CN112876569A - 一种rhTSG6-FNⅢ1-C融合蛋白及其在皮肤护理组合物中的用途和其制备方法 - Google Patents
一种rhTSG6-FNⅢ1-C融合蛋白及其在皮肤护理组合物中的用途和其制备方法 Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及化妆品技术领域,并公开了一种rhTSG6‑FNⅢ1‑C融合蛋白及其在皮肤护理组合物中的用途和其制备方法。所述的rhTSG6‑FNⅢ1‑C融合蛋白通过基因工程原核表达技术制备,具体方法包括:SEQ ID NO:1的基因序列进行密码子优化;构建重组表达载体并进行转化;重组大肠埃希菌进行诱导与表达;表达产物经分离纯化,从而制备rhTSG6‑FNⅢ1‑C融合蛋白。本发明的皮肤护理组合物具有多种剂型,如水溶液、软膏、栓剂、乳膏和面膜,并具有良好的改善皮肤皱纹和保持皮肤弹性,减少皮肤发炎与过敏风险等皮肤护理功效,因此,可应用于功能性化妆品或医学美容领域。
Description
技术领域
本发明涉及一种具有增强皮肤细胞增殖与皮肤组织修复作用的重组人肿瘤坏死因子α诱导蛋白-6-纤连蛋白Ⅲ1-C(recombination human tumor necrosis factorαstimulated gene-6-fibronectinⅢ1-C,rhTSG6-FNⅢ1-C)融合蛋白,以及一种用于改善皮肤皱纹和保持皮肤弹性,减少皮肤发炎与过敏风险的皮肤护理组合物。其包含相同的有效成分。
背景技术
人皮肤由表皮层、真皮层、皮下组织及皮肤附属器官组成,具有保护、调节体温、新陈代谢和感觉等功能。其中表皮层和真皮层的状态与皮肤状态紧密相关,由细胞和胞外基质组成的细胞环境稳态与皮肤状态相关。
纤连蛋白(fibronectin,FN)是一种高分子量(约440kDa)的细胞外基质的二聚体糖蛋白,它与整合素跨膜受体蛋白结合,由两个几乎相同的单体通过一对二硫键连接而成,其基因位于人类2号染色体上,由Ⅰ型、Ⅱ型、Ⅲ型3种同源性序列组成,在结构上该蛋白分为3个区域,即N端的明胶结合区、中间部分的细胞结合区和C端的Ⅱ型肝素结合区。Ⅰ型和Ⅱ型都含有两个二硫键,而Ⅲ型则没有二硫键。RGD序列在细胞的黏附及FN与其他蛋白的相互作用中具有重要作用,该序列位于细胞结合区的第10个Ⅲ型重复中,FN可通过RGD与受体相连,从而发挥调节作用。与整合素相似的是,纤连蛋白结合了细胞外基质成分,如胶原蛋白、纤维蛋白和肝素硫酸盐蛋白聚糖(如多配体聚糖)。FN广泛参与细胞迁移、黏附、增殖、止血及组织修复等过程,调动单核吞噬细胞系统清除损伤组织处有害物质,具有生长因子的作用。FN作为细胞培养的基质,可提高多种细胞的贴壁率、汇合率,缩短细胞汇合时间,使细胞形态结构良好,代谢率增强,DNA、RNA及蛋白质合成速度显著提高;细胞的集落率升高,原代培养成活率提高。FN作为一种胞外基质蛋白能够促进细胞增殖、粘附及其他胞外基质蛋白如胶原蛋白的形成,参与细胞迁移、黏附、增殖、止血及组织修复等过程,能促进单核吞噬细胞系统清除损伤组织处有害物质,具有生长因子的作用。现已知,纤连蛋白除已被用于创伤修复等临床应用外,还是一个重要的美容因子。
肿瘤坏死因子α刺激基因6(tumor necrosis factor-αstimulated gene 6,TSG-6)是一种炎症相关的分泌性蛋白,是由间充质干细胞或基质细胞(MSCs)对炎症信号的反应产生的,在多种炎症性疾病或类炎症性病理过程中呈高表达。现已知,TSG-6介导了许多免疫调节和组织修复过程,具有抗炎和组织保护作用,是一个正调节的功能蛋白。大量研究表明,TSG-6具有抑制炎症因子TNF-α、IL-1β和IL-6等的表达,抑制中性粒细胞浸润等功能,发挥较强的抗炎作用。随着相关研究进展,TSG-6的抑炎功能逐渐受到生物医药领域的日益重视。
目前,市场上具有改善皮肤皱纹和保持皮肤弹性,减少皮肤发炎与过敏风险等功效的产品种类繁多,其有效作用成分包括抗生素、杀菌剂以及中药等。抗生素会对皮肤免疫系统产生影响,导致皮肤脆弱敏感;杀菌剂会破坏皮肤的微生态系统;中药类产品的效果相对温和,但是见效慢,治疗周期长。
在抗皱祛痘等一类功效性化妆品市场上,以具有相应功效的蛋白质作为有效成分的产品越来越多,但还未见有rhTSG6-FNⅢ1-C融合蛋白作为有效成分的产品,也未见有相关专利记载。
发明内容
本发明是针对上述情况而设计的,根据rhTSG6-FNⅢ1-C融合蛋白,本发明的发明人生产了一种rhTSG6-FNⅢ1-C融合蛋白,具有增强皮肤细胞增殖与皮肤组织修复作用,还能减少皮肤发炎或过敏的风险。因此,通过生产含有rhTSG6-FNⅢ1-C融合蛋白作为有效成分的各种皮肤护理组合物配方并进行皮肤试验,通过试验对象证实了改善皮肤皱纹、保持皮肤弹性的效果。本发明也随之完成。
为解决上述问题,本发明提供了一种热稳定的rhTSG6-FNⅢ1-C融合蛋白,具有增强皮肤细胞增殖与皮肤组织修复作用。具体方案如下:
所述融合蛋白为具有SEQ ID NO:2的氨基酸序列编码的蛋白,或者该蛋白的功能当量。
本发明还保护一种编码上述rhTSG6-FNⅢ1-C融合蛋白的基因,所述基因包括SEQID NO:1的核苷酸序列,或者该核苷酸序列的同源物。
本发明还保护一种包含上述基因的重组载体。
优选地,所述载体为细菌质粒、噬菌体、酵母质粒、植物细胞病毒或哺乳动物细胞病毒。
本发明还保护一种用上述的重组载体转化的宿主细胞。
