CN115521373A - 一种三螺旋重组人源化i型胶原蛋白、制备方法及其应用 - Google Patents
一种三螺旋重组人源化i型胶原蛋白、制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种三螺旋重组人源化I型胶原蛋白、制备方法及其应用,所述的重组人源化I型胶原蛋白具有胶原蛋白特征性的三螺旋结构和(Gly‑X‑Y)n重复氨基酸序列,并可自组装形成良好形貌的胶原纤维;本发明制备的重组人源化I型胶原蛋白具有人I型胶原蛋白重要的功能位点,表现出良好的生物相容性和生物活性,可以显著促进人皮肤成纤维细胞的增殖、黏附和迁移;本发明通过基因工程菌表达制备重组人源化I型胶原蛋白,工艺简单方便,易于实现工业化生产;制备的高生物活性的三螺旋重组人源化I型胶原蛋白,可广泛应用于皮肤修复敷料、植入剂、人工皮肤、人工骨、人工软骨、人工眼角膜、生物材料、医疗器械、化妆品、保健品、食品等领域。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种三螺旋重组人源化I型胶原蛋白、制备方法及其应用。
背景技术
胶原蛋白是哺乳动物体内含量最高的蛋白质,约占蛋白质总量的30%。胶原蛋白具有特征性的三螺旋结构,并可以自组装形成良好形貌的胶原纤维。胶原纤维进一步组装形成纤维网格结构,为***提供机械强度,并为细胞生长提供适宜的环境。到目前为止,已发现至少40余种胶原蛋白链的编码基因。这些不同的胶原蛋白链,以不同的方式组合,可以形成20多种不同类型的胶原蛋白分子。其中,I型胶原蛋白是人体含量最丰富的胶原蛋白类型,它是皮肤、骨骼、肌腱等***的主要成分。
I型胶原蛋白为主要原料的生物材料在组织工程和再生医学等领域广泛应用。目前I型胶原蛋白主要通过动物组织提取。已经开发酸、碱或蛋白酶等不同处理方法和工艺,从牛、猪等动物的皮、肌腱、骨等不同组织中提取获得I型胶原蛋白。然而,动物提取方法容易破坏胶原蛋白的三螺旋结构和生物活性,并存在病毒传播隐患等风险。通过基因工程技术制备的重组胶原蛋白可以克服动物源胶原蛋白的批次质量差异、病毒传染隐患等风险,因此逐渐成为研究热点。转基因植物、哺乳动物细胞、细菌、酵母等被利用来生产重组胶原蛋白,但是转基因植物、哺乳动物细胞等表达体系生产成本高、周期长,难以满足产业化需求。相比之下,大肠杆菌和酵母表达***具有成本低、表达量高等优点,是工业化生产的首选方式。
关于胶原蛋白,本领域已经做了诸多研究,例如中国专利CN110903383A公开了一种重组人源I型胶原蛋白、编码基因、工程菌及其在制备胶原制剂中的应用,其氨基酸序列为肽段GSKGDTGEPGPVGVQGPPGPAGEEGKRGARGEP重复9次;中国专利CN113637068A提供了一种重组I型人源化胶原蛋白C1L5T、制备方法及用途,其氨基酸序列包括人I型胶原蛋白的肽段GEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPTGPPGPRGRTGDAGPVGPPGPPGPPGPPGPP以及端序列肽段GAPGPCCGG。中国专利CN105061589A提供了一种重组类人I型胶原蛋白,包括人I型胶原蛋白α1链660-964肽段,N端680-694位的15个氨基酸,以及C端950-964位的15个氨基酸和3组GER氨基酸三联体。但是这些方法制备的重组I型胶原蛋白均缺乏胶原蛋白特征性的三螺旋结构、纤维形貌和生物功能。
针对上述技术问题,本发明提供了一种三螺旋重组人源化I型胶原蛋白、制备方法及其应用。所述重组人源化I型胶原蛋白具有胶原蛋白特征性的三螺旋结构,且100%严格遵守Gly-X-Y重复序列,并可自组装形成良好形貌的胶原纤维;所述重组人源化I型胶原蛋白具有人I型胶原蛋白重要的功能位点,表现出优异的生物相容性和生物活性,可以显著促进人皮肤成纤维细胞的增殖、黏附和迁移;所述重组人源化I型胶原蛋白通过基因工程菌表达以及前体胶原蛋白酶解方法制备,工艺简单,操作方便,易于实现工业化生产;所述重组人源化I型胶原蛋白可广泛应用于皮肤修复敷料、植入剂、人工皮肤、人工骨、人工软骨、人工眼角膜、生物材料、医疗器械、化妆品、保健品、食品等领域。
发明内容
针对上述技术问题,本发明的首要目的是提供了一种三螺旋重组人源化I型胶原蛋白,所述的三螺旋重组人源化I型胶原蛋白为胶原蛋白THRCI-1或胶原蛋白THRCI-2,所述的胶原蛋白THRCI-1的氨基酸序列如SEQ ID NO.1所示,所述的胶原蛋白THRCI-2的氨基酸序列如SEQ ID NO.2所示。
优选的,所述重组人源化I型胶原蛋白由蛋白酶处理前体胶原蛋白后获得;所述的胶原蛋白THRCI-1对应的前体胶原蛋白1的氨基酸序列如SEQ ID NO.3所示,所述的胶原蛋白THRCI-1对应的前体胶原蛋白2的氨基酸序列如SEQ ID NO.4所示。
