CN113292655A - 重组人血管紧张素转化酶2蛋白及其制备方法和应用 - Google Patents

重组人血管紧张素转化酶2蛋白及其制备方法和应用 Download PDF

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CN113292655A
CN113292655A CN202010107512.3A CN202010107512A CN113292655A CN 113292655 A CN113292655 A CN 113292655A CN 202010107512 A CN202010107512 A CN 202010107512A CN 113292655 A CN113292655 A CN 113292655A
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王伯初
郝石磊
庆睿
查国栋
陈海容
欧珍
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Abstract

本发明属于基因工程技术领域，具体涉及重组人血管紧张素转化酶2蛋白及其制备方法和应用。所述重组人血管紧张素转化酶2蛋白可竞争性结合ACE2配体，通过对设计的全长、膜外区、穿膜区的相关氨基酸进行突变获得亲水性的ACE2全长蛋白；所述重组人血管紧张素转化酶2蛋白的氨基酸序列如SEQ ID NO：1或SEQ ID NO：2所示。利用原核表达系统将该基因在大肠杆菌PET‑3C中进行表达，随后利用His标签可溶性蛋白纯化进行蛋白纯化，获得重组人血管紧张素转化酶2蛋白。所制备得到的重组人血管紧张素转化酶2蛋白与膜外重组蛋白具有更高的配体亲和性、稳定性和生物半衰期，从而提高其成药性。

Description

重组人血管紧张素转化酶2蛋白及其制备方法和应用
技术领域
本发明属于基因工程技术领域，具体涉及重组人血管紧张素转化酶2 蛋白及其制备方法和应用。
背景技术
血管紧张素转换酶(ACE)2是羧肽酶ACE的同源物，羧肽酶生成血管紧 张素II，这是肾素-血管紧张素系统(RAS)的主要活性肽。在2000年克隆 ACE2之后，迄今为止已经描述了三种主要的ACE2功能。ACE2是RAS的一 个强力负调节因子，可平衡ACE的多种功能。通过靶向血管紧张素II，ACE2 在心血管系统和许多其他器官中显示出保护作用。
研究发现新型冠状病毒的受体蛋白为血管紧张素转化酶2(ACE2)。在 这项研究基础上，上海同济大学医学院研究团队连日利用高通量单细胞测 序分析技术，研究了共计四万三千多个肺部细胞，进一步发现80％ACE2受 体主要在II型肺泡聚集。表达ACE2的II型肺泡占其总数量的1.4％。在其 他细胞类型如I型肺泡、支气管上皮细胞、成纤维细胞、内皮细胞和巨噬 细胞中零星可见。而且通过对基因功能的分析，研究者还发现，这1.4％的 ACE2+II型肺泡细胞还表达至少20多个与病毒组装复制相关的功能基因。
因此，本发明旨在研发ACE2蛋白药物，可通过空间位阻效应阻断病毒 与ACE2受体的结合，有望起到病毒感染保护作用。同时，该蛋白药物不仅 对2019新型冠状病毒(2019-nCoV)有望起效，对SARS、猪流行性腹泻病 毒等冠状病毒也有感染保护作用。
发明内容
本发明的目的之一在于提供一种重组人血管紧张素转化酶2蛋白，所 述重组人血管紧张素转化酶2蛋白相比膜外重组蛋白具有更高的配体亲和 性、稳定性和生物半衰期，从而提高其成药性。
为实现以上目的，本发明采用以下方案：
所述重组人血管紧张素转化酶2蛋白可竞争性结合ACE2配体，通过对 设计的全长、膜外区、穿膜区的相关氨基酸进行突变获得亲水性的ACE2全 长蛋白；所述重组人血管紧张素转化酶2蛋白的氨基酸序列如SEQ ID NO： 1或SEQ ID NO：2所示。
进一步，所述氨基酸序列SEQ ID NO：1的结构依次由膜外区、穿膜区 和FC组成；所述氨基酸序列SEQ ID NO：2的结构依次由全长和FC组成。
所述重组蛋白与膜外重组蛋白相比，具有更高的配体亲和性、稳定性 和生物半衰期，从而提高重组蛋白的成药性，用于2019新型冠状病毒 等其它冠状病毒的特异性预防和治疗。
本发明的目的之二在于提供所述重组人血管紧张素转化酶2蛋白的制 备方法，所述制备方法的重组人血管紧张素转化酶2蛋白具有可溶性蛋白 产率高及操作简便的特点。
为实现以上目的，本发明采用以下方案：
所述的重组人血管紧张素转化酶2蛋白的制备方法包括以下步骤：
1)人工合成重组人血管紧张素转化酶2基因；
2)重组人血管紧张素转化酶2蛋白的表达：利用原核表达系统将重组人血 管紧张素转化酶2基因在大肠杆菌中进行表达，并获得重组人血管紧张素 转化酶2基工程菌株，经IPTG诱导表达重组人血管紧张素转化酶2蛋白； 3)重组人血管紧张素转化酶2蛋白的纯化：利用His标签可溶性蛋白纯化 进行蛋白纯化。
进一步，所述重组人血管紧张素转化酶2基因的核苷酸序列如SEQ ID NO：3或SEQID NO：4所示。
进一步，步骤2)将重组人血管紧张素转化酶2基因插入原核表达载体 pET3C，构建重组表达质粒pET3C-ACE2，并将质粒转化大肠杆菌 BL21DE3PLYsS，获得重组人血管紧张素转化酶2基工程菌株，将工程菌株 进行培养然后加入IPTG进行诱导表达重组人血管紧张素转化酶2蛋白。
进一步，所述表达载体pET3C含有T7启动子。
具体的，构建原核表达载体的方法为：将全基因合成的重组人血管紧 张素转化酶2蛋白基基因与表达载体pET3c分别用NdeI和BamHI核酸内切 酶进行双酶切，酶切产物经琼脂糖凝胶电泳和DNA纯化试剂盒纯化，然后 将两个纯化产物利用T4DNA连接酶进行连接，构建重组表达载体 pET3C-ACE2，随后利用该载体转化Bl21DE3PLYSS感受态细胞，获得表达菌株Bl21DE3PLYSS-pET3C-ACE2。
进一步，IPTG进行诱导的条件为37℃，200rpm转速条件下将工程菌株 培养3h至菌液的OD600达到0.50.7时加入终浓度为2mM的IPTG诱导表达 4h，诱导表达的重组人结合珠蛋白β亚基为可溶性蛋白。
进一步，步骤3)利用His标签可溶性蛋白纯化进行蛋白纯化。
具体的，诱导表达的重组人血管紧张素转化酶2纯化方法为：按1：10 超声破碎菌体，离心取上清Soluble Binding Buffer到Ni琼脂糖凝胶层 析柱上，先用SolubleBinding Buffer洗柱子，再用含500mM咪唑浓度的 Soluble Elution Buffer洗脱，收集洗脱液。所述的BindingBuffer的组 份是：pH8.5的20mM Tris-HCL、300mM NaCL、10mM咪唑；Soluble Elution Buffer的组份是：pH8.5的20mM Tris-HCL，300mM NaCL，500mM咪唑。
本发明的目的之三在于提供一种药物组合物及其应用。
为实现以上目的，本发明采用以下方案：
所述药物组合物包含目的一所述的重组人血管紧张素转化酶2蛋白。
所述重组人血管紧张素转化酶2蛋白及其药物组合物可用于其结合游 离血红蛋白的功能研究及其在结合并去除血浆游离血红蛋白中的应用，以 及用于竞争性结合冠状病毒进而抑制肺损伤。
本发明的有益效果在于：
本发明所制备得到的重组人血管紧张素转化酶2蛋白，具有亲水性的 ACE2的全长蛋白，水溶性高，其全长结构有助于蛋白的正确折叠；与膜外 重组蛋白具有更高的配体亲和性、稳定性和生物半衰期，从而提高其成药 性；在针对2019新冠病毒等冠状病毒的特异性预防和治疗中将会起到较大 效用；且可用于进一步研究该蛋白体外结合血红蛋白的功能及应用于清除 血浆中游离血红蛋白。
具体实施方式
所举实施例是为了更好地对本发明进行说明，但并不是本发明的内容 仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实 施方案进行非本质的改进和调整，仍属于本发明的保护范围。
实施例1
重组表达载体pET3C-ACE2的构建
1.1利用全基因合成得到人血管紧张素转化酶2基因
合成人血管紧张素转化酶2基因CDNA序列，插入pUC57克隆载体中， 序列长度为3624bp，如SEQ ID NO：3所示。
1.2表达载体pET3C-ACE2的构建
(1)将上述的全基因合成DNA纯化试剂盒纯化后，对pET3C质粒和纯化 后的基因合成得到人血管紧张素转化酶2同时进行NdeI和BamI双酶切， 37℃酶切1h，然后利用OMEMG公司的DNA纯化试剂盒进行纯化并回收酶切 后的DNA片段；使用宝日医(北京)生物工程有限公司快速酶连接试剂将 酶切并纯化后的质粒片段与人血管紧张素转化酶2基因片段进行连接，16 度连接30分钟。连接体系为:pET3C质粒溶液1.5μL，人血管紧张素转化 酶2基因片段溶液3μL，快速连接缓冲液5μL，快速T4DNA连接酶0.5μL。
