CN111423516A - 一种蛋白及其在创口修复及抑菌中的应用 - Google Patents
一种蛋白及其在创口修复及抑菌中的应用 Download PDFInfo
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Abstract
本发明涉及一种蛋白及其在创口修复及抑菌中的应用。所述蛋白的氨基酸序列如SEQ ID No.1所示。其可以在成纤维细胞增殖、组织修复、创口修复及抑菌中的至少一种的应用。
Description
技术领域
本发明涉及一种可用于创口修复及抑菌的蛋白。
背景技术
创面愈合(Wound healing)是指由于致伤因子的作用造成组织缺失后,局部组织通过再生、修复、重建等进行皮肤修补的一系列病理生理过程。创面愈合包括凝血期、炎症期以及修复期,其中,止血、防止细菌的感染以及快速填补组织缺损是促进创面愈合最为关键的问题。
纤连蛋白(fibronectin,FN)是一种广泛存在于血液、体液及各种组织中的高分子糖蛋白,由两个分子质量各为235-270千道尔顿(kDa)的亚基通过分子间二硫键连接而成;其在动物或人体内具有多种生物学功能,广泛参与细胞迁移、黏附、增殖、止血及组织修复等过程。然而,分子量大、分子间二硫键及糖基化后修饰等特点限制了人们利用基因工程技术大规模获得天然结构和活性的纤连蛋白。现时,纤连蛋白的生产主要以动物血为原料经过分级盐析与层析等方法获得。
此外,现有技术中基于对纤连蛋白改造得到的产品主要应用在细胞粘连方面,其存在组织修复效果不佳的技术问题。
发明内容
本发明之一提供了一种蛋白,其氨基酸序列如SEQ ID No.1所示。
本发明的蛋白可以通过与不同类型的标签(例如本发明中使用的组氨酸标签)在其氮端或碳端融合来实现分离纯化等目的。尽管这会导致原设计的蛋白序列发生变化,但这些标签的融合并不***到蛋白序列中的任意位置,而且在其末端接入,而且这些蛋白标签因分子量很小,因此,不会实质性改变原有的蛋白序列结构,因此,其也不会改变原蛋白自身的功能,这是本领域的技术人员公知的,并且实验结果也证明了这一点。而且在本发明实施例中作为阳性对照的商品化重组纤连蛋白III片段(Fibronectin Fragment III1-C,货号:F3542,Sigma-Aldrich)也是含有组氨酸标签。此外,由于蛋白的起始氨基酸为甲硫氨酸或甲酰甲硫氨酸,因此在必要生物合成的情况下,允许该蛋白的第一个氨基酸为甲硫氨酸或甲酰甲硫氨酸,这也不会改变原蛋白自身的功能。
因此,本发明之二提供了一种蛋白,其包括第一氨基酸序列和第二氨基酸序列,其中,所述第一氨基酸序列如SEQ ID No.1所示;所述第二氨基酸序列为标签的氨基酸序列或起始氨基酸。
在一个具体实施方式中,所述标签选自组氨酸标签(HIS)、FLAG标签、AVI标签中的至少一种。其中,HIS标签的氨基酸序列如SEQ ID No.5所示,分子量为0.84千道尔顿(kDa);FLAG标签的氨基酸序列如SEQ ID No.6所示,分子量为1.0千道尔顿;AVI标签的氨基酸序列如SEQ ID No.7所示,分子量为1.8千道尔顿。
需要指出的是,如果所述标签位于所述蛋白的氮端,那么为了顺利表达和使用所述标签,还可以在所述标签的氮端再增加数个任意的氨基酸。例如,在一个具体实施方式中,所述蛋白的氨基酸序列如SEQ ID No.3所示。
在一个具体实施方式中,所述起始氨基酸为甲硫氨酸或甲酰甲硫氨酸。此种情况,甲硫氨酸或甲酰甲硫氨酸只能位于蛋白的氮端。
本发明之三提供了一种编码如本发明之一或本发明之二中任意一项所述的蛋白的核酸。
在一个具体实施方式中,所述核酸的序列如SEQ ID No.2所示,或所述核酸的序列如SEQ ID No.4所示。
本发明之四提供了一种携带有如本发明之三所述的核酸的重组微生物。
在一个具体实施方式中,所述重组微生物的出发菌株选自埃希氏菌属(Escherichia)和酿酒酵母属(Saccharomyce)中的至少一种,
在一个具体实施方式中,所述重组微生物选自大肠杆菌(Escherichia coli)、酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)中的至少一种。
