一种用于解除免疫抑制的融合蛋白及其应用的制作方法
本发明涉及生物医药技术领域，更具体地说，它涉及一种用于解除免疫抑制的融合蛋白及其应用。
背景技术：
嵌合抗原受体t细胞(car-t)疗法在血液系统肿瘤的治疗中取得了显著的疗效，尤其是靶向cd19的car-t疗法，已经有两款产品被fda批准上市。但是car-t疗法在实体瘤治疗中仍未有大的进展，其疗效不太显著的一个重要原因是实体肿瘤的免疫微环境过于复杂，具有很强的抑制t细胞活力的能力，从而导致t细胞在实体肿瘤内不能有效增殖，耗竭过后，导致不能起到杀伤肿瘤组织的能力。
免疫检查点是一类表达在t细胞表面的抑制性受体，其与配体的结合会导致t细胞功能的耗竭。pd-1是当前研究较为深入的免疫检查点之一，通过pd-1抑制剂阻止pd-1与其配体pd-l1的结合，从而拯救t细胞耗竭表型，恢复t细胞杀伤肿瘤的能力。临床结果也显示pd-1抑制剂在多种实体瘤中有疗效，但是有效率仅为20％左右。tgf-β是实体瘤组织中常见抑制性细胞因子，其以自分泌或是旁分泌的方式通过细胞表面的受体信号传导途径参与细胞的调节作用，主要包括抑制免疫作用，使肿瘤细胞逃避免疫监视。tgf-β抑制白细胞介素2的产生，从而抑制t细胞增殖、分化及其细胞毒性，最终促进肿瘤的生长。有研究表明tgf-β能够抑制nk细胞增殖及其受体的表达，从而抑制nk-cell毒性。tgf-β因其具有重要的促肿瘤作用，而被认为是治疗的理想靶点。tgf-β受体有三种类型分别为：tgf-βri、rii、riii，其中只有在i型和ii型同时表达时才能转导信号至细胞内，tgf-βiii型不直接参与信号转导。tgf-β信号转导第一步是tgf-βrii自身被磷酸化，进而激活其氨基酸残基ser213及ser409，而后与tgf-βi相互作用，激活tgf-βi，最终通过smads蛋白将信号直接转导至细胞核内。鉴于tgf-βii在信号转导的关键作用，因此阻断tgf-β信号转导，tgf-βii受体更加理想。tgf-β三种受体均含有胞外区、跨膜区、胞内区，通过在受体胞外区容易阻断tgf-β信号转导，进而抑制tgf-β信号转导，起到解除免疫抑制作用。
鉴于car-t在实体瘤治疗中由于免疫微环境导致疗效受限和pd-1抗体单药使用中的局限性，我们开发出整合了car-t、pd-1抑制剂、tgf-β抑制剂的新型免疫疗法，使得car-t细胞靶向实体瘤的同时，分泌抑制pd-1/pd-l1信号通路和tgf-β信号通路的融合蛋白，可能有更强的解除免疫抑制微环境，治疗肿瘤的能力。
技术实现要素：
针对上述技术问题，本发明的目的在于提供一种用于解除免疫抑制的融合蛋白及其应用，针对肿瘤内部抑制性微环境，设计了新型融合蛋白pta和ptr，pta和ptr能够同时抑制pd-1和tgf-β信号通路，单独使用或者联合car-t用于实体瘤的治疗。
本发明的上述技术目的是通过以下技术方案得以实现的：
一种用于解除免疫抑制的融合蛋白，所述融合蛋白为pta或者ptr中的一种，所述融合蛋白其中一部分为pd-1的抑制剂，另一部分为tgf-β的抑制剂。
进一步地，所述pd-1的抑制剂为pd-1抗体。
进一步地，所述tgf-β的抑制剂为tgf-β抗体或者tgfbrii的胞外区片段。
进一步地，所述融合蛋白为pta，所述pta的一部分为pd-1抗体，另一部分为tgf-β抗体，所述pta氨基酸序列为seqidno.1。
进一步地，所述融合蛋白为ptr，所述ptr的一部分为pd-1抗体，另一部分为tgfbrii的胞外区片段，所述ptr氨基酸序列为seqidno.2。
编码上述氨基酸序列的dna序列。
一种载体，编码上述的dna序列。
一种细胞，稳定或瞬时表达上述的载体。
上述的氨基酸、dna、载体和细胞在人类肿瘤治疗中的应用。
与现有技术相比，本发明的有益效果如下：
本发明解决了car-t细胞在实体瘤治疗中疗效有限、pd-1有效率不高的问题，针对肿瘤内部抑制性微环境，设计了新型融合蛋白pta和ptr，pta和ptr能够同时抑制pd-1和tgf-β信号通路，单独使用或者联合car-t用于实体瘤的治疗，使得car-t细胞靶向实体瘤的同时，分泌抑制pd-1/pd-l1信号通路和tgf-β信号通路的融合蛋白，具有更强的解除免疫抑制微环境，治疗肿瘤的能力。
附图说明
图1为carl6-pta和carl6-ptr分子结构示意图；
图2为car-t细胞阳性率的谱图；
图3为a549细胞tm4sf1阳性率的谱图；
图4为不同car-t细胞杀靶细胞a549能力的谱图；
图5为回输后小鼠外周血car-t细胞数量的谱图；
图6为回输后小鼠瘤体生长曲线。
具体实施方式
下面结合实施例及附图对本发明作进一步的详细说明，但本发明的实施方式不仅限于此。
如图1-6所示，一种用于解除免疫抑制的融合蛋白，融合蛋白为pta或者ptr中的一种，融合蛋白其中一部分为pd-1的抑制剂，另一部分为tgf-β的抑制剂。优选地，pd-1的抑制剂为pd-1抗体；tgf-β的抑制剂为tgf-β抗体或者tgfbrii的胞外区片段。
当融合蛋白为pta时，pta的一部分为pd-1抗体，另一部分为tgf-β抗体，且pta氨基酸序列为seqidno.1。
当融合蛋白为ptr时，ptr的一部分为pd-1抗体，另一部分为tgfbrii的胞外区片段，且ptr氨基酸序列为seqidno.2。
编码上述氨基酸序列的dna序列。一种载体，编码上述的dna序列。一种细胞，稳定或瞬时表达上述的载体。氨基酸、dna、载体和细胞在人类肿瘤治疗中的应用。
实施例一：
本实施例中构建了带有pta或者ptr表达的car分子carl6-pta或carl6-ptr。其中pta为由anti-pd1scfv和anti-tgfβscfv通过柔性gs连接肽链接而成的融合蛋白；ptr为由anti-pd1scfv和stgfbrii通过柔性gs连接肽链接而成的融合蛋白；stgfbrii为tgfbrii的胞外区序列。其具体分子结构示意图如图1所示。
carl6-ptr载体构建：首先我们通过基因合成得到anti-pd1scfv质粒和stgfbrii序列质粒，通过pcr的方式将pd1抗体序列和stgfbrii序列分别扩增出来，再通过搭桥pcr的方式将pd1抗体序列和stgfβrii序列拼接到一起，并在中间添加柔性连接多肽，构建成完整的anti-pd1scfv-stgfbrii序列(标记为ptr)。用clai单酶切编码carl6的慢病毒载体phage-carl6，得到带有粘性末端的载体片段，将ptr用重组酶连接到上述单酶切载体中，50℃反应20分钟，随后转化stbl3感受态细胞。第二天接种单菌落至lb扩增培养，并提取质粒。通过酶切和测序验证其序列，留下正确的质粒命名为phage-carl6-ptr进行下一步实验。
