CN115724924A - 一种可自组装成胶的重组类胶原蛋白及其制备方法与应用 - Google Patents
一种可自组装成胶的重组类胶原蛋白及其制备方法与应用 Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
一种可自组装成胶的重组类胶原蛋白及其制备方法与应用，属于生物工程技术领域。为了解决现有细菌类胶原蛋白需要经复杂的改性方法才能制备一定强度的水凝胶，进而限制其在生物医药方面应用的问题，本发明基于化脓链球菌Scl2.28的特征和主要功能序列重新优化设计获得了一种重组类胶原蛋白，该类胶原蛋白不但具有胶原蛋白的特性，而且不需要经过化学、物理及高分子材料共混等复杂的改性方法，仅通过调整pH值就能迅速自组装成一定强度的水凝胶，还可利用光化学交联的方式来获得物理性能更好的水凝胶。本发明获得的重组类胶原蛋白在医学应用和组织工程等方面具有更加广阔的应用价值。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种可自组装成胶的重组类胶原蛋白及其制备方法与应用。
背景技术
胶原蛋白是动物体内最丰富的蛋白质。经过数十年的研究，人们至少发现28种不同类型的胶原蛋白。尽管这些胶原蛋白的生物功能和物理特性差异巨大，但均具有特征性的三重螺旋结构。该结构由3条α肽链以右手螺旋的方式形成，每条α肽链又由重复的Gly-Xaa-Yaa序列组成，这样的三股螺旋区域被称为胶原区域。在Xaa和Yaa位置上的氨基酸通常为脯氨酸，Yaa位置的脯氨酸被翻译后修饰为羟脯氨酸(Hyp)，从而增强三螺旋结构的稳定性。这些结构特征决定胶原蛋白具有良好的物理和生物学特性，使其在食品、医学、化工以及生物材料等领域有着广泛的应用(CN105285645A、CN106806942A、CN104878473A、CN1556715A)。
在过去的几十年中，胶原蛋白作为生物医用材料在医疗产品和临床中发挥着重要作用(CN105664229A)。由于胶原蛋白主要来源于动物组织，存在着病原体或朊病毒污染的风险，这在一定程度上限制基于天然胶原蛋白生物医用材料的进一步发展。在酵母和植物系统中生产的重组胶原蛋白，虽然能够避免这些问题的发生(CN106256911A)。但是，由于表达过程中必须将脯氨酰4-羟化酶引入到重组的生物中来实现脯氨酸的羟化和重组胶原蛋白的稳定性，这无疑增加了大规模生产的难度。
近年来人们在细菌中发现具有类似动物胶原蛋白特征性三螺旋结构的类胶原蛋白，这类蛋白能够在不存在羟脯氨酸的条件下形成稳定的三螺旋结构。更重要的是，它能够通过重组技术在大肠杆菌系统中高效表达，产生的蛋白产物生物相容性良好且无免疫原性、无细胞毒性。来源于细菌性的化脓链球菌Scl2(Streptococcus pyogenes collagen-like protei n 2gene)便是其中的一种类胶原蛋白(CN105859892A)。但是，目前研究出的细菌性类胶原蛋白需要经过化学、物力及高分子材料共混等复杂的改性方法才能制备一定强度的水凝胶，这使得细菌性类胶原蛋白在生物医药方面的应用受到了一定的限制。因此，亟需开发一种能够不需要经过复杂改性方法就能迅速自组装成一定强度水凝胶的细菌性类胶原蛋白。
发明内容
本发明为解决现有细菌类胶原蛋白需要经复杂的改性方法才能制备一定强度的水凝胶，进而限制其在生物医药方面应用的问题，提供了一种可自组装成胶的重组类胶原蛋白，该重组类胶原蛋白是在scl2类胶原蛋白的基础上将胶原区域替换为SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4或SEQ ID No.5任一所示的氨基酸序列，将非胶原区域替换为SEQ ID No.6所示的氨基酸序列后获得的；所述scl2类胶原蛋白的Gene ID：110660。
本发明还提供了一种上述重组类胶原蛋白的制备方法，该方法包括如下步骤：
(1)根据化脓链球菌(Streptococcuspyogenes)Scl2.28的cDNA序列，合成重组的Scl2的全序列，并以合成后的全序列为模板PCR扩增合成DNA片段，再经酶切、连接、转化大肠杆菌获得Scl2蛋白重组表达载体；