优选地,所述宿主细胞为大肠埃希菌Rosetta、大肠埃希菌JM109、大肠埃希菌BL21、大肠埃希菌RR1、大肠埃希菌LE392、大肠埃希菌B、大肠埃希菌X 1776、大肠埃希菌W3110、枯草芽胞杆菌、苏云金杆菌的杆菌属菌株、鼠伤寒沙门菌、粘质沙雷菌或多种假单胞菌种的肠杆菌科菌株。
本发明还保护一种上述rhTSG6-FNⅢ1-C融合蛋白在皮肤护理组合物中的用途。
本发明还保护一种上述融合蛋白的制备方法,包括以下步骤:
(1)优化目的基因序列中稀有密码子,构建重组表达载体和rhTSG6-FNⅢ1-C融合蛋白重组工程菌;优化后的目的基因序列如SEQ ID NO:1所示;
(2)重组工程菌经发酵罐发酵、菌体破碎,分离rhTSG6-FNⅢ1-C融合蛋白包涵体;
(3)rhTSG6-FNⅢ1-C融合蛋白包涵体经洗涤、变性、复性和亲和层析纯化,制备rhTSG6-FNⅢ1-C融合蛋白原液。
优选的,步骤(1)的具体方法是根据大肠埃希菌对密码子的偏好性使用同义密码子替换目的基因中稀有密码子(AGG、AGA、AUA、CUA、CGA、CGG、CCC、UCG),3’端添加稀有密码子。
优选的,步骤(1)所述优化后的目的基因由下表所示的引物通过重叠延伸PCR技术扩增得到:
进一步优选的,使用pET-32a作为表达载体,使用E.coli BL21(DE3)作为宿主菌。
优选的,步骤(2)的具体方法是先培养种子,然后接种至含培养基的发酵罐中进行培养,再加入1.0mmol/L IPTG诱导表达,最后完成发酵并收菌。
进一步优选的,所述的培养基为LB培养基,使用氨水调节pH至7.0,并加入1ml消泡剂减小发酵过程中泡沫产生的影响。
进一步优选的,待发酵液OD600在4.0以上时加入IPTG诱导表达,发酵6小时后停止发酵,收集发酵液。
优选的,步骤(3)的包涵体变复性具体方法是使用洗涤缓冲液(50mmol/L Tris,100mmol/L NaCl,2mol/L尿素,1mmol/L EDTA,0.5%TritonX-100,pH为8.0)洗涤两次,再使用溶解缓冲液(50mmol/L Tris,100mmol/L NaCl,7mol/L盐酸胍,0.1%β-巯基乙醇,pH为8.0)过夜溶解,最后使用复性缓冲液(50mmol/L Tris,100mmol/L NaCl,1mmol/L GSH,0.2mmol/L GSSG,pH8.0)进行稀释复性,透析换液,过滤除菌制备rhTSG6-FNIII1-C融合蛋白粗纯品。
优选的,步骤(3)的His-tag亲和层析具体方法是使用结合缓冲液(50mmol/LTris,500mmol/L NaCl,50mM咪唑,pH 8.0)过柱平衡,平衡后rhTSG6-FNⅢ1-C融合蛋白粗纯品上样纯化,再使用洗涤缓冲液(50mmol/LTris,500mmol/L NaCl,150mM咪唑,pH 8.0)过柱洗涤去除杂蛋白,最后使用洗脱缓冲液(50mmol/LTris,500mmol/L NaCl,150mM咪唑,pH 8.0)洗脱收集rhTSG6-FNⅢ1-C融合蛋白纯化产物。本发明还保护一种上述皮肤护理组合物的制备方法,包括以下步骤:
(1)优化目的基因序列中稀有密码子,构建重组表达载体和rhTSG6-FNⅢ1-C融合蛋白重组工程菌;优化后的目的基因序列如SEQ ID NO:1所示;
(2)重组工程菌经发酵罐发酵、菌体破碎,分离rhTSG6-FNⅢ1-C融合蛋白包涵体;
(3)rhTSG6-FNⅢ1-C融合蛋白包涵体经洗涤、变性、复性和亲和层析纯化,制备rhTSG6-FNⅢ1-C融合蛋白原液;
(4)配制辅助剂,并与rhTSG6-FNⅢ1-C融合蛋白原液进行混合,经除菌处理,配制所述的皮肤护理组合物。
优选的,步骤(4)的辅助剂为保水剂、防腐剂、增白剂、增稠剂和乳化剂中一种或两种以上的组合。
优选的,所述的皮肤护理组合物具有选自化妆水、柔肤水、爽肤水、收敛水、乳液、牛奶乳液、保湿乳液、营养乳液、按摩霜、营养霜、眼霜、保湿霜、护手霜、精华液、营养精华液、面膜、洁面泡沫、洁面水、洁面乳、洁面霜、身体乳液、身体洁肤露、香皂及粉中的任意一种剂型。
本发明的技术方案具有以下有益效果:
1、本发明的皮肤护理组合物主要有效成分是rhTSG6-FNⅢ1-C融合蛋白,成分简单不含防腐剂,不易造成皮肤过敏,安全性高。
2、本发明的皮肤护理组合物温和清爽不黏腻、肤感较好。
3、本发明制备方法简便,具有生产成本低、产量高等优势,适用于大规模的工业化生产。
附图说明
图1为pET-32a-rhTSG-6-FNⅢ1-C重组质粒PCR鉴定结果,其中M泳道为DL2000marker;泳道1为阴性对照;泳道2-3为pET-32a-rhTSG-6-FNⅢ1-C重组质粒PCR产物。
图2为pET-32a-rhTSG-6-FNⅢ1-C重组质粒双酶切鉴定结果,其中M泳道为DL2000marker;泳道1为pET-32a-rhTSG-6-FNⅢ1-C重组质粒经BamH I和HindⅢ双酶切产物,泳道2、3分别为pET-32a-rhTSG-6-FNⅢ1-C重组质粒经BamH I和HindⅢ单酶切结果,泳道4为pET-32a-rhTSG-6-FNⅢ1-C重组质粒阴性对照。
图3为rhTSG6-FNⅢ1-C融合蛋白纯化产物SDS-PAGE电泳纯度检测结果,其中M为蛋白marker,泳道1为rhTSG6-FNⅢ1-C融合蛋白纯化前样品,泳道2为去除的杂蛋白,泳道3为rhTSG-6-FNⅢ1-C融合蛋白纯化后样品。
图4为rhTSG6-FNⅢ1-C融合蛋白纯化产物Western blot鉴定结果,其中M为蛋白marker;泳道1为阴性对照;泳道2为rhTSG6-FNⅢ1-C融合蛋白纯化产物。
