CN112789063B - 半月板再生用材料 - Google Patents
半月板再生用材料 Download PDFInfo
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- CN112789063B CN112789063B CN201980065130.4A CN201980065130A CN112789063B CN 112789063 B CN112789063 B CN 112789063B CN 201980065130 A CN201980065130 A CN 201980065130A CN 112789063 B CN112789063 B CN 112789063B
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- Prior art keywords
- gly val
- gly
- val pro
- ala
- pro gly
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- 108090000623 proteins and genes Proteins 0.000 claims abstract description 178
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- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000004472 Lysine Substances 0.000 claims abstract description 21
- 239000004475 Arginine Chemical group 0.000 claims abstract description 17
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Chemical group OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract description 17
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- YGHSQRJSHKYUJY-SCZZXKLOSA-N Gly-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN YGHSQRJSHKYUJY-SCZZXKLOSA-N 0.000 description 91
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Abstract
本发明的目的在于提供半月板再生能力高的半月板再生用材料。本发明的半月板再生用材料是包含蛋白质(A)的半月板再生用材料，其中，上述蛋白质(A)具有多肽链(Y)和/或多肽链(Y’)，上述蛋白质(A)中的上述多肽链(Y)与上述多肽链(Y’)的合计个数为1～100个，上述多肽链(Y)是2～200个氨基酸序列(X)连续而成的多肽链，该氨基酸序列(X)为序列号1所示的氨基酸序列即VPGVG序列(1)、序列号4所示的氨基酸序列即GVGVP序列(4)、GPP序列、GAP序列和序列号3所示的氨基酸序列即GAHGPAGPK序列(3)中的至少一者，上述多肽链(Y’)是上述多肽链(Y)中的5％以下的氨基酸被赖氨酸和/或精氨酸置换而得到的多肽链，上述赖氨酸和上述精氨酸的合计个数为1～100个，通过圆二色光谱法求出的上述蛋白质(A)的β‑转角结构和无规卷曲结构的合计比例为60～85％，设上述氨基酸序列(X)中的60％以下的氨基酸被赖氨酸和/或精氨酸置换而得到的氨基酸序列为氨基酸序列(X’)时，以上述蛋白质(A)的总氨基酸数为基准，构成上述蛋白质(A)中包含的上述氨基酸序列(X)和上述氨基酸序列(X’)的氨基酸的合计个数为50～70％。
Description
技术领域
本发明涉及半月板再生用材料。
背景技术
老年人的“关节疾病”中，最多的是变形性关节病(骨关节炎(Osteo Arthritis：OA))。患有OA的人数根据2011年统计为2,530万人(非专利文献1)，其中约800万人有疼痛等症状。膝OA被分类为基于衰老性变化而产生的原发性OA、以及继发于外伤所致的软骨缺损、半月板损伤、前十字韧带损伤等原发疾病而产生的继发性OA。特别是半月板损伤被认为是膝OA的诱因之一，在世界范围内引起了关注。
关于膝OA的治疗，迄今为止聚焦于缺损的关节软骨的再生设计出了各种治疗方法并得以实用化(专利文献1)。另一方面，只要半月板没有修复，即使修复了关节软骨，也非常难以维持良好的状态。因此，为了实现膝OA的最终根治，除了力线矫正以外，还必须确立半月板的修复/再生的治疗法。
半月板在膝关节中除了吸收冲击、稳定关节以外，对于关节的顺滑运动也是非常重要的组织。半月板在组织学上由纤维软骨构成，与关节软骨同样地是缺乏血管的组织，因此修复能力非常低，并且由于其处于力学上的高负荷环境，因此被认为是一旦损伤则非常难以修复的组织。对于症状(物理性的牵拉感和疼痛等)严重的病例，最常进行的是作为所谓对症疗法的关节镜下半月板部分切除，处于受损组织的修复/再生非常困难的状况。但是，为了预防膝OA，在“拯救半月板(Save the Meniscus)”的口号下，在世界范围内尝试了通过半月板缝合术进行修复。但是，缝合术后再次手术的病例达到3成，治愈能力低，对于还存在变性的半月板的治疗具有局限性。作为克服该问题的先进的治疗法，进行了移植自体培养滑膜干细胞的再生医疗、使用胶原蛋白的半月板填补材料的开发研究等(专利文献2)。但是，与软骨损伤的治疗相比，选项非常有限，需要针对半月板损伤的新疗法。
现有技术文献
专利文献
专利文献1：日本专利第6338259号公报
专利文献2：日本特开2012-236112号公报
非专利文献
非专利文献1
変形性関節症治療的国内外的ガイドライン日関病誌(变形性关节病治疗的国内外指南日关病志),35(1):1～9,2016
发明内容
发明所要解决的课题
本发明的目的在于提供半月板再生能力高的半月板再生用材料。
用于解决课题的手段
本发明人为了实现上述目的进行了研究，结果完成了本发明。即，本发明涉及一种半月板再生用材料，其是包含蛋白质(A)的半月板再生用材料，其中，
上述蛋白质(A)具有多肽链(Y)和/或多肽链(Y’)，
上述蛋白质(A)中的上述多肽链(Y)与上述多肽链(Y’)的合计个数为1～100个，
上述多肽链(Y)是2～200个氨基酸序列(X)连续而成的多肽链，该氨基酸序列(X)为序列号1所示的氨基酸序列即VPGVG序列(1)、序列号4所示的氨基酸序列即GVGVP序列(4)、GPP序列、GAP序列和序列号3所示的氨基酸序列即GAHGPAGPK序列(3)中的至少一者，
上述多肽链(Y’)是上述多肽链(Y)中的5％以下的氨基酸被赖氨酸和/或精氨酸置换而成的多肽链，上述赖氨酸和上述精氨酸的合计个数为1～100个，
通过圆二色光谱法求出的上述蛋白质(A)的β-转角结构和无规卷曲结构的合计比例为60％～85％，
设上述氨基酸序列(X)中的60％以下的氨基酸被赖氨酸和/或精氨酸置换而得到的氨基酸序列为氨基酸序列(X’)时，以上述蛋白质(A)的总氨基酸数为基准，构成上述蛋白质(A)中包含的上述氨基酸序列(X)和上述氨基酸序列(X’)的氨基酸的合计个数为50％～70％。
发明的效果
本发明的半月板再生用材料的半月板再生能力优异，能够实现正常的半月板再生。
具体实施方式
本发明的半月板再生用材料是包含蛋白质(A)的半月板再生用材料，其中，
上述蛋白质(A)具有多肽链(Y)和/或多肽链(Y’)，
上述蛋白质(A)中的上述多肽链(Y)与上述多肽链(Y’)的合计个数为1～100个，
上述多肽链(Y)是2～200个氨基酸序列(X)连续而成的多肽链，该氨基酸序列(X)为序列号1所示的氨基酸序列即VPGVG序列(1)、序列号4所示的氨基酸序列即GVGVP序列(4)、GPP序列、GAP序列和序列号3所示的氨基酸序列即GAHGPAGPK序列(3)中的至少一者，
上述多肽链(Y’)是上述多肽链(Y)中的5％以下的氨基酸被赖氨酸和/或精氨酸置换而成的多肽链，上述赖氨酸和上述精氨酸的合计个数为1～100个，
通过圆二色光谱法求出的上述蛋白质(A)的β-转角结构和无规卷曲结构的合计比例为60％～85％，