(2)将连接产物转化到BL21DE3PLYsS感受态细胞中，获得转化菌落。 转化条件为：连接产物10μL加入100μL BL21DE3PLYsS感受态细胞溶液 中，轻弹混匀，在冰浴中静置30分钟，随后42℃热激90s，然后快速转移 到冰浴中，使细胞冷却3min，该过程不要摇动离心管。加入900μL无菌的 LB培养基(不含抗生素)，混匀后置于37℃摇床振荡培养45min(150rpm)，然后吸取100μL已转化的感受态细胞涂布于含氨苄抗性的LB平板，37℃ 倒置培养过夜。
(3)挑取单菌落培养后进行PCR验证，筛选阳性克隆菌落进行测序，测 序结果表明插入人结合珠蛋白基因核苷酸序列正确，从而获得表达 pET3C-ACE2的基因工程菌，即Bl21DE3PLYSS-pET3C-ACE2。
实施例2
重组人人血管紧张素转化酶2蛋白的表达与鉴定
2.1挑取重组基因工程菌Bl21DE3PLYSS-pET3C-ACE2单菌落接种于 10mlLB培养基中(含氨苄100μg/mL)，37℃，150rpm摇床振荡培养过夜。
2.2按1:9的比例将过夜培养的菌液接种到10ml新鲜LB培养基中(含 氨苄100μg/mL)，37℃，200rpm转速条件下培养大约3h至菌液的OD600 达到0.5-0.7，并且OD600最好接近0.6时加入终浓度为2mM的IPTG(异 丙基-β-D-硫代半乳糖苷)诱导表达4h，诱导表达的重组人结合珠蛋白为可 溶性蛋白和包涵体的混合物。同时设立工程菌Bl21DE3PLYSS-pET3C-ACE2 不进行IPTG诱导组进行对照。
2.3诱导培养结束后将培养液于4℃，12,000g离心1min，弃去上清， 收集下层菌体沉淀。用1mLPBS缓冲液重悬菌体，超声破碎至菌液由浑浊变 清亮。4℃，12,000g离心5min，吸取上清到新的离心管中即为上清部分； 沉淀用100μLPBS重悬即为沉淀部分。利用PBS重悬菌体进行10％SDS-PAGE 电泳及考马斯亮蓝染色，结果显示，在约130KDa处有一特异性表达增加的 蛋白条带，与预期的重组人血管紧张素转化酶2蛋白分子量相符，诱导表 达的重组人血管紧张素转化酶2蛋白为可溶性蛋白。
实施例3
重组人血管紧张素转化酶2的纯化
3.1根据实施例2方法，诱导表达2L重组基因工程菌株 Bl21DE3PLYSS-pET3C-ACE2，用适量预冷的PBS缓冲液重悬菌体，冰浴超声 破碎至菌液由浑浊变清亮后于4℃，12,000g离心5min，收集上清。将上清 溶液加入等倍体积预冷的Soluble Binding Buffer(20mM PBS(pH7.9)，300mM NaCL，300mM咪唑)，利用纳微的His标签蛋白纯化试剂盒(可溶性蛋白)进 行纯化。
3.2将含有可溶性蛋白的用Soluble Binding Buffer等倍稀释的上清 液负载到Ni琼脂糖凝胶层析柱上，先用15倍柱体积的Soluble Binding Buffer洗柱子，再用含300mM咪唑浓度的Soluble Elution Buffer(20mM PBS(pH7.9)，300mM NaCL，300mM咪唑。)洗脱，收集洗脱液，即为纯化的 蛋白溶液。经12％SDS-PAGE电泳及考马斯亮蓝凝胶染色，结果显示，在大 约130KDa处有一特异性蛋白条带，与预期的重组人血管紧张素转化酶2蛋 白的分子量大小相符。通过进一步的质谱鉴定，验证该重组人血管紧张素 转化酶2蛋白。最后将纯化蛋白置于-70℃冰箱保存。
实施例4
重组蛋白的理化性质表征及体外亲和性测试
对重组蛋白的溶解性、分子量、稳定性、圆二色光谱等进行表征；采 用微量
热泳动仪检测ACE2重组蛋白与Spike蛋白的亲和性。
实施例5
体内分布及半衰期研究
以自动化多功能合成模块GE TRACERlab FXFN为平台，合成用于蛋白 多肽正电子核素18F标记的辅基N-琥珀酰亚胺-4-[18F]氟苯甲酸酯 (18F-SFB)，用于标记重组蛋白。使用PET观察重组蛋白不同时间点的体 内分布，同时动物麻醉处死后去除内脏测定其活度。构建ACE2重组蛋白 的HPLC-MS检测方法，体内注射重组蛋白后，测定蛋白在大鼠体内的药物 浓度，计算其生物半衰期。
实施例6
肺损伤保护效果研究
构建急性肺损伤模型评价的治疗效果。在肺损伤动物模型构建前30 分钟注射重组蛋白，使用低pH酸吸入动物模型，通过测定血浆 Angiotensin II水平、肺部病例切片观察等观察的治疗效果。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制， 尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应 当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发 明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 海默斯（重庆）医学生物技术有限公司
<120> 重组人血管紧张素转化酶2蛋白及其制备方法和应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1002
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ser Ser Ser Ser Thr Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gly Ser Thr Ile Gly Gly Gly Ala Leu Thr Pro Leu Ala Leu Pro
20 25 30
Ala His Gly Ala Gly Ala Leu Pro Thr Gly Ser Ser Leu Ala Ser Thr
35 40 45
Ala Thr Ala Thr Ala Ile Thr Gly Gly Ala Val Gly Ala Met Ala Ala
50 55 60
Ala Gly Ala Leu Thr Ser Ala Pro Leu Leu Gly Gly Ser Thr Leu Ala
65 70 75 80
Gly Met Thr Pro Leu Gly Gly Ile Gly Ala Leu Thr Val Leu Leu Gly
85 90 95
Leu Gly Ala Leu Gly Gly Ala Gly Ser Ser Val Leu Ser Gly Ala Leu
100 105 110
Ser Leu Ala Leu Ala Thr Ile Leu Ala Thr Met Ser Thr Ile Thr Ser
115 120 125
Thr Gly Leu Val Cys Ala Pro Ala Ala Pro Gly Gly Cys Leu Leu Leu
130 135 140
Gly Pro Gly Leu Ala Gly Ile Met Ala Ala Ser Leu Ala Thr Ala Gly
145 150 155 160
Ala Leu Thr Ala Thr Gly Ser Thr Ala Ser Gly Val Gly Leu Gly Leu
165 170 175
Ala Pro Leu Thr Gly Gly Thr Val Val Leu Leu Ala Gly Met Ala Ala
180 185 190
Ala Ala His Thr Gly Ala Thr Gly Ala Thr Thr Ala Gly Ala Thr Gly
195 200 205
Val Ala Gly Val Ala Gly Thr Ala Thr Ser Ala Gly Gly Leu Ile Gly
210 215 220
Ala Val Gly His Thr Pro Gly Gly Ile Leu Pro Leu Thr Gly His Leu
225 230 235 240
His Ala Thr Val Ala Ala Leu Leu Met Ala Ala Thr Pro Ser Thr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Ala Met Thr Gly
260 265 270
Ala Pro Thr Thr Ala Leu Thr Ser Leu Thr Val Pro Pro Gly Gly Leu
275 280 285
