CN114369585B - 重组幽门螺杆菌的岩藻糖基转移酶突变体及应用 - Google Patents

重组幽门螺杆菌的岩藻糖基转移酶突变体及应用 Download PDF

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CN114369585B
CN114369585B CN202210115274.XA CN202210115274A CN114369585B CN 114369585 B CN114369585 B CN 114369585B CN 202210115274 A CN202210115274 A CN 202210115274A CN 114369585 B CN114369585 B CN 114369585B
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王冀姝
黄滔
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Abstract

本发明公开了一种幽门螺杆菌的岩藻糖基转移酶的突变体，其通过对特定位点和片段进行增减、或删除后，改变岩藻糖基转移活性，显著提高转移分子量大于100KD的含有鸟苷二磷酸岩藻糖(GDP‑Fucose)的供体分子。在酶突变体的作用下，可以将大分子量的供体分子（例如偶联了抗体分子或者重组蛋白的GDP‑Fucose）转移至受体GlcNAc（N‑乙酰氨基葡萄糖）和LacNac(N‑乙酰乳糖胺)，受体可存在于大分子物质和细胞膜表面。本发明还公开了岩藻糖基转移酶突变体在分子标记和活体细胞标记中的应用。

Description

重组幽门螺杆菌的岩藻糖基转移酶突变体及应用
技术领域
本发明涉及一种蛋白突变体，属于多肽技术领域。
背景技术
糖基化是碳水化合物与目标分子（通常为蛋白质和脂质）共价连接的过程，蛋白质分子的糖基化是最丰富的翻译后修饰之一。这种修饰具有多种功能，例如，参与蛋白质分子的正确折叠， 调节蛋白质的热力学和动力学稳定性，参与分子间相互作用和细胞间粘附，参与免疫识别或者免疫逃逸。与DNA转录或蛋白质翻译不同，蛋白质的糖基化过程没有模板，是一种是酶促反应，供体分子通常是一种活化的核苷酸糖，在糖基转移酶的作用下，对受体位点（羟基或其它官能团）进行特异的糖缀合物的反应。岩藻糖作为糖蛋白中糖链的组成部分，广泛存在于各类细胞表面的质膜上。岩藻糖基转移酶是将L-岩藻糖从GDP-岩藻糖（鸟苷二磷酸岩藻糖）供体底物转移到受体底物的酶。据目前的各种文献报道，岩藻糖基转移酶的主要供体底物都是分子量比较小的GDP-岩藻糖（GDP-Fucose）。原核生物的岩藻糖基转移酶，目前经过验证的主要来自幽门螺杆菌（Helicobacter pylori），包括6条序列（Uniprot：http://www.cazy.org/GT10.html）。
细胞表面的分子决定了细胞如何与其它细胞以及周围环境相互作用。肿瘤的治疗性抗体例如抗-CD20, 抗-VEGFR等通过结合T淋巴细胞和NK细胞表面的FcgR，同时结合肿瘤细胞表面的抗原，使得T和NK细胞发挥肿瘤杀伤作用（ADCC）。受其启发，肿瘤免疫疗法近年来有了突变进展 —— 嵌合抗原受体T细胞免疫疗法， Chimeric Antigen Receptor T-Cell Immunotherapy （CAR-T），其中Kymriah是第一款被美国批准用于治疗B细胞前体急性淋巴细胞白血病。这是一种治疗肿瘤的新型精准靶向疗法，用生物技术把辨识癌细胞表面CD19抗原的CAR基因插到T细胞膜表面上，直接识别肿瘤细胞，激活T细胞进行肿瘤杀伤。但CAR-T的技术工艺复杂，价格昂贵。类似CAR-T这样的细胞工程的主要技术挑战是在不干扰细胞内源性功能的情况下赋予被操纵细胞新的特性。作为目前最常见和最稳健的细胞工程方法，首先受到技术复杂性和安全问题的限制，如原代细胞病毒转导效率的再现性不一致、CAR基因的异质表达水平，以及内源性基因被破坏的可能性。因此，使用化学生物学工具直接修饰细胞表面已成为细胞治疗的一种补充和普遍适用的方法，其中包括Bertozzi等人开发的代谢寡糖修饰法（MOE）和细菌转肽酶sortases催化的转肽反应。（Stephan,等.NanoToday 2011, 6, 309−325.；Griffin等. Cell Chem. Biol. 2016, 23, 108−121. ；Hudak等 Chem. Biol. 2014, 21, 16−37. ；Bi, X等. Engineering. Chem. - Eur. J. 2018,24, 8042−8050.））此外，Wu等利用幽门螺杆菌的岩藻糖基转移酶成功地将抗体类大分子蛋白转移到细胞膜表面的多糖上例如LacNAc和α2,3 sialyl LacNAc。利用这种技术，Wu等构建了两种类型的工程细胞−使用自然杀伤细胞系（NK-92MI）和小鼠原代CD8+OT-1T细胞，通过岩藻糖基转移酶将Her2抗体和PD-L1的抗体分别转移至NK-92MI和CD8+OT-1T细胞，并在小鼠模型中展示了特异性肿瘤靶向性和对抗肿瘤细胞产生的抑制信号（参见Li J,等. ACSCent Sci. 2018 Dec 26;4(12):1633-1641. ）。因此利用岩藻糖基转移酶将与供体底物相结合的目的分子标记到携带有受体底物的目的细胞将使得诸如CAR-T这样的细胞治疗的效果得到大幅提升。
糖苷转移酶(glycotransferase)能够在中性条件下，尤其是哺乳细胞无损伤的反应条件下作用，对于细胞学工程是一个无以比拟的长处。但是无论是细菌来源的还是人源的岩藻糖基转移酶对于大分子的供体底物的酶活性并不好。本发明人在研究中发现，相比小分子GDP-Fucose而言，岩藻糖基转移酶对于大分子的供体底物的酶活性降低近千倍，不能满足临床治疗的需求。因此，本发明的目的就是提供一种对于大分子的供体底物转移具有优异酶活性的岩藻糖基转移酶突变体。
发明内容
基于上述目的，本发明首先提供了一种幽门螺杆菌的岩藻糖基转移酶的突变体，所述突变体为序列如SEQ ID NO. 15所示的野生型幽门螺杆菌的岩藻糖基转移酶去除包括435-476位氨基酸残基的疏水末端，以及逐一去除365-434位氨基酸残基的tandem 重复单位D-D-L-R-V-N-Y直至第405位氨基酸形成的突变体。
在一个优选的实施方案中，所述突变体的氨基酸序列如SEQ ID NO. 1、3、5、7、9 、11或13所示。
其次，本发明提供了一种上述重组岩藻糖基转移酶突变体的多核苷酸，所述多核苷酸的序列如SEQ ID NO. 2、4、6、8、10、12或14所示。
第三，本发明提供了一种含有上述多核苷酸的表达载体。
第四，本发明提供了一种含有上述表达载体的宿主细胞。
在一个优选的实施方案中，所述宿主细胞为大肠杆菌。
第五，本发明提供了一种应用上述的重组岩藻糖基转移酶突变体将靶分子标记于目的细胞或者目的蛋白质的方法，所述方法包括：
（1）将供体底物与靶分子相缀合获得连接复合物；
（2）在所述的重组岩藻糖基转移酶突变体存在的条件下，使步骤（1）获得的连接复合物与含有GlcNac受体分子的目的细胞或者目的蛋白质一起孵育，获得被标记了靶分子的目的细胞。
