CN114249839A - 一种ⅲ型胶原蛋白的融合蛋白、表达系统、药物组合物及应用 - Google Patents
一种ⅲ型胶原蛋白的融合蛋白、表达系统、药物组合物及应用 Download PDFInfo
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Abstract
本发明涉及一种Ⅲ型胶原蛋白的融合蛋白、表达系统、药物组合物及应用。本发明首先提供了一种Ⅲ型胶原蛋白与硫氧还蛋白组装的融合蛋白,通过硫氧还蛋白的修饰作用,所述Ⅲ型胶原蛋白实现了更好的水溶性和空间结构稳定性,具备更好的工业加工性能。另外,为了实现上述融合蛋白的工业放大生产,本发明还针对所述目的基因进行了优化,提供了一种适合在大肠杆菌中进行表达的方式,具体提供了上述融合蛋白表达的工程菌。
Description
技术领域
本发明属于重组胶原蛋白技术领域,具体涉及一种Ⅲ型胶原蛋白与硫氧还蛋白的融合蛋白、编码所述融合蛋白的核苷酸序列、包含所述核苷酸序列的表达载体,本发明还涉及表达所述融合蛋白的工程菌株、包含所述融合蛋白的药物组合物及其在制备洗护用品、医疗器械领域的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
Ⅲ型胶原蛋白,是人体皮肤、筋膜、肌腱中主要的胶原蛋白。Ⅲ型胶原蛋白较细小,存在于表皮层和真皮层之间,是撑起表皮的关键,也是肌肤塌陷的第一步。另外,Ⅲ型胶原蛋白也是中空器官(例如大血管、子宫和肠)的主要结构成分。现有研究证实胶原蛋白水解物具有显著生物活性,如抗氧化特性,抗高血压活性,降脂活性,以及修复受损皮肤性质,现已被广泛应用于化妆品、医疗器械等领域,用于实现对组织的修复、再生。Ⅲ型胶原蛋白具有三重螺旋结构,三个相同的Ⅲ型前胶原链在羧基末端连接在一起,并通过形成二硫键使结构稳定,每个单独的链折叠成左手螺旋,然后将三个链缠绕在一起,形成右手超螺旋。每个前胶原链形成过程中需要经过若干翻译后的修饰过程,再在组织的细胞外空间中组装成大分子原纤维,然后聚集成纤维,为需要抗张强度的组织提供了强大的支撑结构。因此,这种三重螺旋结构对维持胶原蛋白的支撑作用具有作用。
目前,采用重组大肠杆菌发酵表达Ⅲ型胶原蛋白是本领域常用的制备方法,对于依靠基因工程技术通过生物发酵法获取的重组胶原蛋白,由于应用领域差异以及技术限制等因素,在表达产物长度与表达水平上都存在巨大差异。重组Ⅲ型胶原蛋白难以复刻天然Ⅲ型胶原蛋白的三重螺旋支撑结构,同时还存水溶性不足、不稳定等缺陷。发明人认为,目前以大肠杆菌作为宿主进行表达的生产方式中,由于物种基因特性及密码子偏嗜性的差异,会出现一些稀有密码子,过多的出现大肠杆菌稀有密码子会影响外源基因的表达,从而影响Ⅲ型胶原蛋白的表达。现有研究方式,通过筛选Ⅲ型胶原蛋白中的保守序列进行表达从而获得性能优化的重组胶原蛋白。
硫氧还蛋白(Thioredoxin,Trx),是一类氢载体的蛋白质(约12 kDa),具有良好的热稳定性,广泛的存在于植物、细菌到哺乳动物的许多生物体中;在许多还原反应中作为氢供体,特别是核苷二磷酸变成相应的脱氧产物和光依赖性的还原反应中,也以结构域的形式出现于二硫键异构酶中。硫氧还蛋白通过半胱氨酸硫醇-二硫键交换促进其他蛋白还原而起抗氧化剂作用。它涉及许多细胞过程,包括脱氧核糖核苷酸合成,氧化损伤蛋白的修复,蛋白质折叠,硫代谢和氧化还原稳态。目前已经鉴定出多种硫氧还蛋白的体外底物,包括核糖核酸酶、绒毛膜促性腺激素、凝血因子、糖皮质激素受体和胰岛素。
发明内容
现有研究中将硫氧还蛋白作为宫颈癌亚单位疫苗的骨架来提高疫苗的免疫原性,在流感病毒、马尔堡病毒和轮状病毒等亚单位疫苗的制备也有尝试,这可能说明硫氧还蛋白能够辅助维持抗原活性。基于上述技术背景,发明人联想到提供一种Ⅲ型胶原蛋白-硫氧还蛋白的融合蛋白,硫氧还蛋白是一种广泛存在于不同生物体中的载体蛋白,应用于不同的宿主细胞中可能展现良好的相容性。