在一个具体实施方式中,所述微生物能够表达如本发明之一或本发明之二中任意一项所述的蛋白。
本发明之五提供了一种组合物,其包括如本发明之一或本发明之二中任意一项所述的蛋白和药学上可接受的载体。
在一个具体实施方式中,所述药学上可接受的载体选自水、生理盐水、生理上可匹配的缓冲液、生理上可匹配的溶液和生理上可匹配的悬浮液中的至少一种。
在一个具体实施方式中,所述蛋白在所述组合物中的质量百分含量为0.01%至20%。
本发明之六提供了本发明之一或本发明之二中任意一项所述的蛋白、本发明之三所述的核酸、本发明之四所述的重组微生物和本发明之五所述的组合物中的至少一种在成纤维细胞增殖、组织修复、创口修复及抑菌中的至少一种的应用。
在一个具体实施方式中,所述应用为在抑制色葡萄糖菌属(Staphylococcus)中的至少一种细菌中的应用。
在一个具体实施方式中,所述应用为在抑制金黄色葡萄糖菌(Staphylococcusaureus)中的应用。
本发明的有益效果:
本发明人工合成了一种DNA序列,并将其克隆到表达质粒中,利用大肠杆菌进行重组表达。然后,经离子交换层析和组氨酸亲和层析方法分离纯化,得到纯度高于95%的蛋白。体外细胞实验证明,本发明的蛋白能有效促进成纤维细胞增殖。糖尿病动物模型中,与对照组相比,该蛋白能显著促进创伤皮肤的增生。此外,该蛋白还能抑制人体致病菌--金黄色葡萄球菌的生长。因此,本发明获得的蛋白,可以在伤口表面形成覆膜、减少有害细菌的数量同时促进皮肤细胞增生,从而降低伤口感染及加快皮肤创口,尤其是难愈性皮肤创口或者大面积伤口的愈合,最终实现皮肤创口快速修复,特别是对患有糖尿病或因辐射引起皮肤病的患者出现的难愈性创伤和/或大面积皮肤创口问题,同时降低患者出现皮肤感染、溃疡以及因此而引起导致的其它严重疾病(如组织坏死等)的几率,从而有效提供病人的生存和生活质量。
本发明的蛋白在不含有标签时的分子量仅为17.0千道尔顿(kDa)(SEQ ID No.1),含有组氨酸标签时的分子量仅为18.25千道尔顿(SEQ ID No.3),相比于天然的纤连蛋白(二聚体,约440千道尔顿)或基于天然纤连蛋白改造的蛋白(70.1千道尔顿,货号:3938-FN-050,R&D公司,美国)具有分子量小、无分子内/间二硫键等方面的优点。其还可以与用于纯化蛋白的标签融合,从而有利于分离纯化。此外,其有利于利用大肠杆菌、酵母等基因工程宿主进行大规模生产,因而生产成本更低,更容易满足临床和日常应用的需要。
附图说明
图1.蛋白FN-P的凝胶电泳检测以及蛋白杂交分析。
A图为蛋白FN-P的凝胶电泳检测。重组大肠杆菌菌株在LB培养基中生长到对数生长期,然后利用终浓度为1毫摩尔的异丙基-β-D-硫代半乳糖苷在37℃下进行诱导表达4小时。收集诱导结束后的菌体,加入裂解缓冲液(250mM Tris-HCl,pH6.8;10%(W/V)十二烷基硫酸钠;0.5%(W/V)溴酚蓝;50%(V/V)甘油;5%(W/V)β-巯基乙醇)重悬菌体,并至于沸水中处理5-10分钟。然后,12000转/分钟(rpm)离心10分钟。取上清液进行12%的聚丙烯酰胺凝胶电泳(SDS-PAGE)。结果显示,蛋白FN-P约为16千道尔顿(kDa)。泳道1:重组菌株诱导前样品,泳道2和泳道3均为表达蛋白FN-P的重组菌株样品;M为低分子量蛋白标记。
B图为利用抗组氨酸标签的抗体对蛋白FN-P进行蛋白杂交分析。结果显示,A图中泳道2和泳道3的蛋白FN-P可以与抗组氨酸标签的抗体呈阳性反应(在B图中分别对应地标记为2和3)。
图2.蛋白FN-P促成纤维细胞增殖分析
用磷酸缓冲液将蛋白FN-P稀释后,加入到96孔细胞培养板中,使其终浓度依次至200纳克/毫升(ng/ml,低浓度),400纳克/毫升(中浓度)及800纳克/毫升(高浓度),以不加入蛋白FN-P的样品为空白对照组。然后将细胞置于37℃培养48小时,用MTT方法进行检测。实验结果显示,蛋白FN-P以剂量依赖的梯度方式促进成纤维细胞增殖。**p<0.01,蛋白FN-P中浓度处理组vs空白对照组;***p<0.001,蛋白FN-P高浓度处理组vs空白对照组。