设上述氨基酸序列(X)中的60％以下的氨基酸被赖氨酸和/或精氨酸置换而得到的氨基酸序列为氨基酸序列(X’)时，以上述蛋白质(A)的总氨基酸数为基准，构成上述蛋白质(A)中包含的上述氨基酸序列(X)和上述氨基酸序列(X’)的氨基酸的合计个数为50％～70％。
本发明的半月板再生用材料包含蛋白质(A)。另外，蛋白质(A)具有多肽链(Y)和/或多肽链(Y’)。因此，能够保持下文详述的凝胶内的适度的湿润环境。
构成多肽链(Y)的氨基酸序列(X)的种类可以为1种，也可以为2种以上。
作为氨基酸序列(X)，从保持下文详述的凝胶内的适度的湿润环境、提高半月板再生能力的方面出发，优选VPGVG序列(1)和GVGVP序列(4)。
作为多肽链(Y)，具体而言，可以举出(VPGVG)b序列、(GVGVP)c序列和(GAHGPAGPK)d序列等。需要说明的是，b～d分别为氨基酸序列(X)的连续个数，为2～200的整数。
在1分子蛋白质(A)中具有2条以上的多肽链(Y)的情况下，多肽链(Y)可以相同、也可以不同，可以具有选自由(VPGVG)b序列、(GVGVP)c序列和(GAHGPAGPK)d序列组成的组中的1种、也可以具有2种以上。
另外，在蛋白质(A)中具有2条以上的多肽链(Y)的情况下，氨基酸序列(X)的连续个数在各条多肽链(Y)中可以相同，也可以不同。即，可以具有2条以上的氨基酸序列(X)的连续个数b～d相同的多肽链(Y)，也可以具有2条以上的b～d不同的多肽链(Y)。
作为多肽链(Y)，从保持下文详述的凝胶内的适度的湿润环境的方面出发，优选为(VPGVG)b序列和(GVGVP)c序列。
多肽链(Y)是2～200个(上述b～d为2～200)氨基酸序列(X)连续而成的多肽链，从保持凝胶内的适度的湿润环境的方面出发，氨基酸序列(X)的连续个数优选为2～100个(上述b～d为2～100)、进一步优选为2～50个(上述b～d为2～50)、特别优选为2～40个(b～d为2～40)。
多肽链(Y’)是多肽链(Y)中的5％以下的氨基酸被赖氨酸和/或精氨酸置换而得到的多肽链，置换的赖氨酸和精氨酸的合计个数为1～100个。
是否为多肽链(Y’)通过将蛋白质(A)序列中的全部赖氨酸(K)和精氨酸(R)置换成其他氨基酸[甘氨酸(G)、丙氨酸(A)、缬氨酸(V)、脯氨酸(P)或组氨酸(H)]时是否成为多肽链(Y)来进行判断。
从保持下文详述的凝胶内的适度的湿润环境的方面出发，多肽链(Y’)中置换的赖氨酸和/或精氨酸的比例优选为0.06％～5％、进一步优选为0.5～5％、特别优选为1～5％。
另外，多肽链(Y’)可以包含氨基酸序列(X)中的60％以下的氨基酸被赖氨酸和/或精氨酸置换而得到的氨基酸序列(X’)。
此外，构成多肽链(Y’)的氨基酸序列(X)和/或氨基酸序列(X’)的种类可以分别为1种，也可以为2种以上。
作为氨基酸序列(X’)，具体而言，可以举出序列号7所示的氨基酸序列即GKGVP序列(7)、序列号8所示的氨基酸序列即GKGKP序列(8)、序列号9所示的氨基酸序列即GKGRP序列(9)、以及序列号10所示的氨基酸序列即GRGRP序列(10)等。
从使半月板缺损面保持适度的湿润环境的方面出发，氨基酸序列(X’)优选为选自由GKGVP序列(7)、GKGKP序列(8)和GRGRP序列(10)组成的组中的至少一种序列，进一步优选为GKGVP序列(7)和GKGKP序列(8)。
1分子蛋白质(A)中的多肽链(Y)和多肽链(Y’)的合计个数为1～100个。另外，它们的合计个数优选为1～80个、更优选为1～60个。
1分子蛋白质(A)中的多肽链(Y)和多肽链(Y’)的合计个数为上述范围时，能够保持下文详述的凝胶内的适度的湿润环境。
需要说明的是，在蛋白质(A)中具有氨基酸序列(X)的种类和/或连续个数不同的多肽链(Y)的情况下，将它们分别计为1个，多肽链(Y)的个数为其合计。对于多肽链(Y’)也是同样的。
本发明的半月板再生用材料中，以蛋白质(A)的总氨基酸数为基准，构成蛋白质(A)中包含的氨基酸序列(X)和氨基酸序列(X’)的氨基酸的合计个数为50～70％。
上述比例小于50％时，下文详述的凝胶化性能变差，超过70％时，水溶性变差。
从提高半月板再生能力的方面出发，上述比例优选为52.5～67.5％、进一步优选为55～65％。
上述比例可以利用蛋白质测序仪求出。具体而言，可以通过下述测定法求出。
<构成氨基酸序列(X)和氨基酸序列(X’)的氨基酸的合计个数相对于蛋白质(A)中的总氨基酸数的比例的测定法>
从能够在特定氨基酸残基处切断的切断方法中选用2种以上，将蛋白质(A)分解至30个残基以下的程度。之后，利用高效液相色谱法(HPLC)进行分离后，利用蛋白质测序仪读取氨基酸序列。由所得到的氨基酸序列进行肽图谱分析，确定蛋白质(A)的全序列。之后，利用下述记载的测定式计算出全部氨基酸序列(X)中的氨基酸数和全部氨基酸序列(X’)中的氨基酸数的合计个数相对于蛋白质(A)中的总氨基酸数的比例。
构成蛋白质(A)的全部氨基酸序列(X)中的氨基酸数和构成蛋白质(A)的全部氨基酸序列(X’)中的氨基酸数的合计数的比例(％)＝[{氨基酸序列(X)的数目}×{氨基酸序列(X)中的氨基酸数}+{氨基酸序列(X’)的数目}×{氨基酸(X’)中的氨基酸数}]/{蛋白质(A)中的总氨基酸数}×100
从使下文详述的蛋白质(A)中的β-转角结构和无规卷曲结构的合计比例为规定范围的方面以及保持凝胶内的适度的湿润环境、提高半月板再生能力的方面出发，蛋白质(A)优选具有2～50个GAGAGS序列(2)连续而成的多肽链(S)。
多肽链(S)中，从保持凝胶内的适度的湿润环境的方面出发，GAGAGS序列(2)的连续个数优选为2～40个、进一步优选为2～30个、特别优选为2～10个。
蛋白质(A)中，从提高半月板再生能力的方面出发，全部GAGAGS序列(2)中的氨基酸数相对于蛋白质(A)中的总氨基酸数的比例[{蛋白质(A)中的GAGAGS序列(2)的数目×6}/{蛋白质(A)中的总氨基酸数}×100]优选为5～50％、进一步优选为10～47.5％、特别优选为20～45％。
全部GAGAGS序列(2)中的氨基酸数相对于蛋白质(A)中的总氨基酸数的比例可以利用蛋白质测序仪求出。具体而言，通过下述测定法求出。
<全部GAGAGS序列(2)中的氨基酸数相对于蛋白质(A)中的总氨基酸数的比例>
从能够在特定氨基酸残基处切断的切断方法中选用2种以上，将蛋白质(A)分解至30个残基以下的程度。之后，利用高效液相色谱法(HPLC)进行分离后，利用蛋白质测序仪读取氨基酸序列。由所得到的氨基酸序列进行肽图谱分析，确定蛋白质(A)的全序列。之后，利用下述记载的测定式计算出全部GAGAGS序列(2)中的氨基酸数相对于蛋白质(A)中的总氨基酸数的比例。
全部GAGAGS序列(2)中的氨基酸数相对于蛋白质(A)中的总氨基酸数的比例(％)＝[{GAGAGS序列(2)的数目×6}/{蛋白质(A)中的总氨基酸数}]×100
在蛋白质(A)具有合计2个以上的选自由多肽链(Y)、多肽链(Y’)和多肽链(S)组成的组中的至少一种多肽链的情况下，在它们之间可以具有间插氨基酸序列(Z)。间插氨基酸序列(Z)是1个或2个以上的氨基酸结合而成且并非GAGAGS序列(2)、氨基酸序列(X)和氨基酸序列(X’)的肽序列。从保持下文详述的凝胶内的适度的湿润环境的方面出发，构成间插氨基酸序列(Z)的氨基酸数优选为1～30个、进一步优选为1～15个、特别优选为1～10个。作为间插氨基酸序列(Z)，具体而言，可以举出序列号11所示的氨基酸序列即VAAGY序列(11)、序列号12所示的氨基酸序列即GAAGY序列(12)、以及LGP序列等。
从保持下文详述的凝胶内的适度的湿润环境的方面出发，全部间插氨基酸序列(Z)中的氨基酸数相对于蛋白质(A)中的总氨基酸数的比例[Σ{(间插氨基酸序列(Z)中的氨基酸数)×(间插氨基酸序列(Z)的数目)}/{蛋白质(A)中的总氨基酸数}×100]优选为0～25％、进一步优选为0～22.5％、特别优选为0.01～15％。
蛋白质(A)中，从在生物体内的分解性的方面出发，除了GAGAGS序列(2)、氨基酸序列(X)、氨基酸序列(X’)和间插氨基酸序列(Z)以外，还可以在末端具有末端氨基酸序列(T)。需要说明的是，末端氨基酸序列(T)可以位于蛋白质(A)的一个末端，也可以位于两末端。
需要说明的是，末端氨基酸序列(T)中不包含后述的纯化标签。
