CN111187347A - 重组胶原蛋白、重组胶原海绵材料及其制备方法和应用 - Google Patents
重组胶原蛋白、重组胶原海绵材料及其制备方法和应用 Download PDFInfo
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- CN111187347A CN111187347A CN202010052422.9A CN202010052422A CN111187347A CN 111187347 A CN111187347 A CN 111187347A CN 202010052422 A CN202010052422 A CN 202010052422A CN 111187347 A CN111187347 A CN 111187347A
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- recombinant collagen
- crosslinking
- sponge material
- drying
- collagen sponge
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Abstract
本发明提供了一种重组胶原蛋白、重组胶原海绵材料及其制备方法和应用。所述重组胶原蛋白的氨基酸序列包括如SEQ ID NO:1所示氨基酸序列。所述重组胶原海绵材料是通过将所述重组胶原蛋白依次经过物理交联和化学交联后获得的;该重组胶原海绵材料的吸湿能力为40~50,孔隙率为90%以上。本发明重组胶原海绵材料具有止血、创面修复、吸湿和血小板聚集能力,其吸湿性高、止血效果显著、生物相容性佳,在外科领域中具有重要得临床意义。
Description
技术领域
本发明属于医疗技术外科技术领域,具体涉及一种重组胶原蛋白、重组胶原海绵材料及其制备方法和应用,尤其涉及一种具有止血、创面修复作用的重组胶原海绵材料及其制备方法和应用。
背景技术
在日常生活中,很多场合例如突发性事故的急救治疗、手术过程中的创伤止血等,因为未受控制的出血是导致突发事故与医疗手术中大出血的主要原因,故患者局部有效的快速止血非常重要。因此,缩短止血材料的止血时间、提高止血质量成为降低患者死亡的最佳策略。临床常用的止血材料(如纤维素类止血纱布、止血纤维、止血绷带)在使用中都有局限性,如止血时间较长、对伤口的感染和化脓无能为力、对伤口无诱导再生能力等。因此一种具有超强止血作用及良好组织修复功效的产品是外科领域亟待需要的临床产品之一。
公开号CN101890179A提供了一种水溶性止血材料,主要成分为氧化再生纤维素醚,止血效果显著,但存在强度不足的问题,且体内难以降解,吸收后可能沉积在人体其它器官中。
公开号CN104558675A提供了一种具有止血作用的微纤维胶原海绵,止血效果良好,然而其仅采用物理热交联难以保障产品的结构和质量可控,同时存在免疫原性和动物病毒风险。
公开号CN103432620A提供了一种防粘连止血膜,主要成分为右旋糖酐,交联后采用静电纺丝技术制备,生产工艺相对复杂,成本较高,且未对其作为临床产品需严格要求的生物相容性指标进行评价。
发明内容
基于现有技术中所存在的缺陷,本发明的第一目的在于提供一种重组胶原蛋白;本发明的第二目的在于提供一种具有止血、创面修复作用的重组胶原海绵材料;本发明的第三目的在于提供该重组胶原海绵材料的制备方法;本发明的第四目的在于提供该重组胶原海绵材料作为止血产品在止血、创面修复中的应用。
本发明的目的通过以下技术方案得以实现:
一方面,本发明提供一种重组胶原蛋白,该重组胶原蛋白的氨基酸序列如SEQ IDNO:1所示氨基酸序列。
SEQ ID NO:1:
GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP
发明人根据人I型胶原蛋白Gly-X-Y重复序列作为最小重复单位,创造性的采用亲水性的Gly-X-Y进行排列组合,设计长度为411氨基酸的胶原蛋白(如SEQ ID NO:1所示),并根据毕赤酵母密码子的偏好性设计相应核苷酸序列(如SEQ ID NO:2所示),然后将设计合成的核苷酸序列插入毕赤酵母的表达载体pPIC9K中,构建获得pPIC9K-COL表达载体,利用电转化法将pPIC9K-COL表达载体转化毕赤酵母宿主菌GS115,并通过抗生素G418筛选获得高拷贝菌株,最后经摇瓶筛选获得高表达毕赤酵母工程菌;采用此工程菌进行大规模生物发酵获得该重组胶原蛋白的原料。本发明的重组胶原蛋白具有优良的细胞黏附性能及亲水性能,是用于制备重组胶原海绵材料的较佳原料。
另一方面,本发明还提供编码上述重组胶原蛋白氨基酸序列的多核苷酸,所述多核苷酸的DNA序列包括SEQ ID NO:2所示DNA序列。
SEQ ID NO:2:
GGTCCTCCCGGCGAACCAGGTAATCCTGGTAAACCTGGTTCTCCCGGCCCAGCTGGTTCCAACGGGGAGCCGGGTCCTGCCGGCTCACCCGGAGAAAAGGGGTCGCAAGGTAGTAATGGCAACCCAGGACCGGCAGGGAATCAGGGTCAACCTGGCAACAAAGGAAGCCCCGGGAATCCAGGTAAGCCGGGCGAGCCTGGATCTAACGGGCCCCAGGGTGAACCAGGCTCCCAAGGAAATCCGGGGAAAAACGGTCAGCCTGGCTCACCCGGATCGCAAGGGAGTCCAGGTAATCAGGGCCAACCGGGAAAGCCTGGGCAGCCCGGTGAGCAAGGCAGCCCAGGAAACCAGGGGCCGGCGGGTAATGAAGGCCCTAAAGGACAACCCGGGCAGAACGGTAAGCCAGGATCCCCGGGTCCTCCCGGCGAACCAGGTAATCCTGGTAAACCTGGTTCTCCCGGCCCAGCTGGTTCCAACGGGGAGCCGGGTCCTGCCGGCTCACCCGGAGAAAAGGGGTCGCAAGGTAGTAATGGCAACCCAGGACCGGCAGGGAATCAGGGTCAACCTGGCAACAAAGGAAGCCCCGGGAATCCAGGTAAGCCGGGCGAGCCTGGATCTAACGGGCCCCAGGGTGAACCAGGCTCCCAAGGAAATCCGGGGAAAAACGGTCAGCCTGGCTCACCCGGATCGCAAGGGAGTCCAGGTAATCAGGGCCAACCGGGAAAGCCTGGGCAGCCCGGTGAGCAAGGCAGCCCAGGAAACCAGGGGCCGGCGGGTAATGAAGGCCCTAAAGGACAACCCGGGCAGAACGGTAAGCCAGGTACCCCAGGTCCTCCCGGCGAACCAGGTAATCCTGGTAAACCTGGTTCTCCCGGCCCAGCTGGTTCCAACGGGGAGCCGGGTCCTGCCGGCTCACCCGGAGAAAAGGGGTCGCAAGGTAGTAATGGCAACCCAGGACCGGCAGGGAATCAGGGTCAACCTGGCAACAAAGGAAGCCCCGGGAATCCAGGTAAGCCGGGCGAGCCTGGATCTAACGGGCCCCAGGGTGAACCAGGCTCCCAAGGAAATCCGGGGAAAAACGGTCAGCCTGGCTCACCCGGATCGCAAGGGAGTCCAGGTAATCAGGGCCAACCGGGAAAGCCTGGGCAGCCCGGTGAGCAAGGCAGCCCAGGAAACCAGGGGCCGGCGGGTAATGAAGGCCCTAAAGGACAACCCGGGCAGAACGGTAAGCCA
再一方面,本发明还提供包含有上述多核苷酸的表达载体。
