CN111494712B - 一种融合有nt3的丝素蛋白神经移植物的制备方法 - Google Patents
一种融合有nt3的丝素蛋白神经移植物的制备方法 Download PDFInfo
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Abstract
本发明属于生物医学材料领域,具体涉及一种融合有NT3的丝素蛋白神经移植物及其制备方法,步骤包括:合成包含有丝素蛋白轻链以及NT‑3的基因片段,连接至pET‑30表达载体,将得到的重组表达载体转入BL21大肠杆菌,得到融合蛋白;将丝素纤维网置于模具中,混合融合蛋白和丝素蛋白溶液,冷冻干燥,丝素蛋白与融合蛋白交联形成神经导管;变形处理后最终获得具有NT‑3活性的丝素蛋白神经移植物。本发明制备的丝素蛋白神经移植物在提供力学支撑的同时能够长时间的发挥神经保护以及促进神经再生的作用,同时可以根据实际情况调整神经导管中NT‑3活性肽的比例,利于神经损伤的修复。
Description
技术领域
本发明属于生物医学材料领域,具体涉及一种具有NT3活性、能够促进神经恢复的丝素蛋白神经移植物,可在周围神经、脊髓损伤、脑损伤修复再生中应用。
背景技术
随着组织工程、生物材料学等新兴学科的发展,越来越多的生物材料被用在神经修复上。优秀的生物材料应该具有良好的生物相容性,支持细胞的黏附、迁移、细胞间的相互作用、增殖以及分化。同时这些材料还需要合适的降解速率,力学性质以及有限的免疫反应,具有多种加工选择,可以根据组织的具体需要改变结构和形态。蛋白质作为天然组织的组成部分,是组织工程应用的理性选择。结构蛋白如胶原蛋白、弹力蛋白、弹性肽、白蛋白和纤维蛋白被用作缝合线、组织支架、止血剂和药物递送剂。
神经损伤包括周围神经损伤和中枢神经损伤,目前长距离周围神经损伤和脊髓损伤的治疗往往需要应用不同类型的移植物,自体神经或者异种移植物等由于来源匮乏,应用范围受限等缺点使得人们开始寻找新的组织工程移植物替代物上。蚕丝蛋白是一种常见的天然生物高分子材料,作为缝合线在人体中有着悠久的应用历史。近年来,由于它的一些特性作为生物材料逐渐受到了人们的关注。与来自同种或异种来源组织的其他蛋白基生物材料相比,丝素蛋白有几个主要优势:良好的生物相容性,优异的机械性能,可控生物降解性,加工工艺简单等等。同时丝素蛋白聚合物表现出不同的加工可塑性,通过制造技术的变化,可以实现多种基质配置,包括三维多孔泡沫、纳米纤维、水凝胶、管和薄膜,可以应用多种组织修复。神经营养因子3是由NTF3基因编码的一种蛋白,是神经生长因子家族的重要成员,可以营养周围和中枢神经系统的神经元,不仅能够有助于现有神经元的存活,还能促进新生神经元的存活和分化以及突触的连接。
目前仅仅使用丝素蛋白构建的组织工程支架功能较为单一,且促进神经组织的恢复作用较为有限。有研究通过将神经移植物浸泡在营养因子(如NGF)中或与丝素蛋白混合后冻干形成导管,在前期具有良好的促神经生长作用,但这种作用会随着时间的延长逐渐衰弱,同时营养因子在体内的不稳定性以及有限的来源使得它们的使用受到了很大的限制。
发明内容
本发明针对现有技术的不足,将神经保护因子3(NT-3)活性肽段与丝素蛋白轻链肽段相结合构成融合蛋白,以此融合蛋白为基础进行丝素蛋白的自组装获得了一种既能提供良好的力学支撑又能长时间稳定发挥神经保护功能的新型神经移植物。
本发明的技术方案如下:
一种融合有NT3的丝素蛋白神经移植物的制备方法,包括以下步骤:
合成包含有丝素蛋白轻链以及NT-3的基因片段,连接至pET-30表达载体,将得到的重组表达载体转入BL21大肠杆菌,得到融合蛋白;
将融合蛋白以及丝素蛋白溶液混合,得到混合蛋白溶液,将丝素纤维网置于模具中,将混合蛋白溶液倒入模具中冷冻干燥,形成神经导管;变性处理后最终获得具有NT-3活性的丝素蛋白神经移植物。进一步的,所述丝素纤维网由丝素纤维用编织机编织而成。
进一步的,所述丝素蛋白溶液的制备方法为:将丝素纤维放入硫氰酸锂溶液中进行溶解,溶解后的溶液装入透析袋,以三蒸水为透析液透析60~80小时,得到丝素蛋白溶液。
