CN113862301A - 一种vwf前肽表达载体及其制备方法和应用 - Google Patents

一种vwf前肽表达载体及其制备方法和应用 Download PDF

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CN113862301A
CN113862301A CN202110982954.7A CN202110982954A CN113862301A CN 113862301 A CN113862301 A CN 113862301A CN 202110982954 A CN202110982954 A CN 202110982954A CN 113862301 A CN113862301 A CN 113862301A
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vwf
expression vector
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gly
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王学锋
周爱武
梁茜
武文漫
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Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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Abstract

本发明涉及一种VWF前肽表达载体及其制备方法和应用，包括VWF信号肽和VWF结构域D1D2区的编码序列以及终止密码子。基于VWF前肽参与VWF多聚体组装的作用机制，本发明的VWF前肽表达载体可以仅仅通过引入正常的VWF前肽，恢复部分先天性VWF多聚体形成障碍，为通过“蛋白质结构编辑”在蛋白水平上纠正先天性缺陷的基因治疗策略铺平了道路。

Description

一种VWF前肽表达载体及其制备方法和应用
技术领域
本发明属于血管性血友病领域，特别涉及一种VWF前肽表达载体及其制备方法和应用。
背景技术
血管性血友病因子(VWF)是出凝血系统中非常重要的高分子量糖蛋白，其主要功能是介导血小板粘附于受损的内皮下暴露的胶原，并保护凝血因子VIII不被过快降解。这些功能依赖于VWF亚基多结构域结构中许多配体的结合位点：D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK。此外，VWF单体可首尾相连形成多聚体，且只有高分子量VWF多聚体才具备相应的生理功能。VWF多聚体形成缺陷会导致2A型血管性血友病。位于VWF前肽区(D1D2区)、D3区、CK区的VWF基因突变可影响VWF多聚体组装，使其不能形成高分子量多聚体，从而导致VWF功能缺失和出血倾向。像其他遗传性出血性疾病一样，基因治疗也是治愈血管性血友病(VWD)的唯一希望。通常基因治疗的方法是把完整的正常VWF基因通过病毒载体输入人体来纠正遗传缺陷，然而由于VWF分子特别大，是人体中最大的基因之一，病毒载体包装制备难度很大，几乎无法实现。
发明内容
本发明所要解决的技术问题是提供一种VWF前肽表达载体及其制备方法和应用，克服了现有技术需要把完整的正常基因通过病毒载体输入人体来纠正遗传缺陷的缺点。
本发明基于VWF多聚体组装机制，提供了一种VWF前肽表达载体，包括VWF信号肽和VWF结构域D1D2的编码序列以及终止密码子；所述编码序列如SEQ NO.1所示。
所述载体包括pCIneo、慢病毒、腺病毒、腺相关病毒等。
本发明还提供了一种VWF前肽表达载体的制备方法，包括：
将全长野生型人VWF cDNA克隆到表达载体pCIneo中，构建了VWF表达载体pCIneoVWFWT；基于pCIneoVWFWT进行定点突变构建突变体表达载体，并通过直接测序进行确认；根据VWF信号肽和VWF结构域D1D2的编码序列，在pCIneoVWFWT上插入一个位点定向突变的终止密码子，得到VWF前肽表达载体pCIneoVWFpp。
用于制备基因治疗药物。
有益效果
通过本发明的VWF前肽表达载体仅仅引入野生型VWF前肽，便可恢复部分先天性VWF多聚体形成障碍，为通过“蛋白质结构编辑”在蛋白水平上纠正先天性缺陷的基因治疗策略铺平了道路。
附图说明
图1为VWF E8del全长质粒单独转染或与野生型VWF全长质粒/野生型VWF前肽质粒共转染后VWF多聚体形成检测；其中，1.野生型VWF全长质粒单独转染；2.E8del突变型VWF全长质粒单独转染；3.E8del突变型VWF全长质粒与野生型VWF前肽质粒1:1摩尔比共转染；4.E8del突变型VWF全长质粒与野生型VWF前肽质粒1:2摩尔比共转染；5.E8del突变型VWF全长质粒与野生型VWF前肽质粒1:4摩尔比共转染；6.E8del突变型VWF全长质粒与野生型VWF前肽质粒1:8摩尔比共转染；7.E8del突变型VWF全长质粒与野生型VWF前肽质粒1:16摩尔比共转染；8.E8del突变型VWF全长质粒与野生型VWF全长质粒共转染；图2为VWF Y87S全长质粒单独转染或与野生型VWF全长质粒/野生型VWF前肽质粒共转染后VWF多聚体形成检测；其中，1.野生型VWF全长质粒单独转染；2.Y87S突变型VWF全长质粒单独转染；3.Y87S突变型VWF全长质粒与野生型VWF前肽质粒1:1摩尔比共转染；4.Y87S突变型VWF全长质粒与野生型VWF前肽质粒1:2摩尔比共转染；5.Y87S突变型VWF全长质粒与野生型VWF前肽质粒1:4摩尔比共转染；6.Y87S突变型VWF全长质粒与野生型VWF前肽质粒1:8摩尔比共转染；7.Y87S突变型VWF全长质粒与野生型VWF前肽质粒1:16摩尔比共转染；8.EY87S突变型VWF全长质粒与野生型VWF全长质粒共转染。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