再一方面,本发明还提供包含有上述多核苷酸的宿主菌,该宿主菌为毕赤酵母工程菌,所述毕赤酵母工程菌已进行保藏,保藏日期:2020.01.08;保藏单位:中国微生物菌种保藏管理委员会普通微生物中心(CGMCC);保藏单位地址:北京市朝阳区北辰西路1号院3号;保藏编号:CGMCC No.19314;分类命名:毕赤酵母Pichia sp.。本发明的重组胶原蛋白是通过该毕赤酵母工程菌发酵获得的。
另一方面,本发明还提供一种重组胶原海绵材料,其是通过将上述重组胶原蛋白依次经过物理交联和化学交联后获得的;该重组胶原海绵材料的吸湿能力为40~50,孔隙率为90%以上。
本发明中,吸湿能力指吸水后的重组胶原海绵材料的重量与吸水前的重组胶原海绵材料的重量的比值。孔隙率指重组胶原海绵材料的孔隙体积与重组胶原海绵材料在自然状态下的总体积的百分比。
本发明通过低度交联和冷冻干燥技术实现高孔隙率,低度交联(10%~20%)可以保证产品具有良好的溶胀性,冷冻干燥可以保证均一稳定的孔隙分布。本发明的高吸水性主要通过重组蛋白设计的氨基酸序列和互通的高孔隙率决定,设具体为计的氨基酸增加了亲水性氨基酸,提高了吸水性,互通的高孔隙率可以提供吸水保水性。
再一方面,本发明还提供上述重组胶原海绵材料的制备方法,其包括以下步骤:
将上述的重组胶原蛋白溶解于水中获得重组胶原蛋白溶液;
采用冷冻干燥法对重组胶原蛋白溶液冻干处理;
对冻干成型后的重组胶原蛋白依次进行物理交联和化学交联,获得重组胶原海绵材料。
本发明的重组胶原蛋白原料是水溶性的,具有优良的细胞黏附性能及亲水性能,经物理交联和化学交联,交联度低,保证海绵机械强度的基础上避免了材料体内残留过可能久引起的炎症风险,经过低度交联和冷冻干燥工艺后获得具有吸水性佳的胶原海绵,特殊氨基酸序列设计提高了血小板黏附能力,同时高吸水性可以浓缩血液中的血小板浓度,进而快速止血。
一般单独物理交联难以获得均匀的交联度,质量不可控;单独化学交联多需要较高的化学交联剂介入,影响终产品生物相容性。本发明采取“物理+化学”交联能够获得低交联度且较佳生物相容性能的重组胶原海绵材料。
上述的方法中,优选地,交联后获得的重组胶原海绵材料还包括进行清洗、干燥和灭菌;
上述的方法中,优选地,所述清洗采用的装置包括转动杆和固定于转动杆上的多孔夹盒;所述多孔夹盒用于夹持重组胶原海绵,所述转动杆用于旋转带动所述多孔夹盒在清洗介质中翻转,进而清洗所述重组胶原海绵材料中的残留试剂。
本发明的清洗设备为自主设计的设备,设计的多孔夹盒对重组海绵具有固定、塑型效果,通过转动杆旋转带动夹盒在清洗介质中翻转,可提高对重组海绵中残留试剂的清洗效率。由于清洗设备的高效率,低度交联剂使用,保证了产品的生物相容性。
上述的方法中,优选地,所述干燥包括烘干、冻干和真空干燥中的一种或多种的组合。
上述的方法中,优选地,所述灭菌采用的为15~25kGy的Co60辐照灭菌。
上述的方法中,优选地,所述物理交联包括热交联、辐照交联和反复冷冻复溶中的一种或多种的组合。
上述的方法中,优选地,采用热交联的温度为110℃,交联时间为2h。
上述的方法中,优选地,采用辐照交联的辐照源包括紫外射线和/或伽马射线。
上述的方法中,优选地,所述化学交联采用添加化学交联剂进行交联,所述化学交联剂包括戊二醛、碳化二亚胺和京尼平中的一种或多种的组合。
上述的方法中,优选地,所述化学交联剂的浓度为0.005~0.015mol/L。
上述的方法中,优选地,所述化学交联剂与所述重组胶原蛋白的质量比为1:1~5。
交联后,通过对氨基酸侧链上的自由氨基进行比例分析确定,本发明具有重组胶原蛋白海绵材料具备低交联度,交联度为10%~20%。
上述的方法中,优选地,所述化学交联的时间为1~5h。
上述的方法中,优选地,所述重组胶原蛋白溶液的浓度为1%~5%。
上述的方法中,优选地,对重组胶原蛋白溶液进行冷冻干燥前还包括将其注入模具成型。
上述的方法中,优选地,对重组胶原蛋白溶液进行冷冻干燥,采取-50~30℃的梯度干燥。
再一方面,本发明还提供上述重组胶原海绵材料作为止血产品在止血、创面修复中的应用。主要适用于医疗外科领域,针对术中急慢性伤口止血及创面组织修复。