carl6-pta载体构建：首先我们通过基因合成得到anti-pd1scfv质粒和anti-tgfβscfv序列质粒，通过pcr的方式将anti-pd1scfv序列和anti-tgfβscfv序列分别扩增出来，再通过搭桥pcr的方式将anti-pd1scfv序列和anti-tgfβscfv序列拼接到一起，并在中间添加柔性连接多肽，构建成完整的anti-pd1scfv-anti-tgfβscfv序列(标记为pta)。用clai单酶切编码carl6的慢病毒载体phage-carl6，得到带有粘性末端的载体片段，将pta用重组酶连接到上述单酶切载体中，50℃反应20分钟，随后转化stbl3感受态细胞。第二天接种单菌落至lb扩增培养，并提取质粒。通过酶切和测序验证其序列，留下正确的质粒命名为phage-carl6-pta进行下一步实验。
实施例二：
通过本实施例构建了自分泌pta或者ptr的carl6-t细胞carl6-pta-t或carl6-ptr-t，在本具体实例中我们通过以下方式实现；
首先获得带有pta或者ptr的car分子慢病毒，在本实例中，所选的重组表达载体为慢病毒载体。进一步地，将实施例一中的质粒phage-carl6、phage-carl6-pta和phage-carl6-ptr在辅助包装质粒pspax2和vsv-g包膜质粒pmd2.g存在下共转染293t细胞以包装为慢病毒载体。具体流程为：提前一天将1.7×107293t细胞接种至t175培养瓶中，待细胞汇合率达到70-90％时，使用lipo-3000将上述质粒共转染至293t细胞中，转染前用50ml培养基换液。转染后48小时和72小时收取细胞上清即为慢病毒粗度液。之后通过离心和过滤去除细胞碎片等杂质，再通过超速离心浓缩，获得带有carl6、carl6-pta及carl6-ptr的慢病毒。
构建car-t细胞：构建前一天包被抗体，用于激活t细胞，在六孔板中加入retronectin、抗人cd3以及cd8抗体，放在4℃条件下过夜。通过常规采血的方法获得血样，用淋巴细胞分离液离心获得pbmc，之后使用stemcellt细胞分选试剂盒，从pbmc中分选出t细胞并计数。用培养基重悬t细胞至密度为1×106/ml，培养基为含有人ab血清和il-2的x-vivo15培养基。取出前一天包被好的六孔板，用pbs洗两遍，将重悬好的t细胞加入到包被的六孔板中培养。细胞培养至24h后加入polybrene和对应慢病毒(carl6、carl6-pta和carl6-ptr)，混合均匀后放回培养箱培养。感染24h后将六孔板中的细胞离心换液，换成含人ab血清和il-2的x-vivo15培养基，之后在培养过程中只需补充培养基，使细胞密度维持在1×106/ml即可。培养至第四天取1mlt或者car-t细胞与fab抗体共孵育、盐水清洗、离心、流式细胞术检测scfv的表达，以确定car-t细胞的转导效率。如图2所示：carl6-t阳性率为48.2％，carl6-pta-t阳性率为42.4％，carl6-ptr-t的阳性率为30.1％大约，t细胞为未转导对照细胞。
实施例三：
通过本实施例证明了carl6、carl6-pta-t和carl6-ptr-t能够在体外有效杀伤靶细胞。
car-t细胞构建好后，我们通过细胞杀伤试验，验证构建的carl6-t、carl6-pta-t和carl6-ptr-t能有效的杀伤肿瘤细胞。通过tm4sf1抗体染色和流式细胞术检测，如图3所示，肺癌细胞系a549呈tm4sf1阳性。在96孔板中将car-t细胞(carl6-t、carl6-pta-t、carl6-ptr-t)以及t细胞分别和l6阳性高表达的靶细胞a549，按照1:1、5:1、10:1的效靶比来设置，同时设置培养基空白组以及靶细胞对照组。在car-t细胞和靶细胞共培养16h后，采用cck8的方法并用酶标仪测定od450的值，计算car-t细胞的杀伤效率。结果如图4所示，我们够建的gar-t细胞与普通的t细胞相比有明显的杀伤优势，且杀伤效率非常高。在效靶比为1:1时，杀伤效率即可达到40％左右，5:1时可达到60-70％，当效靶比为10:1时，大部分靶细胞能被杀死，杀伤效率可达90％左右。这些结果充分说明，我们构建的car-t细胞能够有效的杀伤靶细胞，且carl6-pta-t和carl6-ptr-t杀伤靶细胞的能力显著优于carl6-t。
实施例四：
通过本实施例我们证明了在体内carl6-pta-t和carl6-ptr-t相对于carl6-t具有更强的增殖能力和抑制瘤能力。
在本实验开始前，使用周龄在6周的nod-scid免疫缺陷鼠，经皮下注射肺癌细胞系a549。接种一到两周之后，测瘤体的大小。瘤体大小计算，用瘤体的最长长度乘最长长度垂直方向的长度除以2得到以mm3为单位的瘤体大小。等到瘤体达到100-300mm3大小时，将培养好的car阳性的carl6-pta-t、carl6-ptr-t、carl6-t及t细胞通过尾静脉注射至小鼠体内，5×10e6个细胞/只小鼠。回输细胞后，通过小鼠尾静脉采血来检测car-t细胞在小鼠体内的增殖情况，同时测量瘤体大小变化，并绘制肿瘤变化曲线。
小鼠体内car-t增殖情况：采小鼠尾静脉血20ul加至1.5ml灭菌的ep管，管中提前加有20ul肝素抗凝剂。在每个管中加2ul抗人cd45抗体，室温避光孵育十分钟，在管中加1ml红细胞裂解液裂解5分钟，离心收集细胞沉淀，用生理盐水洗一遍，流式检测car-t细胞数量。结果如图5所示，carl6-pta-t和carl6-ptr-t比carl6-t数量明显高出7到8倍，说明我们构建的carl6-pta-t和carl6-ptr-t在小鼠体内增殖能力优于普通的carl6-t。进一步地，通过测量回输后小鼠瘤体大小变化来确定我们构建的car-t在体内治疗肿瘤的效果。结果如图6所示，与t组对比，我们构建的carl6-t、carl6-pta-t、carl6-ptr-t均能明显的控制并减小瘤体的大小，说明我们构建的car-t细胞在体内对治疗肿瘤有显著的效果。进一步地，对比carl6-pta-t、carl6-ptr-t和普通carl6，结果显示carl6-pta-t和carl6-ptr-t组瘤体平均大小显著低于carl6-t，更进一步地说明我们构建的carl6-pta-t和carl6-ptr-t相比普通的carl6有明显的优势。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
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<120>一种用于解除免疫抑制的融合蛋白及其应用