优选的,所述的胶原蛋白THRCI-1对应的前体胶原蛋白1的基因序列如SEQ IDNO.5所示,所述的胶原蛋白THRCI-2对应的前体胶原蛋白2的基因序列如SEQ ID NO.6所示。
优选的,所述蛋白酶包括胃蛋白酶、胰蛋白酶、木瓜蛋白酶、菠萝蛋白酶、凝血酶。
本发明还提供了一种携带所述基因的重组载体或重组基因工程菌。
优选的,所述载体包括pCold或pET。
优选的,所述工程菌为大肠杆菌。
本发明还提供了所述三螺旋重组人源化I型胶原蛋白的制备方法,步骤如下:
(1)合成编码所述前体胶原蛋白的基因序列;
(2)将步骤(1)所述基因序列与载体连接,转化细菌,构建重组基因工程菌;
(3)表达步骤(2)构建的重组基因工程菌,收集菌体沉淀,破碎得上清液,纯化后获得前体胶原蛋白;
(4)加入蛋白酶对步骤(3)获得的前体胶原蛋白进行处理,纯化后获得三螺旋重组人源化I型胶原蛋白。
优选的,步骤(4)所述的蛋白酶包括胃蛋白酶、胰蛋白酶、木瓜蛋白酶、菠萝蛋白酶、凝血酶。
本发明还提供了所述的三螺旋重组人源化I型胶原蛋白在制备皮肤修复敷料、植入剂、人工皮肤、人工骨、人工软骨、人工眼角膜、生物材料、医疗器械、化妆品、保健品、食品中的应用。
与现有技术相比,本发明的有益效果是:①本发明提供了一种三螺旋重组人源化I型胶原蛋白,所述的胶原蛋白的氨基酸序列源于人I型胶原蛋白α1链,与动物来源的I型胶原蛋白相比,具有无病毒传播隐患、低免疫原性等优点;②本发明提供的重组人源化I型胶原蛋白具有胶原蛋白特征性的三螺旋结构以及(Gly-X-Y)n重复氨基酸序列;③本发明提供的三螺旋重组人源化I型胶原蛋白可自组装形成形貌良好、与天然胶原蛋白类似的胶原纤维;④本发明提供的三螺旋重组人源化I型胶原蛋白具有人I型胶原蛋白重要的功能位点,生物相容性好、生物活性高,可以显著促进人皮肤成纤维细胞(HFF-1)的增殖、黏附和迁移;⑤本发明提供的三螺旋重组人源化I型胶原蛋白的基因工程菌表达体系,制备工艺简单方便,易于实现规模化生产;⑥本发明制备的高生物活性的三螺旋重组人源化I型胶原蛋白,可广泛应用于皮肤修复敷料、植入剂、人工皮肤、人工骨、人工软骨、人工眼角膜、生物材料、医疗器械、化妆品、保健品、食品等领域。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解。
图1为三螺旋重组人源化I型胶原蛋白的SDS-PAGE鉴定图
图2为三螺旋重组人源化I型胶原蛋白的二级结构测定
a,b,c分别为THRCI-1的圆二色谱图、圆二色谱热变曲线及热变曲线的一阶导数(dθ/dT);d,e,f分别为THRCI-2的圆二色谱图、圆二色谱热变曲线及热变曲线的一阶导数(dθ/dT)
图3为三螺旋重组人源化I型胶原蛋白的扫描电子显微镜(SEM)图
图4为三螺旋重组人源化I型胶原蛋白的细胞增殖图
图5为三螺旋重组人源化I型胶原蛋白的细胞黏附图
图6为三螺旋重组人源化I型胶原蛋白的细胞迁移图
具体实施方式
为使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施方式,进一步阐述本发明。但本发明的保护范围并不局限于以下实施例所述。
以下实施例中的实验材料,若无特殊说明,均可在市场上购买得到。
实施例1重组人源化I型胶原蛋白的制备
本发明所述高生物活性的三螺旋重组人源化I型胶原蛋白THRCI-1和胶原蛋白THRCI-2的氨基酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示,该蛋白通过前体胶原蛋白经蛋白酶处理获得;前体胶原蛋白1的氨基酸序列如SEQ ID NO.3所示、基因序列如SEQ IDNO.5所示,前体胶原蛋白2的氨基酸序列如SEQ ID NO.4所示,基因序列如SEQ ID NO.6所示。
1.确定前体胶原蛋白的氨基酸序列如SEQ ID NO.3和SEQ ID NO.4所示;
2.构建前体胶原蛋白的表达菌株:
合成编码前体胶原蛋白的基因序列如SEQ ID NO.5和SEQ ID NO.6所示,构建导入上述核酸的质粒,通过DNA测序确认质粒成功合成;将质粒转化至大肠杆菌BL21-DE3菌株获得前体胶原蛋白表达菌株,将转化成功的菌株加入甘油后,保存于-80度冰箱;
3.胶原蛋白的制备与纯化:
取20μL菌液加到200mL含抗生素的LB液体培养基中,37℃恒温摇床过夜进行增菌培养后,按2%接种量转移到1L含抗生素的LB液体培养基中,在37℃恒温摇床中继续扩增培养;待OD600值达到0.8-1.0范围,将摇床温度调为25℃,加入终浓度为1mM IPTG诱导表达,恒温过夜培养;将菌液在低温离心机中离心,离心条件3200rpm,4℃,30min,收集菌体。
将菌体用缓冲液溶解(20mM磷酸钠缓冲溶液,20mM咪唑,0.5M氯化钠,pH为7.4),利用高压均质机进行细胞破碎,将破碎完的悬浊液再次离心,收集上清液,即粗蛋白溶液,通过镍离子亲和层析柱进一步纯化得到前体胶原蛋白。加入终浓度为0.008mg/mL的胃蛋白酶处理前体胶原蛋白,纯化去除酶切产物,冻干即得高生物活性的三螺旋重组人源化I型胶原蛋白。