图5为MDBK/VSV系统检测rhTSG6-FNⅢ1-C融合蛋白抗病毒活性的CPE结晶紫染色后的观察结果,其中第一行和第二行为rhTSG-6-FNⅢ1-C融合蛋白样品,第三行和第四行为重组人干扰素α国家标准品。
图6为普通光镜下观察的rhTSG6-FNⅢ1-C融合蛋白促细胞结果。图A为细胞对照组;图B为rhTSG6-FNⅢ1-C融合蛋白实验组。
具体实施方式
为实现本发明的目的,本发明提供了一种rhTSG6-FNⅢ1-C融合蛋白,该蛋白由SEQID NO:2的氨基酸序列组成,具有增强皮肤细胞增殖与皮肤组织的修复作用,并且还具有抗炎症的作用。
根据本发明的rhTSG-6-FNⅢ1-C融合蛋白的范围包括具有以SEQ ID NO:2为代表的氨基酸序列的蛋白,以及该蛋白的功能当量。“功能当量”这个词表示一种蛋白质,由于添加、替换,或删除一个氨基酸,至少70%,最好是至少80%,更好是至少90%,甚至最好是至少95%和氨基酸序列SEQ ID NO:2具有同源性,它表明一种蛋白质表现出的生物学活性与SEQ ID NO:2实际上是相同的。“实质上相同的生物学活性”是指改善皮肤皱纹和保持皮肤弹性的活性。此外,包括在rhTSG-6-FNⅢ1-C融合蛋白区域肽段,衍生物,及其类似物。这里使用的术语“肽段”、“衍生物”和“类似物”表示一种多肽,它与本发明的rhTSG-6-FNⅢ1-C融合蛋白具有实质上相同的生理功能或活性。
本发明的rhTSG6-FNⅢ1-C融合蛋白优选由SEQ ID NO:2的349个氨基酸组成,是由1.sup.st到257.sup.th氨基酸组成的rhTSG-6和由273.sup.rd到349.sup.th氨基酸序列组成rhFNⅢ1-C融合产生的一种新型蛋白。
本发明还提供了一种编码具有增强皮肤细胞增殖与皮肤组织的修复作用,并且还具有抗炎症的作用的rhTSG6-FNⅢ1-C融合蛋白的基因。该基因可能由大肠杆菌编码优化的SEQ ID NO:1的核苷酸序列组成,但不限于此。
本发明编码具有增强皮肤细胞增殖与皮肤组织的修复作用,并且还具有抗炎症的作用的rhTSG6-FNⅢ1-C融合蛋白的基因,可能包括SEQ ID NO:1的核苷酸序列。此外,核苷酸序列的同源物也属于本发明的范围。具体地说,上述基因可包括与SEQ ID:1的核苷酸序列具有至少70%、优选至少80%、更优选至少90%和甚至最好是至少95%同源性的核苷酸序列。通过将一个比较区域与两个最佳对齐的序列进行比较,可以确定特定多核苷酸的“序列同源性%”。在这方面,比较区多核苷酸的一部分(包括添加或删除),即(a gap)相对于参考序列(没有任何添加或删除)的两个序列的优化对齐。
“密码子优化”指编码蛋白质的多核苷酸密码子的修改,该密码子在特定生物体中首先使用,以便编码的蛋白质能在其中更有效地表达。因为大多数氨基酸是由几个密码子描述的,这些密码子被称为“同义词”或“同义密码子”,所以遗传密码具有简并性。然而,特定生物体对密码子的使用并不是随机的,它更偏向于特定的密码子三胞胎。这种密码子使用偏差可能与特定基因、具有共同功能或祖先起源的基因、高水平表达的蛋白与低拷贝数的蛋白或生物体基因组的一组蛋白编码区域有关。本发明的SEQ ID NO:1核苷酸序列是对大肠杆菌密码子进行优化的序列,使编码rhTSG6-FNⅢ1-C融合蛋白的基因在大肠杆菌中表达良好。
本发明还提供了包含上述基因的重组载体,以及用该整合载体转化的宿主细胞。
“重组”一词指细胞复制异种核苷酸或表达所述核苷酸、肽、异种肽或由异种核苷酸编码的蛋白的细胞。重组细胞可以将所述细胞在天然形态中没有被发现的基因或基因片段表达为正义或反义形态中的一种。并且,重组细胞可以表达在天然状态的细胞中发现的基因,但是所述基因是经过变形的,是通过人为的手段重新导入于细胞内的。根据本发明,编码rhTSG6-FNⅢ1-C融合蛋白的基因可以插入到重组表达载体中。
本发明中,所述的编码rhTSG6-FNⅢ1-C融合蛋白的基因可以插入到重组表达载体内,术语“重组表达载体”是指细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒、或其他载体。大体地,任意的质粒及载体只要能够在宿主内复制及稳定化即可以使用。所述表达载体的重要的特性为具有复制起点、启动子、标记基因及转译调控因子(translation control element)。
包含编码rhTSG6-FNⅢ1-C融合蛋白基因序列的表达载体以及适当的调控转录/翻译的信号可以通过本领域技术人员公知的方法构建。该方法包括体外重组DNA技术、DNA合成技术和体内重组技术。为了诱导mRNA的合成,DNA序列可以有效地与表达载体中存在的合适的启动子连接。此外,表达载体可以包括核糖体结合位点作为翻译起始位点和转录终止位点。
本发明能够使载体既稳定又连续地克隆及表达于原核细胞的宿主细胞,可以利用本领域公知的任何宿主细胞,例如,大肠埃希菌Rosetta(E.coli Rosetta)、大肠埃希菌JM109(E.coli JM109)、大肠埃希菌BL21(E.coli BL21)、大肠埃希菌RR1(E.coli RR1)、大肠埃希菌LE392(E.coli LE392)、大肠埃希菌B(E.coli B)、大肠埃希菌X 1776(E.coli X1776)、大肠埃希菌W3110(E.coli W3110)、枯草芽胞杆菌、苏云金杆菌的杆菌属菌株,以及鼠伤寒沙门菌、粘质沙雷菌及多种假单胞菌种的肠杆菌科菌株等。