在一个优选的实施方案中，所述供体底物的分子量为500D 至150KD的GDP-Fucose-(PEG4)n，所述n为0-10的整数。
在一个更为优选的实施方案中，所述靶分子为IgG，所述n=2。
第六，本发明提供了一种根据上述方法标记的细胞或者蛋白质。
最后，本发明提供了上述的细胞或者蛋白质在制备疾病治疗药物中的应用。
在一个优选的实施方案中，所述疾病为肿瘤、炎症性疾病、代谢类疾病或需要酶替代治疗的罕见病。
本发明对幽门螺杆菌的岩藻糖基转移酶的特定位点和片段进行替换、增减、或删除后，改变岩藻糖基转移活性，对于含有鸟苷二磷酸岩藻糖(GDP-Fucose)的抗体作为供体分子时，酶活性提高了数十倍。本发明显示利用重组的突变体FucTd可以将分子量500D （例如GDP-Fucose）至 150KD (例如GDP-Fucose-免疫球蛋白)的供体分子，催化转移到含有GlcNac的受体分子上，例如将GDP-Fucose-(PEG4)n-IgG有效地转移至HEK293细胞表面。利用这种方法，不仅可以使得细胞迅速获取新的细胞表面信号分子，通过和效应细胞相互作用，用于激活或者抑制，例如T细胞表面偶联PD-L1的抗体用于拮抗肿瘤细胞传递给T细胞的抑制作用；也可以通过NK细胞表面偶联肿瘤相关抗原（TAAs）的抗体，例如抗Her2，EFGR，VEGFR，CD19等加强NK细胞的靶向性、提高抗肿瘤的效果。此外，还可以通过偶联一种或者数种肿瘤特异识别抗原，改善CAR-T的靶向性，减少CAR-T的脱靶副作用。此外，还可以将代谢相关的酶偶联在血液细胞表面，例如尿酸氧化酶，清除血液和组织中的尿酸，治疗难治性痛风。
附图说明
图1. 重组幽门螺杆菌的岩藻糖基转移酶的突变体表达产物活性检测图；
图2. 重组幽门螺杆菌的岩藻糖基转移酶的突变体FucTd的 液相色谱-质谱法鉴定；
图3. 重组幽门螺杆菌的岩藻糖基转移酶的突变体以小分子为供体底物时Km值的比较；
图4. 重组幽门螺杆菌的岩藻糖基转移酶的突变体以大分子为供体底物时酶活性Km的比较；
图5.岩藻糖基转移酶的突变体催化的供体底物标记HEK293的流式细胞术检测图。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的权利要求所限定的保护范围构成任何限制。
实施例1，alfa-1,3岩藻糖基转移酶变异体的制备
幽门螺杆菌的alfa-1,3岩藻糖基转移酶(strain ATCC 700392 / 26695) （FucT）催化岩藻糖从供体GDP-岩藻糖到受体N-乙酰乳糖胺的糖基, 产生Lewis x和Lewis y表位抗原，以模拟胃上皮细胞的碳水化合物抗原，从而避免被宿主免疫系统检测到。FucT全长476个氨基酸（UniProtKB - O25366），序列如SEQ ID NO. 15所示，365-434是由10个连续的7个氨基酸组成的重复片段D-D-L-R-V-N-Y，435-476是一段疏水的氨基酸。 文献报道，FucT的C末端的80个氨基酸残基都可以删除掉，在结构和酶活性上没有明显变化[ref 2006-Carboxyl Terminus of Helicobacter pylori R1,3-Fucosyltransferase Determinesthe Structure and Stability]，但在大肠杆菌E.coli中的表达量却明显升高了。我们尝试表达时，首先去除了疏水末端包括435-478位（FucT-dM），随后逐一去除tandem repeatof D-D-L-R-V-N-Y，直至第405位氨基酸，形成如下截短体，FucT-d M（序列如SEQ ID NO. 1所示，核苷酸序列如SEQ ID NO. 2所示）,FucT-d1R（序列如SEQ ID NO. 3所示，核苷酸序列如SEQ ID NO. 4所示）, FucT-d2R（序列如SEQ ID NO. 5所示，核苷酸序列如SEQ ID NO. 6所示）, FucT-d3R（序列如SEQ ID NO. 7所示，核苷酸序列如SEQ ID NO. 8所示）, FucT-d4R（序列如SEQ ID NO. 9所示，核苷酸序列如SEQ ID NO. 10所示）, FucT-d5R（序列如SEQID NO. 11所示，核苷酸序列如SEQ ID NO. 12所示）, FucTd（序列如SEQ ID NO. 13所示，核苷酸序列如SEQ ID NO. 14所示）。这7个截短体分别在Ecoli中进行表达，纯化，具体步骤如下文所述。
1. 质粒制备
委托苏州君跻生物科技有限公司合成质粒DNA，并克隆至载体pET41a（MilliporeSigma™ pET-41a(+) DNA Vector，目录号：70-556-3）上，分别用于表达FucT-dM、FucT-d1R、FucT-d2R、FucT-d3R、FucT-d4R、FucT-d5R、FucTd重组蛋白，构建成表达质粒FucT-dM-pET41a、FucT-d1R-pET41a、FucT-d2R-pET41a、FucT-d3R-pET41a、FucT-d4R-pET41a、FucT-d5R-pET41a、FucTd-pET41a。具体操作方法参照《分子克隆实验指南》，质粒先转化DH10B、测序、保菌和菌液培养。按照《Qiagen Mini-prep Kit》和《Qiagen EndofreeMaxi-prep Kit》提到的操作方法，进行质粒的制备。
2.FucT的截短体蛋白表达：
2.1 转化BL21(DE3)
2.1.1 取2 uL质粒加入100 uL BL21(DE3)感受态细胞中（(ThermoFisher, 目录号:EC0114），立即混匀，冰上放置30 min；
2.1.2 42℃热激90 s，迅速冰浴2 min；
2.1.3 加入500 uL LB培养基，37℃，rpm<=200振荡培养60 min；
2.1.4 6000rpm离心1min，弃去大部分上清，留约100-150 uL，重悬菌体后，涂于含Amp的LB平板上，37℃过夜培养。
2.2 小量表达
2.2.1 保菌：挑取1个单克隆于1 mL Amp抗性的LB培养基中，37 ℃，220 rpm振荡培养约5h，加入1mL 40%甘油，分装2管，冻存于-80℃；
2.2.2 在上一步管中加入2.5mL 含Amp的LB液体培养基，37 ℃，220 rpm振荡培养过夜；
2.2.3 将过夜培养的菌液按1:50比例转接到20ml含Amp的LB培养基中，37℃，220rpm，培养至OD600=0.6 (约3h)，加入终浓度0.5mM IPTG，37℃，220 rpm培养3h；
2.3 SDS-PAGE鉴定表达量
2.3.1 测定培养液的OD600，取10OD菌液，10000rpm，2min离心，去掉上清；
2.3.2 用1 mL 裂解液 (10mM Tris-HCl, pH 8.0)重悬菌体，置于冰上进行超声裂解细胞，超声条件：130W、4min、on 3s、off 3s；
2.4 纯化