(2)将步骤(1)获得的Scl2蛋白重组表达载体转化至大肠杆菌中获得阳性菌株；
(3)将步骤(2)获得的阳性菌株于LB培养基中培养至OD600达到0.4～0.6时，向菌液中加入IPTG，继续培养5h～6h，对菌液进行离心或过滤后，去除上清并收集菌体沉淀；
(4)向步骤(3)获得的菌体中加入蛋白提取液和溶菌酶，于冰上静置1h，获得蛋白溶液，经超声后，通过离心或过滤收集上清获得粗蛋白；
(5)利用已装镍的重力柱对步骤(4)获得的粗蛋白进行纯化，经水透析后获得重组后的类胶原蛋白。
进一步地限定，步骤(1)所述PCR扩增使用的上游引物的核苷酸序列如SEQ IDNo.7所示，下游引物的核苷酸序列如SEQ ID No.8所示。
进一步地限定，步骤(1)所述酶切是将扩增后的DNA片段及Pet28a空载体经过NcoI和Xho I限制性内切酶进行双酶切；所述连接使用的连接酶为T4 DNA连接酶；所述转化利用的大肠杆菌为DH5α。
本发明还提供了一种上述重组类胶原蛋白的自组装成胶方法，该方法是将重组类胶原蛋白与0.1～0.2M的盐酸或醋酸混合，制成25～50mg/mL的酸溶液，利用1～2M的NaOH溶液将pH调节至6～8，室温静置1～2min即可成胶。
本发明还提供了另外一种上述重组类胶原蛋白的自组装成胶方法，该方法是将重组类胶原蛋白与0.1～0.2M的盐酸或醋酸混合，制成25～50mg/mL的酸溶液，然后向酸溶液中加入透明质酸或硫酸软骨素，利用1～2M的NaOH溶液将pH调节至6～8，室温静置5～30min即可成胶。
进一步地限定，所述透明质酸和硫酸软骨素的添加量为酸溶液的0.1％～0.5％。
进一步地限定，上述加入透明质酸或硫酸软骨素的成胶方法在静置5～30min后可成胶；以质量分数记透明质酸和硫酸软骨素的添加量为0.1％～0.5％。
本发明还提供了一种基于上述重组类胶原蛋白的可光化学交联水凝胶的制备方法，该方法具体步骤如下：
S1、将重组类胶原蛋白与盐酸溶液或醋酸溶液混合，制成25～50mg/mL的酸溶液；
S2、向S1的酸溶液中加入甲基丙烯酸酐，获得溶液后于4℃条件搅拌8-10h；
S3、将S2获得的溶液置于透析袋中，以水为透析液，于4℃搅拌40-50h，冷冻风干；此步骤全程避光；
S4、将S3获得的固体蛋白与盐酸溶液或醋酸溶液充分混合，配置成浓度为50mg/mL的酸溶液；
S5、加入NaOH溶液调节溶液pH至5～8，然后加入终浓度为0.1g/mL的PEGDA，振荡混匀后加入终浓度为10％的光引发剂，充分溶解后置于紫外灯下照射30min，形成光交联凝胶。
进一步地限定，所述制备方法中S1和S4所述盐酸溶液或醋酸溶液的浓度为0.1～0.2M；S5所述NaOH溶液的浓度为1～2M。
进一步地限定，S2所述甲基丙烯酸酐的添加量根据下式计算：重组胶原蛋白的质量(g)/类胶原蛋白摩尔质量×羧基百分比×甲基丙烯酸酐的摩尔质量/甲基丙烯酸酐的密度。
本发明的有益效果：
1、本发明基于化脓链球菌Scl2.28的特征和主要功能序列重新优化设计，构建得到重组类胶原蛋白。与胶原蛋白类似，本发明获得的重组类胶原蛋白由含重复的Gly-Xaa-Yaa序列的胶原区域和可变的非胶原区域组成，该蛋白同样具备特征性的三重螺旋结构，以及能够利用大肠杆菌进行大规模生产等类胶原蛋白的特点。更加难得的是，该蛋白能够在无任何化学和物理修饰的条件下，在中性条件下自组装成水凝胶。这一特性在之前的类胶原蛋白甚至重组的Scl2类胶原蛋白研究中未见相关报道，这对于研究类胶原蛋白自组装成胶开辟新的研究思路。
2、本发明提供的重组类胶原蛋白制备中的纯化和提取方法，是基于镍柱亲和层析的基础上，选择合适的洗脱液对重组类胶原蛋白进行纯化，纯化过程步骤简单，因其带有特异化标签，易于得到高纯度的产品。
3、本发明通过添加透明质酸和硫酸软骨素以及光化学交联法对重组类胶原蛋白进行改性，使改性后的重组类胶原蛋白更加符合实际生产应用。添加透明质酸和硫酸软骨素主要使重组类胶原蛋白水凝胶的成胶时间得到有效的控制，减缓自组装成胶的时间，便于后续的实际操作。另外，本研究中的光化学交联主要使用MA改性重组类胶原蛋白，再利用紫外光照射的方法制备具有三维结构的PEGDA和改性的Scl2类胶原蛋白复合的水凝胶。该复合水凝胶弹性和延展性等机械性能优良。这些改性使得本发明获得的重组类胶原蛋白在医学应用和组织工程等方面具有更加广阔的应用价值。