Pro Ala Ile Ala Val Thr Ala Ala Met Val Ala Gly Ala Thr Ala Ala
290 295 300
Gly Ala Ile Pro Leu Gly Ala Gly Leu Pro Pro Val Ser Val Gly Leu
305 310 315 320
Pro Ala Met Thr Gly Gly Pro Thr Gly Ala Ser Met Leu Thr Ala Pro
325 330 335
Gly Ala Val Gly Leu Ala Val Cys His Pro Thr Ala Thr Ala Leu Gly
340 345 350
Leu Gly Ala Pro Ala Ile Leu Met Cys Thr Leu Val Thr Met Ala Ala
355 360 365
Pro Leu Thr Ala His His Gly Met Gly His Ile Gly Thr Ala Met Ala
370 375 380
Thr Ala Ala Gly Pro Pro Leu Leu Ala Ala Gly Ala Ala Gly Gly Pro
385 390 395 400
His Gly Ala Val Gly Gly Ile Met Ser Leu Ser Ala Ala Thr Pro Leu
405 410 415
His Leu Leu Ser Ile Gly Leu Leu Ser Pro Ala Pro Gly Gly Ala Ala
420 425 430
Gly Thr Gly Ile Ala Pro Leu Leu Leu Gly Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Pro Thr Thr Met Leu Gly Leu Thr Ala Thr Met Val Pro
450 455 460
Leu Gly Gly Ile Pro Leu Ala Gly Thr Met Leu Leu Thr Thr Gly Met
465 470 475 480
Leu Ala Gly Ile Val Gly Val Val Gly Pro Val Pro His Ala Gly Thr
485 490 495
Thr Cys Ala Pro Ala Ser Leu Pro His Val Ser Ala Ala Thr Ser Pro
500 505 510
Ile Ala Thr Thr Thr Ala Thr Leu Thr Gly Pro Gly Pro Gly Gly Ala
515 520 525
Leu Cys Gly Ala Ala Leu His Gly Gly Pro Leu His Leu Cys Ala Ile
530 535 540
Ser Ala Ser Thr Gly Ala Gly Gly Leu Leu Pro Ala Met Leu Ala Leu
545 550 555 560
Gly Leu Ser Gly Pro Thr Thr Leu Ala Leu Gly Ala Val Val Gly Ala
565 570 575
Leu Ala Met Ala Val Ala Pro Leu Leu Ala Thr Pro Gly Pro Leu Pro
580 585 590
Thr Thr Leu Leu Ala Gly Ala Leu Ala Ser Pro Val Gly Thr Ser Thr
595 600 605
Ala Thr Ser Pro Thr Ala Ala Gly Ser Ile Leu Val Ala Ile Ser Leu
610 615 620
Leu Ser Ala Leu Gly Ala Leu Ala Thr Gly Thr Ala Ala Ala Gly Met
625 630 635 640
Thr Leu Pro Ala Ser Ser Val Ala Thr Ala Met Ala Gly Thr Pro Leu
645 650 655
Leu Val Leu Ala Gly Met Ile Leu Pro Gly Gly Gly Ala Val Ala Val
660 665 670
Ala Ala Leu Leu Pro Ala Ile Ser Pro Ala Pro Pro Val Thr Ala Pro
675 680 685
Leu Ala Val Ser Ala Ile Ile Pro Ala Thr Gly Val Gly Leu Ala Ile
690 695 700
Ala Met Ser Ala Ser Ala Ile Ala Ala Ala Pro Ala Leu Ala Ala Ala
705 710 715 720
Ser Leu Gly Pro Leu Gly Ile Gly Pro Thr Leu Gly Pro Pro Ala Gly
725 730 735
Pro Pro Thr Ser Thr Thr Ala Thr Thr Thr Gly Thr Thr Met Gly Thr
740 745 750
Thr Thr Thr Gly Thr Thr Thr Ala Thr Gly Gly Ser Ser Ala Leu Thr
755 760 765
His Thr Cys Pro Pro Cys Pro Ala Pro Gly Leu Leu Gly Gly Pro Ser
770 775 780
Val Pro Leu Pro Pro Pro Leu Pro Leu Ala Thr Leu Met Ile Ser Ala
785 790 795 800
Thr Pro Gly Val Thr Cys Val Val Val Ala Val Ser His Gly Ala Pro
805 810 815
Gly Val Leu Pro Ala Thr Thr Val Ala Gly Val Gly Val His Ala Ala
820 825 830
Leu Thr Leu Pro Ala Gly Gly Gly Thr Ala Ser Thr Thr Ala Val Val
835 840 845
Ser Val Leu Thr Val Leu His Gly Ala Thr Leu Ala Gly Leu Gly Thr
850 855 860
Leu Cys Leu Val Ser Ala Leu Ala Leu Pro Ala Pro Ile Gly Leu Thr
865 870 875 880
Ile Ser Leu Ala Leu Gly Gly Pro Ala Gly Pro Gly Val Thr Thr Leu
885 890 895
Pro Pro Ser Ala Gly Gly Met Thr Leu Ala Gly Val Ser Leu Thr Cys
900 905 910
Leu Val Leu Gly Pro Thr Pro Ser Ala Ile Ala Val Gly Thr Gly Ser
915 920 925
Ala Gly Gly Pro Gly Ala Ala Thr Leu Thr Thr Pro Pro Val Leu Ala
930 935 940
Ser Ala Gly Ser Pro Pro Leu Thr Ser Leu Leu Thr Val Ala Leu Ser
945 950 955 960
Ala Thr Gly Gly Gly Ala Val Pro Ser Cys Ser Val Met His Gly Ala
965 970 975
Leu His Ala His Thr Thr Gly Leu Ser Leu Ser Leu Ser Pro Gly Leu
980 985 990
His His His His His His His His His His
995 1000
<210> 2
<211> 2048
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ser Ser Ser Ser Thr Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gly Ser Thr Ile Gly Gly Gly Ala Leu Thr Pro Leu Ala Leu Pro
20 25 30
Ala His Gly Ala Gly Ala Leu Pro Thr Gly Ser Ser Leu Ala Ser Thr
35 40 45