作为蛋白质(A)的末端结构，优选为在多肽链(Y)上结合有末端氨基酸序列(T)的结构。末端氨基酸序列(T)是1个或2个以上的氨基酸结合而成且并非GAGAGS序列(2)、氨基酸序列(X)和氨基酸序列(X’)的肽序列。从在生物体内的分解性的方面出发，构成末端氨基酸序列(T)的氨基酸数优选为1～100个、进一步优选为1～50个、特别优选为1～40个。作为末端氨基酸序列(T)，具体而言，可以举出序列号13所示的氨基酸序列即MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM序列(13)等。
从在生物体内的分解性的方面出发，末端氨基酸序列(T)的氨基酸数相对于蛋白质(A)中的总氨基酸数的比例优选为0～25％、进一步优选为0～22.5％、特别优选为0.01～15％。
如下文所述，蛋白质(A)有时使用细菌通过生物工程方法来制造。这种情况下，为了易于进行所表达的蛋白质(A)的纯化或检测，蛋白质(A)中除了末端氨基酸序列(T)以外还可以在N或C末端具有包含特殊氨基酸序列的蛋白质或肽(以下将它们称为“纯化标签”)。作为纯化标签，使用亲和纯化用的标签。作为这样的纯化标签，包括由多组氨酸构成的6×His标签、V5标签、Xpress标签、AU1标签、T7标签、VSV-G标签、DDDDK标签、S标签、CruzTag09TM、CruzTag22TM、CruzTag41TM、Glu-Glu标签、Ha.11标签和KT3标签等。
以下示出各纯化标签(i)与识别并结合该标签的配体(ii)的组合的一例。
(i-1)谷胱甘肽-S-转移酶(GTS)(ii-1)谷胱甘肽
(i-2)麦芽糖结合蛋白(MBP)(ii-2)直链淀粉
(i-3)HQ标签(ii-3)镍
(i-4)Myc标签(ii-4)抗Myc抗体
(i-5)HA标签(ii-5)抗HA抗体
(i-6)FLAG标签(ii-6)抗FLAG抗体
(i-7)6×His标签(ii-7)镍或钴
作为上述纯化标签序列的导入方法，可以举出在表达用载体中的编码蛋白质(A)的核酸的5’或3’末端插入编码纯化标签的核酸的方法、使用市售的纯化标签导入用载体的方法等。
蛋白质(A)中，从在生物体内的分解性的方面出发，以蛋白质(A)中的总氨基酸数为基准，构成蛋白质(A)的全部间插氨基酸序列(Z)中的氨基酸数、构成蛋白质(A)的全部末端氨基酸序列(T)中的氨基酸数以及纯化标签的氨基酸数的合计数的比例优选为0～25％、进一步优选为0～22.5％、特别优选为0.01～15％。
在蛋白质(A)中包含多肽链(Y)和/或多肽链(Y’)以及多肽链(S)的情况下，从使缺损面保持适度的湿润环境的方面出发，优选多肽链(Y)或多肽链(Y’)与多肽链(S)交替地进行化学结合。
从使蛋白质(A)中的β-转角结构和无规卷曲结构的合计比例适度的方面出发，GAGAGS序列(2)的个数与氨基酸序列(X)和氨基酸序列(X’)的合计个数之比(GAGAGS序列(2)：氨基酸序列(X)和氨基酸序列(X’)的合计)优选为1:1.5～1:20、进一步优选为1:1.5～1:6、特别优选为1:2～1:5。
以下例示出一部分优选的蛋白质(A)。
(A1)：氨基酸序列(X)为GVGVP序列(4)的蛋白质
(A11)：具有多肽链(Y’1)的蛋白质，该多肽链(Y’1)是2～200个GVGVP序列(4)连续而成的氨基酸序列中的1个氨基酸被赖氨酸(K)置换而成的
(A11-1)具有多肽链(Y’1)和2～200个GAGAGS序列(2)连续而成的多肽链(S1)的蛋白质
(A11-2)：具有序列号6所示的氨基酸序列即(GVGVP)4GKGVP(GVGVP)3序列(6)(Y’11)和2～200个GAGAGS序列(2)连续而成的多肽链(S1)的蛋白质，该(Y’11)是8个GVGVP序列(4)连续而成的序列号14所示的氨基酸序列即(GVGVP)8序列(14)的多肽链(Y11)中的1个氨基酸被赖氨酸(K)置换而成的
(A11-2-1)：具有多肽链(S1-1)和(GVGVP)4GKGVP(GVGVP)3序列(6)的蛋白质，该多肽链(S1-1)为4个GAGAGS序列(2)连续而成的序列号5所示的氨基酸序列即(GAGAGS)4序列(5)
具体而言，包括下述蛋白质。
(i)在具有12个(GAGAGS)4序列(5)和13个(GVGVP)4GKGVP(GVGVP)3序列(6)且它们交替地化学结合而成的序列上化学结合序列号15所示的氨基酸序列即(GAGAGS)2序列(15)而成的分子量为约80kDa的序列号16所示的氨基酸序列即序列(16)的蛋白质(SELP8K)
(ii)分别具有17个(GAGAGS)2序列(15)和(GVGVP)4GKGVP(GVGVP)3序列(6)且具有它们交替地化学结合而成的结构的分子量为约82kDa的序列号17所示的氨基酸序列即序列(17)的蛋白质(SELP0K)
(iii)分别具有4个(GAGAGS)4序列(5)和(GVGVP)4GKGVP(GVGVP)3序列(6)且它们交替地化学结合而成的分子量为约30kDa的序列号29所示的氨基酸序列即序列(29)的蛋白质(SELP8K4)
(A11-3)：具有序列号18所示的氨基酸序列即(GVGVP)6GKGVP(GVGVP)5序列(18)(Y’12)和2～200个GAGAGS序列(2)连续而成的多肽链(S1)的蛋白质，该(Y’12)是12个GVGVP序列(4)连续而成的多肽链的1个氨基酸被赖氨酸(K)置换而成的
(A11-3-1)：具有4个GAGAGS序列(2)连续而成的序列号19所示的氨基酸序列即(GAGAGS)4序列(19)和(GVGVP)6GKGVP(GVGVP)5序列(18)的蛋白质
(i)在具有12个(GAGAGS)4序列(19)和13个(GVGVP)6GKGVP(GVGVP)5序列(18)且它们交替地化学结合而成的序列上化学结合(GAGAGS)2序列(15)而成的分子量为约105kDa的序列号20所示的氨基酸序列即序列(20)的蛋白质
(A2)：氨基酸序列(X)为VPGVG序列(1)的蛋白质
(A21)：具有2～200个VPGVG序列(1)连续而成的多肽链(Y2)和GAGAGS序列(2)的蛋白质
(i)分别具有40个GAGAGS序列(2)、序列号24所示的氨基酸序列即(VPGVG)4序列(24)和序列号25所示的氨基酸序列即(VPGVG)8序列(25)、且具有这些序列按照(VPGVG)4序列(24)、GAGAGS序列(2)、(VPGVG)8序列(25)的顺序结合而成的40个嵌段化学结合而成的结构的分子量约200kDa的序列号26所示的氨基酸序列即序列(26)的蛋白质(ELP1.1)
(A3)：具有2～200个GVGVP序列(4)连续而成的多肽链(Y1)和2～200个GAGAGS序列(2)连续而成的多肽链(S1)的蛋白质
具体而言，包括下述蛋白质。
(i)分别具有5个序列号21所示的氨基酸序列即(GAGAGS)8序列(21)和序列号22所示的氨基酸序列即(GVGVP)40序列(22)、且具有这些序列交替地化学结合而成的结构的分子量为约110kDa的序列号23所示的氨基酸序列即序列(23)的蛋白质(SELP6.1)
这些之中，优选为序列(16)的蛋白质(SELP8K)、序列(17)的蛋白质(SELP0K)、序列(20)的蛋白质、序列(23)的蛋白质(SELP6.1)、序列(26)的蛋白质(ELP1.1)或序列(29)的蛋白质(SELP8K4)。
另外，蛋白质(A)也可以为具有与序列(16)的蛋白质(SELP8K)、序列(17)的蛋白质(SELP0K)、序列(20)的蛋白质、序列(23)的蛋白质(SELP6.1)、序列(26)的蛋白质(ELP1.1)或序列(29)的蛋白质(SELP8K4)的氨基酸序列的同源性为70％以上的氨基酸序列的蛋白质。
另外，该同源性优选为80％以上、更优选为90％以上。
本发明中的蛋白质(A)中，通过圆二色光谱法求出的蛋白质(A)中的β-转角结构和无规卷曲结构的合计比例为60～85％。即使蛋白质的序列相同，蛋白质中的β-转角结构和无规卷曲结构的合计比例也会根据蛋白质的制作方法、蛋白质的纯化方法、溶解蛋白质的溶剂的pH和溶剂的极性等而有所不同。
β-转角结构和无规卷曲结构的合计比例小于60％时，水溶性变差。另外，超过85％时，下文详述的凝胶化性能变差。
从使缺损面保持适度的湿润环境、促进半月板的再生的方面出发，蛋白质(A)中的β-转角结构和无规卷曲结构的合计比例优选为65～80％、进一步优选为70～75％。
作为控制上述比例的方法没有特别限定，可以通过下述方法使其增加或降低。
在使上述比例增加的情况下，例如可以举出将蛋白质(A)、例如将单独的蛋白质(A)利用过量的缓冲液稀释后进行重折叠的稀释重折叠法(大量稀释法)。
另外，在使上述比例降低的情况下，例如可以举出利用变性剂或热等使蛋白质(A)变性的方法。
蛋白质(A)中的β-转角结构和无规卷曲结构的合计比例通过下述测定法求出。