超声结束后，裂解液12000rpm, 10min离心得到上清(lysis supernatant)；将上清液在4°C下以125000g超速离心。根据使用手册，将上清液应用于HiTrap螯合HP柱，并用20mM咪唑溶液进行洗脱。将洗脱液汇集并通过透析将溶剂更换为50 mM Tris缓冲液（pH8.0），然后再经过凝胶过滤层析（superdex200，GE Healthcare）进行进一步纯化得到纯度较高的蛋白，产生具有98%以上均一性的蛋白质。以牛血清白蛋白为标准，使用基于Bradford法的Bio-Rad蛋白质分析试剂盒测定蛋白质浓度。
图1显示了 FucT的6种截短体，经过纯化后，按照《分子克隆实验指南》描述的方法进行SDS-PAGE分析的图谱。 80uL 经过纯化的重组蛋白分别加入20uL 5×Reduce loadingbuffer，95 ℃加热5 min，每个样品取12.5uL(0.1OD)进行SDS-PAGE电泳。
图2为液相色谱-质谱(LC-MS)法鉴定FucTd。FucTd的理论分子量为48555 Delton，LC-MS测量的完整分子量，与理论分子量相匹配。
仪器：LC: Waters ACQUITY UPLC-Class；
MS: Waters ACQUITY Rda；
柱子： Waters ACQUITY UPLC Protein BEH C4, 300Å, 1.7um, 2.1 x 100mm ;
柱温：70˚C;
流速：0.4ml/min;
检测UV： 215/280nm;
Buffer A: 0.1% FA in distilled water;
Buffer B: 0.1% FA in Acetonitrile;
Gradient: 5%-60% buffer B in 10min;
95% buffer B hold 1min。
质谱选择Full scan模式，阳离子极性，Cone voltage为70V，Mass range为High(400-7000m/z)，Scan rate为1Hz，Capillary voltage为1.5KV，Desolvation temperature550℃。
实施例2. alfa-1,3岩藻糖基转移酶变异体的酶活性检测
分别以GDP-Fucose和Fucose-PEG4×2-IgG为岩藻糖供体，胎球蛋白为受体进行酶活检测。其中，Fucose-PEG4×2-IgG制备方法：TCO-PEG4-NHS（购自Sigma）与抗体IgG在室温下反应30分钟，获得TCO-PEG4-IgG。GDP-Azido-Fucose（购自R&D system），与Methyltetrazine-PEG4-azide或Alkyne-PEG4-NHS Ester (购自Click Chemistry Tools）在30°C反应6小时，获得GDP-Fucose-PEG4-Methyltetrazine和GDP-Fucose-PEG4-NHSEster，分别与TCO-PEG4-IgG和IgG在室温下反应得到GDP-Fucose-PEG4×2-IgG和GDP-Fucose-PEG4-IgG。
结果显示，FucT-dM表达量最低，FucT-d5R表达量最高。酶活检测显示，小分子GDP-Fucose作为供体底物，所有截短体的酶活性并无明显差异。而当大分子GDP-Fucose-PEG4×2-IgG作为供体底物，FucT-dM, FucT-d1R, FucT-d2R 的酶活性都降低超过50倍。而FucT-d3R, FucT-d4R, FucT-d5R, FucTd, 虽然酶活性也有降低，但相比仅去除了疏水末端包括435-478位的FucT-dM，针对于大分子Fucose-PEG4×2-IgG，都显示出了良好的岩藻糖转移活性。FucTd能够有效催化以Fucose-PEG4×2-IgG为岩藻糖供体的反应。
以GDP-Fucose作为供体底物，胎球蛋白作为受体底物，测定FucT截短体的Km。结果显示，7个FucT的截短体Km值在5~7 µM之间，无明显差别（参见图3）。
以GDP-Fucose-(PEG4)n-IgG（n=1, 2）作为供体底物，测定FucT截短体的Km。以FucT-dM的Km作为标准，比较各个截短体的酶活性。结果显示，C末端的D-D-L-R-V-N-Y的重复片段逐渐去除后，岩藻糖转移活性逐渐增加，其中去除了3个、4个、5个D-D-L-R-V-N-Y的重复片段后，相对于没有去除D-D-L-R-V-N-Y的重复片段FucT-dM，酶活性增加了10倍；而FucTd则增加了30倍（参见图4）。
实施例3岩藻糖基转移酶的突变体催化的供体底物标记HEK293活细胞
FucTd（序列如SEQ ID NO. 13所示）在大肠杆菌进行表达纯化，用于活细胞的标记。将HEK293活细胞（~100万）悬浮在100μL含有 20 mM MgSO4（例如CPDA-1）和0.5%FBS的溶液中，pH值调节至5-6， 并依次加入0.1 mg/mL GDP-Fucose-（PEG4）n-IgG和0.04 mg/mLFucTd-HIS×6, FUT6-Mut6, FUT6-Mut17，在4度或者室温下，孵育20–30分钟后，用CPDA-1（citrate phosphate dextrose adenine）清洗细胞两次。利用流式细胞仪和针对IgG的荧光抗体分析细胞标记的效率，结果显示，在30分钟内，超过90%的细胞被有效地标记了IgG。
在100万HEK293活细胞中加入供体底物GDP-Fucose-(PEG4)n-IgG，并加入岩藻糖转移酶FucTd，在室温下孵育30分钟后对细胞进行检测。没有进行处理的HEK293细胞（Mock）作为阴性对照，同时转染了hIgG和mIgG的HEK293细胞作为阳性对照（HEK transfected w/surface-hIgG and surface-mIgG）。用anti-human IgG二抗检测hIgG在细胞表面的呈现，用anti-mouse IgG二抗检测mIgG在细胞表面的呈现。 结果显示在岩藻糖转移酶FucTd的作用下，供体底物GDP-Fucose-(PEG4)n-IgG转移至HEK293细胞表面，并能够被anti-hIgG二抗识别（参见图5）。
序列表
<110> 北京睿脉医药科技有限公司
<120> 重组幽门螺杆菌的岩藻糖基转移酶突变体及应用
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305 310 315 320
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actgcaccgt acaaactgaa agataactct ctgtacgcgc tgaagaaacc atcccatcac 480
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<210> 5
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Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Glu
1 5 10 15
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Asn Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Lys Ser Val Leu
35 40 45