在一例2A型血管性血友病患者身上发现了c.875-5T>G突变，该剪切突变可导致VWF基因8号外显子缺失(VWF E8del)。VWF基因8号外显子编码VWF前肽D1区293-333位氨基酸。患者表现为血浆中血管性血友病因子抗原水平的轻度下降以及VWF高分子量多聚体的显著减少。
将全长野生型人VWF cDNA克隆到表达载体pCIneo中，构建了VWF表达载体pCIneoVWFWT；基于pCIneoVWFWT序列扩增VWF信号肽和VWF结构域D1D2的编码序列，并在763位氨基酸后面插入终止密码子，得到VWF前肽表达载体pCIneoVWFpp，通过直接测序进行确认。VWF前肽突变体以及VWF全长质粒突变体(包括VWF E8del突变及Y87S突变)通过定点突变的方式构建。对VWF前肽结构模型的研究表明，VWF 8号外显子缺失可能导致由VWF前肽D1区41个氨基酸残基组成的环状结构的缺失。由VWF E8del引起的结构变化，就像VWF前肽点突变Y87S一样，使不同VWF分子的前肽之间以及VWF前肽与成熟VWF分子的D3区之间的亲和性降低，导致VWF多聚体组装障碍。因此，通过共表达野生型VWF前肽便可竞争性地取代有缺陷的内源性VWF前肽，从而恢复VWF E8del或Y87S突变体的多聚化障碍。如图1和图2所示，单独转染VWF E8del或VWFY87S质粒均表现为显著的VWF多聚体形成障碍，上清中仅可见二聚体和少量四聚体；当野生型VWF前肽与突变型VWF全长质粒共转染时，可检测到四聚体、八聚体，甚至是高分子量VWF多聚体。
本发明发现，由于VWF前肽区突变导致的VWF多聚体组装障碍可通过共转染野生型VWF前肽而部分纠正。该现象提示可尝试导入正常的VWF前肽对部分VWD患者进行基因治疗。
序列表
<110> 上海交通大学医学院附属瑞金医院
<120> 一种VWF前肽表达载体及其制备方法和应用
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
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<211> 763
<212> PRT
<213> 人工序列(Artificial Sequence)
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Met Ile Pro Ala Arg Phe Ala Gly Val Leu Leu Ala Leu Ala Leu Ile
1 5 10 15
Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr
20 25 30
Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly
35 40 45
Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly
50 55 60
Cys Gln Lys Arg Ser Phe Ser Ile Ile Gly Asp Phe Gln Asn Gly Lys
65 70 75 80
Arg Val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp Ile His Leu
85 90 95
Phe Val Asn Gly Thr Val Thr Gln Gly Asp Gln Arg Val Ser Met Pro
100 105 110
Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys
115 120 125
Leu Ser Gly Glu Ala Tyr Gly Phe Val Ala Arg Ile Asp Gly Ser Gly
130 135 140
Asn Phe Gln Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly
145 150 155 160
Leu Cys Gly Asn Phe Asn Ile Phe Ala Glu Asp Asp Phe Met Thr Gln
165 170 175
Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala
180 185 190
Leu Ser Ser Gly Glu Gln Trp Cys Glu Arg Ala Ser Pro Pro Ser Ser
195 200 205
Ser Cys Asn Ile Ser Ser Gly Glu Met Gln Lys Gly Leu Trp Glu Gln
210 215 220
Cys Gln Leu Leu Lys Ser Thr Ser Val Phe Ala Arg Cys His Pro Leu
225 230 235 240
Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu
245 250 255
Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala
260 265 270
Arg Thr Cys Ala Gln Glu Gly Met Val Leu Tyr Gly Trp Thr Asp His
275 280 285
Ser Ala Cys Ser Pro Val Cys Pro Ala Gly Met Glu Tyr Arg Gln Cys
290 295 300
Val Ser Pro Cys Ala Arg Thr Cys Gln Ser Leu His Ile Asn Glu Met
305 310 315 320
Cys Gln Glu Arg Cys Val Asp Gly Cys Ser Cys Pro Glu Gly Gln Leu