此外,将本发明的载体转化至真核细胞中的情况下,作为宿主细胞可以利用酵母(酿酒酵母(Saccharomyce cerevisiae))、昆虫细胞、人类细胞(例如,CHO细胞株(中国仓鼠卵巢(Chinese hamster ovary))、WI-38、BHK-21、C0S-7、293、HepG2、3T3、RIN及MDCK、MDBK细胞株)及植物细胞等。
本发明的一个具体实施例由重组载体转化的宿主细胞可以为大肠埃希菌E.coliRosetta2(DE3)pLysS,但并不限定于此。
就本发明的将载体运至宿主细胞中的方法而言,当宿主细胞为原核细胞时,可以通过CaCl2方法、Hanahan方法(Hanahan,D.,1983J.Mo1.Biol.166,557-580)及电穿孔方法等来实施。此外,当宿主细胞为真核细胞时,可以通过显微注射法、磷酸钙沉淀法、电穿孔法、脂质体介导转染法、DEAE-葡聚糖处理法及基因枪等将载体注入于宿主细胞内。
此外,本发明提供在宿主细胞中生rhTSG6-FNⅢ1-C融合蛋白的基因的方法,其包括通过使用所述重组载体转化宿主细胞来过表达编码rhTSG6-FNⅢ1-C融合蛋白的基因的步骤。
此外,本发明提供用于改善皮肤皱纹及维持皮肤弹性的皮肤护理组合物,其包含由SEQ ID NO:2的氨基酸序列组成的rhTSG6-FNⅢ1-C融合蛋白作为有效成分。
本发明的一个具体实施例的化妆料组合物中,相对于化妆料组合物的总重量,所述rhTSG6-FNⅢ1-C融合蛋白的含量可以为0.000001~0.02重量%,但并不限定于此。
本发明的皮肤护理组合物除了包含所述有效成分以外,还包含化妆品组合物中通常利用的成分,例如,包含诸如脂肪物质、有机溶剂、增溶剂、浓缩剂及凝胶化剂、软化剂、抗氧化剂、悬浮剂、稳定剂、发泡剂(foaming agent)、芳香剂、表面活性剂、水、离子型或非离子型乳化剂、填充剂、金属离子屏蔽剂及螯合剂、保存剂、维生素、阻断剂、湿润剂、精油、染料、颜料、亲水性或亲油性活性剂、脂质囊泡的通常的助剂以及载体。
本发明的组合物可以制备成本领域通常制备的任何剂型,例如,可以剂型化为溶液、悬浮液、乳浊液、膏、凝胶、霜、乳液、粉、油、粉状粉底、乳浊液粉底、蜡状粉底及喷雾等,但并不限定于此。更详细而言,可以制备成化妆水、柔肤水、爽肤水、收敛水、乳液、牛奶乳液、保湿乳液、营养乳液、按摩霜、营养霜、眼霜、保湿霜、护手霜、精华液、营养精华液、面膜、洁面泡沫、洁面水、洁面乳、洁面霜、身体乳液、身体洁肤露、香皂及粉的化妆品剂型。
本发明的皮肤护理组合物的剂型为膏、霜或凝胶的情况下,可以利用动物性油、植物性油、蜡、石蜡、淀粉、黄芪胶、纤维素衍生物、聚乙二醇、硅、膨润土、硅石、滑石或氧化锌等作为载体成分。
本发明的皮肤护理组合物的剂型为粉或喷雾的情况下,可以利用乳糖、滑石、硅石、氢氧化铝、硅酸钙或聚酰胺粉末作为载体成分,尤其剂型为喷雾的情况下,可以进一步包含诸如氯氟烃、丙烷/丁烷或二甲醚的推进剂。
本发明的皮肤护理组合物的剂型为溶液或乳浊液的情况下,利用溶剂、增溶剂或乳浊剂作为载体成分,例如有水、乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苄醇、苯甲酸苄酯、丙二醇、1,3-丁基乙二醇油、脂肪族甘油酯、聚乙二醇或失水山梨糖醇脂肪酸酯。
本发明的皮肤护理组合物的剂型为悬浮液的情况下,可以利用诸如水、乙醇或丙二醇的液态稀释剂,诸如乙氧基化异硬脂醇、聚氧乙烯山梨糖醇酯及聚氧乙烯失水山梨糖醇酯的悬浮剂,微晶纤维素、偏氢氧化铝、膨润土、琼脂或黄芪胶等作为载体成分。
以下,通过实施例对本发明进行详细说明。但是,下述实施例仅用于例示本发明,本发明的内容并不限定于下述实施例。
实施例1
用于生产rhTSG6-FNⅢ1-C融合蛋白的重组表达载体及转化重组微生物的制备
通过如下所述的方法来制作了编码rhTSG6-FNⅢ1-C融合蛋白的优化基因、重组表达载体及转化重组微生物。
将编码rhFNⅢ1-C蛋白和作为伴侣蛋白所使用的rhTSG-6的基因作为模板,制作及合成了经过优化而能够在宿主微生物内表达的、编码由349个氨基酸组成的rhTSG6-FNⅢ1-C融合蛋白的基因(SEQ ID NO:1)片段,该基因由表1所示的引物通过重叠延伸PCR技术扩增得到,经BamHⅠ和HindⅢ双酶切处理后,与同样双酶切处理的pET-32a载体连接。
表1 PCR扩增引物
从而制作了pET-32a-rhTSG-6-FNⅢ1-C重组质粒。重组质粒鉴定结果见图1,图2。
将所制备的重组质粒转化至大肠埃希菌E.coli BL21(DE3)中,从而从宿主微生物获得了大量的基因构建体。
之后,将所制备的重组质粒转化至大肠埃希菌中,从而制作了用于生产rhTSG6-FNⅢ1-C融合蛋白的重组微生物。
实施例2
rhTSG6-FNⅢ1-C融合蛋白的表达诱导、分离及纯化
发酵罐发酵:配制LB培养基2瓶,高压蒸汽灭菌后接种,37℃摇瓶培养过夜;次日,配制20L体系发酵罐培养基,溶解;安装好pH电极、溶氧电极,加入培养基,待培养基加热至37℃时,加入氨水调节pH至7.0,以及1mL的消泡剂减小发酵过程中泡沫产生对实验的影响;将“温度T”选项调至灭菌模式(121℃,20min),同时紫外线照射房间,建立一个相对密闭和无菌的环境;待罐内灭菌结束,温度降至37℃时,酒精喷壶喷洒罐顶灼烧,在火圈内将进料口打开,火圈接种倒入种子液;关闭进料口,维持溶氧量在30%以上,培养,每间隔1h记录发酵的pH、OD值、溶氧等指数;称量IPTG和无水葡萄糖溶解,而后用0.22μm滤头过滤除菌。待发酵液OD600在4.0以上,将发酵体系通气管道关闭,而在火圈内打开进料口,倒入IPTG诱导表达,而后将温度调节至30℃培养,在诱导后每间隔1h取1mL样于EP管中备用;发酵6小时后菌液的OD600开始平稳,停止发酵,收集菌液,在高速离心机中配平离心收集菌体,7000r/min离心20min,弃上清,收集菌体沉淀于平皿中,称重记录;用离心法收集菌体,在4℃条件下以7000r/min离心15分钟,弃上清,湿菌体称重,-20℃冻存,并同时在容器上标明批号、罐别、重量和日期。发酵液灭菌后弃去。