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50 55 60
Asn Glu Ser Ser Asp Leu Val Phe Ser Asn Pro Leu Gly Ala Ala Arg
65 70 75 80
Lys Ile Leu Ser Tyr Gln Asn Thr Lys Arg Val Phe Tyr Thr Gly Glu
85 90 95
Asn Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp
100 105 110
Glu Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Ala
115 120 125
His Leu His Tyr Glu Ala Glu Leu Val Asn Asp Thr Thr Ala Pro Tyr
130 135 140
Lys Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His His
145 150 155 160
Phe Lys Glu Asn His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser
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Asp Leu Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Ala Asn
180 185 190
Ala Pro Met Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro
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Val Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Lys Val Gly
210 215 220
Asn Lys Ser Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu
225 230 235 240
Asn Ser Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Leu Asp Ala Tyr
245 250 255
Phe Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys
260 265 270
Asp Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Asn Asn Phe
275 280 285
Asp Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Pro Asn Ala
290 295 300
Tyr Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys
305 310 315 320
Ala Tyr Phe Tyr Gln Asp Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe
325 330 335
Lys Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asn Asn Pro Phe Ile
340 345 350
Phe Tyr Arg Asp Leu His Glu Pro Leu Ile Ser Ile Asp Asp Leu Arg
355 360 365
Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn
370 375 380
Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp
385 390 395 400
Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu
405 410 415
Arg Val Asn Tyr His His His His His His
420 425
<210> 6
<211> 1278
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgttccagc cgctcctgga cgcgttcatt gaatctgcgt ctattgaaaa aatggtatct 60
aaatccccac cgccaccgct gaaaatcgcc gtagccaact ggtggggcga cgaggaaatc 120
aaggagttca agaaatccgt actgtatttc atcctctctc agcgttatgc gatcacgctg 180
catcagaacc caaacgagtc tagcgacctg gttttttcca acccgctggg cgcggcccgt 240
aagatcctgt cctaccaaaa caccaaacgc gtgttttaca ccggtgaaaa cgaatctcca 300
aacttcaacc tgttcgacta tgcaatcggc tttgacgaac tggacttcaa tgatcgttac 360
ctccgcatgc cactgtatta cgcgcatctg cattacgagg ctgagctggt aaacgatacc 420
actgccccgt acaaactcaa ggacaacagc ctgtatgctc tgaagaaacc gtcccaccat 480
ttcaaggaaa accacccgaa cctgtgtgcc gttgtgaacg atgaatctga tctcctgaaa 540
cgtggttttg cgtccttcgt agcatctaac gcgaatgctc cgatgcgtaa tgctttctat 600
gacgcgctga acagcatcga gccggttacc ggtggcggta gcgttcgcaa taccctgggc 660
tacaaagttg gtaacaaaag cgaattcctg tctcagtata aattcaacct gtgcttcgaa 720
aattctcagg gctatggtta cgttactgaa aagatcctgg atgcctactt cagccacacc 780
atcccaattt actggggctc cccgagcgtt gccaaggact ttaacccgaa atccttcgtg 840
aacgttcacg atttcaataa ctttgatgaa gcgatcgatt atatcaagta cctgcacact 900
cacccgaacg cgtatctcga catgctgtac gaaaacccgc tgaacaccct ggacggtaaa 960