Ala Thr Ala Thr Ala Ile Thr Gly Gly Ala Val Gly Ala Met Ala Ala
50 55 60
Ala Gly Ala Leu Thr Ser Ala Pro Leu Leu Gly Gly Ser Thr Leu Ala
65 70 75 80
Gly Met Thr Pro Leu Gly Gly Ile Gly Ala Leu Thr Val Leu Leu Gly
85 90 95
Leu Gly Ala Leu Gly Gly Ala Gly Ser Ser Val Leu Ser Gly Ala Leu
100 105 110
Ser Leu Ala Leu Ala Thr Ile Leu Ala Thr Met Ser Thr Ile Thr Ser
115 120 125
Thr Gly Leu Val Cys Ala Pro Ala Ala Pro Gly Gly Cys Leu Leu Leu
130 135 140
Gly Pro Gly Leu Ala Gly Ile Met Ala Ala Ser Leu Ala Thr Ala Gly
145 150 155 160
Ala Leu Thr Ala Thr Gly Ser Thr Ala Ser Gly Val Gly Leu Gly Leu
165 170 175
Ala Pro Leu Thr Gly Gly Thr Val Val Leu Leu Ala Gly Met Ala Ala
180 185 190
Ala Ala His Thr Gly Ala Thr Gly Ala Thr Thr Ala Gly Ala Thr Gly
195 200 205
Val Ala Gly Val Ala Gly Thr Ala Thr Ser Ala Gly Gly Leu Ile Gly
210 215 220
Ala Val Gly His Thr Pro Gly Gly Ile Leu Pro Leu Thr Gly His Leu
225 230 235 240
His Ala Thr Val Ala Ala Leu Leu Met Ala Ala Thr Pro Ser Thr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Ala Met Thr Gly
260 265 270
Ala Pro Thr Thr Ala Leu Thr Ser Leu Thr Val Pro Pro Gly Gly Leu
275 280 285
Pro Ala Ile Ala Val Thr Ala Ala Met Val Ala Gly Ala Thr Ala Ala
290 295 300
Gly Ala Ile Pro Leu Gly Ala Gly Leu Pro Pro Val Ser Val Gly Leu
305 310 315 320
Pro Ala Met Thr Gly Gly Pro Thr Gly Ala Ser Met Leu Thr Ala Pro
325 330 335
Gly Ala Val Gly Leu Ala Val Cys His Pro Thr Ala Thr Ala Leu Gly
340 345 350
Leu Gly Ala Pro Ala Ile Leu Met Cys Thr Leu Val Thr Met Ala Ala
355 360 365
Pro Leu Thr Ala His His Gly Met Gly His Ile Gly Thr Ala Met Ala
370 375 380
Thr Ala Ala Gly Pro Pro Leu Leu Ala Ala Gly Ala Ala Gly Gly Pro
385 390 395 400
His Gly Ala Val Gly Gly Ile Met Ser Leu Ser Ala Ala Thr Pro Leu
405 410 415
His Leu Leu Ser Ile Gly Leu Leu Ser Pro Ala Pro Gly Gly Ala Ala
420 425 430
Gly Thr Gly Ile Ala Pro Leu Leu Leu Gly Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Pro Thr Thr Met Leu Gly Leu Thr Ala Thr Met Val Pro
450 455 460
Leu Gly Gly Ile Pro Leu Ala Gly Thr Met Leu Leu Thr Thr Gly Met
465 470 475 480
Leu Ala Gly Ile Val Gly Val Val Gly Pro Val Pro His Ala Gly Thr
485 490 495
Thr Cys Ala Pro Ala Ser Leu Pro His Val Ser Ala Ala Thr Ser Pro
500 505 510
Ile Ala Thr Thr Thr Ala Thr Leu Thr Gly Pro Gly Pro Gly Gly Ala
515 520 525
Leu Cys Gly Ala Ala Leu His Gly Gly Pro Leu His Leu Cys Ala Ile
530 535 540
Ser Ala Ser Thr Gly Ala Gly Gly Leu Leu Pro Ala Met Leu Ala Leu
545 550 555 560
Gly Leu Ser Gly Pro Thr Thr Leu Ala Leu Gly Ala Val Val Gly Ala
565 570 575
Leu Ala Met Ala Val Ala Pro Leu Leu Ala Thr Pro Gly Pro Leu Pro
580 585 590
Thr Thr Leu Leu Ala Gly Ala Leu Ala Ser Pro Val Gly Thr Ser Thr
595 600 605
Ala Thr Ser Pro Thr Ala Ala Gly Ser Ile Leu Val Ala Ile Ser Leu
610 615 620
Leu Ser Ala Leu Gly Ala Leu Ala Thr Gly Thr Ala Ala Ala Gly Met
625 630 635 640
Thr Leu Pro Ala Ser Ser Val Ala Thr Ala Met Ala Gly Thr Pro Leu
645 650 655
Leu Val Leu Ala Gly Met Ile Leu Pro Gly Gly Gly Ala Val Ala Val
660 665 670
Ala Ala Leu Leu Pro Ala Ile Ser Pro Ala Pro Pro Val Thr Ala Pro
675 680 685
Leu Ala Val Ser Ala Ile Ile Pro Ala Thr Gly Val Gly Leu Ala Ile
690 695 700
Ala Met Ser Ala Ser Ala Ile Ala Ala Ala Pro Ala Leu Ala Ala Ala
705 710 715 720
Ser Leu Gly Pro Leu Gly Ile Gly Pro Thr Leu Gly Pro Pro Ala Gly
725 730 735
Pro Pro Thr Ser Thr Thr Ala Thr Thr Thr Gly Thr Thr Met Gly Thr
740 745 750
Thr Thr Thr Gly Thr Thr Thr Ala Thr Gly Gly Ser Ser Ala Leu Thr
755 760 765
His Thr Cys Pro Pro Cys Pro Ala Pro Gly Leu Leu Gly Gly Pro Ser
770 775 780
Val Pro Leu Pro Pro Pro Leu Pro Leu Ala Thr Leu Met Ile Ser Ala
785 790 795 800
Thr Pro Gly Val Thr Cys Val Val Val Ala Val Ser His Gly Ala Pro
805 810 815
Gly Val Leu Pro Ala Thr Thr Val Ala Gly Val Gly Val His Ala Ala