进一步的,所述丝素纤维的制备方法为:将桑蚕丝置于碳酸钠溶液中煮沸不少于20分钟,取出后用三蒸水充分洗涤,重复此步骤2~4次,得到脱去外部丝胶的丝素纤维。
进一步的,融合蛋白和丝素蛋白溶液的浓度为5%-40%;融合蛋白与丝素蛋白的质量比例为1:99~50:50。
进一步的,变形处理为浸泡在60%乙醇中处理10~14h。
进一步的,透析袋的截留分子量为12~16kDa。
进一步的,冷冻干燥的温度为-70℃。
进一步的,硫氰酸锂溶液的浓度为9mol/L。
本发明还提供一种融合有NT3的丝素蛋白神经移植物,由上述制备方法制备而成。
有益效果
现有技术主要采用神经营养因子吸附或共价连接结合到支架的方法,本发明采用NT-3的生物活性片段和轻链丝素蛋白同时表达在融合蛋白,该融合蛋白是以丝素蛋白为主体融合NT-3的有效成分,能够随着丝素蛋白一同被固定在丝素导管中。能够在不改变丝素蛋白的成分的前提下,能够长时间的发挥神经保护以及促进神经再生的作用。
本发明中所用到的主要材料为丝素蛋白,辅以融合NT-3活性肽段的丝素蛋白轻链,在加工过程中没有加入例如交联剂、表面活性剂等任何有毒、副作用的物质,因此具有良好的生物相容性。
本发明所述的丝素融合蛋白在体外与神经组织细胞进行共培养时,通过形态学观察,NT-3相关因子的表达测定,均显示出明显的促生长作用,其中FIBL-NT3-L-NT3的活性最强。
本发明中所述的丝素导管,在丝素蛋白结晶化之前引入了融合有NT-3功能肽段的丝素蛋白轻链,因此在丝素蛋白自组装的过程中,NT-3活性多肽将随着丝素轻链一同被固定在丝素导管中,在提供力学支撑的同时能够长时间的发挥神经保护以及促进神经再生的作用,同时可以根据实际情况调整神经导管中NT-3活性肽的比例,利于神经损伤的修复。
附图说明
图1为不同设计的含NT3的融合蛋白对体外培养的背根节神经元突起生长的影响;
图2为不同设计的含NT3的融合蛋白对体外培养的背根节神经元突起生长的影响;
具体实施方式
实施例1
一种融合有NT3的丝素蛋白神经移植物的制备方法,包括以下步骤:
1.丝素融合蛋白的表达及纯化:构建重组表达载体,表达目的蛋白后进行纯化;
2.蚕丝丝素纤维的获取:取桑蚕丝生丝,去除丝胶,获得桑蚕丝丝素纤维;
3.丝素蛋白溶液的制备;
4.丝素纤维网的制备;
5.将丝素纤维网放置在模具当中,将步骤1和3获得的蛋白溶液混合后导入模具当中,冷冻干燥使丝素蛋白进行自组装后形成神经导管。
实施例2
一种融合有NT3的丝素蛋白神经移植物的制备方法,包括以下步骤:
1.重组表达载体的构建:合成包含有丝素蛋白轻链以及NT-3的基因片段,连接至pET-30表达载体,完成重组表达载体的构建,含有标签tag和linker的不同融合蛋白序列如下所示。
融合蛋白序列(包含N端His和柔性Linker):
FIBL Protein Length=253MW=26898.8pI=6.10
MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNG NDATGLVANAQRYIAQAASQVHV
FIBL-NT3 Protein Length=372MW=40503.7pI=7.39
MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT
FIBL-linker-NT3 Protein Length=382MW=41134.1pI=7.39
MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIG RT
FIBL-(NT3)2Protein Length=501MW=54739.0pI=8.28
MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRTGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRA LTSENNKLVGWRWIRIDTSCVCALSRKIGRT
2.融合蛋白的表达及纯化:将步骤1获得的重组表达载体转入BL21大肠杆菌,37℃诱导表达后超声破碎,使用Ni-NTA纯化融合蛋白并鉴定,得到融合蛋白。