325 330 335
Leu Asp Glu Gly Leu Cys Val Glu Ser Thr Glu Cys Pro Cys Val His
340 345 350
Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn
355 360 365
Thr Cys Ile Cys Arg Asn Ser Gln Trp Ile Cys Ser Asn Glu Glu Cys
370 375 380
Pro Gly Glu Cys Leu Val Thr Gly Gln Ser His Phe Lys Ser Phe Asp
385 390 395 400
Asn Arg Tyr Phe Thr Phe Ser Gly Ile Cys Gln Tyr Leu Leu Ala Arg
405 410 415
Asp Cys Gln Asp His Ser Phe Ser Ile Val Ile Glu Thr Val Gln Cys
420 425 430
Ala Asp Asp Arg Asp Ala Val Cys Thr Arg Ser Val Thr Val Arg Leu
435 440 445
Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly Val
450 455 460
Ala Met Asp Gly Gln Asp Val Gln Leu Pro Leu Leu Lys Gly Asp Leu
465 470 475 480
Arg Ile Gln His Thr Val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu
485 490 495
Asp Leu Gln Met Asp Trp Asp Gly Arg Gly Arg Leu Leu Val Lys Leu
500 505 510
Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn
515 520 525
Gly Asn Gln Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro
530 535 540
Arg Val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gln
545 550 555 560
Asp Leu Gln Lys Gln His Ser Asp Pro Cys Ala Leu Asn Pro Arg Met
565 570 575
Thr Arg Phe Ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe
580 585 590
Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys
595 600 605
Arg Tyr Asp Val Cys Ser Cys Ser Asp Gly Arg Glu Cys Leu Cys Gly
610 615 620
Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly Val Arg Val
625 630 635 640
Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gln
645 650 655
Val Tyr Leu Gln Cys Gly Thr Pro Cys Asn Leu Thr Cys Arg Ser Leu
660 665 670
Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe
675 680 685
Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp Cys Val Pro Lys
690 695 700
Ala Gln Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gln Pro Glu Asp
705 710 715 720
Ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met
725 730 735
His Cys Thr Met Ser Gly Val Pro Gly Ser Leu Leu Pro Asp Ala Val
740 745 750
Leu Ser Ser Pro Leu Ser His Arg Ser Lys Arg
755 760

Claims (4)

1.一种VWF前肽表达载体，其特征在于：包括VWF信号肽和VWF结构域D1D2的编码序列以及终止密码子；所述编码序列如SEQ NO.1所示。
2.根据权利要求1所述的表达载体，其特征在于：所述载体包括pCIneo、慢病毒、腺病毒或腺相关病毒。
3.一种VWF前肽表达载体的制备方法，包括：
将全长野生型人VWF cDNA克隆到表达载体pCIneo中，构建了VWF表达载体pCIneoVWFWT；基于pCIneoVWFWT表达载体，扩增VWF信号肽和VWFD1D2结构域序列，并在763位氨基酸后面插入终止密码子，构建VWF前肽表达载体pCIneoVWFpp，通过直接测序进行确认。
4.一种如权利要求1所述的VWF前肽表达载体的应用，其特征在于：用于制备基因治疗药物。
CN202110982954.7A 2021-08-25 2021-08-25 一种vwf前肽表达载体及其制备方法和应用 Pending CN113862301A (zh)

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