将实施例1中制备的高生物活性的三螺旋重组人源化I型胶原蛋白进行SDS-PAGE鉴定。
图1显示制备得到的重组人源化I型胶原蛋白THRCI-1和重组人源化I型胶原蛋白THRCI-2均为单一条带,表明成功制备高纯度的重组人源化I型胶原蛋白。由于胶原蛋白的特殊氨基酸序列和结构,在SDS-PAGE上的迁移速率小于同分子量的球形蛋白。
实施例2重组人源化I型胶原蛋白的二级结构测定
圆二色谱是用于表征蛋白质结构的常用方法。将实施例1中制备的重组人源化I型胶原蛋白配成1mg/mL的溶液,用1mm的比色皿,在4℃下进行圆二色谱全波长(190-260nm)扫描,波长间隔1nm,每个波长下停留5s。热变曲线在220nm下测定,温度从4℃-70℃。温度递增速度如下:4℃-20℃(1℃/min);20℃-50℃(0.3℃/min);50℃-70℃(1℃/min)。
如图2所示,所述重组人源化I型胶原蛋白THRCI-1和重组人源化I型胶原蛋白THRCI-2在225nm附近均显示胶原蛋白特征性的吸收峰,表明所述重组人源化I型胶原蛋白均形成了三螺旋结构。热变曲线研究进一步表明,所述重组人源化I型胶原蛋白THRCI-1和重组人源化I型胶原蛋白THRCI-2形成了稳定的三螺旋结构,其热变温度分别为40℃和42℃。
实施例3重组人源化I型胶原蛋白的组装形貌
将实施例1中制备的重组人源化I型胶原蛋白THRCI-1和重组人源化I型胶原蛋白THRCI-2用20mM PB溶解成终浓度为3mg/mL的溶液,4℃下放置至少12hrs,取少量滴在硅片至完全干燥,用Hitachi S-4800扫描电子显微镜(SEM)表征前将干燥的样品进行2分钟喷金处理。
如图3所示,所述重组人源化I型胶原蛋白THRCI-1和重组人源化I型胶原蛋白THRCI-2自组装形成形貌良好、与天然胶原蛋白类似的胶原纤维。
实施例4重组人源化I型胶原蛋白的生物活性
(1)细胞增殖实验
向96孔板中加入100μL,细胞密度为1×105个/mL的人皮肤成纤维细胞HFF-1,37℃,5%CO2孵育24h,24h后吸出96孔板中的培养液,将实施例1中制备的重组人源化I型胶原蛋白和组织提取牛I型胶原蛋白用DMEM高糖培养基配成0.1mg/mL的胶原蛋白溶液加于96孔板中,对照组只加DMEM高糖培养基,继续于37℃,5%CO2培养箱中孵育1、3、5天。利用cck-8检测所设计重组人源化I胶原蛋白促人皮肤成纤维细胞HFF-1增殖效果。
如图4所示,所述重组人源化I型胶原蛋白THRCI-1和重组人源化I型胶原蛋白THRCI-2均具有显著的促人皮肤成纤维细胞HFF-1增殖的效果,且优于组织提取牛I型胶原蛋白。
(2)细胞黏附实验
将实施例1中制备的重组人源化I型胶原蛋白用PBS配成0.1mg/mL的胶原蛋白溶液,热变性1%BSA以及0.1mg/mL牛I型胶原蛋白标准品分别作为阴性对照和阳性对照,将上述各蛋白溶液加入24孔板中于4℃吸附24h,然后吸出溶液。用DMEM高糖培养基将人皮肤成纤维细胞HFF-1细胞稀释成密度为1×105个/mL,向每孔中加入500μL细胞,6h后在显微镜下观察细胞黏附情况。
如图5所示,相比于BSA,所述重组人源化I型胶原蛋白THRCI-1和重组人源化I型胶原蛋白THRCI-2均表现出良好的细胞黏附效果,具有优异的细胞粘附性能。
(3)细胞迁移实验
采用细胞划痕法测定实施例1中制备的重组人源化I型胶原蛋白促细胞迁移效果。用marker笔在6孔板背后每孔横向画3条线,线间距为0.5-1cm。向6孔板中接种2mL细胞密度为2.5×105个/mL的HFF-1细胞,以保证细胞培养24h后能达到95%-100%汇合率。24h后,用10μL枪头比着直尺对准孔板,向下轻推纵向形成划痕,并将实施例1中制备的重组人源化I型胶原蛋白用DMEM高糖培养基配成0.1mg/mL的胶原蛋白溶液,DMEM高糖培养基和0.1mg/mL牛I型胶原蛋白分别作为阴性对照和阳性对照。倒置荧光显微镜下记录0h和24h划痕面积的变化。
如图6所示,相比于空白对照组,所述重组人源化I型胶原蛋白THRCI-1和重组人源化I型胶原蛋白THRCI-2均表现出良好的促细胞迁移效果。
以上实验结果表明,本发明制备的重组人源化I型胶原蛋白具有胶原蛋白特征性的三螺旋结构和(Gly-X-Y)n重复氨基酸序列,并可自组装形成良好形貌的胶原纤维;本发明制备的重组人源化I型胶原蛋白具有人I型胶原蛋白重要的功能位点,表现出良好的生物相容性和生物活性,可以显著促进人皮肤成纤维细胞的增殖、黏附和迁移;本发明通过基因工程菌表达制备重组人源化I型胶原蛋白,工艺简单方便,易于实现工业化生产;本发明制备的高生物活性的三螺旋重组人源化I型胶原蛋白,可广泛应用于皮肤修复敷料、植入剂、人工皮肤、人工骨、人工软骨、人工眼角膜、生物材料、医疗器械、化妆品、保健品、食品等领域。