gcgtattttt accaggacct gtcttttaaa aagattctgg attttttcaa aaccatcctg 1020
gaaaacgata cgatctacca caataacccg tttatcttct accgtgatct gcatgaaccg 1080
ctgatctcta tcgatgacct gcgtgtaaac tatgacgatc tgcgtgtgaa ctatgacgat 1140
ctgcgtgtca actacgatga cctgcgcgtt aactacgatg acctgcgtgt caactatgat 1200
gacctccgcg tcaactacga cgatctgcgt gtaaactacg atgacctgcg tgttaactac 1260
catcaccatc accaccac 1278
<210> 7
<211> 419
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Glu
1 5 10 15
Lys Met Val Ser Lys Ser Pro Pro Pro Pro Leu Lys Ile Ala Val Ala
20 25 30
Asn Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Lys Ser Val Leu
35 40 45
Tyr Phe Ile Leu Ser Gln Arg Tyr Ala Ile Thr Leu His Gln Asn Pro
50 55 60
Asn Glu Ser Ser Asp Leu Val Phe Ser Asn Pro Leu Gly Ala Ala Arg
65 70 75 80
Lys Ile Leu Ser Tyr Gln Asn Thr Lys Arg Val Phe Tyr Thr Gly Glu
85 90 95
Asn Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp
100 105 110
Glu Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Ala
115 120 125
His Leu His Tyr Glu Ala Glu Leu Val Asn Asp Thr Thr Ala Pro Tyr
130 135 140
Lys Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His His
145 150 155 160
Phe Lys Glu Asn His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser
165 170 175
Asp Leu Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Ala Asn
180 185 190
Ala Pro Met Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro
195 200 205
Val Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Lys Val Gly
210 215 220
Asn Lys Ser Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu
225 230 235 240
Asn Ser Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Leu Asp Ala Tyr
245 250 255
Phe Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys
260 265 270
Asp Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Asn Asn Phe
275 280 285
Asp Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Pro Asn Ala
290 295 300
Tyr Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys
305 310 315 320
Ala Tyr Phe Tyr Gln Asp Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe
325 330 335
Lys Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asn Asn Pro Phe Ile
340 345 350
Phe Tyr Arg Asp Leu His Glu Pro Leu Ile Ser Ile Asp Asp Leu Arg
355 360 365
Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn
370 375 380
Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp
385 390 395 400
Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr His His His
405 410 415
His His His
<210> 8
<211> 1257
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgtttcagc cgctcctgga tgccttcatc gaatctgcaa gcatcgaaaa aatggttagc 60
aaaagcccgc caccgccact gaaaatcgct gttgccaatt ggtggggcga cgaggaaatc 120
aaggaattca agaaatctgt tctgtatttc atcctgtccc agcgctacgc aatcaccctg 180
catcaaaacc caaacgaatc ttccgacctg gtcttctcta acccgctggg tgcagcgcgt 240
aaaatcctgt cttatcagaa cactaaacgt gtcttctata ctggtgaaaa tgaatctccg 300
aacttcaatc tgttcgacta cgcgatcggt ttcgatgaac tggactttaa cgatcgctac 360
ctgcgcatgc cgctgtacta tgcacatctg cactacgaag ccgaactggt taatgacacg 420
accgcgccgt ataaactgaa agacaactct ctgtacgcgc tgaaaaagcc atcccatcac 480
ttcaaagaaa accacccaaa cctgtgtgcc gtggtaaacg atgaatctga tctcctgaag 540