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435440445
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450455460
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465470475480
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485490495
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500505510
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515520525
cysalacyscysthralacysalacysthrglyglyalacysalagly
530535540
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545550555560
alacysthrthrcysalaalaglyglyglyalacysglyglythrthr
565570575
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580585590
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595600605
cyscysthralathrthrthrglycysalaglyalathrcysalaala
610615620
cysalaalacyscysthrcysalaalaalaalaalathrglyalagly
625630635640
glyalacysalathrglyglycysthralacysalathralathrthr
645650655
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660665670
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675680685
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690695700
glycyscysalaalaglyglycysalacyscysalacysthrcysthr
705710715720
cysalacysalaglythrcysthrcyscysthrcysalaglyglyala
725730735
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740745750
thralaglyglyalathrglythrglycysalaglycyscysthrthr
755760765
thrglythrthrglythralaglythrcysthrglycysalathrthr
770775780
thrthrglyglyglyalathrglycysalathralacysthrthrala
785790795800
thrthrthrglythrthrglyglycysthrthralacysalaalaala
805810815
alaalaalaglyalaalaglythralathrthrcysalathrcyscys
820825830
alaglythrglythrglycysalacysglyalacyscyscysthrala
835840845
alacysglyglythrglyalaalathralacysalathrglythrthr
850855860
cysalathrglyalaglyalaglycysalaglythrglyalaalacys
865870875880
alacysalaglycyscysalaalaalaalaalaalathrcysthrala
885890895
glyalacysthrcysalacysalaglyalathrglythrglyalacys
900905910
cyscysthralathrcysthralaglyalaglyalaglyglyglycys
915920925
alaglyalaglyglyalaalaglythrcysthrthrcysthralaala
930935940
cysalathrglycysglyglythrglyalacysglythrglyglyala
945950955960
glyglyalaglyalaalathrcyscyscysglyglycyscyscysthr
965970975
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980985990
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99510001005
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101010151020
glyalacysalacysalaalacyscysglyglycysglyalaglyala
1025103010351040
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104510501055
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106010651070
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107510801085
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109010951100
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1105111011151120
glythrglythrcysalathrcysthrthralacyscysthrcysgly
112511301135
cysthrthrglyglythralathrcysalaglycysalaglyalaala
114011451150