<蛋白质(A)中的β-转角结构和无规卷曲结构的合计比例的测定方法>
将蛋白质以0.3mg/ml溶解在去离子水(4℃)中，制作蛋白质的水溶液。利用圆二色光谱测定器(日本分光株式会社制造，“J-820”)对所制作的蛋白质的水溶液进行测定(测定温度：4℃)，利用二级结构解析程序(JWSSE型：日本分光株式会社制造)计算出β-转角结构的比例和无规卷曲结构的比例，将它们的合计作为β-转角结构和无规卷曲结构的合计比例。
从在生物体内的分解性的方面出发，蛋白质(A)的利用SDS-PAGE(SDS聚丙烯酰胺凝胶电泳)法得到的分子量优选为15～200kDa、进一步优选为30～150kDa、特别优选为70～120kDa。
本发明中，从提高半月板再生能力的方面出发，蛋白质(A)的疏水度优选为0.2～1.2、进一步优选为0.4～1.0、特别优选为0.42～0.80。
蛋白质(A)的疏水度表示蛋白质(A)分子的疏水性的程度，可以通过将构成蛋白质(A)分子的各氨基酸残基的数目(Mα)、各氨基酸的疏水度(Nα)和1分子蛋白质(A)中的氨基酸残基的总数(MT)代入到下述数学式中来算出。需要说明的是，各氨基酸的疏水度使用非专利文献(Albert L.Lehninger,David L.Nelson,Lehninger的新生物化学上,广川书店,2010年9月,p.346-347)中记载的下述数值。
疏水度＝Σ(Mα×Nα)/(MT)
Mα：1分子人工蛋白质(A)中的各氨基酸残基的数目
Nα：各氨基酸的疏水度
MT：1分子人工蛋白质(A)中的氨基酸残基的总数
A(丙氨酸)：1.8
R(精氨酸)：-4.5
N(天冬酰胺)：-3.5
D(天冬氨酸)：-3.5
C(半胱氨酸)：2.5
Q(谷氨酰胺)：-3.5
E(谷氨酸)：-3.5
G(甘氨酸)：-0.4
H(组氨酸)：-3.2
I(异亮氨酸)：4.5
L(亮氨酸)：3.8
K(赖氨酸)：-3.9
M(蛋氨酸)：1.9
F(苯丙氨酸)：2.8
P(脯氨酸)：-1.6
S(丝氨酸)：-0.8
T(苏氨酸)：-0.7
W(色氨酸)：-0.9
Y(酪氨酸)：-1.3
V(缬氨酸)：4.2
例如，在人工蛋白质(A)为(GVGVP)4GKGVP(GVGVP)3序列(6)的情况下，人工蛋白质(A)的疏水度＝{16(G的数目)×(-0.4)+15(V的数目)×4.2+8(P的数目)×(-1.6)+1(K的数目)×(-3.9)}/40(氨基酸残基的总数)＝1.0。
本发明的半月板再生用材料含有包含上述氨基酸序列的蛋白质(A)，因而可被生物体内的酶分解，因此生物降解性优异。
本发明中，蛋白质(A)通过从天然物中提取、有机合成法(酶法、固相合成法和液相合成法等)和基因重组法等而得到。关于有机合成法，可以应用“生物化学实验讲座1、蛋白质的化学IV(1981年7月1日、日本生化学会编、株式会社东京化学同人发行)”中记载的方法以及“续生物化学实验讲座2、蛋白质的化学(下)(1987年5月20日、日本生物化学会编、株式会社东京化学同人发行)”中记载的方法等。关于基因重组法，可以应用日本专利第3338441号公报中记载的方法等。从天然物中提取、有机合成法和基因重组法均可得到蛋白质(A)，从能够简便地变更氨基酸序列、能够低成本地大量生产等方面出发，优选基因重组法。
从提高半月板再生能力的方面出发，本发明的半月板再生用材料优选含有半月板组织片(B)。
作为上述半月板组织片(B)，可以通过采集半月板(可以是患者本人的半月板，也可以不是患者本人而是他人的半月板)的一部分并将其微细地粉碎而得到。
作为微细地粉碎的方法，可以举出利用手术刀进行剪裁等方法。
从提高半月板再生能力的方面出发，半月板组织片(B)的每1片的数均体积优选为0.001～1,000mm3、进一步优选为0.008～970mm3、特别优选为1～500mm3。
本发明的半月板再生用材料中，除了上述的蛋白质(A)和半月板组织片(B)以外，还可以包含水、无机盐和/或磷酸(盐)。
作为无机盐，具体而言，可以举出氯化钠、氯化钾、氯化钙、氯化镁、硫酸钠、硫酸钾、硫酸钙、硫酸镁、碳酸氢钠、碳酸氢钾、碳酸氢钙和碳酸氢镁等。需要说明的是，本说明书中的磷酸(盐)不包括在无机盐中。
从提高半月板再生能力的方面出发，以半月板再生用材料的重量为基准，半月板再生用材料中的无机盐的含量(重量％)优选为0～3重量％、进一步优选为0～1重量％、特别优选为0.001～0.5重量％。
磷酸(盐)是指磷酸和/或磷酸盐。
作为磷酸盐，可以举出磷酸的碱金属盐和碱土金属盐，具体而言，可以举出钠盐、钾盐、钙盐和镁盐等。
从提高半月板再生能力的方面出发，以半月板再生用材料的重量为基准，半月板再生用材料中的磷酸(盐)的含量优选为0.001～2重量％、进一步优选为0.001～0.5重量％、特别优选为0.001～0.05重量％。
从提高半月板再生能力的方面出发，以半月板再生用材料的重量为基准，本发明的半月板再生用材料所含有的蛋白质(A)的重量比例优选为5～25重量％、进一步优选为10～20重量％。
从提高半月板再生能力的方面出发，以半月板再生用材料的重量为基准，本发明的半月板再生用材料所含有的半月板组织片(B)的重量比例优选为5～50重量％、进一步优选为10～40重量％。
另外，从进一步提高半月板再生能力的方面出发，本发明的半月板再生用材料所含有的蛋白质(A)的重量与半月板组织片(B)的重量的比例[蛋白质(A)的重量/半月板组织片(B)的重量]优选为0.30～0.90、进一步优选为0.40～0.75。
本发明的半月板再生用材料可以通过将半月板组织片(B)加入到溶解有蛋白质(A)的水溶液中并通过吹打等进行混合调整等的方法来制造。此时，从维持细胞活性的方面出发，优选在4～10℃实施上述操作。
作为本发明的半月板再生用材料的使用方法，可以举出下述方法等。
(1)通过使本发明的半月板再生用材料为4～40℃(优选20～40℃)，可以使其凝胶化。
通过将该凝胶化的半月板再生用材料施用到经切开等而露出的半月板损伤部位，可以促进半月板的再生。需要说明的是，在施用半月板再生用材料后，可以利用胶原蛋白膜或患者的滑膜等将切开部缝合/封闭。
(2)通过将本发明的半月板再生用材料在关节镜下施用至半月板损伤部，利用患者的体温将半月板再生用材料凝胶化，可以与上述(1)同样地促进半月板的再生。
这些方法也是本发明的半月板损伤部的治疗方法。
本发明的半月板再生用材料可用于变形性关节病、软骨缺损和半月板损伤(外伤性半月板损伤等)等。
另外，上述半月板再生用材料在选自由变形性关节病、软骨缺损和半月板损伤(外伤性半月板损伤)组成的组中的至少一种疾病中的应用也是本发明的半月板再生用材料的应用。
[实施例]
下面以实施例的形式进一步详细地说明本发明，但本发明并不仅限于这些实施例。
<制造例A1：SELP8K(A11-2-1(i))的制作>
○SELP8K(A11-2-1(i))的生产
按照日本专利第4088341号公报的实施例中记载的方法，制作编码SELP8K的质粒pPT0345。
将所制作的质粒转化到大肠杆菌中，得到SELP8K生产株。下面示出使用该SELP8K生产株生产具有序列(16)的蛋白质(A)的方法。
○SELP8K生产株的培养
使用在30℃生长得到的SELP8K生产株的过夜培养液，接种在250mL烧瓶中的LB培养基50mL中。加入卡那霉素使最终浓度为50μg/mL，在30℃搅拌的同时(200rpm)对该培养液进行培养。在培养液的浊度OD600＝0.8(使用吸光度计UV1700：岛津制作所制)时，将培养液40mL转移到事先加温到42℃的烧瓶中，在同样的温度下培养约2小时。将培养得到的培养液在冰上冷却，测定培养液的浊度OD600，通过离心分离进行大肠杆菌的集菌。
○SELP8K(A11-2-1(i))的纯化
使用集菌得到的大肠杆菌，通过下述1：菌体裂解、2：通过离心分离除去不溶性碎片、3：硫酸铵沉淀、4：超滤、5：阴离子交换层析、6：超滤、7：冷冻干燥从大肠杆菌生物质中纯化蛋白质。这样得到分子量为约80kDa的具有序列(16)的蛋白质的纯化物即制造例A1的蛋白质(A)(SELP8K)。
1：菌体裂解
在集菌得到的大肠杆菌100g中加入去离子水200g，利用高压均质机(55MPa)进行菌体裂解，得到含有裂解后的菌体的菌体裂解液。然后，利用冰乙酸将菌体裂解液调整为pH4.0。
2：通过离心分离除去不溶性碎片
进一步将菌体裂解液进行离心分离(6300rpm、4℃、30分钟)，回收上清。
3：硫酸铵沉淀
在回收的上清中投入饱和硫酸铵溶液使硫酸铵浓度达到25重量％。之后，静置8～12小时后，通过离心分离回收沉淀物。将回收的沉淀物溶解在去离子水中。向溶解得到的液体中同样地投入饱和硫酸铵溶液使硫酸铵浓度达到25重量％。之后，静置8～12小时后，通过离心分离回收沉淀物。将回收的沉淀物溶解在去离子水中，得到溶液。