本发明的重组胶原蛋白原料是水溶性的,具有优良的细胞黏附性能及亲水性能,经过低度交联和冷冻干燥工艺后获得具有吸水性佳的胶原海绵;由于清洗设备的高效率,低度交联剂使用,保证了产品的生物相容性;特殊氨基酸序列设计提高了血小板黏附能力,同时高吸水性可以浓缩血液中的血小板浓度,进而快速止血。总之,本发明重组胶原海绵材料具有止血、创面修复、吸湿和血小板聚集能力,其吸湿性高、止血效果显著、生物相容性佳,在外科领域中具有重要得临床意义。
附图说明
图1为本发明不同菌株蛋白表达量电泳检测结果图;
图2为本发明实施例中所采用的清洗设备的结构示意图;
图3为本发明实施例中重组胶原海绵材料的细胞增殖测试对比实验图;
图4为本发明实施例中重组胶原海绵材料针对新西兰兔肝脏止血20s止血性能测试对比实验图。
图5为本发明实施例中重组胶原蛋白氨基酸成分液相色谱成分分析图谱。
图6为本发明实施例中重组胶原蛋白分子量质谱检测图谱。
用于专利程序的培养物保藏:
本发明的毕赤酵母工程菌;
保藏日期:2020.01.08
保藏单位:中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)
保藏单位地址:北京市朝阳区北辰西路1号院3号
保藏编号:CGMCC No.19314
分类命名:毕赤酵母Pichia sp.
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解,现对本发明的技术方案进行以下详细说明,但不能理解为对本发明的可实施范围的限定。
以下实施例中所采用的重组胶原蛋白原料是通过构建基因工程菌,采用微生物发酵法生产分子量为38Kda的重组胶原蛋白,经纯化后冻干,获得重组胶原蛋白原料。
具体为:
根据人I型胶原蛋白Gly-X-Y重复序列作为最小重复单位,创造性的采用亲水性的Gly-X-Y进行排列组合,设计长度为411氨基酸的胶原蛋白(如SEQ ID NO:1所示),并根据毕赤酵母密码子的偏好性设计相应核苷酸序列(如SEQ ID NO:2所示),然后将设计合成的核苷酸序列插入毕赤酵母的表达载体pPIC9K中,构建获得pPIC9K-COL表达载体,利用电转化法将pPIC9K-COL表达载体转化毕赤酵母宿主菌GS115,通过不断提高遗传霉素G418在培养基中的浓度,相应筛选较高拷贝数的转化子,分别挑取高拷贝的转化子,利用摇瓶进行表达,选取表达量高的菌株作为基因工程菌用于生产,在相同电泳条件下,B31号菌株表达量相对较高(如图1所示),故以B31号菌株作为保藏的工程菌(保藏编号CGMCC No.19314)进行放大生产。采用此工程菌进行大规模生物发酵获得该重组胶原蛋白的原料。
该重组胶原蛋白的氨基酸序列如SEQ ID NO:1所示。
SEQ ID NO:1:
GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGK
本发明的重组胶原蛋白具有优良的细胞黏附性能及亲水性能,是用于制备重组胶原海绵材料的较佳原料。
以下实施例中所采用的清洗设备如图2所示,该清洗设备包括转动杆和固定于转动杆上的多孔夹盒;所述多孔夹盒用于夹持重组胶原海绵,所述转动杆用于旋转带动所述多孔夹盒在清洗介质中翻转,进而清洗所述重组胶原海绵材料中的残留试剂。
实施例1:
本实施例提供一种重组胶原海绵材料及其制备方法,该方法如下:
(1)配制3%的重组胶原蛋白溶液(分子量38Kda),搅拌均匀;将重组胶原蛋白溶液倒入模具成型,采用冷冻干燥法进行冻干处理,冻干参数为-50℃5h、-30℃3h、-20℃3h、-10℃2h、0℃1h、10℃5h、20℃20h、30℃60h;
(2)先采用紫外光进行物理交联,照射距离20cm,时间6h,之后采用0.01mol/L的碳化二亚胺进行化学交联反应(所述碳化二亚胺与所述重组胶原蛋白的质量比为1:1),反应温度4℃,时间5h;
(3)采用本发明的清洗设备进行清洗,每次20min,清洗5次;
(4)清洗后的重组胶原海绵进行干燥处理,温度60℃,时间3h;样品封装后采用15kGy Co60辐照灭菌,获得重组胶原海绵材料。