超声破碎:收集的菌体,使用200mL的PBS重悬菌体,4℃超声破碎细菌沉淀,超声的条件为:功率:400W,工作3S,间隔3S,超声6min,重复3~4次;12000r/min离心20min分离上清和沉淀,分离出的沉淀即为rhTSG-6-FNⅢ1-C包涵体。
包涵体变复性:
(1)洗涤:使用洗涤缓冲液(50mmol/L Tris,100mmol/L NaCl,2mol/L尿素,1mmol/L EDTA,0.5%TritonX-100,pH为8.0)以1∶20的湿重体积比对包涵体(10g)进行重悬,洗涤2h,12000r/min离心20min取沉淀,再重复洗涤一次;
(2)变性:称量洗涤后沉淀湿重(5.0g),再用溶解缓冲液(50mmol/L Tris,100mmol/L NaCl,7mol/L盐酸胍,1mmol/L GSH,0.2mmol/L GSSG,pH为8.0)以1∶50的湿重体积比重悬沉淀(250mL),置于磁力搅拌器上过夜,充分溶解;
(3)稀释复性:配制复性缓冲液(50mmol/L Tris,100mmol/L NaCl,1mmol/L GSH,0.2mmol/L GSSG,pH8.0)进行复性。取溶解后的蛋白溶液,12000r/min离心20min取上清,加入等体积的复性缓冲液(加250mL复性缓冲液),4℃静置3h;再加入复性缓冲液稀释至原体积的4倍(加500mL复性缓冲液),4℃静置3h;最后加入复性缓冲液稀释至原体积的5倍(加250mL复性缓冲液),4℃静置3h。透析换液制备出rhTSG-6-FNⅢ1-C融合蛋白粗制品。
纯化:rhTSG6-FNⅢ1-C融合蛋白粗制品过滤处理后过His-tag亲和层析柱,使用结合缓冲液(50mmol/LTris,500mmol/L NaCl,50mM咪唑,pH 8.0)过柱平衡,平衡后rhTSG-6-FNⅢ1-C融合蛋白粗纯品上样纯化,再使用洗涤缓冲液(50mmol/LTris,500mmol/L NaCl,150mM咪唑,pH 8.0)过柱洗涤去除杂蛋白,最后使用洗脱缓冲液(50mmol/LTris,500mmol/LNaCl,150mM咪唑,pH8.0)洗脱收集rhTSG-6-FNⅢ1-C融合蛋白纯化产物。纯化产物进行SDS-PAGE电泳检测其纯度,结果见图3。
实施例3
rhTSG6-FNⅢ1-C融合蛋白Western blot鉴定
取实施例2制备的rhTSG6-FNⅢ1-C融合蛋白纯化产物,用聚丙烯酰胺凝胶电泳(SDS-PAGE)进行分离,采用湿转法进行转膜。将完成转膜后的PVDF膜浸入封闭液,放于37℃封闭60分钟。将封闭后的PVDF膜浸入已经封闭液稀释的一抗(Abcam公司兔抗人TSG-6多克隆抗体,1︰2000稀释)溶液中,4℃过夜孵育。用TBST缓冲液在摇床上漂洗PVDF膜3次,每次5分钟。将洗涤后的PVDF浸入已经封闭液稀释的二抗(辣根过氧化物酶标记山羊抗兔IgG,1︰50000稀释)溶液中,37℃孵育1小时。再用TBST缓冲液浸洗PVDF膜3次,每次5分钟。新鲜配制ECL工作液:ECL显色液(购自Thermo公司,货号PD197918),试剂A和试剂B按1︰1混合,常规配制200μL即可。将PVDF膜蛋白质面朝上,将ECL工作液均匀滴加于目的蛋白条带大小处,进行曝光。
鉴定结果显示,被检测蛋白能与兔抗人TSG-6多克隆抗体特异性结合,具有免疫学活性,见图4。
实施例4
检测rhTSG6-FNⅢ1-C融合蛋白在MDBK中的抗VSV病毒活性
(1)将rhTSG6-FNⅢ1-C融合蛋白经稀释液依次进行2倍梯度稀释,做10个稀释梯度,稀释后rhTSG6-FNⅢ1-C融合蛋白按100μL/孔加入96孔细胞板中,并设置细胞对照组和病毒对照组,细胞对照和病毒对照只加稀释液,所述稀释液为10%FBS的DMEM培养基;细胞对照组和病毒对照组只加稀释液;
(2)采用常规技术将MDBK单层细胞制备成细胞密度4.0~6.0×105/mL的单细胞悬液,按每孔100μL加入至含rhTSG6-FNⅢ1-C融合蛋白的细胞板孔中,培养长至均匀的单层细胞;
(3)弃上清,向每孔中按照100TCID50/孔加入VSV病毒液,细胞对照孔加维持液,于37℃、5%CO2条件下培养18~24小时;
(4)在倒置显微镜下观察细胞病变,结果为图5,病变程度用以下符号表示:“-”表示无细胞病变;“+”表示25%以下的细胞有病变;“++”表示25%~50%的细胞有病变;“+++”表示50%~75%的细胞有病变;“++++”表示75%~100%的细胞有病变;结果如表2所示。
表2 Reed-Muench法计算rhTSG6-FNⅢ1-C融合蛋白的抗病毒活性
(5)根据Reed-Muench法计算rhTSG6-FNⅢ1-C融合蛋白的抗病毒活性。能保护半数细胞免受攻击病毒破坏的rhTSG6-FNⅢ1-C融合蛋白最高稀释度的倒数,即为rhTSG-6-FNⅢ1-C融合蛋白抗病毒活性。计算方法如下:
距离比例=(高于50%保护百分率-50%)/(高于50%保护百分率-低于50%保护百分率)=(84.6-50)/(84.6-41.6)=0.8