4：超滤
将“3：硫酸铵沉淀”中得到的溶液供给到截止分子量为30,000Da的超滤装置(中空纤维：GE Healthcare公司制造)中。使用相对于“3：硫酸铵沉淀”中得到的溶液为10倍量的去离子水实施超滤，得到超滤后的蛋白质。
5：阴离子交换层析
将超滤后的蛋白质溶解在10mM乙酸钠缓冲液中制成20g/L，供给至设有阴离子交换柱HiPrepSP XL16/10(GE Healthcare公司制造)的AKTAPrime(GE Healthcare公司制造)中。作为洗脱液，使用500mM 10mM乙酸钠缓冲液，回收洗脱级分。
6：超滤
将“5：阴离子交换层析”中得到的溶液与上述“4：超滤”同样地进行处理，得到超滤后的蛋白质。
7：冷冻干燥
将蛋白质溶解在去离子水中制成5g/L，按照水位为15mm以下的方式装入不锈钢制托盘中。之后，放入冷冻干燥机(日本Techno Service株式会社制)中，在-40℃用时16小时进行冷冻。冷冻后，在真空度为8Pa以下在-20℃用时90小时进行一次干燥，在真空度为8Pa以下在20℃用时24小时进行二次干燥。这样得到制造例A1的蛋白质(A)(SELP8K)。
○SELP8K(A11-2-1(i))的鉴定
按照下述过程对制造例A1的蛋白质(A)进行鉴定。
使用兔抗SELP8K抗体和针对C末端序列的6×His标签的兔抗6×His抗体(Roland公司制造)通过蛋白免疫印迹法进行分析。蛋白免疫印迹法的过程如下所述。在表观分子量80kDa的位置观察到对各抗体显示出抗体反应性的条带。
另外，将使用氨基酸分析系统(Prominence岛津制作所制)通过氨基酸组成分析得到的制造例A1的蛋白质(A)的氨基酸的组成比率(实测值)、以及由合成基因序列推测出的SELP8K的氨基酸的组成比率(理论值)示于表1。
由此确认，制造例A1的蛋白质(A)是具有13个多肽链(Y’2)和12个多肽链(S1-1)且它们交替地化学结合而成的、具有序列(16)的蛋白质(SELP8K)，该多肽链(Y’2)具有8个GVGVP序列(4)连续而成的多肽链(Y)中的1个缬氨酸(V)被赖氨酸(K)置换而成的(GVGVP)4GKGVP(GVGVP)3序列(6)，该多肽链(S1-1)具有4个GAGAGS序列(2)连续而成的(GAGAGS)4序列(5)。
[表1]
| 氨基酸 | 实测 | 理论 |
| 组成比率(％) | 组成比率(％) | |
| Ala | 12.3 | 12.2 |
| Asx | 0.9 | 0.8 |
| Glx | n.d. | 0.4 |
| Phe | 0.4 | 0.1 |
| Gly | 43.7 | 41.5 |
| His | 0.4 | 0.8 |
| Ile | 0.3 | 0 |
| Lys | 1.5 | 1.5 |
| Leu | 0.3 | 0.5 |
| Met | 0.3 | 0.3 |
| Pro | 11.7 | 12.4 |
| Arg | 0.5 | 0.6 |
| Ser | 5.3 | 6.1 |
| Thr | n.d. | 0.1 |
| Val | 21.2 | 22.4 |
| Tyr | 1.1 | 0.1 |
<蛋白免疫印迹法>
在蛋白质印迹用样品20μL中添加3×SDS处理缓冲液[包含150mM Tris HCl(pH6.8)、300mM二硫苏糖醇、6％十二烷基硫酸钠(SDS)、0.3％溴酚蓝以及30％甘油]10μL，在95℃加热5分钟，制备电泳用试样。使用该电泳用试样15μL进行SDS-PAGE。将电泳后的凝胶转印到聚偏氟乙烯膜(下文中简称为“膜”)上，将其浸渍在封闭缓冲液[包含20mM Tris(pH7.6)、137mM NaCl、0.1％Tween20以及5％脱脂乳]中，在室温振荡1小时，由此进行膜的封闭处理。封闭处理后，将膜用TBS-T[包含20mM Tris(pH7.6)、137mM NaCl以及0.1％Tween20]清洗2分钟。接着，将膜浸渍在一次抗体溶液(将一次抗体：抗SELP8K抗体和抗His标签抗体(Rockland公司制造)用TBS-T稀释成1/500而得到的溶液)中，在4℃静置过夜，进行抗体反应。反应后，将该膜用TBS-T进行5分钟、4次的清洗后，将膜浸渍在能够与一次抗体结合并且结合有作为标记酶的辣根过氧物酶的二次抗体的溶液(将二次抗体：偶联有ECL抗兔IgG HRP的F(ab’)2片段(GE Healthcare公司制造)用TBS-T稀释成1/2000而得到的溶液)中，在室温静置30分钟，进行抗体反应。反应后，将膜用TBS-T进行5分钟、4次的清洗后，利用ECL-Advance蛋白免疫印迹检测试剂盒(GE Healthcare公司制造)进行酶反应。使用ECL化学发光仪(GE Healthcare公司制造)使高灵敏度即显黑白胶片(Fuji Film株式会社制造)感光，使条带可视化。
○β-转角结构和无规卷曲结构的合计比例的测定
使用制造例A1的蛋白质(A)，按照下述过程测定β-转角结构和无规卷曲结构的合计比例。
<β-转角结构和无规卷曲结构的合计比例的测定>
将制造例A1的蛋白质(A)以0.3mg/ml溶解在去离子水(4℃)中，制作制造例A1的蛋白质(A)水溶液。利用圆二色光谱测定器(日本分光：J-820)对所制作的制造例A1的蛋白质(A)水溶液进行测定(测定温度：4℃)，使用二级结构解析程序(JWSSE型：日本分光株式会社制造)计算出β-转角结构和无规卷曲结构的合计比例。将结果示于
表2。
<制造例A2>
在制造例A1的“SELP8K(A11-2-1(i))的制作”中，在“SELP8K(A11-2-1(i))的纯化”的“5：阴离子交换层析”与“6：超滤”之间进行下述“5-2：重折叠(大量稀释法)”，除此以外同样地制作制造例A2的蛋白质(A)，测定β-转角结构和无规卷曲结构的合计比例。将结果示于表2。
5-2：重折叠(大量稀释法)
利用作为蛋白质变性剂的10M脲溶液将阴离子交换层析的洗脱级分制备成6M脲溶液，在4℃静置12小时。将所制备的溶液投入到透析膜(Viskase Companies,Inc.公司制造)中，利用洗脱级分的10倍容量的去离子水进行12小时的透析。之后弃掉去离子水，重新利用洗脱级分的10倍容量的去离子水进行12小时的透析。再进行3次该操作、总计反复进行5次后，回收透析膜中的溶液。
<制造例A3>
在制造例A1的“SELP8K(A11-2-1(i))的制作”中，在“SELP8K(A11-2-1(i))的纯化”的“5：阴离子交换层析”与“6：超滤”之间进行下述“5-3：重折叠(大量稀释法)”，除此以外同样地制作制造例A3的蛋白质(A)，测定β-转角结构和无规卷曲结构的合计比例。将结果示于表2。
5-3：重折叠(大量稀释法)
利用作为蛋白质变性剂的10M脲溶液将阴离子交换层析的洗脱级分制备成6M脲溶液，在4℃静置12小时。将所制备的溶液投入到透析膜(Viskase Companies,Inc.公司制造)中，利用洗脱级分的10倍容量的去离子水进行12小时的透析。之后弃掉去离子水，重新利用洗脱级分的3倍容量的去离子水进行12小时的透析。利用洗脱级分的3倍容量的去离子水再反复进行5次透析，之后回收透析膜中的溶液。
<制造例A4>
在制造例A1的“SELP8K(A11-2-1(i))的制作”中，将“SELP8K(A11-2-1(i))的纯化”的“5：阴离子交换层析”替换为下述“5’：亲和层析”，除此以外同样地制作制造例A4的蛋白质(A)，测定β-转角结构和无规卷曲结构的合计比例。将结果示于表2。
5’：亲和层析
将“4：超滤”后的蛋白质通过使用His标签的亲和层析(GE Healthcare公司制造、Ni Sepharose 6Fast Flow)进行纯化，回收洗脱级分。
<制造例A5>
在制造例A1中，使用“编码SELP8K4的质粒pPT0345-4”来代替“编码SELP8K的质粒pPT0345”，除此以外同样地制作分子量为约30kDa的序列(29)的制造例A5的蛋白质(A)，测定β-转角结构和无规卷曲结构的合计比例。将结果示于表2。
<比较制造例A1>
在制造例A1的“SELP8K(A11-2-1(i))的制作”中，将“SELP8K(A11-2-1(i))的纯化”的“5：阴离子交换层析”替换为下述“5”：亲和层析”，除此以外同样地制作比较制造例A1的蛋白质(A’)，测定β-转角结构和无规卷曲结构的合计比例。将结果示于表2。
5”：亲和层析