附图说明
图1为本发明所述重组类胶原蛋白纯化的电泳图；
图2为本发明获得的重组类胶原蛋白的圆二色谱图；
图3为基于本发明获得的重组类胶原蛋白制备的自组装水凝胶状态图；
图4为本发明获得的重组类胶原蛋白的扫描电镜图；
图5为基于本发明获得的重组类胶原蛋白的自组装水凝胶的机械强度测试图；
图6为基于本发明获得的重组类胶原蛋白的光交联后水凝胶的机械强度测试图。
具体实施方式
以下结合具体实施例和附图，对本发明作进一步的详细说明，实施本发明的过程、条件、试剂、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。
实施例1：一种可自组装成胶的重组类胶原蛋白
一种具有较高热稳定性的重组Scl2类胶原蛋白，由胶原区域和非胶原区域组成，非胶原区域位于胶原区域的羧基端。该重组Scl2类胶原蛋白是在scl2类胶原蛋白的基础上将胶原区域替换为SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4或SEQ ID No.5任一所示的氨基酸序列，将非胶原区域替换为SEQ ID No.6所示的氨基酸序列后获得的；所述scl2类胶原蛋白的Gene ID：110660。
胶原区域的氨基酸序列根据生物特性和物理性能的不同划分为以下5种：
胶原区域-1的氨基酸序列为SEQ ID No.1：
5’-GQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPLEHHHHHH-3’
胶原区域-2的氨基酸序列为SEQ ID No.2：
5’-GQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPLEHHHHHH-3’
胶原区域-3的氨基酸序列为SEQ ID No.3：
5’-GQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGLAGQRGIVGLPGQRGERGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPLEHHHHHH-3’
胶原区域-4的氨基酸序列为SEQ ID No.4：
5’-GQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGLAGQRGIVGLPGQRGERGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGDKGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPLEHHHHHH-3’
胶原区域-5的氨基酸序列为SEQ ID No.5：
5’-GQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGLAGQRGIVGLPGQRGERGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGDKGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGLAGQRGIVGLPGQRGERGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGDKGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPLEHHHHHH-3’
非胶原区域的氨基酸序列为SEQ ID No.6：
5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
实施例2：实施例1所述重组类胶原蛋白的制备方法
实施例1所述的重组类胶原蛋白的制备方法包括如下步骤：