cgcggtttcg cgtctttcgt agcgagcaat gctaacgcac caatgcgcaa tgccttctac 600
gacgctctga attccattga accggttacc ggtggcggtt ctgtgcgtaa cactctgggc 660
tacaaagtag gtaacaagtc tgaattcctg agccagtaca agtttaacct gtgtttcgaa 720
aactctcagg gttacggtta cgtgaccgaa aaaatcctgg acgcctactt ctcccatact 780
atcccaatct attggggcag cccgtctgtt gcaaaagact ttaacccgaa atctttcgtt 840
aatgtacatg acttcaataa cttcgatgag gctatcgatt acatcaagta tctgcacacc 900
catccgaacg cctatctgga catgctgtat gaaaatccgc tgaacaccct ggacggcaaa 960
gcgtacttct accaggatct gtctttcaag aaaattctgg attttttcaa gaccatcctg 1020
gagaatgaca ccatctacca caataaccca ttcatctttt accgcgatct ccatgaaccg 1080
ctgatttcca ttgatgacct gcgtgttaac tatgatgacc tgcgcgttaa ctatgacgat 1140
ctgcgcgtaa actacgacga tctgcgtgtt aattacgatg acctgcgtgt taattacgat 1200
gacctgcgtg taaactacga cgatctgcgt gtcaactatc atcaccatca ccaccac 1257
<210> 9
<211> 412
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Glu
1 5 10 15
Lys Met Val Ser Lys Ser Pro Pro Pro Pro Leu Lys Ile Ala Val Ala
20 25 30
Asn Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Lys Ser Val Leu
35 40 45
Tyr Phe Ile Leu Ser Gln Arg Tyr Ala Ile Thr Leu His Gln Asn Pro
50 55 60
Asn Glu Ser Ser Asp Leu Val Phe Ser Asn Pro Leu Gly Ala Ala Arg
65 70 75 80
Lys Ile Leu Ser Tyr Gln Asn Thr Lys Arg Val Phe Tyr Thr Gly Glu
85 90 95
Asn Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp
100 105 110
Glu Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Ala
115 120 125
His Leu His Tyr Glu Ala Glu Leu Val Asn Asp Thr Thr Ala Pro Tyr
130 135 140
Lys Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His His
145 150 155 160
Phe Lys Glu Asn His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser
165 170 175
Asp Leu Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Ala Asn
180 185 190
Ala Pro Met Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro
195 200 205
Val Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Lys Val Gly
210 215 220
Asn Lys Ser Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu
225 230 235 240
Asn Ser Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Leu Asp Ala Tyr
245 250 255
Phe Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys
260 265 270
Asp Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Asn Asn Phe
275 280 285
Asp Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Pro Asn Ala
290 295 300
Tyr Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys
305 310 315 320
Ala Tyr Phe Tyr Gln Asp Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe
325 330 335
Lys Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asn Asn Pro Phe Ile
340 345 350
Phe Tyr Arg Asp Leu His Glu Pro Leu Ile Ser Ile Asp Asp Leu Arg
355 360 365
Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn
370 375 380
Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp
385 390 395 400
Asp Leu Arg Val Asn Tyr His His His His His His
405 410
<210> 10
<211> 1236
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgtttcaac cgctcctgga cgctttcatc gagagcgcgt ctatcgaaaa aatggtctcc 60
aaatcccctc cgcctccgct caaaatcgca gttgcgaact ggtggggcga cgaagagatc 120