820 825 830
Leu Thr Leu Pro Ala Gly Gly Gly Thr Ala Ser Thr Thr Ala Val Val
835 840 845
Ser Val Leu Thr Val Leu His Gly Ala Thr Leu Ala Gly Leu Gly Thr
850 855 860
Leu Cys Leu Val Ser Ala Leu Ala Leu Pro Ala Pro Ile Gly Leu Thr
865 870 875 880
Ile Ser Leu Ala Leu Gly Gly Pro Ala Gly Pro Gly Val Thr Thr Leu
885 890 895
Pro Pro Ser Ala Gly Gly Met Thr Leu Ala Gly Val Ser Leu Thr Cys
900 905 910
Leu Val Leu Gly Pro Thr Pro Ser Ala Ile Ala Val Gly Thr Gly Ser
915 920 925
Ala Gly Gly Pro Gly Ala Ala Thr Leu Thr Thr Pro Pro Val Leu Ala
930 935 940
Ser Ala Gly Ser Pro Pro Leu Thr Ser Leu Leu Thr Val Ala Leu Ser
945 950 955 960
Ala Thr Gly Gly Gly Ala Val Pro Ser Cys Ser Val Met His Gly Ala
965 970 975
Leu His Ala His Thr Thr Gly Leu Ser Leu Ser Leu Ser Pro Gly Leu
980 985 990
His His His His His His His His His His Met Ser Ser Ser Ser Thr
995 1000 1005
Leu Leu Leu Ser Leu Val Ala Val Thr Ala Ala Gly Ser Thr Ile Gly
1010 1015 1020
Gly Gly Ala Leu Thr Pro Leu Ala Leu Pro Ala His Gly Ala Gly Ala
1025 1030 1035 1040
Leu Pro Thr Gly Ser Ser Leu Ala Ser Thr Ala Thr Ala Thr Ala Ile
1045 1050 1055
Thr Gly Gly Ala Val Gly Ala Met Ala Ala Ala Gly Ala Leu Thr Ser
1060 1065 1070
Ala Pro Leu Leu Gly Gly Ser Thr Leu Ala Gly Met Thr Pro Leu Gly
1075 1080 1085
Gly Ile Gly Ala Leu Thr Val Leu Leu Gly Leu Gly Ala Leu Gly Gly
1090 1095 1100
Ala Gly Ser Ser Val Leu Ser Gly Ala Leu Ser Leu Ala Leu Ala Thr
1105 1110 1115 1120
Ile Leu Ala Thr Met Ser Thr Ile Thr Ser Thr Gly Leu Val Cys Ala
1125 1130 1135
Pro Ala Ala Pro Gly Gly Cys Leu Leu Leu Gly Pro Gly Leu Ala Gly
1140 1145 1150
Ile Met Ala Ala Ser Leu Ala Thr Ala Gly Ala Leu Thr Ala Thr Gly
1155 1160 1165
Ser Thr Ala Ser Gly Val Gly Leu Gly Leu Ala Pro Leu Thr Gly Gly
1170 1175 1180
Thr Val Val Leu Leu Ala Gly Met Ala Ala Ala Ala His Thr Gly Ala
1185 1190 1195 1200
Thr Gly Ala Thr Thr Ala Gly Ala Thr Gly Val Ala Gly Val Ala Gly
1205 1210 1215
Thr Ala Thr Ser Ala Gly Gly Leu Ile Gly Ala Val Gly His Thr Pro
1220 1225 1230
Gly Gly Ile Leu Pro Leu Thr Gly His Leu His Ala Thr Val Ala Ala
1235 1240 1245
Leu Leu Met Ala Ala Thr Pro Ser Thr Ile Ser Pro Ile Gly Cys Leu
1250 1255 1260
Pro Ala His Leu Leu Gly Ala Met Thr Gly Ala Pro Thr Thr Ala Leu
1265 1270 1275 1280
Thr Ser Leu Thr Val Pro Pro Gly Gly Leu Pro Ala Ile Ala Val Thr
1285 1290 1295
Ala Ala Met Val Ala Gly Ala Thr Ala Ala Gly Ala Ile Pro Leu Gly
1300 1305 1310
Ala Gly Leu Pro Pro Val Ser Val Gly Leu Pro Ala Met Thr Gly Gly
1315 1320 1325
Pro Thr Gly Ala Ser Met Leu Thr Ala Pro Gly Ala Val Gly Leu Ala
1330 1335 1340
Val Cys His Pro Thr Ala Thr Ala Leu Gly Leu Gly Ala Pro Ala Ile
1345 1350 1355 1360
Leu Met Cys Thr Leu Val Thr Met Ala Ala Pro Leu Thr Ala His His
1365 1370 1375
Gly Met Gly His Ile Gly Thr Ala Met Ala Thr Ala Ala Gly Pro Pro
1380 1385 1390
Leu Leu Ala Ala Gly Ala Ala Gly Gly Pro His Gly Ala Val Gly Gly
1395 1400 1405
Ile Met Ser Leu Ser Ala Ala Thr Pro Leu His Leu Leu Ser Ile Gly
1410 1415 1420
Leu Leu Ser Pro Ala Pro Gly Gly Ala Ala Gly Thr Gly Ile Ala Pro
1425 1430 1435 1440
Leu Leu Leu Gly Ala Leu Thr Ile Val Gly Thr Leu Pro Pro Thr Thr
1445 1450 1455
Met Leu Gly Leu Thr Ala Thr Met Val Pro Leu Gly Gly Ile Pro Leu
1460 1465 1470
Ala Gly Thr Met Leu Leu Thr Thr Gly Met Leu Ala Gly Ile Val Gly
1475 1480 1485
Val Val Gly Pro Val Pro His Ala Gly Thr Thr Cys Ala Pro Ala Ser
1490 1495 1500
Leu Pro His Val Ser Ala Ala Thr Ser Pro Ile Ala Thr Thr Thr Ala
1505 1510 1515 1520
Thr Leu Thr Gly Pro Gly Pro Gly Gly Ala Leu Cys Gly Ala Ala Leu
1525 1530 1535
His Gly Gly Pro Leu His Leu Cys Ala Ile Ser Ala Ser Thr Gly Ala
1540 1545 1550
Gly Gly Leu Leu Pro Ala Met Leu Ala Leu Gly Leu Ser Gly Pro Thr
1555 1560 1565
Thr Leu Ala Leu Gly Ala Val Val Gly Ala Leu Ala Met Ala Val Ala