实施例2:
本实施例提供一种重组胶原海绵材料及其制备方法,该方法如下:
(1)配制5%的重组胶原蛋白溶液(分子量38Kda),搅拌均匀;将重组胶原蛋白溶液倒入模具成型,采用冷冻干燥法进行冻干处理,冻干参数为-50℃6h、-30℃2h、-20℃2h、-10℃2h、0℃2h、10℃10h、20℃20h、30℃50h;
(2)先采用高温进行物理热交联,温度110℃,时间2h,之后采用0.005mol/L的京尼平溶液进行化学交联反应(所述京尼平与所述重组胶原蛋白的质量比为1:4),反应温度25℃,时间2h;
(3)采用本发明的清洗设备进行清洗,每次15min,清洗6次;
(4)清洗后的重组胶原海绵进行冷冻干燥处理,冻干参数同第一次清洗,样品封装后采用25kGy的Co60辐照灭菌,获得重组胶原海绵材料。
实施例3:
本实施例提供一种重组胶原海绵材料及其制备方法,该方法如下:
(1)配制1%的重组胶原蛋白溶液(分子量38Kda),搅拌均匀;将重组胶原蛋白溶液倒入模具成型,采用冷冻干燥法进行冻干处理,冻干参数为-50℃6h、-30℃2h、-10℃4h、0℃2h、10℃5h、20℃20h、30℃50h;
(2)先采用γ射线进行辐射物理交联,辐射剂量35kGy,之后采用0.015mol/L的戊二醛溶液进行化学交联反应(所述戊二醛与所述重组胶原蛋白的质量比为1:4),反应温度25℃,时间1h;
(3)采用专利所述清洗设备进行清洗,每次10min,清洗9次;
(4)清洗后的重组胶原海绵进行冷冻干燥处理,冻干参数同清洗,样品封装后采用25kGy Co60辐照灭菌,获得重组胶原海绵材料。
测试实验:
1、重组胶原海绵材料的细胞增殖测试
将适量大小样品材料(实施例1)放入孔板中,将处于对数生长期的L929细胞以2×105cells的密度接种至置于孔板中的材料上,1h后每孔补加1ml培养基继续培养。20h后将材料用PBS轻柔漂洗3遍移入新的孔中用CCK-8法检测细胞量。每孔加入含10%(体积分数)CCK-8试剂的培养基1ml,培养箱孵育2h后用酶标仪于450nm处检测吸光度;增殖试验时接种细胞后培养1d、3d、5d、7d后采用CCK-8法检测细胞数量,观察细胞在材料中的生长情况。实验结果如图3所示。
由图3可以看出:空白对照组3d后开始细胞量下降,牛胶原海绵对照组5d后开始下降,试验组1~7d均呈增加趋势,说明重组胶原海绵材料可以为细胞增殖提供更有效的空间和生长环境,具有良好的引导组织修复效果。
2、重组胶原海绵材料的吸水性能测试
称重样品(实施例2)质量记为m1,生理盐水中充分吸湿(10s)后质量记为m2,根据公式:吸水倍率=(m2-m1)/m1计算每个样品的吸水倍数。结果如下表1所示,表1为两组样品吸水倍数比较。
表1:
组别 | 吸湿倍数 |
对照组(牛胶原海绵) | 22.6±2.1 |
试验组(实施例2重组胶原海绵) | 44.3±5.8* |
由表1可知,试验组的吸湿倍数显著高于对照组,差异具有统计下意义(P<0.05)。
3、重组胶原海绵材料的止血性能测试
采用新西兰兔体内肝脏止血试验进行评价,具体为,将新西兰兔逐层开腹后暴露肝脏,用刀片在兔子的肝叶作一个0.5cm*1.0cm的出血创面,立即在出血部位分别用重组胶原海绵及天然胶原海绵两种材料进行止血处理,观察止血效果及局部止血时间。观察一定时间后拿去止血材料,观察是否继续出血。实验结果如图4所示。
由图4可以看出:分别采用对照组(牛胶原海绵)和试验组(重组胶原海绵)进行止血效果观察,止血时间20s,移除材料后,试验组完全止血,对照组依然有渗血情况,未完成止血。
由以上检测评价结果可知,本发明制备的重组胶原海绵材料具有良好的临床有效性,可应用于医疗外科领域的止血及伤口修复。
4、重组胶原蛋白原料的验证实验
采用本发明的毕赤酵母工程菌进行大规模生物发酵获得重组胶原蛋白原料,分别采用N端测序、氨基酸分析和质谱法检测蛋白原料的序列和分子量,结果显示:
①N端测序