(1)根据化脓链球菌(Streptococcuspyogenes)Scl2.28的cDNA序列，在华大青兰生物科技(无锡)有限公司合成重组的Scl2的全序列，并以合成后的全序列为模板，使用核苷酸序列如SEQ ID No.7(5’-ATATCTCGAGAGGTTTACCAGGCTGGCCATCT-3’)所示的上游引物和核苷酸序列如SEQ ID No.8(5’-TATACCATGGGCAGCAGCCATCATCATCA-3’)所示的下游引物PCR扩增合成DNA片段；扩增后的DNA片段及Pet28a空载体经过Nco I和Xho I限制性内切酶进行双酶切得到酶切产物，用1％的琼脂糖凝胶电泳分离，切取目的片段，再用凝胶DNA回收试剂盒(TIANGEN)纯化；纯化后的片段和载体在T4 DNA连接酶的作用下进行16℃过夜连接，获得连接产物；利用热激转化法转入大肠杆菌DH5α中，涂布至含有卡纳抗生素的固体LB培养基平板中，挑取单克隆，转移至含有预先加入卡纳抗生素培养基的试管中，37℃震荡过夜培养，收集菌液用质粒小提试剂盒(TIANGEN)制备质粒，并经DNA测序确认其序列；
(2)将步骤(1)测序正确的Scl2蛋白重组表达载体转化至大肠杆菌BL21(DE3)菌株中，涂布至含有卡纳抗生素的固体LB培养基平板中，挑取单克隆；
(3)将步骤(2)获得的单克隆菌株转移至含有预先加入卡纳抗生素培养基的试管中，37℃震荡过夜培养，获得菌液，再将菌液转移至预先加入卡纳抗生素的LB培养基中，37℃震荡培养至OD600达到0.4～0.6时，向菌液中加入IPTG，继续培养5h～6h，对菌液进行离心或过滤后，去除上清并收集菌体沉淀；
(4)向步骤(3)获得的菌体中加入蛋白提取液和溶菌酶，于冰上静置1h，获得蛋白溶液，经超声破碎后，通过离心或过滤收集上清获得粗蛋白；
(5)利用已装镍的重力柱对步骤(4)获得的粗蛋白进行纯化，镍柱无剩余粗蛋白溶液时，加入BufferA(50mM Tris，250mM NaCl，30mM咪唑)进行杂蛋白的清洗，再向镍柱中加入Buffer B(50mM Tris，250mM NaCl，400mM咪唑)将目的蛋白洗脱下来，将洗脱下来的目的蛋白直接用蒸馏水进行透析，透析时间为每次6h，共2次，透析后的溶液进行冷冻风干即可获得重组后的细菌性类胶原蛋白。目的蛋白的电泳图如图1所示，获得的重组后的细菌性类胶原蛋白的扫面电镜图和圆二色谱图分别如图2和图4所示。
实施例3：实施例1所述重组类胶原蛋白的自组装成胶方法
实施例1所述重组类胶原蛋白的自组装成胶方法，包括但不限于一下方式：
第一种：称取重组的类胶原蛋白，将其与0.1～0.2M HCl充分混合，配制成浓度为25mg/mL的酸溶液；之后缓慢加入2M NaOH调节溶液pH，使最终的pH在6～8；最后，室温静置1min内即可成胶。
第二种：称取重组的类胶原蛋白，将其与0.1～0.2M HCl充分混合，配制成浓度为50mg/mL的酸溶液；之后缓慢加入2M NaOH调节溶液pH，使最终的pH在6～8；最后，室温静置1min内即可成胶。该方法制备获得的自组装水凝胶状态如图3所示，机械强度测试结果如图5所示。
第三种：称取重组的类胶原蛋白，将其与0.1～0.2M CH3COOH充分混合，配制成浓度为25mg/mL的酸溶液；之后缓慢加入1MNaOH调节溶液pH，使最终的pH在6～8；最后，室温静置1～2min即可成胶。
第四种：称取重组的类胶原蛋白，将其与0.1～0.2M CH3COOH充分混合，配制成浓度为50mg/mL的酸溶液；之后缓慢加入1MNaOH调节溶液pH，使最终的pH在6～8；最后，室温静置1～2min即可成胶。
实施例4：实施例1所述重组类胶原蛋白的自组装成胶方法
实施例1所述重组类胶原蛋白的自组装成胶方法，包括但不限于一下方式：
第一种：称取重组的类胶原蛋白，将其与0.1～0.2M HCl充分混合，配制成浓度为25mg/mL的酸溶液，然后加入0.1％～0.5％的透明质酸；溶解均匀后缓慢加入2M NaOH调节溶液pH，使最终的pH在6～8；最后，室温静置5～30min即可成胶。
第二种：称取重组的类胶原蛋白，将其与0.1～0.2M HCl充分混合，配制成浓度为50mg/mL的酸溶液，然后加入0.1％～0.5％的透明质酸；溶解均匀后缓慢加入2M NaOH调节溶液pH，使最终的pH在6～8；最后，室温静置5～30min即可成胶。