rhTSG6-FNⅢ1-C融合蛋白抑制50%的细胞病变的最高稀释度对数=高于或等于50%的细胞病变抑制率的稀释度的对数+距离比例×稀释系数的对数;
即:lg(抑制50%的细胞病变的最高稀释度)=-0.9+0.8×(-0.3)=-1.14
可抑制50%的细胞病变的最高稀释度=10-1.14=14-1
即此批次rhTSG-6-FNⅢ1-C融合蛋白稀释到1︰14时,能保护半数细胞免受“攻击病毒”侵害,在加入96板之前干扰素被稀释了100倍,所以实际效价为1400U/mL。同理可求算得重组人干扰素α国家标准品的工作单位为1100U/mL。根据重组人干扰素α国家标准品来进行修正。修正方法:
R1:R2=X1:X2
R1:重组人干扰素α国家标准品的标准工作单位
R2:在相同条件下测定的重组人干扰素α国家标准品的工作单位
X2:rhTSG6-FNⅢ1-C融合蛋白测定的工作单位
X1:修正后rhTSG6-FNⅢ1-C融合蛋白的工作单位
如上所示:R1=1000IU/mL,R2=1100IU/mL,X2=1400IU/mL;
则按上式可得出:X1=1272IU/mL。
rhTSG6-FNⅢ1-C融合蛋白修正后所得效价为1272IU/mL。
实施例5
rhTSG6-FNⅢ1-C融合蛋白抗VSV病毒活性评价
按照实施例4中的步骤,对10批次的rhTSG6-FNⅢ1-C融合蛋白在MDBK细胞中的抗VSV病毒活性进行测试。
10批次rhTSG6-FNⅢ1-C融合蛋白对VSV在宿主细胞MDBK中的半数抑制浓度IC50(ng/mL)如表3所示。
表3 10批次rhTSG6-FNⅢ1-C融合蛋白IC50(ng/mL)
如上表表明,制备多批次rhTSG6-FNIII1-C融合蛋白蛋白,其对VSV病毒的抑制作用均较稳定,活性较高。其结果可以表明rhTSG6-FNⅢ1-C融合蛋白具有体外抑制病毒致细胞病变效应(cytopathic effect,CPE)现象,提示其具有良好的抗炎症功能。
实施例6
rhTSG6-FNⅢ1-C融合蛋白的促皮肤成纤维细胞粘附试验
(1)将实施例2得到的精制rhTSG6-FNⅢ1-C和阳性对照纤连蛋白(购自上海纤连生物技术有限公司)加入到96孔细胞板上用无菌PBS按2倍梯度进行稀释;为了避免边缘效应,四周边缘孔不作实验,并设立阴性对照(阴性对照只加入100μL PBS缓冲液);4℃孵育过夜。
(2)每孔加入100μL PBS洗涤,共洗涤3次;洗涤结束后,每孔加入100μL30g/L BSA封闭,即置37℃温箱孵育1h。孵育完成后,弃去板中液体。
(3)制备生长状态良好的MDBK细胞单细胞悬液。将MDBK细胞单细胞单细胞悬液密度调整为1.0×105个/mL,每孔接种100μL,温箱孵育5h后,置显微镜下观察细胞贴壁和粘壁现象等。
(4)在加入细胞孵育3h后,显微镜下观察四批次蛋白对细胞促粘附的效果。
对MDBK细胞(图6),观察细胞对照组,大量细胞还悬浮的圆形的状态存在板孔中,仅有少量细胞贴壁并分化(图6,A);
观察实验组,存在细胞出现贴壁并分化并形成三角形和多角形(图6,B);经计算促粘附活性为64U/mL,可见本发明研制的rhTSG-6-FNⅢ1-C较对照组具有更好的促细胞贴壁和粘附的生物学活性。
上述参照实施例是对一种rhTSG6-FNⅢ1-C融合蛋白及其制备方法和应用进行的详细描述,是说明性的而不是限定性的,可按照所限定范围列举出若干个实施例,因此在不脱离本发明总体构思下的变化和修改,均应属本发明的保护范围之内。
实验例:改善皮肤肤色、达到美白的效果的官能实验
将在所述实施例2中经过分离、精制的rhTSG6-FNⅢ1-C融合蛋白作为有效成分制备了制备例1、2、3和比较例1、2、3的化妆料组合物,并实施了官能试验。
具体而言,为了确认改善皮肤肤色的情况,将30岁以上且未满60岁的女性和男性共30名(30岁年龄段10名、40岁年龄段10名、50岁年龄段10名)作为对象,将左侧面部使用比较例(对照组),将右侧面部使用制备例(试验组),一天使用一次并持续使用两周。评价是对左右面部的皮肤改善程度进行了评价。评价标准是基于下述的5分法标准实施:非常优异(5分);优异(4分);普通(3分);差(2分);非常差(1分)。
<制备例1及比较例1>
添加rhTSG6-FNⅢ1-C融合蛋白作为有效成分,并按照下述表4中记载的成分和含量制备了制备例1的化妆水。此外,没有添加rhTSG6-FNⅢ1-C融合蛋白作为有效成分,并按照下述表4中记载的成分和含量制备了比较例1的化妆水。
表4化妆水组合物
成分 | 制备例1(重量%) | 比较例1(重量%) |
rhTSG-6-FNⅢ1-C融合蛋白 | 0.01 | — |
氨基酸原液 | 0.1 | 0.1 |
矿物混合物 | 0.0007 | 0.0007 |
精制水 | 余量 | 余量 |
制备例1及比较例1的官能实验结果如下述表5所示。
表5制备例1及比较例1官能实验结果
<制备例2及比较例2>
添加rhTSG6-FNⅢ1-C融合蛋白作为有效成分,并按照下述表6中记载的成分和含量制备了制备例2的精华液。此外,没有添加rhTSG6-FNⅢ1-C融合蛋白作为有效成分,并按照下述表6中记载的成分和含量制备了比较例2的精华液。
表6精华液组合物
所示制备例2及比较例2的官能实验结果如下述表7所示。
表7制备例2及比较例2官能实验结果
<制备例3及比较例3>
添加rhTSG6-FNⅢ1-C融合蛋白作为有效成分,并按照下述表8中记载的成分和含量制备了制备例3的霜。此外,没有添加rhTSG6-FNⅢ1-C融合蛋白作为有效成分,并按照下述表8中记载的成分和含量制备了比较例3的霜。