经Edman降解法检测样品的N端序列,结果为:
NH2-Gly-Pro-Pro-Gly-Glu-Pro-Gly-Asn-Pro-Gly-Lys-Pro-Gly-Ser-Pro,与设计序列一致。
②氨基酸分析
由图5可知,制备样品的氨基酸成分为G、E、S、T、A、P、K、N,与设计氨基酸成分一致;
③质谱检测:
由质谱检测图6可知,样品的分子量约为38Kda,与设计结果一致。
结论:所制备的重组胶原蛋白与所设计的要求结果一致,为38Kda的重组胶原蛋白。
序列表
<110> 陕西慧康生物科技有限责任公司
<120> 重组胶原蛋白、重组胶原海绵材料及其制备方法和应用
<130> GAI19CN6072
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 411
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<213> 人工序列()
<400> 1
Gly Pro Pro Gly Glu Pro Gly Asn Pro Gly Lys Pro Gly Ser Pro Gly
1 5 10 15
Pro Ala Gly Ser Asn Gly Glu Pro Gly Pro Ala Gly Ser Pro Gly Glu
20 25 30
Lys Gly Ser Gln Gly Ser Asn Gly Asn Pro Gly Pro Ala Gly Asn Gln
35 40 45
Gly Gln Pro Gly Asn Lys Gly Ser Pro Gly Asn Pro Gly Lys Pro Gly
50 55 60
Glu Pro Gly Ser Asn Gly Pro Gln Gly Glu Pro Gly Ser Gln Gly Asn
65 70 75 80
Pro Gly Lys Asn Gly Gln Pro Gly Ser Pro Gly Ser Gln Gly Ser Pro
85 90 95
Gly Asn Gln Gly Gln Pro Gly Lys Pro Gly Gln Pro Gly Glu Gln Gly
100 105 110
Ser Pro Gly Asn Gln Gly Pro Ala Gly Asn Glu Gly Pro Lys Gly Gln
115 120 125
Pro Gly Gln Asn Gly Lys Pro Gly Ser Pro Gly Pro Pro Gly Glu Pro
130 135 140
Gly Asn Pro Gly Lys Pro Gly Ser Pro Gly Pro Ala Gly Ser Asn Gly
145 150 155 160
Glu Pro Gly Pro Ala Gly Ser Pro Gly Glu Lys Gly Ser Gln Gly Ser
165 170 175
Asn Gly Asn Pro Gly Pro Ala Gly Asn Gln Gly Gln Pro Gly Asn Lys
180 185 190
Gly Ser Pro Gly Asn Pro Gly Lys Pro Gly Glu Pro Gly Ser Asn Gly
195 200 205
Pro Gln Gly Glu Pro Gly Ser Gln Gly Asn Pro Gly Lys Asn Gly Gln
210 215 220
Pro Gly Ser Pro Gly Ser Gln Gly Ser Pro Gly Asn Gln Gly Gln Pro
225 230 235 240
Gly Lys Pro Gly Gln Pro Gly Glu Gln Gly Ser Pro Gly Asn Gln Gly
245 250 255
Pro Ala Gly Asn Glu Gly Pro Lys Gly Gln Pro Gly Gln Asn Gly Lys
260 265 270
