CN114592023A - 一种细胞裂解自组装多肽复合物、自组装方法、自组装多肽制剂及应用 - Google Patents
一种细胞裂解自组装多肽复合物、自组装方法、自组装多肽制剂及应用 Download PDFInfo
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Abstract
本申请涉及一种细胞裂解自组装多肽复合物、自组装方法、自组装多肽制剂及应用，涉及生物技术的领域。细胞裂解多肽复合物自组装方法，将嗜热栖热菌进行细胞裂解、离心、蛋白酶酶解后得到上清酶解液，将上清酶解液加入到嗜热栖热菌种中或嗜热栖热菌发酵液中进行发酵自组装，得到细胞裂解自组装多肽复合物。本申请具有提升多肽自组装效率，提升细胞裂解多肽活性和抗菌消炎的效果。
Description
技术领域
本申请涉及生物技术的领域，尤其是涉及一种细胞裂解自组装多肽复合物、自组装方法、自组装多肽制剂及应用。
背景技术
多肽分子的自组装是在构成系统的分子在热力学平衡的条件下，通过自身一系列的非共价作用力而自发地组装、排列、整合，并最终形成均一有序的自组装体的过程。多肽的自组装主要依靠分子内部多种非共价作用力相互制约、达到平衡后达成的结果，这些作用包括疏水作用、氢键、静电作用、兀-兀共轭作用等等。这些非共价键作用力虽然微弱，但依靠多肽分子数目上的优势，仍能有效地驱动自组装。多肽分子自组装经过了三十多年的发展，多肽分子自组装系统已经成为分子生物学、化学、材料学和工程技术等学科的交汇热点，有着广阔的应用前景。
自组装多肽被应用于多种用途，包括三维细胞培养支架，止血剂，鼻窦手术后止血和防粘连剂，黏膜填充剂，3D打印和组织工程支架，药物控释等领域。
目前，较多的研究针对于自组装单肽的设计，而细菌发酵产生的各种多肽复合物的自组装还需要进一步研究以促进其应用，鉴于此，特提出本申请。
发明内容
为了提高细胞裂解多肽活性和抗菌消炎的效果，本申请提供一种细胞裂解自组装多肽复合物、自组装方法、自组装多肽制剂及应用。
第一方面，本申请提供一种细胞裂解多肽复合物自组装方法，采用如下的技术方案：
一种细胞裂解多肽复合物自组装方法，将嗜热栖热菌进行细胞裂解、离心、蛋白酶酶解后得到上清酶解液，将上清酶解液加入到嗜热栖热菌种中或嗜热栖热菌发酵液中进行发酵自组装，得到细胞裂解自组装多肽复合物。
通过采用上述方案，嗜热栖热菌发酵液或嗜热栖热菌种中的酶和发酵物对上清酶解液中的多肽促组装，降低多肽自组装临界浓度，从而形成多形态的多肽复合物。相比于未进行自组装的多肽复合物，自组装后的多肽复合物具有更好的抗水解稳定性，也产生了更强的抗菌消炎、抗病毒作用。
另外多肽分子内部非共价作用力达到平衡时，它可以吸收和固定大量水分子，形成水凝胶，而组装后的电荷分布变化及二级结构变化等因素，大大改善了细胞毒性。
在自组装后，电荷分布和二级结构都可能发生变化，这都直接影响多肽后续与细胞膜的相互作用，使多肽的细胞活性增强。
可选的，将上清酶解液与嗜热栖热菌种混合，加入发酵底物进行发酵自组装。
可选的，所述发酵底物包括可溶性钙盐、可溶性锰盐、硫酸铜和/或氨基酸。
可选的，所述发酵底物包括氯化钙、硫酸锰、硫酸铜和/或精氨酸和/或赖氨酸和/或谷氨酸。
通过采用上述方案，氯化钙、硫酸锰不仅可作为嗜热栖热菌种的发酵底物，同时也具有多肽促组装，降低多肽自组装临界浓度。同添加硫酸铜和/或氨基酸，可进一步对多肽促组装，降低多肽自组装临界浓度，提升多肽自组装的效率。
可选的，将上清酶解液、嗜热栖热菌种和发酵底物混合，在温度65-85℃，pH 7.0-8.5的条件下，发酵1-4小时，除去菌液；在65-85℃，pH 7.0-8.5条件下组装12-18小时。