基于上述技术思路,本发明提供以下技术方案:
本发明第一方面,提供一种Ⅲ型胶原蛋白的融合蛋白,所述融合蛋白:
(1)具有SEQ ID NO.1所示的氨基酸序列;
(2)对SEQ ID NO.1所示的序列进行一个或多个氨基酸的添加、取代或缺失所形成的衍生多肽,所述衍生多肽与SEQ ID NO.1所示多肽具有相同或基本相同的功能;
(3)对(1)或(2)中所述多肽进行修饰后的衍生多肽。
本发明技术方案首先提供了一种Ⅲ型胶原蛋白-硫氧还蛋白的融合蛋白(SEQ IDNO.1所示),所述融合蛋白由硫氧还蛋白和Ⅲ型胶原蛋白组成。经验证,上述融合蛋白相比单一表达Ⅲ型胶原蛋白具有更好的稳定性和水溶解效果,硫氧还蛋白能够促进Ⅲ型胶原蛋白中二硫键的形成和蛋白质正确的折叠,确保表达的蛋白处于一个正确的构象,还可以阻止宿主蛋白酶对蛋白的降解。
优选的,上述技术方案(2)中,所述“一个或多个氨基酸的添加、取代或缺失”是指SEQ ID NO.1所示氨基酸序列的融合蛋白进行一个或多个氨基酸的添加、取代或缺失后仍然能够发挥相同或相似的功能,所述“发挥相同或相似的功能”表示能够基于基因重组的方式在宿主细胞中表达,并且表达物还能够发挥与SEQ ID NO.1所示序列融合蛋白相似的生理活性、水溶性或稳定性;所述“一个或多个氨基酸”是1-7个氨基酸,优先1-6个氨基酸;更为优选的,为 1-3个氨基酸残基的取代、缺失或添加而形成的且具有本发明所述的衍生多肽。
另外,应当说明的是,本发明所述融合蛋白相比现有Ⅲ型胶原蛋白具有更好的水溶性及稳定性,应用于化妆品或医疗器械的制备具有更好的工业加工性能。基于该融合蛋白的上述特性,所述融合蛋白通过化学或遗传修饰后获得衍生多肽也属于本发明相同的技术构思,所述修饰方法包括聚乙二醇(PEG)、链霉亲和素、各种分子,例如生物素、放射性同位素、荧光剂、酶、细胞毒性物质、抗肿瘤剂等,还包括所述融合蛋白与功能性多肽连接后的衍生多肽,所述功能性基团包括但不限于靶向肽、穿膜肽等,具体的实例中,所述穿膜肽为H7R8、HIV-I TAT (48-60)、TAT (47-57)、TAT (48-57)、R9-Tat中的一种。
其中,上述功能性基团的修饰方法可以通过公知的方式进行,如固相合成或液相合成等方式。
本发明第二方面,提供编码第一方面所述Ⅲ型胶原蛋白融合蛋白的核苷酸序列。
所述核酸序列包括由于密码子简并性能够翻译得到第一方面所述融合蛋白或衍生多肽的核酸序列。
所述编码核酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA,DNA可以是单链的或是双链的,可以是编码链或非编码链。
本发明的一种实施方式中,提供一种适合在大肠杆菌表达的核苷酸序列,根据大肠杆菌密码子偏嗜性,选用大肠杆菌优势密码子,优化外源基因密码子。由于外源目的基因中G+C含量过高过低都不利于基因的表达,应根据大肠杆菌基因组中的G+C含量做相应的调整,另外,基因中AT富含重复区往往导致转录的提前终止。本发明对编码融合蛋白的核苷酸序列进行了调整,包括对其中的AT进行适当减少提前终止的概率,调整后G+C含量为56.97%,并且消除了AT富含区,优化后所述融合蛋白的核苷酸序列如下:
经验证,该编码序列导入大肠杆菌进行表达,能够有效的防止转录的提前终止,并且提高了潜在表达量。
本发明第三方面,提供一种表达载体,所述表达载体中含有第二方面所述的核苷酸序列。
优选的,第二方面所述编码核酸序列可插入所述表达载体。