glycyscysthrglyglyglycysalaglyglycysthrcyscysala
115511601165
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117011751180
alathrglycysalathrcysthralaalacysalaglyalaglycys
1185119011951200
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120512101215
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122012251230
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123512401245
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125012551260
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1265127012751280
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128512901295
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130013051310
alaalacysthrglyglycyscysthralaglyalaalacysalathr
131513201325
thrcysglyglycyscysalaglyglyglyalaalacyscysalaala
133013351340
glyglythrglyglyalaglyalathrthralaalaglyglyglyala
1345135013551360
glyglyalaglyglycysglyglyglythrcyscysglyglycysgly
136513701375
glyalaglyglycysglyglyalaalaglycysglyglyalaglygly
138013851390
cysglyglyalaglyglyalaalaglythrglyglycysglyglyala
139514001405
glyglyalathrcysthrglyglycysglyglycysglyglyalathr
141014151420
cysthrcysalaglyglythrglycysalaglycysthrcysglythr
1425143014351440
glyglyalaglythrcyscysglyglyalaglyglycysglyglyala
144514501455
glythrglyglythrglycysalaglycyscysthrglyglyalacys
146014651470
glyglythrcyscyscysthrcysalaglyalacysthrcysglyala
147514801485
cysthrglycysalaalaglyglycysthrthrcyscysglyglycys
149014951500
alathrcysalacysalathrthrcysthrcysthralaalacysthr
1505151015151520
cysthrglyglythralathrglycysalacysthrglyglyglythr
152515301535
glyalaglyalacysalaglyglycysalacyscysalaglyglyala
154015451550
alaalaglyglyglycysthrthralaglyalaglythrglyglygly
155515601565
thrglyglycysthrglythrglyalathrthrthrglyglythrala
157015751580
cysglyalacysglyglyglythrcysglyalaalaglyalaglyala
1585159015951600
thralacysthralacysglycyscysglyalacysalaglycysgly
160516101615
thrglyalaalaglyglyglyalacysglyglythrthrcysalacys
162016251630
alaalathrthralaglycysalaglyalaglyalacysalaalacys
163516401645
thrcyscysalaalaglyalaalacysalacyscyscysthrglythr
165016551660
thrcyscysthrcyscysalaglyalathrglyalaalacysthrcys
1665167016751680
thrcysthrglycysglycysglycyscysglyalaglyglyalacys
168516901695
alacyscysglycyscysglythrglythralacysthralacysthr
170017051710
glycysglycysthralacysalaalaalacysglyalacysglyala
171517201725
cysthralacysthrglyglyglyglyalacysalaglyglyglycys
173017351740
alacysalacysthrcysglythrglyalacyscysglythrglythr
1745175017551760
cysthralaglycysglyglycysglyglycysglyglycysthrcys
176517701775
glyglyglycysglyglycysglyglyglythrcyscysglyglycys
178017851790
glyglyalaglyglyalaglyglythrthrcysglyglyalaalaala
179518001805
cysglyglythralacysthrcysalacysglycysalaglythrcys
181018151820
thrcyscysalaglyglythralacyscyscysthrglythrcysthr
1825183018351840
thrthrglythrcysthrcyscysalaglyglyglyglyalaalaala
184518501855
glyalaglycyscysalacyscyscysthrcysthrcyscysthrgly