可选的，将上清酶解液与嗜热栖热菌发酵液混合，在温度65-85℃，pH 7.0-8.5的条件下组装12-18小时。
通过采用上述方案，在上述温度和pH条件下进行自组装，多肽复合物可形成较优的表征组合。
可选的，所述蛋白酶包括巯基蛋白酶、碱性蛋白酶、中性蛋白酶、酸性蛋白酶中的一种或多种的组合。
可选的，所述蛋白酶包括木瓜蛋白酶、菠萝蛋白酶、无花果蛋白酶中的一种或多种的组合。
可选的，所述蛋白酶包括木瓜蛋白酶、菠萝蛋白酶、无花果蛋白酶按照质量比为1：（2-3）：2混合而成。
第二方面，本申请提供一种自装多肽复合物，采用如下的技术方案：
一种自装多肽复合物，采用上述细胞裂解多肽复合物自组装方法制成。
第三方面，本申请提供一种自装多肽复合物制剂，采用如下的技术方案：
一种自装多肽复合物制剂，包括上述细胞裂解自组装多肽复合物。
第四方面，本申请提供上述细胞裂解自组装多肽复合物或上述自装多肽复合物制剂在制备消炎、舒敏、抗衰老、抗皱、修复、紧致的化妆品、护肤品或保健品的应用，采用如下技术方案。
上述细胞裂解自组装多肽复合物或上述自装多肽复合物制剂在制备消炎、舒敏、抗衰老、抗皱、修复、紧致的化妆品、护肤品或保健品的应用。
综上所述，本申请包括以下至少一种有益技术效果：
1．采用细胞裂解发酵进行多肽自组装，裂解肽的自组装临界浓度大大降低，且提高了组装效率。
1.自组装后的多肽，具有更好的抗水解稳定性，也产生了更强的抗菌消炎、抗病毒作用；
2.多个多肽分子内部非共价作用力达到平衡时，它可以吸收和固定大量水分子，形成水凝胶，而组装后的电荷分布变化及二级结构变化等因素，大大改善了细胞毒性；
3.多肽的细胞活性增强：在自组装后，电荷分布和二级结构都可能发生变化，这都直接影响多肽后续与细胞膜的相互作用，最终影响了其生物活性。
附图说明
图1是本申请荧光探针1,8-ANS的测试结果图。
具体实施方式
本申请所使用的嗜热栖热菌为嗜热栖热菌HB27，购于美国模式菌种收集中心(ATCC)，保藏号是BAA-163的菌株。
其中，嗜热菌的发酵方法如下，单独的嗜热栖热菌发酵产物的具体实施方式如下：
培养液配制：20L培养液的配比为60g蛋白胨、4g麦芽汁、7g无水硫酸镁、2 .4g磷酸二氢钾、40g硫酸铵、1g无水氯化铁、5g氯化钠，pH值用酸或碱进行调节控制，水补足到20L，不同量的培养液按照配比比例进行缩小或放大。
1.菌种扩大培养
在超净工作台上取嗜热栖热菌HB27冻存液2mL接入装有2L培养液(pH为7 .8±0.2)的2 .5L三角瓶中，通气量维持在5m3/h/50L ,150rpm，65℃培养12h左右，菌体处于对数生长期后，将30L菌液接种到300L培养液的500L二级500L种子罐中。
2.发酵
将300L嗜热栖热菌增菌液转移到含有2700L培养液的发酵罐中，通气量维持在5m3/h/50L ,150rpm搅拌，pH值调节到7 .8±0 .2，150rpm，65℃培养18h左右，得到嗜热栖热菌发酵液。
实施例
实施例1
细胞裂解多肽复合物自组装方法，包括以下步骤：
S1、细胞裂解
取发酵液在4,000rpm，30min，4℃条件下离心收集菌体；用匀浆缓冲液(20mmol/LTris-Cl pH8.0，100mmol/L NaCl, 2mmol/L MgCl2，1mmol/ L DTT)重悬菌体，在6,000rpm，10min，4℃条件下离心去上清；加入匀浆缓冲液重悬沉淀（1g沉淀加入10ml匀浆缓冲液），再加入终浓度为1mmol/L的PMSF，冰浴超声破碎30min（振幅为55%，超声9s，停5s），得到细胞裂解液。
S2、离心
将细胞裂解液在4,000rpm，30min，4℃条件下离心，得到上清液。