第三种：称取重组的类胶原蛋白，将其与0.1～0.2M CH3COOH充分混合，配制成浓度为25mg/mL的酸溶液，然后加入0.1％～0.5％的透明质酸；溶解均匀后缓慢加入1MNaOH调节溶液pH，使最终的pH在6～8；最后，室温静置5～30min即可成胶。
第四种：称取重组的类胶原蛋白，将其与0.1～0.2M CH3COOH充分混合，配制成浓度为50mg/mL的酸溶液，然后加入0.1％～0.5％的透明质酸；溶解均匀后缓慢加入1MNaOH调节溶液pH，使最终的pH在6～8；最后，室温静置5～30min即可成胶。
第五种：称取重组的类胶原蛋白，将其与0.1～0.2M HCl充分混合，配制成浓度为25mg/mL的酸溶液，然后加入0.1％～0.5％的硫酸软骨素；溶解均匀后缓慢加入2M NaOH调节溶液pH，使最终的pH在6～8；最后，室温静置5～30min即可成胶。
第六种：称取重组的类胶原蛋白，将其与0.1～0.2M HCl充分混合，配制成浓度为50mg/mL的酸溶液，然后加入0.1％～0.5％的硫酸软骨素；溶解均匀后缓慢加入2M NaOH调节溶液pH，使最终的pH在6～8；最后，室温静置5～30min即可成胶。
第七种：称取重组的类胶原蛋白，将其与0.1～0.2M CH3COOH充分混合，配制成浓度为25mg/mL的酸溶液，然后加入0.1％～0.5％的硫酸软骨素；溶解均匀后缓慢加入1MNaOH调节溶液pH，使最终的pH在6～8；最后，室温静置5～30min即可成胶。
第八种：称取重组的类胶原蛋白，将其与0.1～0.2M CH3COOH充分混合，配制成浓度为50mg/mL的酸溶液，然后加入0.1％～0.5％的硫酸软骨素；溶解均匀后缓慢加入1MNaOH调节溶液pH，使最终的pH在6～8；最后，室温静置5～30min即可成胶。
实施例5：基于实施例1所述重组类胶原蛋白的可光化学交联水凝胶的制备方法
一种基于权利要求1所述重组类胶原蛋白的可光化学交联水凝胶的制备方法包括如下步骤：
S1、用0.1～0.2M的盐酸溶液或醋酸溶液充分溶解重组类胶原蛋白，配置成浓度为25～50mg/ml的酸溶液；
S2、向S1的酸溶液中加入甲基丙烯酸酐(MA)，获得溶液后于4℃条件搅拌8-10h，甲基丙烯酸酐的添加量根据下式计算：重组胶原蛋白的质量(g)/类胶原蛋白摩尔质量×羧基百分比×甲基丙烯酸酐的摩尔质量/甲基丙烯酸酐的密度；
S3、将S2获得的溶液置于透析袋中，以水为透析液，于4℃搅拌40-50h，冷冻风干；此步骤全程避光；
S4、将S3获得的固体蛋白与0.1～0.2M盐酸溶液或醋酸溶液充分混合，配置成浓度为50mg/ml的酸溶液；
S5、加入1～2M NaOH溶液调节溶液pH至5～8，然后加入终浓度为0.1g/mL的PEGDA，振荡混匀后加入终浓度约为10％的光引发剂，充分溶解后置于紫外灯下照射30min，形成光交联凝胶。
本实施例获得的光交联后水凝胶的机械强度测试图如图6所示。
SEQUENCE LISTING
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Claims (10)
1.一种可自组装成胶的重组类胶原蛋白，其特征在于，所述重组类胶原蛋白是在scl2类胶原蛋白的基础上将胶原区域替换为SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ IDNo.4或SEQ ID No.5任一所示的氨基酸序列，将非胶原区域替换为SEQ ID No.6所示的氨基酸序列后获得的；所述scl2类胶原蛋白的GeneID：110660。
2.一种权利要求1所述重组类胶原蛋白的制备方法，其特征在于，包括如下步骤：
(1)根据化脓链球菌(Streptococcuspyogenes)Scl2.28的cDNA序列，合成重组的Scl2的全序列，并以合成后的全序列为模板PCR扩增合成DNA片段，再经酶切、连接、转化大肠杆菌获得Scl2蛋白重组表达载体；
(2)将步骤(1)获得的Scl2蛋白重组表达载体转化至大肠杆菌中获得阳性菌株；
(3)将步骤(2)获得的阳性菌株于LB培养基中培养至OD600达到0.4～0.6时，向菌液中加入IPTG，继续培养5h～6h，对菌液进行离心或过滤后，去除上清并收集菌体沉淀；