186018651870
cysalaglyglyglycyscysalaglythrcysalaglyalaglythr
187518801885
cysthrthrglyglycysalaglycysalaglycysthralacysthr
189018951900
thralaglycyscysthrglyglythralathrcysalaglycysala
1905191019151920
glyalaalaalacyscysthrglyglythrcysalaglyglycysthr
192519301935
cyscyscysalaglyglycysthrcyscysthrcysalathrcysthr
194019451950
alathrglyglythrglycysalathrcyscysalaglycysalagly
195519601965
glyglycysalacyscysthrglyglycysalathrcyscyscysala
197019751980
glyalacysalaglyglythrthrcysalaglythrglyglycysala
1985199019952000
glythrglyglyglythrcysthrglyglythralacyscysglyala
200520102015
cysthrthrcysalacysthrcysthrcysalacyscysalathrcys
202020252030
alaglycyscysglyalacysthrglyglyalaglycyscysthrgly
203520402045
alaalaglyalathrthrthrthrglycysalaglythrthrthrala
205020552060
thrthralacysthrglythrcysalaglycysalaglythralathr
2065207020752080
glycysthrglyalacysthrcysalacyscysglyalathrcysala
208520902095
cyscysthrthrcysglyglycyscysalaalaglyglyglyalacys
210021052110
alacysglyalacysthrglyglyalaglyalathrthralaalaala
211521202125
glyglycysthrcyscysalacyscysthrcysthrglyglyalathr
213021352140
cyscysglyglycysalaalaglycyscyscysglyglyalathrcys
2145215021552160
thrglyglycysglyalaglyglyglyalathrcyscysalacyscys
216521702175
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218021852190
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219522002205
thrglyalaglyglythrglyalaalaglyalaalaglycyscysthr
221022152220
glyglyglythrcyscysthrcysglyglythrglyalaalaglygly
2225223022352240
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224522502255
thrglyglyalathralacysalacyscysthrthrcysalaglythr
226022652270
alaglycysalaalathrglythrthralathrcysalaglycysthr
227522802285
glyglyglythrglycysglycyscysalaglyglycyscyscyscys
229022952300
thrglyglyalacysalaalaglyglyglycysthrcysglyalagly
2305231023152320
thrglyglyalathrglyglyglyglyglyglyglyglythrcysala
232523302335
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234023452350
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235523602365
alaglyalathrthrcysalaalaglyglyglycysalaglyalagly
237023752380
thrcysalacysglyalathrthralacyscysglycysglyglyala
2385239023952400
cysglyalaalathrcyscysalacysthralaglythralacysala
240524102415
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242024252430
glycysalaglycyscysthrglyalaglyglythrcysthrglyala
243524402445
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245024552460
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2465247024752480
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248524902495
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250025052510
glyglythrcysalaglyglyglythralacysglythrthrglygly
251525202525
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253025352540
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151015
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202530