S3、酶解
上清液中加入0.5%wt的蛋白酶，在55℃下进行酶解，得到上清酶解液；具体的，蛋白酶包括木瓜蛋白酶、菠萝蛋白酶、无花果蛋白酶按照质量比为1：2：2混合而成。
S4、发酵组装
将上清酶解液与嗜热栖热菌种按体积比1:20混合，加入发酵底物进行发酵反向自组装，发酵底物包括可溶性钙盐、可溶性锰盐、硫酸铜和/或氨基酸，具体的为0.1mM氯化钙，0.1mM硫酸锰，0.1mM硫酸铜，0.01mM谷氨酸；在68℃，pH 7.5的条件下，发酵3小时，除去菌液；在68℃，pH 7.5的条件下继续组装16小时，得到细胞裂解自组装多肽复合物。
实施例2-11
与实施例1的区别在于，发酵底物的原料和用量有所不同，具体见表1。
实施例1-11发酵底物的原料和用量表
| 氯化钙/mM | 硫酸锰/ mM | 硫酸铜/ mM | 精氨酸/ mM | 赖氨酸/ mM | 谷氨酸/ mM | |
| 实施例1 | 0.1 | 0.1 | 0.1 | / | / | 0.01 |
| 实施例2 | 10 | 0.1 | 0.1 | / | / | / |
| 实施例3 | 50 | 0.1 | 0.1 | / | / | / |
| 实施例4 | 10 | 50 | 0.1 | / | / | / |
| 实施例5 | 10 | 100 | 0.1 | / | / | 0.1 |
| 实施例6 | 10 | 50 | 30 | / | / | / |
| 实施例7 | 10 | 50 | 60 | / | / | / |
| 实施例8 | 10 | 50 | / | 0.01 | / | 0.2 |
| 实施例9 | 10 | 50 | / | / | 0.01 | / |
| 实施例10 | 10 | 50 | / | / | 50 | 10 |
| 实施例11 | 10 | 50 | / | / | 100 | / |
实施例12
与实施例1的区别在于，S1、细胞裂解的方式不同，具体地，取嗜热栖热菌，加1倍量PBS使菌液混悬，在8℃，80MPa超高压下，循环3次，使细胞破碎，得到细胞裂解液。
实施例13
与实施例1的区别在于，S1、细胞裂解的方式不同，在4℃，10000g×下离心30分钟，弃上清，加1倍量PBS清洗，加入10-50ul浓度为100mg/ml的溶菌酶，冰浴超声破碎30min（振幅为55 %，超声9s，停5s），得到细胞裂解液。
实施例14
与实施例1的区别在于，S4、发酵组装方式不同，具体地，将上清酶解液与嗜热栖热菌发酵液按体积比1:2混合，在85℃，pH8.5条件下静态自组装18小时，得到细胞裂解自组装多肽复合物。
实施例15
与实施例1的区别在于，S4、发酵组装方式不同，具体地，将上清酶解液与嗜热栖热菌发酵液按照体积比1:10混合，在50℃，pH 5条件下静态组装12小时，在调节PH至7，即得细胞裂解自组装多肽复合物。
实施例16
与实施例1的区别在于，蛋白酶采用枯草杆菌蛋白酶。
实施例17
与实施例1的区别在于，蛋白酶采用经地衣芽孢杆菌蛋白酶。
实施例18
与实施例1的区别在于，蛋白酶采用胃蛋白酶。
对比例
对比例1
与实施例1的区别在于，不包括步骤S4、发酵组装。
检测试验
一、表征试验
采用荧光探针1,8-ANS检测多肽的自组装性质。（8-苯胺-1-萘磺酸(1，8-ANS)是一种疏水的荧光染料，其内部含有一个硫酸根和一个氨基，可用作分子探针，检测多肽或蛋白的自组装能力。l，8-ANS可以与多肽自组装过程中形成的疏水核心相结合，插入到疏水核心内部，并且造成自身荧光性质的改变。一般来说，l，8-ANS在与自组装多肽结合后，荧光强度会明显上升，且发生蓝移现象，以此反映出多肽的自组装进程；而对于自组装不明显的多肽，则其强度和位移程度则都不够明显。