1570 1575 1580
Pro Leu Leu Ala Thr Pro Gly Pro Leu Pro Thr Thr Leu Leu Ala Gly
1585 1590 1595 1600
Ala Leu Ala Ser Pro Val Gly Thr Ser Thr Ala Thr Ser Pro Thr Ala
1605 1610 1615
Ala Gly Ser Ile Leu Val Ala Ile Ser Leu Leu Ser Ala Leu Gly Ala
1620 1625 1630
Leu Ala Thr Gly Thr Ala Ala Ala Gly Met Thr Leu Pro Ala Ser Ser
1635 1640 1645
Val Ala Thr Ala Met Ala Gly Thr Pro Leu Leu Val Leu Ala Gly Met
1650 1655 1660
Ile Leu Pro Gly Gly Gly Ala Val Ala Val Ala Ala Leu Leu Pro Ala
1665 1670 1675 1680
Ile Ser Pro Ala Pro Pro Val Thr Ala Pro Leu Ala Val Ser Ala Ile
1685 1690 1695
Ile Pro Ala Thr Gly Val Gly Leu Ala Ile Ala Met Ser Ala Ser Ala
1700 1705 1710
Ile Ala Ala Ala Pro Ala Leu Ala Ala Ala Ser Leu Gly Pro Leu Gly
1715 1720 1725
Ile Gly Pro Thr Leu Gly Pro Pro Ala Gly Pro Pro Thr Ser Thr Thr
1730 1735 1740
Ala Thr Thr Thr Gly Thr Thr Met Gly Thr Thr Thr Thr Gly Thr Thr
1745 1750 1755 1760
Thr Ala Thr Pro Thr Gly Ile Ala Ala Ala Leu Leu Leu Ala Leu Ala
1765 1770 1775
Ala Ser Gly Gly Ala Pro Thr Ala Ser Ile Ala Ile Ser Leu Gly Gly
1780 1785 1790
Ala Ala Pro Gly Pro Gly Ala Thr Ala Ala Val Gly Thr Ser Pro Gly
1795 1800 1805
Gly Ser Ser Ala Leu Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly
1810 1815 1820
Leu Leu Gly Gly Pro Ser Val Pro Leu Pro Pro Pro Leu Pro Leu Ala
1825 1830 1835 1840
Thr Leu Met Ile Ser Ala Thr Pro Gly Val Thr Cys Val Val Val Ala
1845 1850 1855
Val Ser His Gly Ala Pro Gly Val Leu Pro Ala Thr Thr Val Ala Gly
1860 1865 1870
Val Gly Val His Ala Ala Leu Thr Leu Pro Ala Gly Gly Gly Thr Ala
1875 1880 1885
Ser Thr Thr Ala Val Val Ser Val Leu Thr Val Leu His Gly Ala Thr
1890 1895 1900
Leu Ala Gly Leu Gly Thr Leu Cys Leu Val Ser Ala Leu Ala Leu Pro
1905 1910 1915 1920
Ala Pro Ile Gly Leu Thr Ile Ser Leu Ala Leu Gly Gly Pro Ala Gly
1925 1930 1935
Pro Gly Val Thr Thr Leu Pro Pro Ser Ala Gly Gly Met Thr Leu Ala
1940 1945 1950
Gly Val Ser Leu Thr Cys Leu Val Leu Gly Pro Thr Pro Ser Ala Ile
1955 1960 1965
Ala Val Gly Thr Gly Ser Ala Gly Gly Pro Gly Ala Ala Thr Leu Thr
1970 1975 1980
Thr Pro Pro Val Leu Ala Ser Ala Gly Ser Pro Pro Leu Thr Ser Leu
1985 1990 1995 2000
Leu Thr Val Ala Leu Ser Ala Thr Gly Gly Gly Ala Val Pro Ser Cys
2005 2010 2015
Ser Val Met His Gly Ala Leu His Ala His Thr Thr Gly Leu Ser Leu
2020 2025 2030
Ser Leu Ser Pro Gly Leu His His His His His His His His His His
2035 2040 2045
<210> 3
<211> 3018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
catatgtcgt ctagctcttg gttattgttg tctttagttg cagtgaccgc agcccagtca 60
accattgaag aacaggcgaa aacgtttctc gataaattta atcatgaagc agaggacctg 120
ttttatcagt catcactggc ctcttggaat tataatacca atattaccga agaaaatgtt 180
cagaatatga ataatgccgg cgataaatgg agtgcctttc tgaaagaaca gtcaaccctg 240
gcccagatgt atccgttaca ggaaattcag aatctgaccg ttaaactcca gttacaggcc 300
ttacagcaga atggctctag cgtcctttcg gaagataaat ctaaacgtct gaataccatt 360
ctgaatacca tgtcaaccat ttatagtacc ggcaaagttt gtaatccgga taatccgcag 420
gaatgtctgc tgttagaacc gggcttaaat gaaattatgg ccaatagctt agattataat 480
gaacgtctgt gggcctggga atcttggcgt agcgaagtgg gcaaacagtt acgcccgctg 540
tatgaagaat atgtggtgct gaaaaatgaa atggcacgcg ccaatcatta tgaagattat 600
ggcgattatt ggcgcggcga ttatgaagtt aatggtgttg atggctatga ttatagtcgc 660
ggccagctga ttgaagatgt ggaacatacc tttgaagaaa ttaaaccgct gtatgaacat 720
ttacatgcct atgttcgtgc caaactgatg aatgcctatc cgagctatat tagcccgatt 780
ggttgtctgc cggcacattt actgggtgat atgtggggtc gcttttggac caatctgtat 840
tcactgaccg ttccgtttgg ccagaaaccg aatattgatg tgaccgatgc gatggttgat 900
caggcctggg atgcacagcg catttttaaa gaagcagaaa aattttttgt gagcgtgggt 960
ctgccgaata tgacccaggg cttttgggaa aatagtatgc tgaccgatcc gggtaatgtg 1020
cagaaagcag tttgtcatcc gaccgcctgg gatctgggca aaggcgattt tcgcattctg 1080