Pro Gly Thr Pro Gly Pro Pro Gly Glu Pro Gly Asn Pro Gly Lys Pro
275 280 285
Gly Ser Pro Gly Pro Ala Gly Ser Asn Gly Glu Pro Gly Pro Ala Gly
290 295 300
Ser Pro Gly Glu Lys Gly Ser Gln Gly Ser Asn Gly Asn Pro Gly Pro
305 310 315 320
Ala Gly Asn Gln Gly Gln Pro Gly Asn Lys Gly Ser Pro Gly Asn Pro
325 330 335
Gly Lys Pro Gly Glu Pro Gly Ser Asn Gly Pro Gln Gly Glu Pro Gly
340 345 350
Ser Gln Gly Asn Pro Gly Lys Asn Gly Gln Pro Gly Ser Pro Gly Ser
355 360 365
Gln Gly Ser Pro Gly Asn Gln Gly Gln Pro Gly Lys Pro Gly Gln Pro
370 375 380
Gly Glu Gln Gly Ser Pro Gly Asn Gln Gly Pro Ala Gly Asn Glu Gly
385 390 395 400
Pro Lys Gly Gln Pro Gly Gln Asn Gly Lys Pro
405 410
<210> 2
<211> 1233
<212> DNA
<213> 人工序列()
<400> 2
ggtcctcccg gcgaaccagg taatcctggt aaacctggtt ctcccggccc agctggttcc 60
aacggggagc cgggtcctgc cggctcaccc ggagaaaagg ggtcgcaagg tagtaatggc 120
aacccaggac cggcagggaa tcagggtcaa cctggcaaca aaggaagccc cgggaatcca 180
ggtaagccgg gcgagcctgg atctaacggg ccccagggtg aaccaggctc ccaaggaaat 240
ccggggaaaa acggtcagcc tggctcaccc ggatcgcaag ggagtccagg taatcagggc 300
caaccgggaa agcctgggca gcccggtgag caaggcagcc caggaaacca ggggccggcg 360
ggtaatgaag gccctaaagg acaacccggg cagaacggta agccaggatc cccgggtcct 420
cccggcgaac caggtaatcc tggtaaacct ggttctcccg gcccagctgg ttccaacggg 480
gagccgggtc ctgccggctc acccggagaa aaggggtcgc aaggtagtaa tggcaaccca 540
ggaccggcag ggaatcaggg tcaacctggc aacaaaggaa gccccgggaa tccaggtaag 600
ccgggcgagc ctggatctaa cgggccccag ggtgaaccag gctcccaagg aaatccgggg 660
aaaaacggtc agcctggctc acccggatcg caagggagtc caggtaatca gggccaaccg 720
ggaaagcctg ggcagcccgg tgagcaaggc agcccaggaa accaggggcc ggcgggtaat 780
gaaggcccta aaggacaacc cgggcagaac ggtaagccag gtaccccagg tcctcccggc 840