图3.蛋白FN-P对金黄色葡萄球菌增长的分析
将金黄色葡萄糖菌(Staphylococcus aureus)培养至对数生长期,用培养基稀释至OD600=0.001后,接种到96孔板中。用磷酸缓冲液将蛋白FN-P重溶加入到96孔板中,稀释成低、中、高浓度。将培养板置于细菌培养箱中,37℃培养20小时。在培养过程不同时间点分别取样,利用酶标仪进行光密度检测。结果显示,与商品化重组纤连蛋白相比,蛋白FN-P能以浓度梯度依赖的方式显著抑制金黄色葡萄球菌的增长。
图4.蛋白FN-P对糖尿病小鼠皮肤创口的修复的分析
在2型糖尿病小鼠背部形成开放式创口,每天加入50纳克蛋白FN-P,以商品化重组纤连蛋白(阳性对照)以及生理盐水(阴性对照)作为对照。实验结果显示,蛋白FN-P处理仅需6天即可使皮肤创口愈合,显著优于商品化重组纤连蛋白及生理盐水处理组。A为生理盐水处理组;B为蛋白FN-P处理组;C为商品化重组纤连蛋白处理组;day表示处理日;标尺中每个间隔为1毫米。
具体实施方式
以下通过优选的实施案例的形式对本发明的上述内容作进一步的详细说明,但其不构成对本发明的限制。
需要说明的是,本领域技术人员应该理解,下述实施例中所用的试剂、酶类等除特别说明外,均为可从试剂公司商购分析纯级别的试剂或酶类。
大肠杆菌pET-21a表达载体(货号:69740)以及重组表达宿主菌BL21(DE3)(货号:69450)购自Sigma-Aldrich公司。
纯化重组融合蛋白所用到的组氨酸亲和层析凝胶(Ni-agarose,货号:17526802)和离子层析凝胶(CM Sepharose Fast Flow,货号:17071901)购自美国GE公司。
金黄色葡萄球菌购自广东省微生物菌种保藏中心(货号:GDMCC No.1.178,中国)。
商品化重组纤连蛋白(Fibronectin Fragment III1-C,货号:F3542)购自美国Sigma-Aldrich公司。
实施例1
表达重组蛋白FN-P表达质粒的构建、表达以及分离纯化
本发明设计的目标蛋白的氨基酸序列如SEQ ID No.1,其可以通过如SEQ ID No.2所示的核酸编码。为了方便分离纯化该目标蛋白的目前,在其氮端引入组氨酸标签(6×HIS),同时为了表达顺利和易于纯化,在起始氨基酸甲硫氨酸和组氨酸标签之间引入KSS三个氨基酸,如此形成融合蛋白,该融合蛋白的氨基酸序列如SEQ ID No.3,其可以通过如SEQID No.4所示的核酸编码。
由南京金斯瑞生物科技有限公司合成上述融合蛋白(其序列如SEQ ID No.3所示)的核酸(其序列如SEQ ID No.4所示),然后利用基因工程方法将该核酸片段克隆到大肠杆菌表达***pET-21a中。将重组质粒转化到大肠杆菌宿主BL21(DE3)中表达出含有组氨酸标签的重组融合蛋白(命名为FN-P)。十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecylsulfate polyacrylamide gel electrophoresis,SDS-PAGE)检测分析显示蛋白FN-P的分子量约为18千道尔顿(kDa)(图1A)。通过组氨酸标记凝胶亲和层析以及CM离子交换层析凝胶联用的方法获得纯化的蛋白FN-P,并利用抗组氨酸标签抗体(货号:ab18184,Abcam公司,美国)进行蛋白杂交分析,结果显示,蛋白FN-P能与抗体成阳性反应(图1B)。将纯化后的蛋白FN-P通过低温真空干燥的方法进行冻干并保存。上述所有实验的具体操作方法参考Sambrook et al.,Molecular Cloning:A LaboratoryManual,Cold Spring HarborLaboratory Press,Cold Spring Harbor,NY,1989。
实施例2
蛋白FN-P促成纤维细胞(NIH/3T3)增殖分析