以上所述仅为本发明的个别示范性实施案例的细节,对于本领域的技术人员来说,本发明在实际应用过程中根据具体的制备条件可以有各种更改和变化,并不用于限制本发明。凡在本发明的精神和原则之内,均应包含在本发明的保护范围之内。
序列表
<110> 胶原蛋白(武汉)生物科技有限公司
<120> 一种三螺旋重组人源化I型胶原蛋白、制备方法及其应用
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Gly Gln Met Gly Pro Arg Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly
50 55 60
Ala Pro Gly Pro Ala Gly Ala Arg Gly Glu Pro Gly Ala Pro Gly Ser
65 70 75 80
Lys Gly Asp Thr Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln
85 90 95
Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys Arg Gly Ala Arg Gly
100 105 110
Glu Pro Gly Pro Thr Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu
115 120 125
Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro Gly Pro Pro Gly Pro Pro
130 135 140
Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly
145 150 155 160
Pro Pro Gly Pro Pro Gly Pro Pro
165
<210> 3
<211> 253
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met His His His His His His Ala Asp Glu Gln Glu Glu Lys Ala Lys
1 5 10 15
Val Arg Thr Glu Leu Ile Gln Glu Leu Ala Gln Gly Leu Gly Gly Phe
20 25 30
Glu Lys Lys Asn Phe Pro Thr Leu Gly Asp Glu Asp Leu Asp His Thr
35 40 45
Tyr Met Thr Lys Leu Leu Thr Tyr Leu Gln Glu Arg Glu Gln Ala Glu
50 55 60
Asn Ser Trp Arg Lys Arg Leu Leu Lys Gly Ile Gln Asp His Ala Leu
65 70 75 80
Asp Leu Val Pro Arg Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro
85 90 95
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
100 105 110
Gly Pro Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly
115 120 125
Leu Pro Gly Ala Pro Gly Gln Met Gly Pro Arg Gly Leu Pro Gly Glu
130 135 140
Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Glu Pro
145 150 155 160
Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys Gly Phe Pro Gly
165 170 175
Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys
180 185 190
Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Pro Ala Gly Pro Lys
195 200 205
Gly Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro Gly
210 215 220
Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro
225 230 235 240
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