具体的，将细胞裂解自组装多肽复合物储液(10 mM，10％DMSO／水)用10 mM ，pH7.4的PBS稀释至所需浓度后，与20μM的1,8-ANS共孵育15～25 min。
参见图1，多肽自组装后与1,8-ANS结合会造成1,8-ANS荧光强度和位置的变化，以369 nm为激发波长，检测440nm～550 nm的发射光谱。l，8-ANS在与自组装多肽结合后，荧光强度会明显上升，且发生蓝移现象，以此反映出了多肽的自组装进程。
二、消炎检测试验
嗜热栖热菌裂解肽自组装前、后的消炎能力对比（RAW细胞NO抑制率）：
将RAW264.7细胞培养24h后，吸出培养基，以脂多糖（LPS）培养基溶液为对照，分别加入含药培养基（包括1%和5%的不同浓度药物、30ng/m脂多糖（LPS）），继续培养72小时后，析出每孔吸出培养基至不同离心管中，1000rmp离心5min，在540nm波长进行测定一氧化氮含量, 结果发现组装后的组装肽消炎能力显著增强。
表1 消炎检测结果表
| NO抑制率（1%） | NO抑制率（5%） | |
| 实施例1 | 32.24% | 45.27% |
| 实施例2 | 34.45% | 48.77% |
| 实施例3 | 36.25% | 48.63% |
| 实施例4 | 36.15% | 46.66% |
| 实施例5 | 36.27% | 57.33% |
| 实施例6 | 37.54% | 57.83% |
| 实施例7 | 37.34% | 56.75% |
| 实施例8 | 36.83% | 57.33% |
| 实施例9 | 36.54% | 57.21% |
| 实施例10 | 37.83% | 56.54% |
| 实施例11 | 37.54% | 57.83% |
| 实施例12 | 32.54% | 45.97% |
| 实施例13 | 32.14% | 45.27% |
| 实施例14 | 32.33% | 45.23% |
| 实施例15 | 32.54% | 43.33% |
| 实施例16 | 32.75% | 45.35% |
| 实施例17 | 32.45% | 45.64% |
| 实施例18 | 32.34% | 45.54% |
| 对比例1 | 28.75% | 40.19% |
三、采用透射电镜TEM来观察多肽样品的微观结构，样品经磷钨酸负染色后再进行观察。磷钨酸浓度20mg/mL，用1M的氢氧化钠溶液将pH调节至7左右用于染色。取少量样品溶液(50μL)，小心滴在铜网上，静置约90S,自然干燥晾干，滴入适量的磷钨酸，染色时间90S左右,用滤纸吸去残余的染色液，将铜网充分晾干后观察。
以下，以本申请嗜热菌裂解多肽组学（前12的肽序列）为例：
| 序号 | 氨基酸序列 |
| SEQ ID NO.1 | AC-V-F-Y-Y-I-amide |
| SEQ ID NO.2 | AC-E-K-R-A-I-A-amide |
| SEQ ID NO.3 | AC-L-K-R-A-L-L-amide |
| SEQ ID NO.4 | AC-R-K-R-A-L-S--amide |
| SEQ ID NO.5 | AC-Q-K-R-A-L-E-amide |
| SEQ ID NO.6 | AC-A-K-R-A-L-E--amide |
| SEQ ID NO.7 | AC-G-P-L-L-F-V-E-amide |
| SEQ ID NO.8 | AC-E-V-A-K-K-L-amide |
| SEQ ID NO.9 | AC-E-K-G-L-K-V-amide |