表8霜组合物
成分 | 制备例2(重量%) | 比较例2(重量%) |
rhTSG6-FNIII1-C融合蛋白 | 0.01 | — |
氨基酸原液 | 0.1 | 0.1 |
矿物混合物 | 0.0007 | 0.0007 |
甘油 | 5 | 5 |
橄榄油乳化蜡 | 3 | 3 |
鲸蜡醇 | 2 | 2 |
精制水 | 余量 | 余量 |
所示制备例3及比较例3的官能实验结果如下述表9所示。
表9制备例3及比较例3官能实验结果
通过所述官能试验结果可以确认,包含本发明的rhTSG6-FNⅢ1-C融合蛋白作为有效成分的制备例1、2、3相比于所对应的比较例具有改善皮肤肤色、达到变白的效果。
SEQUENCE LISTING
<110> 安徽绿珏生物科技有限公司
<120> 一种rhTSG6-FNⅢ1-C融合蛋白及其在皮肤护理组合物中的用途和其制备方法
<130> 2020
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1050
<212> DNA
<213> 人工序列
<400> 1
tggggtttta aagatggcat ttttcataat agcatctggc tggaacgcgc cgccggcgtt 60
tatcatcgtg aagcccgtag tggcaaatat aaactgacct atgcagaagc caaagcagtg 120
tgcgaatttg aaggcggtca tctggcaacc tataaacagc tggaagcagc ccgtaaaatt 180
ggctttcatg tgtgcgcagc cggttggatg gccaaaggtc gcgtgggcta tccgattgtg 240
aaaccgggcc cgaattgcgg ttttggcaaa accggtatta ttgattatgg tattcgtctg 300
aatcgtagtg aacgttggga tgcctattgt tataatccgc atgcaaaaga atgcggtggc 360
gtttttaccg atccgaaaca gatttttaaa agcccgggct ttccgaatga atatgaagat 420
aatcagatct gctactggca tattcgtctg aaatatggtc agcgcattca tctgagtttt 480
ctggattttg atctggaaga tgatccgggc tgcctggcag attatgttga aatctatgat 540
agctatgacg atgttcatgg ttttgtgggt cgctattgtg gtgacgaact gccggatgat 600
attattagta ccggcaatgt gatgaccctg aaatttctga gtgatgccag cgtgaccgca 660
ggcggttttc agattaagta tgttgcaatg gaccctgtga gcaaaagcag ccagggcaaa 720
aataccagta ccaccagtac cggtaataag aattttctgg ccggtcgctt tagtcatctg 780
ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttctaacgc ccctcagccg 840
agtcatatta gtaaatatat tctgcgctgg cgcccgaaaa atagcgtggg tcgctggaaa 900
gaagccacca ttccgggtca tctgaatagc tataccatta agggtctgaa accgggtgtg 960
gtttatgaag gtcagctgat tagtattcag cagtatggtc atcaggaagt gacccgcttt 1020
gattttacca ccaccagtac cagtaccccg 1050
<210> 2
<211> 350
<212> PRT
<213> 人工序列
<400> 2
Trp Gly Phe Lys Asp Gly Ile Phe His Asn Ser Ile Trp Leu Glu Arg Ala Ala
1 5 10 15
Gly Val Tyr His Arg Glu Ala Arg Ser Gly Lys Tyr Lys Leu Thr Tyr Ala
20 25 30 35
Glu Ala Lys Ala Val Cys Glu Phe Glu Gly Gly His Leu Ala Thr Tyr Lys
40 45 50
Gln Leu Glu Ala Ala Arg Lys Ile Gly Phe His Val Cys Ala Ala Gly Trp
55 60 65
Met Ala Lys Gly Arg Val Gly Tyr Pro Ile Val Lys Pro Gly Pro Asn Cys
70 75 80 85
Gly Phe Gly Lys Thr Gly Ile Ile Asp Tyr Gly Ile Arg Leu Asn Arg Ser Glu
90 95 100
Arg Trp Asp Ala Tyr Cys Tyr Asn Pro His Ala Lys Glu Cys Gly Gly Val
105 110 115 120