将“4：超滤”后的蛋白质通过使用His标签的亲和层析(Clontech公司制造、TALON(注册商标)一步法纯化柱)进行纯化，回收洗脱级分。
<比较制造例A2>
在制造例A1的“SELP8K(A11-2-1(i))的制作”中，不实施“SELP8K(A11-2-1(i))的纯化”的“3：硫酸铵沉淀、4：超滤、5：阴离子交换层析”，而进行上述“5’：亲和层析”，除此以外同样地制作比较制造例A2的蛋白质(A’)，测定β-转角结构和无规卷曲结构的合计比例。将结果示于表2。
<比较制造例A3>
在制造例A1的“SELP8K(A11-2-1(i))的制作”中，不实施“SELP8K(A11-2-1(i))的纯化”的“5：阴离子交换层析”，除此以外同样地制作比较制造例A3的蛋白质(A’)，测定β-转角结构和无规卷曲结构的合计比例。将结果示于表2。
<比较制造例A4>
在制造例A1中，使用“编码ELP1.2的质粒pPT0102-2”来代替“编码SELP8K的质粒pPT0345”，除此以外同样地制作分子量为约37kDa的序列号27所示的氨基酸序列即序列(27)的比较制造例A4的蛋白质(A’)，测定β-转角结构和无规卷曲结构的合计比例。将结果示于表2。
<比较制造例A5>
在制造例A1中，使用“编码SLP4.1的pSY1398-1”来代替“编码SELP8K的质粒pPT0345”，除此以外同样地制作分子量为约93kDa的序列号28所示的氨基酸序列即序列(28)的比较制造例A5的蛋白质(A’)，测定β-转角结构和无规卷曲结构的合计比例。将结果示于表2。
<制造例B1：半月板组织片(B)的制作>
将由日本白色家兔(体重：3.0kg)采集的半月板组织固定于预先经高压釜灭菌的载台上。
之后使用将手术刀片以200μm间隔配置成棋盘格状的手术刀片保持器对固定化的半月板组织进行切割。之后，使用上述手术刀片保持器再次进行切割以形成边长为约200μm的立方体形状，得到制造例B1的半月板组织片(B)。
对于制造例B1的半月板组织片(B)中的任意的100个组织片，在光学显微镜下测定每1边的长度，计算出每1片的体积。其结果，制造例B1的半月板组织片(B)的每1片的数均体积为0.008mm3。
<制造例B2>
在制造例B1中，将手术刀片的间隔由200μm变更为1,000μm，除此以外与制造例B1同样地得到切割成边长为约1,000μm的立方体形状的制造例B2的半月板组织片(B)。
对于制造例B2的半月板组织片(B)中的任意的100个组织片，在光学显微镜下测定每1边的长度，计算出每1片的体积。其结果，制造例B2的半月板组织片(B)的每1片的数均体积为1.141mm3。
<制造例B3>
在制造例B1中，将手术刀片的间隔由200μm变更为9,900μm，除此以外与制造例B1同样地得到细切成边长为约9,900μm的立方体形状的制造例B3的半月板组织片(B)。
对于制造例B3的半月板组织片(B)中的任意的100个组织片，在光学显微镜下测定每1边的长度，计算出每1片的体积。其结果，制造例B3的半月板组织片(B)的每1片的数均体积为970mm3。
<制造例B4>
在制造例B1中，将手术刀片的间隔从200μm变更为90μm，除此以外与制造例B1同样地得到细切成边长为约90μm的立方体形状的制造例B4的半月板组织片(B)。
对于制造例B4的半月板组织片(B)中的任意的100个组织片，在光学显微镜下测定每1边的长度，计算出每1片的体积。其结果，制造例B4的半月板组织片(B)的每1片的数均体积为0.0007mm3。
<制造例B5>
在制造例B1中，将手术刀片的间隔从200μm变更为10,500μm，除此以外与制造例B1同样地得到细切成边长为约10,500μm的立方体形状的制造例B5的半月板组织片(B)。
对于制造例B5的半月板组织片(B)中的任意的100个组织片，在光学显微镜下测定每1边的长度，计算出每1片的体积。其结果，制造例B5的半月板组织片(B)的每1片的数均体积为1152mm3。
<实施例1～18和比较例1～5：半月板再生用材料和比较用的半月板再生用材料的制作>
按照表3所示的比例(重量份)将各制造例和各比较制造例的蛋白质与磷酸缓冲生理盐水(以下有时简称为PBS。pH：7.2)混合，使蛋白质溶解在PBS中。接着，以表3所示的比例(重量份)通过颠倒混合将各制造例的半月板组织片进行混合，制作各实施例的半月板再生用材料和各比较例的半月板再生用材料。
<使用半月板再生用材料的半月板再生试验>
通过下述方法将各实施例的半月板再生用材料或各比较例的半月板再生用材料应用于使用兔的半月板损伤模型中。
将麻醉下的日本白色家兔(体重：3.0kg)的膝部切开，在半月板的前角处制作直径2mm的圆柱状的缺损模型。
将各半月板再生用材料施用于所制作的缺损部，用缝合线缝合切开的膝部。
4周后，对兔充分进行深麻醉，之后在充满二氧化碳的容器内确认心脏跳动和呼吸的停止。之后摘除包含缺损部的组织。将摘除的组织浸泡在10％缓冲福尔马林中，实施福尔马林固定。
之后将组织进行石蜡包埋，使用切片机[产品名：Retratome REM-710、大和光机工业株式会社制]，在垂直于圆柱状缺损的形成圆的面的方向上以得到厚度为约4μm的截面的方式进行切片，反复进行该操作，采集切片。
将所采集的切片中包含圆柱状缺损的形成圆的面的中心的切片作为评价用试验片，实施苏木精曙红染色(评价项目1和2)、番红O染色(评价项目3)。为了利用下述方法实施评价，使用数字显微镜[VHX-2000、株式会社KEYENCE制]对组织图像进行拍摄。
<评价项目1：所结合的修复组织>
利用光学显微镜对染色后的缺损部进行观察，按下述基准进行评价。试验中使用3只兔子来进行试验，将其分数的平均值示于表3。
各项目分数越高，表示半月板再生能力越高。
2：在试验片的截面中，从圆柱状缺损的两端再生出修复组织，从两端再生的修复组织彼此结合。
1：在试验片的截面中，从圆柱状缺损的两端再生出修复组织，但从两端再生的修复组织未彼此结合。
0：在试验片的截面中，未从圆柱状缺损的两端再生出修复组织。
<评价项目2：纤维软骨细胞的存在>
利用光学显微镜对染色后的缺损部进行观察，按下述基准进行评价。各项目分数越高，表示半月板再生能力越高。试验中使用3只兔子进行试验，将其分数的平均值示于表3。
2：在试验片的截面中，纤维软骨细胞广泛地存在于修复组织中(每单位面积的纤维软骨细胞数：2,000细胞/mm2以上)。
1：在试验片的截面中，纤维软骨细胞局部地存在于修复组织中(每单位面积的纤维软骨细胞数：100细胞/mm2以上且小于2,000细胞/mm2)。
0：在试验片的截面中，在修复组织中不存在纤维软骨细胞(每单位面积的纤维软骨细胞数：小于100细胞/mm2)。
需要说明的是，“每单位面积的纤维软骨细胞数”通过下述方法算出。利用荧光显微镜[BIOREVO BZ-9000、株式会社KEYENCE制]观察试验片的缺损部，测量0.5mm见方的面积中存在的纤维软骨细胞数。对缺损部内的中心和四角的5处进行测量，将其平均值作为“每单位面积的纤维软骨细胞数”。
<评价项目3：番红O染色>
利用光学显微镜对染色后的缺损部进行观察，按下述基准进行评价。各项目分数越高，暗示来自纤维软骨细胞的软骨基质产生量越多，表示半月板再生能力高。试验中使用3只兔子进行试验，将其分数的平均值示于表3。
2：在试验片的截面中，高密度地进行了染色(染色强度比例：65％以上)。
1：在试验片的截面中，微弱地进行了染色(染色强度比例：33％以上且小于65％)。
0：在试验片的截面中，无法确认到染色(染色强度比例：小于33％)。
需要说明的是，“染色强度比例”利用下述方法算出。利用荧光显微镜[BIOREVOBZ-9000、株式会社KEYENCE制]观察试验片的缺损部，对0.5mm见方的面积进行直方图分析，得到红色、绿色和蓝色的亮度分布。将所得到的分布中的亮度150以上作为对象，计算出红色的亮度相对于红色、绿色和蓝色的合计亮度的比例(％)(将亮度小于150作为亮度0进行计算)。对缺损部内的中心和四角的5处进行计算，将其平均值作为“染色强度比例”。
评价的结果，在实施例的病例中，利用修复组织使缺损部再生，并且纤维软骨细胞广泛地定位于修复组织内。此外，在对被认为由纤维软骨细胞产生的软骨基质染色的番红O染色中也为良好的结果。
工业实用性
本发明的半月板再生用材料通过在半月板损伤的再生治疗中填充在膝关节的半月板的缺损部分而促进半月板的再生，并且可造出优质的软骨组织，因此作为半月板再生用的基材是有用的。
序列表
<110> 国立大学法人广岛大学(HIROSHIMA UNIVERSITY)
三洋化成工业株式会社(SANYO CHEMICAL INDUSTRIES, LTD.)