所述“表达载体”具体实例包括:本领域熟知的细菌质粒、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。总之,只要能在宿主体内复制和稳定,任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员可采用熟知的方法构建含本发明的融合蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体,包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上,以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外,表达载体优选包含一个或多个选择性标记基因,以提供用于选择转化的宿主细胞的表型性状,如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP),或用于大肠杆菌的卡那霉素或氨苄青霉素抗性。
本发明提供的一个实施方式中,所述表达载体为质粒载体,可使用pBR型、pUC型、pBluescriptII型、pGEM型、pTZ型、pCL型或pET型;具体的实例中,所述表达载体为pET28a。
本发明第四方面,提供一种重组胶原蛋白表达的工程菌,所述工程菌表达第一方面所述Ⅲ型胶原蛋白的融合蛋白。
所述工程菌中包括第三方面所述表达载体,或所述工程菌中整合了第二方面所述的编码核酸。
优选的,所述工程菌可以是原核细胞,如细菌细胞;或是低等真核细胞,如酵母细胞。具体的实例中,所述菌株包括但不限于:大肠杆菌(E .Coli) 、谷氨酸棒杆菌(Coryne bacterium glutamicum)、蜂房哈夫尼菌(Hafniaalvei)、枯草芽孢杆菌(Bacillus subtilis)。更为优选的,所述菌株为大肠杆菌。
其中,重组DNA转化工程菌的出发菌株可用本领域技术人员熟知的常规技术进行。当出发菌株为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaCl2法处理,所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要,转化也可用电穿孔的方法进行。当出发菌株是真核生物,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔、脂质体包装等。
本发明一种具体的实施方式中,所述工程菌为导入第三方面所述表达载体的大肠杆菌,构建方法如下:
(1)重组表达载体的构建:将SEQ ID NO.2所示序列的目的基因插入到表达载体pET28a的酶切位点EcoR1和Not1之间,得到重组表达载体pET28a-III;
(2)转化体的构建:将重组表达载体pET28a-III转化入感受态细胞BL21(DE3),筛选获得重组基因工程菌;
(3)诱导表达:将获得的重组基因工程菌接入抗性LB培养基中培养过夜,筛选生长转况良好的菌落接种至LB培养基中培养至对数生长中期,加入IPTG进行诱导表达;
(4)蛋白纯化:收集重组基因工程菌的培养菌液,通过亲和柱层次纯化获得目的融合蛋白。
本发明第五方面,提供一种药物组合物,其特征在于,所述组合物中包括第一方面所述融合蛋白。
优选的,所述组合物中,还包括药学上所必需的载体。
本发明第六方面,提供第一方面所述融合蛋白、第五方面所述药物组合物在制备洗护用品或医疗器械中的应用。
目前Ⅲ型胶原蛋白作为一种生物材料广泛应用于皮肤修复的护肤品及医疗器械等领域。
上述第六方面中,所述洗护用品为包括但不限于衣物洗涤剂、个人卫生清洁剂、家庭日用洗涤剂或化妆品中的一种。
所述医疗器械包括药物输送和组织工程方面的应用:所述药物输送的应用包括所述融合蛋白、药物组合物作为一种药物递送基质方面的应用,具体实例如作为一种医用贴剂、创面敷料或生物补片;在组织工程方面的应用,包括用于制备植入式生物支架材料、人工皮肤、人工血管、人工骨材料等组织替代材料。
