酸酯,上海源叶生物科技有限公司)作为阳性对照,平行测定3次。保证混合体系中rhFEB的质量浓度梯度均为2.26、22.6μg/mL。
结果如图11所示:弹性蛋白作为皮肤组成中重要的结构蛋白,在皮肤老化过程中由于弹性蛋白酶等基质金属蛋白酶含量的升高而被降解,导致皮肤失去弹性。rhFEB对弹性蛋白酶有较好的抑制作用,可增强肌肤抗拉伸的能力,保持弹性和紧致。
实施例6:促巨噬细胞表型转换实验
选择生长状态良好、细胞形态正常的RAW264.7细胞铺板,按照每孔5×105个细胞接种于六孔板,贴壁过夜后分别添加诱导试剂给药24h。Normal:含10%FBS无双抗的H-DMEM完全培养基,M1阳性组:完全培养基+100ng/ml LPS+20ng/ml IFN-γ;M2阳性组:完全培养基+20ng/ml IL-4;Normal+rhFEB:Normal+10μg/ml rhFEB;M1+rhFEB:M1培养基+10μg/mlrhFEB;M2+rhFEB:M2培养基+10μg/ml rhFEB。诱导结束后提取RNA,进行iNOS(M1型标记物)基因水平的检测。
结果如图12所示:Q-PCR结果显示,rhFEB促进了巨噬细胞转换为M1型,表明rhFEB能促进巨噬细胞释放促炎细胞因子,激活其他免疫细胞,启动炎症反应,是机体对外来损伤(如UV光皮肤刺激损伤)的一种防御方式。
实施例7:促管腔形成实验
将Matrigel胶、200μL枪盒、96孔板置于4℃冰箱中过夜预冷。将胶冻状的Matrigel胶分别与rhFN溶液和EGF蛋白溶液按50μL/孔体积比混匀(1:4)铺入预冷的96孔板的孔中,同时设置Control组为无血清RMPI.1640培养基与Matrigel胶按1:4体积比混匀,4℃冰箱静置2h,使其成为一小丘的形状且观察到无气泡后,置于37℃孵箱中16h使其固化。
将ECV304细胞消化计数,稀释至细胞密度为15万个/mL的细胞悬液,滴加至Matrigel胶的表面置于37℃孵箱继续培养,每孔100μL,每组两个复孔。置于37℃孵箱中12h,取出,荧光显微镜拍照。
结果如图13所示:Ctrl组中无成形管环,rhFEB促进了ECV304血管环形成,效果与EGF一致。本发明的rhFEB可促进微小血管形成。
实施例8:db/db小鼠创面愈合观察
糖尿病db/db小鼠,10只雌性,体重40±2g。实验当天,3%戊巴比妥钠腹腔注射麻醉,背部脱毛膏脱毛,脱毛面积约3cm×3cm。动物在麻醉状态下取俯卧位,背部皮肤消毒,于肩胛骨以下及脊柱中线左、右侧旁开约2mm处,用直径为10mm角膜环钻制造深达筋膜的全程皮肤缺损创面,创面面积为0.8cm2,并拍照记录。造模完成后在创面部位涂布对应样品(rhFEB和aFGF的浓度为200μg/g),0.1g/只。第28d给药24h后,对动物进行相应的评分及拍照记录后,安死术处死小鼠,距创缘3~4mm正常皮肤剪开,深达肌层完整切取创面组织,取材切片做HE染色。
结果如图14,图15,图16所示:rhFEB组比aFGF组愈合性能强,而切片HE染色中,给药组rhFEB毛囊形成情况最好,各组之间表皮厚度没有显著性差异;
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (7)
1.一种重组人源化纤连蛋白rhFEB,其特征在于,其氨基酸序列如SEQ ID NO:1所示。
2.一种编码权利要求1所述的重组人源化纤连蛋白rhFEB的核酸,其特征在于,其核苷酸序列如SEQ ID NO:2所示。
3.一种融合多肽,其特征在于,其氨基酸序列如SEQ ID NO:3所示。
4.一种编码权利要求3所述的融合多肽的核酸,其特征在于,其核苷酸序列如SEQ IDNO:4所示。
5.一种重组表达载体,其特征在于,包含权利要求2所述核酸。
6.一种重组菌,其特征在于,包含权利要求5所述重组表达载体。
7.权利要求1所述的重组人源化纤连蛋白rhFEB、权利要求2所述的核酸、权利要求3所述的融合多肽在制备a~d任一种产品中应用:
a.用于细胞抗光老化的化妆品或护肤品;
b.促细胞修复的化妆品或护肤品;
c.促细胞修复的细胞培养基质;
d.抗皮肤衰老和修复的制剂。
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