(4)向步骤(3)获得的菌体中加入蛋白提取液和溶菌酶，于冰上静置1h，获得蛋白溶液，经超声后，通过离心或过滤收集上清获得粗蛋白；
(5)利用已装镍的重力柱对步骤(4)获得的粗蛋白进行纯化，经水透析后获得重组后的类胶原蛋白。
3.根据权利要求2所述的制备方法，其特征在于，步骤(1)所述PCR扩增使用的上游引物的核苷酸序列如SEQ ID No.7所示，下游引物的核苷酸序列如SEQ ID No.8所示。
4.根据权利要求2所述的制备方法，其特征在于，步骤(1)所述酶切是将扩增后的DNA片段及Pet28a空载体经过NcoI和XhoI限制性内切酶进行双酶切；所述连接使用的连接酶为T4DNA连接酶；所述转化利用的大肠杆菌为DH5α。
5.一种权利要求1所述重组类胶原蛋白的自组装成胶方法，其特征在于，将重组类胶原蛋白与0.1～0.2M的盐酸或醋酸混合，制成25～50mg/mL的酸溶液，利用1～2M的NaOH溶液将pH调节至6～8，室温静置1～2min即可成胶。
6.一种权利要求1所述的重组胶原蛋白的自组装成胶方法，其特征在于，将重组类胶原蛋白与0.1～0.2M的盐酸或醋酸混合，制成25～50mg/mL的酸溶液，然后向酸溶液中加入透明质酸或硫酸软骨素，利用1～2M的NaOH溶液将pH调节至6～8，室温静置5～30min即可成胶。
7.根据权利要求6所述的自组装成胶方法，其特征在于，所述透明质酸和硫酸软骨素的添加量为酸溶液的0.1％～0.5％。
8.一种基于权利要求1所述重组类胶原蛋白的可光化学交联水凝胶的制备方法，其特征在于，具体步骤如下：
S1、将重组类胶原蛋白与盐酸溶液或醋酸溶液混合，制成25～50mg/mL的酸溶液；
S2、向S1的酸溶液中加入甲基丙烯酸酐，获得溶液后于4℃条件搅拌8-10h；
S3、将S2获得的溶液置于透析袋中，以水为透析液，于4℃搅拌40-50h，冷冻风干；此步骤全程避光；
S4、将S3获得的固体蛋白与盐酸溶液或醋酸溶液充分混合，配置成浓度为50mg/mL的酸溶液；
S5、加入NaOH溶液调节溶液pH至5～8，然后加入终浓度为0.1g/mL的PEGDA，振荡混匀后加入终浓度为10％的光引发剂，充分溶解后置于紫外灯下照射30min，形成光交联凝胶。
9.根据权利要求8所述的制备方法，其特征在于，所述制备方法中S1和S4所述盐酸溶液或醋酸溶液的浓度为0.1～0.2M；S5所述NaOH溶液的浓度为1～2M。
10.根据权利要求8所述的制备方法，其特征在于，S2所述甲基丙烯酸酐的添加量根据下式计算：重组胶原蛋白的质量(g)/类胶原蛋白摩尔质量×羧基百分比×甲基丙烯酸酐的摩尔质量/甲基丙烯酸酐的密度。
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| CN101148479A (zh) * | 2007-09-11 | 2008-03-26 | 浙江理工大学 | 一种重组类人胶原蛋白及生物合成方法 |
| US20140163205A1 (en) * | 2009-07-17 | 2014-06-12 | The Texas A&M University System | Designer collagens and use thereof |
| CN104039965A (zh) * | 2011-11-16 | 2014-09-10 | 联邦科学工业研究组织 | 胶原蛋白样丝基因 |
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| CN101148479A (zh) * | 2007-09-11 | 2008-03-26 | 浙江理工大学 | 一种重组类人胶原蛋白及生物合成方法 |
| US20140163205A1 (en) * | 2009-07-17 | 2014-06-12 | The Texas A&M University System | Designer collagens and use thereof |
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