以上一个或多个技术方案的有益效果是:
1、Ⅲ型胶原蛋白是一种重要的生物制品原料,为了获得具有更好工业加工性能的Ⅲ型胶原蛋白产品,本发明通常对Ⅲ型胶原蛋白的重组蛋白表达方式进行优化以获得性能优化的融合蛋白产品。本发明首先联想到采用硫氧还蛋白作为载体与Ⅲ型胶原蛋白融合表达。经本发明设计并表达的重组Ⅲ型胶原蛋白-硫氧还蛋白融合蛋白,不仅增加了胶原蛋白的稳定性和可溶性,还可以促进二硫键的形成和蛋白质正确的折叠,确保表达的蛋白处于一个正确的构象,同时可以阻止宿主蛋白酶对蛋白的降解。
2、本发明还针对上述融合蛋白的重组表达方式进行了优化,针对目的基因与宿主细胞表达系统存在冲突导致目的基因表达量不高的问题,本发明通过Ⅲ型胶原蛋白基因的优化提高目的基因与宿主表达系统之间的符合度,提高潜在表达量,提供了一种融合蛋白可高效表达的基因工程菌株,有望作为一种工程菌株应用于Ⅲ型胶原蛋白的工业化生产。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为实施例1中所述融合蛋白SDS-PAGE中1-10泳道图;
其中,泳道1:Marker 2:全菌 3:上样液 4:离心后沉淀 5:穿过峰
6:20mM咪唑 7:20mM咪唑 8:100mM咪唑 9:100mM咪唑 10:250mM咪唑。
图2为实施例1中所述融合蛋白SDS-PAGE中11-12泳道图;
其中,11:250mM咪唑 12:1M咪唑。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
正如背景技术所介绍的,Ⅲ型胶原蛋白具有良好的生物活性,是一种重要的洗护产品、化妆品或医疗器械制品原料。Ⅲ型胶原蛋白的三重螺旋结构对于发挥生物活性具有重要作用,但现有重组Ⅲ型胶原蛋白较难维持上述活性结构。为了解决如上的技术问题,本发明提出了一种Ⅲ型胶原蛋白-硫氧还蛋白的融合蛋白,通过硫氧还蛋白的修饰巩固了Ⅲ型胶原蛋白的稳定性,提高了水溶性。进一步的,本发明还提供了一种上述融合蛋白的表达系统,通过对目的基因的优化,提高了目的基因与表达系统符合度,实现了融合蛋白的高效表达。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例与对比例详细说明本发明的技术方案。
实施例1
1.1试剂与耗材
EcoR1和Not1限制性内切酶,T4连接酶购自于NEB公司;表达载体pET28a由本实验室保存;菌株BL21(DE3)和DH5α购自于北京全式金生物技术有限公司;山羊抗小鼠IgG购自于达科为生物技术有限公司;
LB(A+)板:将10 g胰蛋白胨、5 g酵母提取物、10 g Nacl和15 g琼脂粉溶解于800mL去离子水中,用1 mol/L的NaOH调节pH为7.0,定容至1000 mL。高压灭菌,待温度降至55℃,加入1 mL 100 mg/mL氨苄,倒板。
LB 培养基:配制每升培养基,应在950mL去离子水中加入胰蛋白胨10g,酵母提取物5g,氯化钠10g,摇动容器直至溶质溶解,用5mol/L (约0.2mL) NaOH 调pH值至7.0,用去离子水定容至1L,高压灭菌20min后即可使用,保存于4℃。
1.2 蛋白表达
本实施例中,提供了一种Ⅲ型胶原蛋白与硫氧还蛋白(Trx)融合表达,所述融合蛋白的序列如下: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSEFMGEPGNPGSPGNQGQPGNKGSPGNPGQPGNEGQPGQPGQNGQPGEPGSNGPQGSQGNPGKNGQPGSPGSQGSPGNQGSPGQPGNPGQPGEQGKPGNQGPAHHHHHH.