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354045
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505560
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65707580
cyscysalaglycysthrcysalaalaglythrglythralaalagly
859095
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100105110
cysalaglycysalaglyalaalaglycyscysalaglyglyalathr
115120125
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130135140
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145150155160
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165170175
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180185190
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195200205
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210215220
cysalaalathrcysalaglycysalaglyalaglythrglyglyala
225230235240
glyglycysthrglyalaalaglyalathrglycysthrglycyscys
245250255
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260265270
alaglythrglyglyalaalathralaglythralaalacyscyscys
275280285
alacysthrcysalacysglythrthrcysglyglythrglycysthr
290295300
glyglyglyalacyscysalaalaglycysthrglyglyalaglycys
305310315320
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325330335
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340345350
glyglycysthrcysthrglyglythrglyglythrglyglycysgly
355360365
glyalathrcyscyscysalaglyalathrcyscysalaglythrthr
370375380
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385390395400
glyalaglycysthrglyalaalaglyalaalaglycyscysthrgly
405410415
glyalaglyalaglyalacysalaglythrcysalaalaglyalathr
420425430
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435440445
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450455460
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465470475480
glyglythrglyalaalaglycysalaglyglycysthrcyscysala
485490495
glyglyalaalaalaglyglyglythrthrthralaalaalaglythr
500505510
glyglyalathrglyglyglycysthrglyglyalathralaalaala
515520525
cysalacyscysthralacysalacysthrglyglyalacysalagly
530535540
cyscysalaalacysalathralathrglycysthrglyalathrgly
545550555560
alacysthrthrcysalaalaglyglyglyalacysglyglythrthr
565570575
thrglycyscysthrthrcysthrcysthrthrthrglyglyalaala
580585590
alacyscysthrcysthrglycyscysthralacysalacysthrgly
595600605
cyscysthralathrthrthrglycysalaglyalathrcysalaala
610615620
cysalaalacyscysthrcysalaalaalaalaalathrglyalagly
625630635640
glyalacysalathrglyglycysthralacysalathralathrthr
645650655
thrcysthrglythrglycysalaalaglyalathrthrthralagly
660665670
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675680685
thralacysglycysthrglyalacysthralacysthrglyglygly
690695700
glycyscysalaalaglyglycysalacyscysalacysthrcysthr
705710715720
cysalacysalaglythrcysthrcyscysthrcysalaglyglyala
725730735
thrcyscysthrthrcysthrglyglythrthralacyscyscysala
740745750
thralaglyglyalathrglythrglycysalaglycyscysthrthr
755760765
thrglythrthrglythralaglythrcysthrglycysalathrthr