Phe Thr Asp Pro Lys Gln Ile Phe Lys Ser Pro Gly Phe Pro Asn Glu Tyr
125 130 135
Glu Asp Asn Gln Ile Cys Tyr Trp His Ile Arg Leu Lys Tyr Gly Gln Arg Ile
140 145 150 155
His Leu Ser Phe Leu Asp Phe Asp Leu Glu Asp Asp Pro Gly Cys Leu Ala
160 165 170
Asp Tyr Val Glu Ile Tyr Asp Ser Tyr Asp Asp Val His Gly Phe Val Gly
175 180 185 190
Arg Tyr Cys Gly Asp Glu Leu Pro Asp Asp Ile Ile Ser Thr Gly Asn Val
195 200 205
Met Thr Leu Lys Phe Leu Ser Asp Ala Ser Val Thr Ala Gly Gly Phe Gln
210 215 220
Ile Lys Tyr Val Ala Met Asp Pro Val Ser Lys Ser Ser Gln Gly Lys Asn Thr
225 230 235 240
Ser Thr Thr Ser Thr Gly Asn Lys Asn Phe Leu Ala Gly Arg Phe Ser His
245 250 255
Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn
260 265 270 275
Ala Pro Gln Pro Ser His Ile Ser Lys Tyr Ile Leu Arg Trp Arg Pro Lys Asn
280 285 290
Ser Val Gly Arg Trp Lys Glu Ala Thr Ile Pro Gly His Leu Asn Ser Tyr Thr
295 300 305 310
Ile Lys Gly Leu Lys Pro Gly Val Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln
315 320 325 330
Gln Tyr Gly His Gln Glu Val Thr Arg Phe Asp Phe Thr Thr Thr Ser Thr
335 340 345
Ser Thr Pro
350
Claims (10)
1.一种rhTSG6-FNⅢ1-C融合蛋白,其特征在于:所述融合蛋白为具有SEQ ID NO:2的氨基酸序列编码的蛋白,或者该蛋白的功能当量。
2.一种编码权利要求1所述rhTSG6-FNⅢ1-C融合蛋白的基因,其特征在于:所述基因包括SEQ ID NO:1的核苷酸序列,或者该核苷酸序列的同源物。
3.一种包含权利要求2所述基因的重组载体。
4.一种用权利要求3所述的重组载体转化的宿主细胞。
5.一种权利要求1所述的rhTSG6-FNⅢ1-C融合蛋白在皮肤护理组合物中的用途。
6.一种权利要求1所述的rhTSG6-FNⅢ1-C融合蛋白的制备方法,其特征在于,包括以下步骤:
(1)优化目的基因序列中稀有密码子,构建重组表达载体和rhTSG6-FNⅢ1-C融合蛋白重组工程菌,优化后的目的基因序列如SEQ ID NO:1所示;
(2)重组工程菌经发酵罐发酵、菌体破碎,分离rhTSG6-FNⅢ1-C融合蛋白包涵体;
(3)rhTSG6-FNⅢ1-C融合蛋白包涵体经洗涤、变性、复性和亲和层析纯化,制备rhTSG6-FNⅢ1-C融合蛋白原液。
7.根据权利要求6所述的rhTSG6-FNⅢ1-C融合蛋白的制备方法,其特征在于:步骤(1)所述优化是根据大肠埃希菌对密码子的偏好性使用同义密码子替换目的基因中的稀有密码子,其中稀有密码子包括AGG、AGA、AUA、CUA、CGA、CGG、CCC、UCG,且3’端含有TAA终止密码子。
9.根据权利要求7所述的rhTSG6-FNⅢ1-C融合蛋白的制备方法,其特征在于:
步骤(1)所述表达载体为pET-32a,宿主菌为E.coli BL21(DE3);以rhTSG6-FNⅢ1-C融合蛋白作为有效成分,且蛋白质含量为80μg/mL;
步骤(2)所述发酵罐发酵使用IPTG作为诱导剂,浓度为1.0mmol/L,30℃诱导6小时;
步骤(3)所述亲和层析纯化使用的缓冲液为Tris-HCl缓冲液,pH8.0;所述的亲和层析纯化是使用150mmol/L咪唑进行洗涤去除杂蛋白,使用500mmol/L咪唑进行洗脱收集目的蛋白。
10.一种利用权利要求6-9任一项所述的rhTSG6-FNⅢ1-C融合蛋白的制备方法制备出的rhTSG6-FNⅢ1-C融合蛋白在制备皮肤护理组合物的方法,其特征在于,包括以下步骤:
(1)优化目的基因序列中稀有密码子,构建重组表达载体和rhTSG6-FNⅢ1-C融合蛋白重组工程菌,优化后的目的基因序列如SEQ ID NO:1所示;
(2)重组工程菌经发酵罐发酵、菌体破碎,分离rhTSG6-FNⅢ1-C融合蛋白包涵体;
(3)rhTSG6-FNⅢ1-C融合蛋白包涵体经洗涤、变性、复性和亲和层析纯化,制备rhTSG6-FNⅢ1-C融合蛋白原液;
(4)配制辅助剂,并与rhTSG6-FNⅢ1-C融合蛋白原液进行混合,经除菌处理,配制所述的皮肤护理组合物。
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