aaagaattca agaaatccgt gctctacttc atcctgtctc aacgttatgc aattaccctg 180
caccagaacc cgaacgaatc ttccgacctg gtgttctcca acccgctggg cgctgcccgc 240
aaaattctgt cttatcagaa cactaaacgt gtcttctaca ccggcgagaa cgaatctcca 300
aatttcaacc tgttcgacta cgcaattggt tttgacgaac tggacttcaa cgatcgttac 360
ctgcgtatgc cgctgtacta tgctcacctg cactacgaag cagaactggt gaacgacacg 420
actgctccgt acaaactgaa ggacaactcc ctgtacgcac tgaagaaacc gtcccaccat 480
ttcaaagaaa accatccgaa cctctgtgcg gtcgttaacg acgaaagcga tctcctgaaa 540
cgtggttttg catccttcgt tgcgagcaac gctaacgcgc cgatgcgtaa cgcgttctac 600
gatgcactga acagcatcga gccggttacc ggtggcggtt ctgtacgcaa cactctgggt 660
tacaaagttg gcaacaaatc tgagtttctg tcccagtaca aattcaacct ctgtttcgaa 720
aattctcaag gttacggtta cgtcaccgaa aaaattctgg atgcctattt ctctcacact 780
atcccgattt actggggtag cccgtctgtt gcgaaagatt ttaatccgaa atccttcgtg 840
aacgttcatg acttcaataa cttcgatgaa gctatcgact acatcaaata cctgcacacc 900
catccaaacg cttatctgga tatgctgtac gaaaaccctc tgaataccct ggatggcaag 960
gcatatttct accaggacct gtctttcaag aaaatcctgg atttctttaa aaccatcctg 1020
gagaacgaca ccatttacca caacaatcct tttatctttt atcgcgatct gcacgaaccg 1080
ctgatttcca ttgacgatct gcgtgtgaac tacgatgacc tgcgtgttaa ctacgatgac 1140
ctgcgcgtta actatgacga tctgcgtgtc aactacgacg atctgcgtgt caactacgat 1200
gacctgcgtg tgaactatca ccatcaccat caccac 1236
<210> 11
<211> 405
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Glu
1 5 10 15
Lys Met Val Ser Lys Ser Pro Pro Pro Pro Leu Lys Ile Ala Val Ala
20 25 30
Asn Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Lys Ser Val Leu
35 40 45
Tyr Phe Ile Leu Ser Gln Arg Tyr Ala Ile Thr Leu His Gln Asn Pro
50 55 60
Asn Glu Ser Ser Asp Leu Val Phe Ser Asn Pro Leu Gly Ala Ala Arg
65 70 75 80
Lys Ile Leu Ser Tyr Gln Asn Thr Lys Arg Val Phe Tyr Thr Gly Glu
85 90 95
Asn Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp
100 105 110
Glu Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Ala
115 120 125
His Leu His Tyr Glu Ala Glu Leu Val Asn Asp Thr Thr Ala Pro Tyr
130 135 140
Lys Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His His
145 150 155 160
Phe Lys Glu Asn His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser
165 170 175
Asp Leu Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Ala Asn
180 185 190
Ala Pro Met Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro
195 200 205
Val Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Lys Val Gly
210 215 220
Asn Lys Ser Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu
225 230 235 240
Asn Ser Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Leu Asp Ala Tyr
245 250 255
Phe Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys
260 265 270
Asp Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Asn Asn Phe
275 280 285
Asp Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Pro Asn Ala
290 295 300
Tyr Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys
305 310 315 320
Ala Tyr Phe Tyr Gln Asp Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe
325 330 335
Lys Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asn Asn Pro Phe Ile
340 345 350
Phe Tyr Arg Asp Leu His Glu Pro Leu Ile Ser Ile Asp Asp Leu Arg