3.取适量的桑蚕丝,置于0.2%的碳酸钠溶液中煮沸30分钟,取出后用三蒸水充分洗涤,重复此步骤三次,得到脱去外部丝胶的丝素纤维,置于超净台晾干备用。
4.将丝素纤维放入9M的硫氰酸锂溶液中进行溶解,溶解后的溶液装入透析袋(截留分子量约为14kDa),以三蒸水为透析液透析72小时,得到丝素蛋白溶液。
5.将步骤3获得的丝素纤维用编织机编织丝素纤维网;
6.将步骤2得到的融合蛋白以及步骤4得到的丝素蛋白溶液按照一定比例混合,得到混合蛋白溶液,将混合蛋白溶液的浓度配置为5%-40%,融合蛋白与丝素蛋白的质量比例为1:99~50:50,将步骤5获得的丝素纤维网置于模具中,将混合蛋白溶液倒入模具中置于-70℃冷冻干燥,丝素蛋白、融合蛋白和丝素纤维网交联形成神经导管。
7.浸泡在60%乙醇中进行变形处理12h,三蒸水洗涤后晾干,最终获得了具有NT-3活性的丝素蛋白神经移植物。
融合NT-3,FIBL,FIBL-NT3,FIBL-linker-NT3,FIBL-(NT3)2的丝素蛋白促进DRG轴突的生长图(图1)和统计图(图2),统计图数据来自各组30个神经元轴突长度计数的平均值。从图中可以看出,相对空白组,NT3能够促进细胞的轴突生长,FIBL-NT3,FIBL-linker-NT3,FIBL-(NT3)能够更加有效促进轴突生长。
Claims (10)
1.一种融合有NT3的丝素蛋白神经移植物的制备方法,其特征在于,包括以下步骤:
合成包含有丝素蛋白轻链以及NT-3的基因片段,连接至pET-30表达载体,将得到的重组表达载体转入BL21大肠杆菌,得到融合蛋白;
所述融合蛋白的序列如下所示:
MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRTGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRA LTSENNKLVGWRWIRIDTSCVCALSRKIGRT;
将融合蛋白以及丝素蛋白溶液混合,得到混合蛋白溶液,将丝素纤维网置于模具中,将混合蛋白溶液倒入模具中冷冻干燥,形成神经导管;变形处理后最终获得具有NT-3活性的丝素蛋白神经移植物。
2.根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法,其特征在于,所述丝素纤维网由丝素纤维用编织机编织而成。
3.根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法,其特征在于,所述丝素蛋白溶液的制备方法为:将丝素纤维放入硫氰酸锂溶液中进行溶解,溶解后的溶液装入透析袋,以三蒸水为透析液透析60~80小时,得到丝素蛋白溶液。
4.根据权利要求2或3所述的融合有NT3的丝素蛋白神经移植物的制备方法,其特征在于,所述丝素纤维的制备方法为:将桑蚕丝置于碳酸钠溶液中煮沸不少于20分钟,取出后用三蒸水充分洗涤,重复此步骤2~4次,得到脱去外部丝胶的丝素纤维。
5.根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法,其特征在于,混合蛋白溶液的浓度为5%-40%;融合蛋白与丝素蛋白的质量比例为1:99~50:50。
6.根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法,其特征在于,变形处理为浸泡在60%乙醇中处理10~14h。
7.根据权利要求3所述的融合有NT3的丝素蛋白神经移植物的制备方法,其特征在于,透析袋的截留分子量为12~16kDa。
8.根据权利要求1所述的融合有NT3的丝素蛋白神经移植物的制备方法,其特征在于,冷冻干燥的温度为-70℃。
9.根据权利要求3所述的融合有NT3的丝素蛋白神经移植物的制备方法,其特征在于,硫氰酸锂溶液的浓度为9mol/L。
10.一种融合有NT3的丝素蛋白神经移植物,其特征在于,由权利要求1~9任一项所述的制备方法制备而成。
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