根据大肠杆菌密码子偏嗜性在蛋白质序列不变的前提下,选用大肠杆菌优势密码子,优化外源基因密码子,优化后 G+C 含量为56.97%,消除AT 富含区。优化后基因由上海生工合成,其核苷酸序列如SEQ ID NO.2所示。应用EcoR1和Not1两个酶切位点连在pET28a载体上,得到的载体命名为pET28a-III。
构建好的表达质粒pET28a-III转化表达菌株BL21(DE3),均匀涂布卡那霉素抗性LB培养基平板,过夜培养(约12h)。挑取2-4个生长良好的单菌落,用20mL试管分别接种于3mL LB液体培养基中,37℃、250rpm振荡培养至对数生长中期(OD600约0.4~0.8),取出2mL培养物作为诱导前对照,在剩余培养物中加入IPTG,使终浓度为1mM,继续培养。诱导3小时后,取1mL诱导后的菌液,离心后弃上清,加入1mLPBS重悬菌体然后超声破壁,离心后对上清和沉淀跑SDS-PAGE,并做WB验证。确定诱导表达条件后,进行大量培养。
1.3蛋白的纯化
收集大肠杆菌液,5000rpm室温离心10min,弃上清,留沉淀;用PBS重悬沉淀后超声重悬液,工作时间3s,间隙时间5s,全程时间20min;12000rpm,10min,收集上清样品过柱,上清用于亲和层析纯化。
1)组装XK16/20柱子(组装前需先用自来水冲洗,再用ddH2O 冲洗),取20mLChelating Sepharose Fast Flow(GE Healthcare)填料摇匀倒入柱子中(贴壁加入填料,避免产生空气,影响柱效);2) 用5倍柱体积ddH2O 洗柱子以去除酒精(测pH 应为中性),准备好平衡缓冲液和洗脱液;3) 用3倍柱体积平衡缓冲液,3mL/min 平衡柱子,然后将系统紫外吸光度读值调零;4) 样品的处理、上样及穿柱液收集:将上清用0.45 um 针头式过滤器过滤, 3mL/min上样,直至泵完全部样品,待样品全部泵完后,继续泵入平衡缓冲液直至系统紫外吸光度读值再次降为零;5) 用150mL含30 mM咪唑的洗脱液洗脱柱子,洗去杂蛋白;然后用50mL含200 mM咪唑的洗脱液洗脱柱子,洗目的蛋白;6) 待纯化完成后,先泵入50mL含500 mM咪唑的洗脱液洗脱柱子冲洗柱子,然后再泵入50mL H2O 清洗柱子,按照装柱的顺序拆卸柱子,倒出填料后,加入20% 的酒精4℃长期保存。将纯化过程中收集到的各种样品加入2×SDS Loading Buffer后,沸水煮10min,SDS-PAGE和WB进行验证。
根据本实施验证,野生型Ⅲ型胶原蛋白采用上述构建方式的表达量很低,约为5mg/mL,采用本发明密码子优化后表达量显著提高,可以达到50mg/mL。
另外,硫氧还蛋白的使用增加了胶原蛋白的可溶性,不含硫氧还蛋白的胶原蛋白约90%以包涵体形式存在的,增加硫氧还蛋白后胶原蛋白的可溶性显著增强,95%的目的蛋白以可溶性表达。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 山东林森生物制品股份有限公司
<120> 一种Ⅲ型胶原蛋白的融合蛋白、表达系统、药物组合物及应用
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Claims (10)
1.一种Ⅲ型胶原蛋白的融合蛋白,其特征在于,所述融合蛋白:
(1)具有如SEQ ID NO.1所示的氨基酸序列;
(2)对SEQ ID NO.1所示的序列进行一个或多个氨基酸的添加、取代或缺失所形成的衍生多肽,所述衍生多肽与SEQ ID NO.1所示多肽具有相同或基本相同的功能;
(3)对(1)或(2)中所述多肽进行修饰后的衍生多肽。