将NIH/3T3胚胎成纤维细胞(货号:CRL-1658,ATCC,美国)接种到改良杜氏伊格尔培养基(Dulbecco's Modified Eagle 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(137毫摩尔氯化钠,2.7毫摩尔氯化钾,10毫摩尔磷酸二氢钠,2毫摩尔磷酸二氢钾,10国际单位凝血酶,1毫摩尔氯化钙以及0.5毫摩尔氯化镁,pH=7.2)稀释蛋白FN-P冻干粉至2毫克/毫升(mg/ml),然后按比例加入到96孔细胞培养板,使蛋白FN-P的终浓度依次至200纳克/毫升(ng/ml,低浓度),400纳克/毫升(中浓度)以及800纳克/毫升(高浓度),以不加入蛋白FN-P的样品为空白对照组。每种浓度处理各5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150微升的二甲基亚砜(DMSO),然后置于摇床上缓慢振荡10分钟。最后,利用酶标仪(Multiskan,MK3酶标仪,上海)测定各孔的吸光度(测定波长为490纳米),结果见图2。结果显示,蛋白FN-P可以显著促进成纤维细胞增殖(图2),其中高浓度组的促增殖能力为空白对照组的1.5倍。
实施例3
蛋白FN-P抑制金黄色葡萄球菌的增长
将金黄色葡萄球菌(Staphylococcus aureus)接种到LB液体培养基中(蛋白胨10克/升,酵母提取物5克/升,氯化钠10克/升,用1摩尔/升的氢氧化钠溶液调pH至7.0),37℃振荡培养14-16小时。然后,按1:1000(体积/体积)的比例将蛋白FN-P接种到新鲜LB培养基中,混匀后接种到96孔细胞培养板(90微升/孔)。具体如下:
将蛋白FN-P冻干粉用磷酸缓冲液(137毫摩尔氯化钠,2.7毫摩尔氯化钾,10毫摩尔磷酸二氢钠,2毫摩尔磷酸二氢钾,10国际单位凝血酶,1毫摩尔氯化钙以及0.5毫摩尔氯化镁,pH=7.2)溶解,配制成终浓度为5毫克/毫升的蛋白水溶液。分别取2.5微升(低浓度)、5微升(中浓度)和10微升(高浓度)蛋白水溶液,分别加入到上述96孔板中,以不加入蛋白FN-P的样品为空白对照组,以加入商品化重组纤连蛋白(货号:F3542,Sigma-Aldrich;用相同的缓冲液并稀释成5毫克/毫升的蛋白水溶液,10微升/孔)的样品为阳性对照组。每种浓度分别重复5个孔。然后,用LB培养基补充至100微升(每孔),置于37℃培养箱孵育20小时。最后,用酶标仪(Multiskan,MK3酶标仪,上海)在光波长600纳米(OD=600nm)的条件下分析细菌生长的密度,结果见图3。结果显示,商品化的重组纤连蛋白对金黄色葡萄菌的生长没有显著抑制作用,而蛋白FN-P,虽然其不能完全抑制该细菌的生长,但具有明显降低该细菌增殖的能力,并呈剂量依赖现象(图3)。
实施例4
2型糖尿病小鼠模型的建立
购买3月龄成年小鼠12只,饲养在环境温度为恒温24±2℃,相对湿度为50%-70%,L:D=12:12的昼夜规律的环境中。将小鼠分为两组,其中第一组按30毫克每公斤体重(mg/kg)剂量腹腔注射链脲佐霉素(在冰浴、避光以及干燥环境中将链脲佐霉素溶解于柠檬酸缓冲液(0.1毫摩尔/升(mM/L),pH=4.4)中,5分钟内使用完毕),而作为正常对照组的第二组则注射等比例的柠檬酸缓冲液(0.1毫摩尔/升(mM/L),pH=4.4)。4天后,鼠尾采血,用血糖仪测量注射链脲佐霉素动物个体的空腹血糖浓度。以血糖浓度达到16毫摩尔/升(mM/L)以上的小鼠视为造模成功。对血糖浓度不达标的大鼠个体,以30mg/kg剂量的链脲佐霉素腹腔注射补注,4天后再次检测血糖,直至血糖含量达到或超过16毫摩尔/升(mM/L)。
监测结果如下:处理组小鼠平均空腹血糖含量高达18.05±1.22毫摩尔/升(mM/L),而正常对照组动物的空腹血糖为5.58±0.86毫摩尔/升(mM/L)。该结果表明2型糖尿病小鼠模型构建成功。
实施例5
蛋白FN-P治疗2型糖尿病小鼠皮肤创伤