770775780
thrthrglyglyglyalathrglycysalathralacysthrthrala
785790795800
thrthrthrglythrthrglyglycysthrthralacysalaalaala
805810815
alaalaalaglyalaalaglythralathrthrcysalathrcyscys
820825830
alaglythrglythrglycysalacysglyalacyscyscysthrala
835840845
alacysglyglythrglyalaalathralacysalathrglythrthr
850855860
cysalathrglyalaglyalaglycysalaglythrglyalaalacys
865870875880
alacysalaglycyscysalaalaalaalaalaalathrcysthrala
885890895
glyalacysthrcysalacysalaglyalathrglythrglyalacys
900905910
cyscysthralathrcysthralaglyalaglyalaglyglyglycys
915920925
alaglyalaglyglyalaalaglythrcysthrthrcysthralaala
930935940
cysalathrglycysglyglythrglyalacysglythrglyglyala
945950955960
glyglyalaglyalaalathrcyscyscysglyglycyscyscysthr
965970975
alathrglyglyalaglyalacysalacyscysalaglycysthrcys
980985990
alaglycysthrglycysthrglythrthrcyscysthrcyscysthr
99510001005
cyscysthrglycysthrglythrglyglycysthrcyscyscysthr
101010151020
glyalacysalacysalaalacyscysglyglycysglyalaglyala
1025103010351040
thrthrglythrglycysthrglyalacyscyscysalaglythrcys
104510501055
thrcyscyscysglycysthralacysalacysthrglythrcysthr
106010651070
cysthrglyalaglycyscyscysthrglyglycysglyalaglycys
107510801085
glycysglycyscysalacyscyscysthrglythrcysthrthrgly
109010951100
cyscysglycysglycysthrthrcysthrcysalaglythrcysthr
1105111011151120
glythrglythrcysalathrcysthrthralacyscysthrcysgly
112511301135
cysthrthrglyglythralathrcysalaglycysalaglyalaala
114011451150
glycyscysthrglyglyglycysalaglyglycysthrcyscysala
115511601165
cysglycyscysthrglycysthrcysalathrthrthralacysgly
117011751180
alathrglycysalathrcysthralaalacysalaglyalaglycys
1185119011951200
cysalacysalaglyglyglyalathrthrcyscyscysglycysthr
120512101215
alaglyglythrthrcysalaglycysglyglyalathrcysthrgly
122012251230
glyglythrcyscysglyglycysalacyscysglyalacysthrthr
123512401245
cysalacysalacysthrglyalacyscysalathrthrthrcysthr
125012551260
alaglycyscysthrcysglyalaalacyscyscysglyalaglygly
1265127012751280
alacysthrthrcysglycyscysglythrglythralacysthrala
128512901295
cysthrglycyscysalaglycysalaglythrcysthrthrcysthr
130013051310
alaalacysthrglyglycyscysthralaglyalaalacysalathr
131513201325
thrcysglyglycyscysalaglyglyglyalaalacyscysalaala
133013351340
glyglythrglyglyalaglyalathrthralaalaglyglyglyala
1345135013551360
glyglyalaglyglycysglyglyglythrcyscysglyglycysgly
136513701375
glyalaglyglycysglyglyalaalaglycysglyglyalaglygly
138013851390
cysglyglyalaglyglyalaalaglythrglyglycysglyglyala
139514001405
glyglyalathrcysthrglyglycysglyglycysglyglyalathr
141014151420
cysthrcysalaglyglythrglycysalaglycysthrcysglythr
1425143014351440
glyglyalaglythrcyscysglyglyalaglyglycysglyglyala
144514501455
glythrglyglythrglycysalaglycyscysthrglyglyalacys
146014651470
glyglythrcyscyscysthrcysalaglyalacysthrcysglyala
147514801485
cysthrglycysalaalaglyglycysthrthrcyscysglyglycys
149014951500
alathrcysalacysalathrthrcysthrcysthralaalacysthr