245 250
<210> 4
<211> 253
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met His His His His His His Ala Asp Glu Gln Glu Glu Lys Ala Lys
1 5 10 15
Val Arg Thr Glu Leu Ile Gln Glu Leu Ala Gln Gly Leu Gly Gly Phe
20 25 30
Glu Lys Lys Asn Phe Pro Thr Leu Gly Asp Glu Asp Leu Asp His Thr
35 40 45
Tyr Met Thr Lys Leu Leu Thr Tyr Leu Gln Glu Arg Glu Gln Ala Glu
50 55 60
Asn Ser Trp Arg Lys Arg Leu Leu Lys Gly Ile Gln Asp His Ala Leu
65 70 75 80
Asp Leu Val Pro Arg Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro
85 90 95
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
100 105 110
Gly Pro Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly
115 120 125
Leu Pro Gly Ala Pro Gly Gln Met Gly Pro Arg Gly Leu Pro Gly Glu
130 135 140
Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Glu Pro
145 150 155 160
Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Glu Arg Gly Phe Pro Gly
165 170 175
Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys
180 185 190
Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Pro Ala Gly Pro Lys
195 200 205
Gly Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro Gly
210 215 220
Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro
225 230 235 240
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
245 250
<210> 5
<211> 759
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgcatcacc accaccacca tgctgatgaa caagaagaga aggcaaaggt gcgtaccgaa 60
ctgatccaag agctggcaca gggtcttggt ggtttcgaaa agaagaactt tccgacgctg 120
ggtgatgaag atctggacca tacctacatg accaaactgt tgacctatct ccaagaacgt 180
gaacaggcag aaaatagctg gcgtaaacgc ctgttaaaag gcattcaaga ccacgccctg 240
gatttggttc cgagaggccc gccgggcccg cccggcccgc cgggcccgcc gggtccgccg 300
ggcccgccgg gaccgccggg tcctccgggc ccaccgggcc cgccggggga gcgcggtccg 360
ccgggcccac agggcgcccg tggtttgccg ggcgcgcctg gtcagatggg tccgcgtggc 420
ctgccgggcg agcgcggtcg tccgggcgcg ccaggcccag cgggtgcgcg tggtgaaccg 480
ggcgctccgg gctccaaagg cgacaccggt gcgaagggct tcccgggtga gcgcggtgtc 540
cagggcccac cgggcccggc gggtgaggaa ggtaaacgtg gtgctcgcgg tgagccgggt 600
ccgactggtc cggctggtcc gaaaggtagc ccgggggagg cgggtcgtcc gggtgaggcg 660