atgtgtacga aggtcactat ggatgatttt ctgaccgcac atcatgaaat gggtcatatt 1140
cagtatgata tggcctatgc agcacagccg tttctgctgc gcaatggtgc caatgaaggc 1200
tttcatgaag cagtgggcga aattatgtct ttaagtgcag caaccccgaa acatctgaaa 1260
tctattggtc tgctgagtcc ggattttcag gaagataatg aaaccgaaat taattttctg 1320
ctgaaacagg ccctgaccat tgtgggcacc ctgccgttta cctatatgct ggaaaaatgg 1380
cgctggatgg tgtttaaagg tgaaattccg aaagatcagt ggatgaaaaa atggtgggaa 1440
atgaaacgcg aaattgttgg cgtggtggaa ccggttccgc atgatgaaac ctattgtgat 1500
ccggcctcac tgtttcatgt gtctaatgat tatagcttta ttcgctatta tacccgtacc 1560
ctgtatcagt ttcagtttca ggaagccctg tgtcaggcag ccaaacatga aggtccgtta 1620
cataaatgtg atatttctaa ttctaccgaa gccggtcaga aactgtttaa tatgctgcgc 1680
ttaggcaaaa gcgaaccgtg gaccttagcc ttagaaaatg ttgtgggtgc caaaaatatg 1740
aatgttcgtc cgttactgaa ttattttgaa ccgctgttta cctggttaaa agatcagaat 1800
aaaaatagct ttgttggttg gagtaccgat tggagtccgt atgcagatca gagtattaaa 1860
gtgcgcattt cactgaaaag cgccttaggc gataaagcgt atgaatggaa tgataatgaa 1920
atgtatctgt ttcgtagctc agttgcctat gccatgcgtc agtattttct gaaagttaaa 1980
aatcagatga ttctgtttgg cgaagaagat gttcgcgttg ccaatttaaa accgcgcatt 2040
agctttaatt tttttgtgac cgccccgaaa aatgtgagcg atattattcc gcgcaccgaa 2100
gtggaaaaag ccattcgcat gtctcgtagt cgtattaatg atgcatttcg cttaaatgat 2160
aattcactgg aatttttagg tattcagccg accttaggcc cgccgaatca gccgccgacc 2220
tcaacctgga ataccaccta tggcaccaca atgggcacca ccaccaccgg taccaccacc 2280
aataccggcg gctcaagtga taaaacccat acctgtccgc cgtgtccggc cccggaactg 2340
ctgggcggtc cgagcgtgtt tctgtttccg ccgaaaccga aagataccct gatgattagt 2400
cgtaccccgg aagtgacctg tgttgtggtg gatgtgtcac atgaagatcc gcaggttaaa 2460
tttaattggt atgttgatgg cgtgcaggtt cataatgcca aaaccaaacc gcgcgaacag 2520
cagtataata gtacctatcg cgttgtgagc gtgctgaccg ttctgcatca gaattggtta 2580
gatggtaaag aatataaatg taaagtgagt aataaagcac tgccggcccc gattgaaaaa 2640
accattagta aagccaaagg tcagccgcgc gaaccgcagg tgtataccct gccgccgtct 2700
cgcgaagaaa tgaccaaaaa tcaggtgtca ctgacctgtc tggttaaagg cttttatccg 2760
agcgatattg cagtggaatg ggaatctaat ggccagccgg aaaataatta taaaaccacc 2820
ccgccggtgt tagatagtga tggctctttt tttctgtata gcaaactgac cgtggataaa 2880
tctcgttggc agcagggcaa tgtgtttagt tgtagcgtga tgcatgaagc cttacataat 2940
cattataccc agaaatctct gtcactgtct ccgggtaaac atcatcatca tcatcatcat 3000
catcatcatt aaggatcc 3018
<210> 4
<211> 3150
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
catatgtcgt ctagctcttg gttattgttg tctttagttg cagtgaccgc agcccagtca 60
accattgaag aacaggcgaa aacgtttctc gataaattta atcatgaagc agaggacctg 120
ttttatcagt catcactggc ctcttggaat tataatacca atattaccga agaaaatgtt 180
cagaatatga ataatgccgg cgataaatgg agtgcctttc tgaaagaaca gtcaaccctg 240
gcccagatgt atccgttaca ggaaattcag aatctgaccg ttaaactcca gttacaggcc 300
ttacagcaga atggctctag cgtcctttcg gaagataaat ctaaacgtct gaataccatt 360
ctgaatacca tgtcaaccat ttatagtacc ggcaaagttt gtaatccgga taatccgcag 420
gaatgtctgc tgttagaacc gggcttaaat gaaattatgg ccaatagctt agattataat 480
gaacgtctgt gggcctggga atcttggcgt agcgaagtgg gcaaacagtt acgcccgctg 540
tatgaagaat atgtggtgct gaaaaatgaa atggcacgcg ccaatcatta tgaagattat 600
ggcgattatt ggcgcggcga ttatgaagtt aatggtgttg atggctatga ttatagtcgc 660
ggccagctga ttgaagatgt ggaacatacc tttgaagaaa ttaaaccgct gtatgaacat 720
ttacatgcct atgttcgtgc caaactgatg aatgcctatc cgagctatat tagcccgatt 780
ggttgtctgc cggcacattt actgggtgat atgtggggtc gcttttggac caatctgtat 840
tcactgaccg ttccgtttgg ccagaaaccg aatattgatg tgaccgatgc gatggttgat 900
caggcctggg atgcacagcg catttttaaa gaagcagaaa aattttttgt gagcgtgggt 960
ctgccgaata tgacccaggg cttttgggaa aatagtatgc tgaccgatcc gggtaatgtg 1020
cagaaagcag tttgtcatcc gaccgcctgg gatctgggca aaggcgattt tcgcattctg 1080
atgtgtacga aggtcactat ggatgatttt ctgaccgcac atcatgaaat gggtcatatt 1140
cagtatgata tggcctatgc agcacagccg tttctgctgc gcaatggtgc caatgaaggc 1200