1505151015151520
cysthrglyglythralathrglycysalacysthrglyglyglythr
152515301535
glyalaglyalacysalaglyglycysalacyscysalaglyglyala
154015451550
alaalaglyglyglycysthrthralaglyalaglythrglyglygly
155515601565
thrglyglycysthrglythrglyalathrthrthrglyglythrala
157015751580
cysglyalacysglyglyglythrcysglyalaalaglyalaglyala
1585159015951600
thralacysthralacysglycyscysglyalacysalaglycysgly
160516101615
thrglyalaalaglyglyglyalacysglyglythrthrcysalacys
162016251630
alaalathrthralaglycysalaglyalaglyalacysalaalacys
163516401645
thrcyscysalaalaglyalaalacysalacyscyscysthrglythr
165016551660
thrcyscysthrcyscysalaglyalathrglyalaalacysthrcys
1665167016751680
thrcysthrglycysglycysglycyscysglyalaglyglyalacys
168516901695
alacyscysglycyscysglythrglythralacysthralacysthr
170017051710
glycysglycysthralacysalaalaalacysglyalacysglyala
171517201725
cysthralacysthrglyglyglyglyalacysalaglyglyglycys
173017351740
alacysalacysthrcysglythrglyalacyscysglythrglythr
1745175017551760
cysthralaglycysglyglycysglyglycysglyglycysthrcys
176517701775
glyglyglycysglyglycysglyglyglythrcyscysglyglycys
178017851790
glyglyalaglyglyalaglyglythrthrcysglyalacysglyala
179518001805
thrcyscyscysalacyscysglycysalacysglythrthrcysala
181018151820
glyalaalaglythrcysglyglyalathrglythrglyglyalaala
1825183018351840
alathrglyglyalaglyglycyscyscysalaglyalaalaalagly
184518501855
alathrglyalaalaalathrcysalathrcysthrglycyscyscys
186018651870
cysalaglycysthrglythralaalathralaglyglyalacysthr
187518801885
glycyscyscysalathrcyscysalacysthrglyalaglyalacys
189018951900
alathralathrthralaalathralaalacysglyalacysalathr
1905191019151920
glyalathralaglythrcysalacysthrglyalacysalaalacys
192519301935
alaalacysglyglythrglycysalaglythrcysalaalaglythr
194019451950
thrthrcyscysalacysalaalacysthrglythrglythralaala
195519601965
alathrthrthrthrglythrglyalathrglythrglyalaglyala
197019751980
thrthrthrthrcyscysalacyscysthrglythrglyalacysala
1985199019952000
alacyscysalaglyalaalaalathrcyscysthrglycysalathr
200520102015
glyalaglycysalaalacysthrglycysalaglycysalathrcys
202020252030
alacyscysthrcyscysalathrcysthrglythrglyalaglyala
203520402045
alaglycyscysalacysalaglyglyalaalaglythrcysthrgly
205020552060
thrglythrglyglycysthrglythralathrglyglyalaglyala
2065207020752080
alaalaglyalaalathrglyalacysglyalaglyalaalacysala
208520902095
thralaalacysalacysthralaglyalaglyalacysalaglythr
210021052110
thrthrglycyscysalathrglyalacyscyscyscysalaalagly
211521202125
cysthrcyscyscyscysthralacyscysalathrglyalacysthr
213021352140
thrthralathrthrcysthrglyglyalaalaglyalathrglycys
2145215021552160
thrglycysthrthrcysthrcyscysalaalaalaglythrglycys
216521702175
alathrthralathrglyalaalaglyglyalaalaalaalaalaala
218021852190
alaalaalaalaglycyscysthrglyglythrglyalaglyalacys
219522002205
thrthrthrcysthrthrcysthralaala
22102215
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