| SEQ ID NO.10 | AC-Y-I-V-V-F-M-amide |
| SEQ ID NO.11 | AC-A-I-L-V-V-S-A-amide |
| SEQ ID NO.12 | AC-A-I-L-L-P-P-Q-amide |
其中，以SEQ ID NO.1 AC-V-F-Y-Y-I-amide为例，苯丙氨酸（F）和络氨酸（Y)通过共轭堆积成瓦片形式的组装体。
SEQ ID NO.2 AC-E-K-R-A-I-A-amide中，赖氨酸(K)、精氨酸（R)残基带正电荷,谷氨酸(E)残基带负电荷，带电残基通过静电交替结合,而由于异亮氨酸(I)残基疏水而不带电荷, 在该活性肽中组成了典型的亲疏水交替肽，从而以β折叠式自组装成交替堆积排列的纤维状态。
SEQ ID NO.12 AC-A-I-L-L-P-P-Q-amide中，由于丙氨酸(A)和谷氨酰胺（G）显著的亲疏水性不同，从而自组装成了纳米管状的表征结构。
应用例
应用例1
本申请提供一种自装多肽复合物制剂，包括上述细胞裂解自组装多肽复合物。具体可以制备为多种制剂剂型进行使用。在本申请提供的自组装多肽制剂中，自组装多肽可以根据实际需要调整为任意浓度，例如0 .1％-99％，本发明对此不作限定。在一些优选的实施方式中，自组装多肽的浓度不大于5％，但不限于5%、4％、3％、2％、1％或分布于上述数值中的其他数值。
应用例2
自装多肽复合物制剂在制备消炎消炎、舒敏、抗衰老、抗皱、修复、紧致的化妆品、护肤品或保健品的应用。
以上均为本申请的较佳实施例，并非依此限制本申请的保护范围，故：凡依本申请的结构、形状、原理所做的等效变化，均应涵盖于本申请的保护范围之内。
Claims (10)
1.一种细胞裂解多肽复合物自组装方法，其特征在于：将嗜热栖热菌进行细胞裂解、蛋白酶酶解后得到上清酶解液，将上清酶解液加入到嗜热栖热菌种中或嗜热栖热菌发酵液中进行发酵自组装，得到细胞裂解自组装多肽复合物。
2.根据权利要求1所述的细胞裂解多肽复合物自组装方法，其特征在于：将上清酶解液与嗜热栖热菌种混合，加入发酵底物进行发酵自组装。
3.根据权利要求2所述的细胞裂解多肽复合物自组装方法，其特征在于：所述发酵底物包括可溶性钙盐、可溶性锰盐、硫酸铜和/或氨基酸。
4.根据权利要求3所述的细胞裂解多肽复合物自组装方法，其特征在于：所述发酵底物包括氯化钙、硫酸锰、硫酸铜和/或精氨酸和/或赖氨酸和/或谷氨酸。
5.根据权利要求2或3所述的细胞裂解多肽复合物自组装方法，其特征在于：将上清酶解液、嗜热栖热菌种和发酵底物混合，在温度65-85℃，pH 7.0-8.5的条件下，发酵1-4小时，除去菌液；在65-85℃，pH 7.0-8.5条件下组装12-18小时。
6.根据权利要求1所述的细胞裂解多肽复合物自组装方法，其特征在于：将上清酶解液与嗜热栖热菌发酵液混合，在温度65-85℃，pH 7.0-8.5的条件下组装12-18小时。
7.根据权利要求1所述的细胞裂解多肽复合物自组装方法，其特征在于：所述蛋白酶包括巯基蛋白酶、碱性蛋白酶、中性蛋白酶、酸性蛋白酶中的一种或多种的组合。
8.一种自装多肽复合物，其特征在于：采用权利要求1-6任一项所述的细胞裂解多肽复合物自组装方法制成。
9.一种自装多肽复合物制剂，其特征在于：包括权利要求8所述的细胞裂解自组装多肽复合物。
10.如权利要求8所述的细胞裂解自组装多肽复合物或权利要求9所述的自装多肽复合物制剂在制备消炎消炎、舒敏、抗衰老、抗皱、修复、紧致的化妆品、护肤品或保健品的应用。
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