ggtctgccgg gtccaccggg tccgccgggc cccccgggcc ctccgggccc accgggtccg 720
ccaggcccgc cgggtccgcc gggcccgcct ggcccgccg 759
<210> 6
<211> 759
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgcatcacc accaccacca tgctgatgaa caagaggaaa aggcgaaagt tcgtaccgaa 60
ctgattcaag aactggctca gggtttaggt ggtttcgaga agaagaattt tccgaccctg 120
ggtgatgaag atctggacca cacctacatg accaagctgt tgacctatct gcaagagcgt 180
gaacaggcag aaaacagctg gcgtaaacgt ctgttgaagg gcatccaaga tcatgcactg 240
gacttggtcc cgagaggtcc gccgggcccg ccgggtccgc cgggtccgcc gggtccgccg 300
ggtcccccgg gtccgccagg cccgccgggt ccgccgggcc cgccgggcga gcgtggtccg 360
ccgggcccac agggcgctcg cggtcttccg ggtgcccctg gccagatggg tccgcgtggt 420
ctgccgggtg agcgcggtcg cccaggcgcg ccaggcccgg ctggtgcgcg tggtgaaccg 480
ggcgcgccgg gcagcaaagg cgacactggt gagcgcggat tcccgggtga gcgcggggtg 540
cagggtccgc cgggcccggc cggtgaggaa ggtaaacgtg gcgcgcgtgg tgagccgggc 600
ccaacgggtc cggcgggtcc gaaaggttcc ccgggcgaag caggcagacc tggcgaggcg 660
ggtctcccgg gccctccggg tccgccgggt cctccgggcc cgccgggccc gccgggcccg 720
ccgggcccac caggcccgcc aggtccgccg ggcccgccg 759
Claims (10)
1.一种三螺旋重组人源化I型胶原蛋白,其特征在于,所述的三螺旋重组人源化I型胶原蛋白为胶原蛋白THRCI-1或胶原蛋白THRCI-2,所述的胶原蛋白THRCI-1的氨基酸序列如SEQ ID NO.1,所述的胶原蛋白THRCI-2的氨基酸序列如SEQ ID NO.2所示。
2.如权利要求1所述的三螺旋重组人源化I型胶原蛋白,其特征在于,所述重组人源化I型胶原蛋白由蛋白酶处理前体胶原蛋白后获得;所述的胶原蛋白THRCI-1对应的前体胶原蛋白的氨基酸序列如SEQ ID NO.3所示,所述的胶原蛋白THRCI-2对应的前体胶原蛋白的氨基酸序列如SEQ ID NO.4所示。
3.根据权利要求2所述三螺旋重组人源化I型胶原蛋白,其特征在于,所述的胶原蛋白THRCI-1对应的前体胶原蛋白1的基因序列如SEQ ID NO.5所示,所述的胶原蛋白THRCI-2对应的前体胶原蛋白2的基因序列如SEQ ID NO.6所示。
4.根据权利要求2的三螺旋重组人源化I型胶原蛋白,其特征在于,所述蛋白酶包括胃蛋白酶、胰蛋白酶、木瓜蛋白酶、菠萝蛋白酶、凝血酶。
5.一种携带权利要求4所述基因的重组载体或重组基因工程菌。
6.根据权利要求5所述的重组载体或重组基因工程菌,其特征在于,所述载体包括pCold或pET。
7.根据权利要求5所述的重组载体或重组基因工程菌,其特征在于,所述工程菌为大肠杆菌。
8.如权利要求1所述三螺旋重组人源化I型胶原蛋白的制备方法,其特征在于,步骤如下:
(1)合成编码权利要求3所述前体胶原蛋白的基因序列;
(2)将步骤(1)所述基因序列与载体连接,转化细菌,构建重组基因工程菌;
(3)表达步骤(2)构建的重组基因工程菌,收集菌体沉淀,破碎得上清液,纯化后获得前体胶原蛋白;
(4)加入蛋白酶对步骤(3)获得的前体胶原蛋白进行处理,纯化后获得三螺旋重组人源化I型胶原蛋白。
9.如权利要求8所述的制备方法,其特征在于,步骤(4)所述的蛋白酶包括胃蛋白酶、胰蛋白酶、木瓜蛋白酶、菠萝蛋白酶、凝血酶。
10.如权利要求1所述的三螺旋重组人源化I型胶原蛋白在制备皮肤修复敷料、植入剂、人工皮肤、人工骨、人工软骨、人工眼角膜、生物材料、医疗器械、化妆品、保健品、食品中的应用。
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