gaaccaggta atcctggtaa acctggttct cccggcccag ctggttccaa cggggagccg 900
ggtcctgccg gctcacccgg agaaaagggg tcgcaaggta gtaatggcaa cccaggaccg 960
gcagggaatc agggtcaacc tggcaacaaa ggaagccccg ggaatccagg taagccgggc 1020
gagcctggat ctaacgggcc ccagggtgaa ccaggctccc aaggaaatcc ggggaaaaac 1080
ggtcagcctg gctcacccgg atcgcaaggg agtccaggta atcagggcca accgggaaag 1140
cctgggcagc ccggtgagca aggcagccca ggaaaccagg ggccggcggg taatgaaggc 1200
cctaaaggac aacccgggca gaacggtaag cca 1233
Claims (10)
1.一种重组胶原蛋白,该重组胶原蛋白的氨基酸序列包括SEQ ID NO:1所示氨基酸序列。
2.一种编码SEQ ID NO:1所示氨基酸序列的多核苷酸,优选地,所述多核苷酸的DNA序列包括SEQ ID NO:2所示DNA序列。
3.一种含有权利要求2所述多核苷酸的表达载体。
4.一种含有权利要求2所述多核苷酸的宿主菌;该宿主菌为毕赤酵母工程菌,所述毕赤酵母工程菌的菌种保藏编号为:CGMCC No.19314。
5.一种重组胶原蛋白,其是采用权利要求4所述的宿主菌发酵获得的。
6.一种重组胶原海绵材料,其是通过将权利要求1或5所述重组胶原蛋白依次经过物理交联和化学交联后获得的;该重组胶原海绵材料的吸湿能力为40~50,孔隙率为90%以上。
7.权利要求6所述重组胶原海绵材料的制备方法,其包括以下步骤:
将权利要求1所述的重组胶原蛋白溶解于水中获得重组胶原蛋白溶液;
采用冷冻干燥法对重组胶原蛋白溶液冻干处理;
对冻干成型后的重组胶原蛋白依次进行物理交联和化学交联,获得重组胶原海绵材料;
优选地,交联后获得的重组胶原海绵材料还包括进行清洗、干燥和灭菌;
优选地,所述清洗采用的装置包括转动杆和固定于转动杆上的多孔夹盒;所述多孔夹盒用于夹持重组胶原海绵,所述转动杆用于旋转带动所述多孔夹盒在清洗介质中翻转,进而清洗所述重组胶原海绵材料中的残留试剂;
优选地,所述干燥包括烘干、冻干和真空干燥中的一种或多种的组合;
优选地,所述灭菌采用的为15~25kGy的Co60辐照灭菌。
8.根据权利要求7所述的方法,其中,所述物理交联包括热交联、辐照交联和反复冷冻复溶中的一种或多种的组合;
优选地,采用热交联的温度为110℃,交联时间为2h;
优选地,采用辐照交联的辐照源包括紫外射线和/或伽马射线。
9.根据权利要求7所述的方法,其中,所述化学交联采用添加化学交联剂进行交联,所述化学交联剂包括戊二醛、碳化二亚胺和京尼平中的一种或多种的组合;
优选地,所述化学交联剂的浓度为0.005~0.015mol/L;
优选地,所述化学交联剂与所述重组胶原蛋白的质量比为1:1~5;
优选地,所述化学交联的时间为1~5h;
优选地,所述重组胶原蛋白溶液的浓度为1%~5%;
优选地,对重组胶原蛋白溶液进行冷冻干燥前还包括将其注入模具成型;
优选地,对重组胶原蛋白溶液进行冷冻干燥,采取-50~30℃的梯度干燥。
10.权利要求6所述的重组胶原海绵材料作为止血产品在止血、创面修复中的应用。
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CN114681662A (zh) * | 2020-12-30 | 2022-07-01 | 江苏江山聚源生物技术有限公司 | 重组胶原蛋白在制备止血材料中的应用 |
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