2.如权利要求1所述Ⅲ型胶原蛋白的融合蛋白,其特征在于,所述“一个或多个氨基酸”是1-7个氨基酸,优先1-6个氨基酸;更为优选的,为 1-3个氨基酸残基的取代、缺失或添加而形成的衍生多肽;
或,所述融合蛋白还包括通过化学或遗传修饰后获得衍生多肽,所述修饰方法包括采用聚乙二醇、链霉亲和素、生物素、放射性同位素、荧光剂、酶、细胞毒性物质或抗肿瘤剂进行修饰,还包括所述融合蛋白与功能性多肽连接后的衍生多肽,所述功能性基团包括但不限于靶向肽、穿膜肽,所述穿膜肽为H7R8、HIV-I TAT (48-60)、TAT (47-57)、TAT (48-57)、R9-Tat中的一种。
3.编码权利要求1或2所述Ⅲ型胶原蛋白融合蛋白的核苷酸序列;
优选的,所述核酸序列包括由于密码子简并性能够翻译得到权利要求1或2所述融合蛋白或衍生多肽的核酸序列;
优选的,所述核苷酸序列如SEQ ID NO.2所示。
4.一种表达载体,其特征在于,所述表达载体中含有权利要求3所述的核苷酸序列。
5.如权利要求4所述表达载体,其特征在于,所述编码核酸序列可插入所述表达载体;
所述表达载体为包括但不限于细菌质粒、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;
进一步的,所述表达载体为质粒载体,包括pBR型、pUC型、pBluescriptII型、pGEM型、pTZ型、pCL型或pET型;
具体的,所述表达载体为pET28a。
6.一种重组胶原蛋白表达的工程菌,其特征在于,所述工程菌表达权利要求1或2所述Ⅲ型胶原蛋白的融合蛋白。
7.如权利要求6所述重组胶原蛋白表达的工程菌,其特征在于,所述工程菌中包含权利要求4或5所述表达载体,或所述工程菌中整合了权利要求3所述的编码核酸;
优选的,所述工程菌为原核细胞或低等真核细胞;进一步的,为大肠杆菌(E .Coli) 、谷氨酸棒杆菌(Coryne bacterium glutamicum)、蜂房哈夫尼菌(Hafniaalvei)或枯草芽孢杆菌(Bacillus subtilis);更为优选的,所述菌株为大肠杆菌。
8.如权利要求6所述重组胶原蛋白表达的工程菌,其特征在于,所述工程菌为导入权利要求4或5所述表达载体的大肠杆菌,构建方法如下:
(1)重组表达载体的构建:将SEQ ID NO.2所示序列的目的基因插入到表达载体pET28a的酶切位点EcoR1和Not1之间,得到重组表达载体pET28a-III;
(2)转化体的构建:将重组表达载体pET28a-III转化入感受态细胞BL21(DE3),筛选获得重组基因工程菌;
(3)诱导表达:将获得的重组基因工程菌接入抗性LB培养基中培养过夜,筛选生长转况良好的菌落接种至LB培养基中培养至对数生长中期,加入IPTG进行诱导表达;
(4)蛋白纯化:收集重组基因工程菌的培养菌液,通过亲和柱层次纯化获得目的融合蛋白。
9.一种药物组合物,其特征在于,所述组合物中包括权利要求1或2所述融合蛋白;
优选的,所述药物组合物中,还包括药学上所必需的载体。
10.权利要求1或2所述融合蛋白、权利要求9所述药物组合物在制备洗护用品或医疗器械中的应用;
优选的,所述洗护用品为包括但不限于衣物洗涤剂、个人卫生清洁剂、家庭日用洗涤剂或化妆品中的一种;
优选的,所述医疗器械包括药物输送和组织工程方面的应用:
所述药物输送的应用包括所述融合蛋白、药物组合物作为一种药物递送基质方面的应用,包括但不限于医用贴剂、创面敷料或生物补片;
在组织工程方面的应用包括用于制备组织替代材料,包括但不限于植入式生物支架材料、人工皮肤、人工血管或人工骨材料。
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