tttcatgaag cagtgggcga aattatgtct ttaagtgcag caaccccgaa acatctgaaa 1260
tctattggtc tgctgagtcc ggattttcag gaagataatg aaaccgaaat taattttctg 1320
ctgaaacagg ccctgaccat tgtgggcacc ctgccgttta cctatatgct ggaaaaatgg 1380
cgctggatgg tgtttaaagg tgaaattccg aaagatcagt ggatgaaaaa atggtgggaa 1440
atgaaacgcg aaattgttgg cgtggtggaa ccggttccgc atgatgaaac ctattgtgat 1500
ccggcctcac tgtttcatgt gtctaatgat tatagcttta ttcgctatta tacccgtacc 1560
ctgtatcagt ttcagtttca ggaagccctg tgtcaggcag ccaaacatga aggtccgtta 1620
cataaatgtg atatttctaa ttctaccgaa gccggtcaga aactgtttaa tatgctgcgc 1680
ttaggcaaaa gcgaaccgtg gaccttagcc ttagaaaatg ttgtgggtgc caaaaatatg 1740
aatgttcgtc cgttactgaa ttattttgaa ccgctgttta cctggttaaa agatcagaat 1800
aaaaatagct ttgttggttg gagtaccgat tggagtccgt atgcagatca gagtattaaa 1860
gtgcgcattt cactgaaaag cgccttaggc gataaagcgt atgaatggaa tgataatgaa 1920
atgtatctgt ttcgtagctc agttgcctat gccatgcgtc agtattttct gaaagttaaa 1980
aatcagatga ttctgtttgg cgaagaagat gttcgcgttg ccaatttaaa accgcgcatt 2040
agctttaatt tttttgtgac cgccccgaaa aatgtgagcg atattattcc gcgcaccgaa 2100
gtggaaaaag ccattcgcat gtctcgtagt cgtattaatg atgcatttcg cttaaatgat 2160
aattcactgg aatttttagg tattcagccg accttaggcc cgccgaatca gccgccgacc 2220
tcaacctgga ataccaccta tggcaccaca atgggcacca ccaccaccgg taccaccacc 2280
aataccttta ccggcattcg cgatcgcaaa aaaaaaaata aagcacgctc aggcgaaaat 2340
ccgtatgcct caattgatat tagcaaaggc gaaaataatc cgggctttca gaataccgat 2400
gatgtgcaga cctcttttgg cggctcaagt gataaaaccc atacctgtcc gccgtgtccg 2460
gccccggaac tgctgggcgg tccgagcgtg tttctgtttc cgccgaaacc gaaagatacc 2520
ctgatgatta gtcgtacccc ggaagtgacc tgtgttgtgg tggatgtgtc acatgaagat 2580
ccgcaggtta aatttaattg gtatgttgat ggcgtgcagg ttcataatgc caaaaccaaa 2640
ccgcgcgaac agcagtataa tagtacctat cgcgttgtga gcgtgctgac cgttctgcat 2700
cagaattggt tagatggtaa agaatataaa tgtaaagtga gtaataaagc actgccggcc 2760
ccgattgaaa aaaccattag taaagccaaa ggtcagccgc gcgaaccgca ggtgtatacc 2820
ctgccgccgt ctcgcgaaga aatgaccaaa aatcaggtgt cactgacctg tctggttaaa 2880
ggcttttatc cgagcgatat tgcagtggaa tgggaatcta atggccagcc ggaaaataat 2940
tataaaacca ccccgccggt gttagatagt gatggctctt tttttctgta tagcaaactg 3000
accgtggata aatctcgttg gcagcagggc aatgtgttta gttgtagcgt gatgcatgaa 3060
gccttacata atcattatac ccagaaatct ctgtcactgt ctccgggtaa acatcatcat 3120
catcatcatc atcatcatca ttaaggatcc 3150

Claims (10)

1.重组人血管紧张素转化酶2蛋白，其特征在于，所述重组人血管紧张素转化酶2蛋白可竞争性结合ACE2配体，通过对设计的全长、膜外区、穿膜区的相关氨基酸进行突变获得亲水性的ACE2全长蛋白；所述重组人血管紧张素转化酶2蛋白的氨基酸序列如SEQ ID NO：1或SEQ ID NO：2所示。
2.根据权利要求1所述的重组人血管紧张素转化酶2蛋白，其特征在于，所述氨基酸序列SEQ ID NO：1的结构依次由膜外区、穿膜区和FC组成；所述氨基酸序列SEQ ID NO：2的结构依次由全长和FC组成。
3.权利要求1或2所述的重组人血管紧张素转化酶2蛋白的制备方法，其特征在于，所述制备方法包括以下步骤：
1)人工合成重组人血管紧张素转化酶2基因；
2)重组人血管紧张素转化酶2蛋白的表达：利用原核表达系统将重组人血管紧张素转化酶2基因在大肠杆菌中进行表达，并获得重组人血管紧张素转化酶2基工程菌株，经IPTG诱导表达重组人血管紧张素转化酶2蛋白；
3)重组人血管紧张素转化酶2蛋白的纯化：利用His标签可溶性蛋白纯化进行蛋白纯化。
4.根据权利要求3所述的制备方法，其特征在于，所述重组人血管紧张素转化酶2基因的核苷酸序列如SEQ ID NO：3或SEQ ID NO：4所示。
5.根据权利要求3所述的制备方法，其特征在于，步骤2)将重组人血管紧张素转化酶2基因插入原核表达载体pET3C，构建重组表达质粒pET3C-ACE2，并将质粒转化大肠杆菌BL21DE3PLYsS，获得重组人血管紧张素转化酶2基工程菌株，将工程菌株进行培养然后加入IPTG进行诱导表达重组人血管紧张素转化酶2蛋白。
6.根据权利要求5所述的制备方法，其特征在于，所述表达载体pET3C含有T7启动子。
7.根据权利要求5所述的制备方法，其特征在于，IPTG进行诱导的条件为37℃，200rpm转速条件下将工程菌株培养3h至菌液的OD600达到0.50.7时加入终浓度为2mM的IPTG诱导表达4h。
8.根据权利要求3所述的制备方法，其特征在于，步骤3)利用His标签可溶性蛋白纯化进行蛋白纯化后采用Ni琼脂糖凝胶层析柱洗脱纯化。
9.一种药物组合物，其特征在于，所述药物组合物包含权利要求1或2所述的重组人血管紧张素转化酶2蛋白。
10.权利要求9所述的药物组合物在制备抗冠状病毒药物中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114525365A (zh) * 2021-12-23 2022-05-24 东莞市松山湖中心医院(东莞市石龙人民医院、东莞市第三人民医院、东莞市心血管病研究所) 一种作用于SARS-Cov-2诱导的急性肺损伤的PRCP试剂盒

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114525365A (zh) * 2021-12-23 2022-05-24 东莞市松山湖中心医院(东莞市石龙人民医院、东莞市第三人民医院、东莞市心血管病研究所) 一种作用于SARS-Cov-2诱导的急性肺损伤的PRCP试剂盒

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