用40%的二氧化碳气体处理2型糖尿病小鼠1-2分钟,待该小鼠失去行动能力后,快速将其背部的毛去掉,然后用灭菌手术剪将皮肤剪出2个大概6毫米*8毫米的创口(左右各一,图4)。然后将其置于实验笼中直至完全苏醒。每天早上,将蛋白FN-P溶于0.9%的生理盐水中,配制成终浓度为5毫克/毫升,然后在小鼠背部右边的创口上滴加10微升,均匀覆盖整个伤口,每天处理1次,连续处理6天。该小鼠背部左边创口用相同体积的生理盐水进行处理,作为阴性对照。此外,使用商品化的重组纤连蛋白(货号:F3542,Sigma-Aldrich)以相同的处理方法治疗2型糖尿病小鼠背部的一个创口,并相同体积的生理盐水进行处理该小鼠背部的另一个创口,作为阴性对照。实验期间,动物自由饮食。
结果见图4,实验结果显示,处理后第一天,由于蛋白FN-P和商品化重组纤连蛋白两种蛋白可以覆盖伤口,因此,两种蛋白在创口表面均形成保护膜;仅仅2天,经蛋白FN-P处理的伤口出现快速愈合的迹象,而且创口大小明显减少;处理4天后,创口表面已经完全被新的上皮覆盖,且没有明显的炎症反应;处理第6天,创口已经完全结痂,新皮肤已经形成。而作为阳性对照组的商品化重组纤连蛋白与生理盐水处理组相比,处理前4天,其变化基本与阴性对照组相当,并且出现明显的炎症现象;直到第6天,创口的愈合情况才优于阴性对照组,但没有完全痊愈;处理14天后,才能使创口愈合达到蛋白FN-P处理6天的水平(该处理14天的结果未显示)。可见,蛋白FN-P能够产生更优的技术效果。
序列表
<110> 广州佰斯伦医疗器械有限公司
广州佰斯伦生物科技有限公司
<120> 一种蛋白及其在创口修复及抑菌中的应用
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Claims (10)
1.一种蛋白,其氨基酸序列如SEQ ID No.1所示。
2.一种蛋白,其包括第一氨基酸序列和第二氨基酸序列,其中,所述第一氨基酸序列如SEQ ID No.1所示;所述第二氨基酸序列为标签的氨基酸序列或起始氨基酸。
3.根据权利要求1或2所述的蛋白,其特征在于,所述标签选自组氨酸标签、FLAG标签、AVI标签中的至少一种;
优选地,所述蛋白的氨基酸序列如SEQ ID No.3所示。
4.一种编码如权利要求1至3中任意一项所述的蛋白的核酸。
5.根据权利要求4所述的核酸,其特征在于,所述核酸的序列如SEQ ID No.2所示,或所述核酸的序列如SEQ ID No.4所示。
6.一种携带有如权利要求4或5所述的核酸的重组微生物;
优选地,所述重组微生物的出发菌株选自埃希氏菌属(Escherichia)和酿酒酵母属(Saccharomyce)中的至少一种;
优选地,所述重组微生物选自大肠杆菌(Escherichia coli)、酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)中的至少一种。
7.根据权利要求6所述的重组微生物,其特征在于,所述微生物能够表达如权利要求1至3中任意一项所述的蛋白。
8.一种组合物,其包括如权利要求1至3中任意一项所述的蛋白和药学上可接受的载体。
9.根据权利要求8所述的组合物,其特征在于,所述药学上可接受的载体选自水、生理盐水、生理上可匹配的缓冲液、生理上可匹配的溶液和生理上可匹配的悬浮液中的至少一种;
优选地,所述蛋白在所述组合物中的质量百分含量为0.01%至20%。
10.权利要求1至3中任意一项所述的蛋白、权利要求4或5所述的核酸、权利要求6或7所述的重组微生物和权利要求8或9所述的组合物中的至少一种在成纤维细胞增殖、组织修复、创口修复及抑菌中的至少一种的应用;
优选地,所述应用为在抑制色葡萄糖菌属(Staphylococcus)中的至少一种细菌中的应用;
优选地,所述应用为在抑制金黄色葡萄糖菌(Staphylococcus aureus)中的应用。
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CN117069864A (zh) * | 2023-09-22 | 2023-11-17 | 英特菲尔(成都)生物制品有限责任公司 | 一种具有修复活性的重组纤连-胶原融合蛋白及其制备方法与应用 |
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