<120> 半月板再生用材料(Material of meniscus repair)
<130> 595-PCT
<150> JP2018-190168
<151> 2018-10-05
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Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly
805 810 815
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val
820 825 830
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly
835 840 845
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
850 855 860
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
865 870 875 880
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
885 890 895
Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
900 905 910
Gly Val Gly Val Pro Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln
915 920 925
Asp Leu Arg Ser His His His His His His
930 935
<210> 18
<211> 60
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 多肽链(Polypeptide chain)(Y')
<400> 18
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1 5 10 15
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys
20 25 30
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
35 40 45
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
50 55 60
<210> 19
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 多肽链(Polypeptide chain)(S)
<400> 19
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
1 5 10 15
Gly Ser Gly Ala Gly Ala Gly Ser
20
<210> 20
<211> 1134
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质(Protein)
<400> 20
Met Asp Pro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val
1 5 10 15
Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro
20 25 30
Met Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
35 40 45
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
50 55 60
Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
65 70 75 80
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly
85 90 95
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
100 105 110
Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
115 120 125
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
130 135 140
Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
145 150 155 160
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
165 170 175
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
180 185 190
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val
195 200 205
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
210 215 220
Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val
225 230 235 240
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
245 250 255
Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
260 265 270
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
275 280 285
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
290 295 300
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro
305 310 315 320
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
325 330 335
Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
340 345 350
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
355 360 365
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
370 375 380
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
385 390 395 400
Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
405 410 415
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly
420 425 430
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
435 440 445
Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
450 455 460
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
465 470 475 480
Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
485 490 495
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
500 505 510
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
515 520 525
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val
530 535 540
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
545 550 555 560
Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val
565 570 575
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
580 585 590
Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
595 600 605
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
610 615 620
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
625 630 635 640
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro
645 650 655
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
660 665 670
Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
675 680 685
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
690 695 700
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
705 710 715 720
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
725 730 735
Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
740 745 750
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly
755 760 765
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
770 775 780
Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
785 790 795 800
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
805 810 815
Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
820 825 830
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
835 840 845
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
850 855 860
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val
865 870 875 880
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
885 890 895
Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val
900 905 910
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
915 920 925
Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
930 935 940
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
945 950 955 960
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
965 970 975
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro
980 985 990
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
995 1000 1005
Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser
1010 1015 1020
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
1025 1030 1035
Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1040 1045 1050
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1055 1060 1065
Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val
1070 1075 1080
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1085 1090 1095
Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
1100 1105 1110
Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln Asp Leu Arg Ser
1115 1120 1125
His His His His His His
1130
<210> 21
<211> 48
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 多肽链(Polypeptide chain)(S)
<400> 21
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
1 5 10 15
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala
20 25 30
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
35 40 45
<210> 22
<211> 200
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 多肽链(Polypeptide chain)(Y)
<400> 22
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1 5 10 15
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
20 25 30
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
35 40 45
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
50 55 60
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
65 70 75 80
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
85 90 95
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
100 105 110
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
115 120 125
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
130 135 140
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
145 150 155 160
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
165 170 175
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
180 185 190
Gly Val Pro Gly Val Gly Val Pro
195 200
<210> 23
<211> 1294
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> SELP6.1
<400> 23
Met Asp Pro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val
1 5 10 15
Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro
20 25 30
Met Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
35 40 45
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
50 55 60
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
65 70 75 80
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
85 90 95
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
100 105 110
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
115 120 125
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
130 135 140
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
145 150 155 160
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
165 170 175
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
180 185 190
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
195 200 205
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
210 215 220
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
225 230 235 240
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
245 250 255
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
260 265 270
Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
275 280 285
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
290 295 300
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
305 310 315 320
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val
325 330 335
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
340 345 350
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
355 360 365
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
370 375 380
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
385 390 395 400
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
405 410 415
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
420 425 430
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
435 440 445
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
450 455 460
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
465 470 475 480
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
485 490 495
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
500 505 510
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
515 520 525
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
530 535 540
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
545 550 555 560
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
565 570 575
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
580 585 590
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
595 600 605
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
610 615 620
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
625 630 635 640
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
645 650 655
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
660 665 670
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
675 680 685
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
690 695 700
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
705 710 715 720
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
725 730 735
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
740 745 750
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
755 760 765
Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
770 775 780
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
785 790 795 800
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
805 810 815
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val
820 825 830
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
835 840 845
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
850 855 860
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
865 870 875 880
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
885 890 895
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
900 905 910
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
915 920 925
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
930 935 940
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
945 950 955 960
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
965 970 975
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
980 985 990
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
995 1000 1005
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
1010 1015 1020
Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
1025 1030 1035
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
1040 1045 1050
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
1055 1060 1065
Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro
1070 1075 1080
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1085 1090 1095
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1100 1105 1110
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1115 1120 1125
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1130 1135 1140
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1145 1150 1155
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1160 1165 1170
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1175 1180 1185
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1190 1195 1200
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1205 1210 1215
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1220 1225 1230
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1235 1240 1245
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1250 1255 1260
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Met
1265 1270 1275
Asp Pro Gly Arg Tyr Gln Asp Leu Arg Ser His His His His His
1280 1285 1290
His
<210> 24
<211> 20
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 多肽链(Polypeptide chain)(Y)
<400> 24
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1 5 10 15
Pro Gly Val Gly
20
<210> 25
<211> 40
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 多肽链(Polypeptide chain)(Y)
<400> 25
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1 5 10 15
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
20 25 30
Gly Val Gly Val Pro Gly Val Gly
35 40
<210> 26
<211> 2694
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> ELP1.1
<400> 26
Met Asp Pro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val
1 5 10 15
Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro
20 25 30
Met Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
35 40 45
Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val Gly
50 55 60
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
65 70 75 80
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
85 90 95
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
100 105 110
Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly
115 120 125
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
130 135 140
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
145 150 155 160
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
165 170 175
Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val
180 185 190
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
195 200 205
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
210 215 220
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
225 230 235 240
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly
245 250 255
Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
260 265 270
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
275 280 285
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
290 295 300
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly
305 310 315 320
Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
325 330 335
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
340 345 350
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
355 360 365
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly
370 375 380
Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val
385 390 395 400
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
405 410 415
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
420 425 430
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
435 440 445
Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val
450 455 460
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
465 470 475 480
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
485 490 495
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
500 505 510
Gly Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro
515 520 525
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
530 535 540
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
545 550 555 560
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
565 570 575
Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val Gly
580 585 590
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
595 600 605
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
610 615 620
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
625 630 635 640
Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly
645 650 655
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
660 665 670
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
675 680 685
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
690 695 700
Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val
705 710 715 720
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
725 730 735
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
740 745 750
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
755 760 765
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly
770 775 780
Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
785 790 795 800
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
805 810 815
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
820 825 830
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly
835 840 845
Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
850 855 860
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
865 870 875 880
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
885 890 895
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly
900 905 910
Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val
915 920 925
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
930 935 940
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
945 950 955 960
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
965 970 975
Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val
980 985 990
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
995 1000 1005
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
1010 1015 1020
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
1025 1030 1035
Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val
1040 1045 1050
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1055 1060 1065
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1070 1075 1080
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1085 1090 1095
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala
1100 1105 1110
Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1115 1120 1125
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1130 1135 1140
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1145 1150 1155
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1160 1165 1170
Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro
1175 1180 1185
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1190 1195 1200
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1205 1210 1215
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1220 1225 1230
Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val
1235 1240 1245
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1250 1255 1260
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1265 1270 1275
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1280 1285 1290
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly
1295 1300 1305
Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly
1310 1315 1320
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
1325 1330 1335
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
1340 1345 1350
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
1355 1360 1365
Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val
1370 1375 1380
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1385 1390 1395
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1400 1405 1410
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1415 1420 1425
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala
1430 1435 1440
Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1445 1450 1455
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1460 1465 1470
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1475 1480 1485
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1490 1495 1500
Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro
1505 1510 1515
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1520 1525 1530
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1535 1540 1545
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1550 1555 1560
Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val
1565 1570 1575
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1580 1585 1590
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1595 1600 1605
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1610 1615 1620
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly
1625 1630 1635
Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly
1640 1645 1650
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
1655 1660 1665
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
1670 1675 1680
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
1685 1690 1695
Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val
1700 1705 1710
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1715 1720 1725
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1730 1735 1740
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
1745 1750 1755
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala
1760 1765 1770
Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1775 1780 1785
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1790 1795 1800
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1805 1810 1815
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1820 1825 1830
Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro
1835 1840 1845
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1850 1855 1860
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1865 1870 1875
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
1880 1885 1890
Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val
1895 1900 1905
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1910 1915 1920
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1925 1930 1935
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
1940 1945 1950
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly
1955 1960 1965
Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly
1970 1975 1980
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
1985 1990 1995
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
2000 2005 2010
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
2015 2020 2025
Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val
2030 2035 2040
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
2045 2050 2055
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
2060 2065 2070
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
2075 2080 2085
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala
2090 2095 2100
Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
2105 2110 2115
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
2120 2125 2130
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
2135 2140 2145
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
2150 2155 2160
Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro
2165 2170 2175
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
2180 2185 2190
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
2195 2200 2205
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
2210 2215 2220
Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val
2225 2230 2235
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
2240 2245 2250
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
2255 2260 2265
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
2270 2275 2280
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly
2285 2290 2295
Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly
2300 2305 2310
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
2315 2320 2325
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
2330 2335 2340
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
2345 2350 2355
Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val
2360 2365 2370
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
2375 2380 2385
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
2390 2395 2400
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
2405 2410 2415
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala
2420 2425 2430
Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
2435 2440 2445
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
2450 2455 2460
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
2465 2470 2475
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
2480 2485 2490
Val Gly Gly Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro
2495 2500 2505
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
2510 2515 2520
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
2525 2530 2535
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
2540 2545 2550
Gly Val Gly Val Pro Gly Val Gly Gly Ala Gly Ala Gly Ser Val
2555 2560 2565
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
2570 2575 2580
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
2585 2590 2595
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
2600 2605 2610
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Gly
2615 2620 2625
Ala Gly Ala Gly Ser Val Pro Gly Val Gly Val Pro Gly Val Gly
2630 2635 2640
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
2645 2650 2655
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
2660 2665 2670
Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln Asp Leu Arg Ser
2675 2680 2685
His His His His His His
2690
<210> 27
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> ELP1.2
<400> 27
Met Asp Pro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val
1 5 10 15
Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro
20 25 30
Met Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
35 40 45
Val Pro Gly Val Gly Val Val Val Pro Gly Val Gly Val Pro Gly Val
50 55 60
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Val Val Pro Gly
65 70 75 80
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
85 90 95
Gly Val Val Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
100 105 110
Val Gly Val Pro Gly Val Gly Val Val Val Pro Gly Val Gly Val Pro
115 120 125
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Val Val
130 135 140
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
145 150 155 160
Gly Val Gly Val Val Val Pro Gly Val Gly Val Pro Gly Val Gly Val
165 170 175
Pro Gly Val Gly Val Pro Gly Val Gly Val Val Val Pro Gly Val Gly
180 185 190
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
195 200 205
Val Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
210 215 220
Val Pro Gly Val Gly Val Val Val Pro Gly Val Gly Val Pro Gly Val
225 230 235 240
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Val Val Pro Gly
245 250 255
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
260 265 270
Gly Val Val Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
275 280 285
Val Gly Val Pro Gly Val Gly Val Val Val Pro Gly Val Gly Val Pro
290 295 300
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Val Val
305 310 315 320
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
325 330 335
Gly Val Gly Val Val Val Pro Gly Val Gly Val Pro Gly Val Gly Val
340 345 350
Pro Gly Val Gly Val Pro Gly Val Gly Val Val Val Pro Gly Val Gly
355 360 365
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
370 375 380
Val Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
385 390 395 400
Val Pro Gly Val Gly Val Val Val Pro Gly Val Gly Val Pro Gly Val
405 410 415
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Val Val Pro Gly
420 425 430
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
435 440 445
Gly Val Val Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
450 455 460
Val Gly Val Pro Gly Val Gly Val Val Gly Ala Gly Ala Met Asp Pro
465 470 475 480
Gly Arg Tyr Gln Asp Leu Arg Ser His His His His His His
485 490
<210> 28
<211> 1388
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> SLP4.1
<400> 28
Met Asp Pro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val
1 5 10 15
Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro
20 25 30
Met Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
35 40 45
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
50 55 60
Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
65 70 75 80
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
85 90 95
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
100 105 110
Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly
115 120 125
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
130 135 140
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
145 150 155 160
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly
165 170 175
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
180 185 190
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
195 200 205
Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
210 215 220
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
225 230 235 240
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
245 250 255
Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly
260 265 270
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
275 280 285
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro
290 295 300
Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
305 310 315 320
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
325 330 335
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val
340 345 350
Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
355 360 365
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
370 375 380
Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val
385 390 395 400
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
405 410 415
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
420 425 430
Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
435 440 445
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
450 455 460
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
465 470 475 480
Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly
485 490 495
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
500 505 510
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
515 520 525
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly
530 535 540
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
545 550 555 560
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
565 570 575
Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
580 585 590
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
595 600 605
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
610 615 620
Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly
625 630 635 640
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
645 650 655
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro
660 665 670
Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
675 680 685
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
690 695 700
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val
705 710 715 720
Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
725 730 735
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
740 745 750
Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val
755 760 765
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
770 775 780
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
785 790 795 800
Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
805 810 815
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
820 825 830
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
835 840 845
Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly
850 855 860
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
865 870 875 880
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
885 890 895
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly
900 905 910
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
915 920 925
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
930 935 940
Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
945 950 955 960
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
965 970 975
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
980 985 990
Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly
995 1000 1005
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
1010 1015 1020
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
1025 1030 1035
Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala
1040 1045 1050
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
1055 1060 1065
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val
1070 1075 1080
Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
1085 1090 1095
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
1100 1105 1110
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
1115 1120 1125
Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser
1130 1135 1140
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
1145 1150 1155
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
1160 1165 1170
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly
1175 1180 1185
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala
1190 1195 1200
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
1205 1210 1215
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala
1220 1225 1230
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
1235 1240 1245
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
1250 1255 1260
Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly
1265 1270 1275
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
1280 1285 1290
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
1295 1300 1305
Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala
1310 1315 1320
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
1325 1330 1335
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala
1340 1345 1350
Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
1355 1360 1365
Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln Asp Leu Arg Ser His
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His His His His His
1385
<210> 29
<211> 310
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> SELP8K4
<400> 29
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1 5 10 15
Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro
20 25 30
Met Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
35 40 45
Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly
50 55 60
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65 70 75 80
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85 90 95
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
100 105 110
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115 120 125
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130 135 140
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
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Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
165 170 175
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180 185 190
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195 200 205
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
210 215 220
Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
225 230 235 240
Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly
245 250 255
Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
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Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
275 280 285
Ser Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln Asp Leu Arg Ser
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His His His His His His
305 310
Claims (5)
1.一种半月板再生用材料，其是包含蛋白质(A)的半月板再生用材料，其中，
所述蛋白质(A)具有多肽链(Y)和/或多肽链(Y’)，
所述蛋白质(A)中的所述多肽链(Y)与所述多肽链(Y’)的合计个数为1～100个，
所述多肽链(Y)是2～200个氨基酸序列(X)连续而成的多肽链，该氨基酸序列(X)为序列号1所示的氨基酸序列即VPGVG序列(1)、序列号4所示的氨基酸序列即GVGVP序列(4)、GPP序列、GAP序列和序列号3所示的氨基酸序列即GAHGPAGPK序列(3)中的至少一者，
所述多肽链(Y’)是所述多肽链(Y)中的5％以下的氨基酸被赖氨酸和/或精氨酸置换而成的多肽链，所述赖氨酸和所述精氨酸的合计个数为1～100个，
通过圆二色光谱法求出的所述蛋白质(A)的β-转角结构和无规卷曲结构的合计比例为60％～85％，
设所述氨基酸序列(X)中的60％以下的氨基酸被赖氨酸和/或精氨酸置换而得到的氨基酸序列为氨基酸序列(X’)时，以所述蛋白质(A)的总氨基酸数为基准，构成所述蛋白质(A)中包含的所述氨基酸序列(X)和所述氨基酸序列(X’)的氨基酸的合计个数为50％～70％，
所述蛋白质(A)具有序列号16所示的氨基酸序列、序列号17所示的氨基酸序列、序列号20所示的氨基酸序列、序列号23所示的氨基酸序列、序列号26所示的氨基酸序列、序列号29所示的氨基酸序列、或者与这些氨基酸序列的同源性为70％以上的氨基酸序列。
2.如权利要求1所述的半月板再生用材料，其中，所述蛋白质(A)的利用SDS-PAGE即SDS聚丙烯酰胺凝胶电泳法得到的分子量为15kDa～200kDa。
3.如权利要求1或2所述的半月板再生用材料，其中，进一步含有半月板组织片(B)。
4.如权利要求3所述的半月板再生用材料，其中，所述半月板组织片(B)的每1片的数均体积为0.001mm3～1,000mm3。
5.如权利要求3所述的半月板再生用材料，其中，以半月板再生用材料的重量为基准，所述蛋白质(A)的重量比例为5重量％～25重量％，所述半月板组织片(B)的重量比例为5重量％～50重量％。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018-190168 | 2018-10-05 | ||
| JP2018190168 | 2018-10-05 | ||
| PCT/JP2019/037589 WO2020071208A1 (ja) | 2018-10-05 | 2019-09-25 | 半月板再生用材料 |
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| Publication Number | Publication Date |
|---|---|
| CN112789063A CN112789063A (zh) | 2021-05-11 |
| CN112789063B true CN112789063B (zh) | 2022-10-25 |
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| CN201980065130.4A Active CN112789063B (zh) | 2018-10-05 | 2019-09-25 | 半月板再生用材料 |
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| US (1) | US20210338890A1 (zh) |
| EP (1) | EP3862030A4 (zh) |
| JP (1) | JP7429929B2 (zh) |
| KR (1) | KR20210060587A (zh) |
| CN (1) | CN112789063B (zh) |
| WO (1) | WO2020071208A1 (zh) |
Family Cites Families (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61150746A (ja) | 1984-12-25 | 1986-07-09 | Honda Motor Co Ltd | シリンダブロツク素材用鋳型 |
| US5830713A (en) * | 1986-11-04 | 1998-11-03 | Protein Polymer Technologies, Inc. | Methods for preparing synthetic repetitive DNA |
| KR0176966B1 (ko) | 1988-11-09 | 1999-04-01 | 제니스 케이. 맥밀리언 | 재조합 방법으로 제조한 기능적 합성 단백질 폴리머 |
| US5817303A (en) | 1995-05-05 | 1998-10-06 | Protein Polymer Technologies, Inc. | Bonding together tissue with adhesive containing polyfunctional crosslinking agent and protein polymer |
| GB0516846D0 (en) | 2005-08-17 | 2005-09-21 | Knight David P | Meniscal repair device |
| ES2439943T3 (es) | 2005-09-02 | 2014-01-27 | ED. Geistlich Söhne AG für Chemische Industrie | Método de reparación de desgarros de menisco |
| ES2403733T3 (es) * | 2005-10-05 | 2013-05-21 | Commonwealth Scientific And Industrial Research Organisation | Proteínas de seda |
| EP2676683B1 (en) * | 2011-02-18 | 2016-09-21 | Kyoto University | Aqueous protein solution containing a material for tissue regeneration |
| GB201118000D0 (en) | 2011-10-19 | 2011-11-30 | Orthox Ltd | An implantable repair device |
| JP6077316B2 (ja) | 2012-02-02 | 2017-02-08 | 三洋化成工業株式会社 | 組織再生用材料及び組織再生用タンパク質溶液 |
| JP6383721B2 (ja) * | 2013-02-22 | 2018-08-29 | 三洋化成工業株式会社 | 創傷治癒剤 |
| JP6326326B2 (ja) * | 2013-08-30 | 2018-05-16 | 三洋化成工業株式会社 | 創傷治癒剤 |
| JP6754982B2 (ja) * | 2015-05-20 | 2020-09-16 | 国立大学法人京都大学 | 発育障害抑制材 |
| JP6730834B2 (ja) | 2016-04-04 | 2020-07-29 | 三洋化成工業株式会社 | タンパク質組成物 |
| JP6982395B2 (ja) * | 2017-03-16 | 2021-12-17 | 三洋化成工業株式会社 | インターロイキン１０の産生量増加剤 |
| CN107496990A (zh) * | 2017-08-15 | 2017-12-22 | 广东泰宝医疗器械技术研究院有限公司 | 一种可注射的抗菌软骨修复水凝胶及其制备方法 |
| CN108404205A (zh) * | 2018-07-04 | 2018-08-17 | 上海交通大学医学院附属第九人民医院 | 一种负载软骨细胞抗炎半月板支架的生物三维打印制备方法 |
-
2019
- 2019-09-25 CN CN201980065130.4A patent/CN112789063B/zh active Active
- 2019-09-25 JP JP2020550335A patent/JP7429929B2/ja active Active
- 2019-09-25 US US17/282,577 patent/US20210338890A1/en active Pending
- 2019-09-25 KR KR1020217011599A patent/KR20210060587A/ko active IP Right Grant
- 2019-09-25 WO PCT/JP2019/037589 patent/WO2020071208A1/ja active Application Filing
- 2019-09-25 EP EP19869881.3A patent/EP3862030A4/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP3862030A1 (en) | 2021-08-11 |
| US20210338890A1 (en) | 2021-11-04 |
| EP3862030A4 (en) | 2022-08-31 |
| CN112789063A (zh) | 2021-05-11 |
| JPWO2020071208A1 (ja) | 2021-09-02 |
| JP7429929B2 (ja) | 2024-02-09 |
| KR20210060587A (ko) | 2021-05-26 |
| WO2020071208A1 (ja) | 2020-04-09 |
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