355 360 365
Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn
370 375 380
Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr His
385 390 395 400
His His His His His
405
<210> 12
<211> 1215
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgtttcagc ctctcctgga cgcattcatc gaatccgcta gcatcgagaa aatggtttcc 60
aagtctccac cgccaccgct gaaaatcgcg gtggcaaact ggtggggtga cgaggaaatc 120
aaagaattca aaaagtctgt cctgtacttc atcctgtctc agcgttacgc catcaccctg 180
caccaaaacc ctaacgaatc ctctgacctg gttttcagca acccgctggg tgctgcccgt 240
aagatcctga gctaccagaa cacgaaacgc gtgttttata cgggcgaaaa cgaatccccg 300
aacttcaacc tgtttgatta tgctatcggc ttcgacgagc tggatttcaa cgaccgttac 360
ctgcgcatgc cgctgtatta cgctcacctg cactacgaag cggaactggt taacgatact 420
accgcgccgt acaaactgaa agataactcc ctgtatgccc tgaagaaacc gtctcatcac 480
ttcaaagaaa accatccgaa cctctgtgca gtcgtaaatg atgaaagcga tctcctgaag 540
cgtggtttcg cttccttcgt tgctagcaac gcgaacgcgc cgatgcgtaa cgccttctac 600
gatgcactga acagcatcga accggtaacc ggcggtggct ccgtgcgtaa taccctgggt 660
tataaagtcg gcaacaaatc cgagttcctg tcccagtaca aattcaacct gtgcttcgaa 720
aactctcagg gttatggtta tgtgaccgaa aagattctgg acgcctactt ctctcatact 780
atcccgatct attggggcag cccgtctgtg gctaaggatt tcaacccgaa atctttcgta 840
aatgtgcatg acttcaataa ctttgacgaa gcgatcgact acatcaaata cctgcacacc 900
catccgaacg cttacctgga tatgctctac gagaacccgc tgaataccct cgacggtaaa 960
gcttacttct atcaggatct gtcttttaag aaaatcctgg actttttcaa gactatcctg 1020
gaaaacgata ccatctacca caataacccg tttatcttct accgtgacct gcacgagcct 1080
ctgatcagca tcgacgatct gcgtgtcaac tacgacgatc tgcgcgtaaa ctacgatgac 1140
ctgcgtgtca actacgacga tctgcgtgta aactatgatg acctgcgtgt aaactaccac 1200
catcaccatc accac 1215
<210> 13
<211> 417
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Glu
1 5 10 15
Lys Met Val Ser Lys Ser Pro Pro Pro Pro Leu Lys Ile Ala Val Ala
20 25 30
Asn Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Lys Ser Val Leu
35 40 45
Tyr Phe Ile Leu Ser Gln Arg Tyr Ala Ile Thr Leu His Gln Asn Pro
50 55 60
Asn Glu Ser Ser Asp Leu Val Phe Ser Asn Pro Leu Gly Ala Ala Arg
65 70 75 80
Lys Ile Leu Ser Tyr Gln Asn Thr Lys Arg Val Phe Tyr Thr Gly Glu
85 90 95
Asn Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp
100 105 110
Glu Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Ala
115 120 125
His Leu His Tyr Glu Ala Glu Leu Val Asn Asp Thr Thr Ala Pro Tyr
130 135 140
Lys Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His His
145 150 155 160
Phe Lys Glu Asn His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser
165 170 175
Asp Leu Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Ala Asn
180 185 190
Ala Pro Met Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro
195 200 205
Val Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Lys Val Gly
210 215 220
Asn Lys Ser Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu
225 230 235 240
Asn Ser Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Leu Asp Ala Tyr
245 250 255
Phe Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys
260 265 270
Asp Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Asn Asn Phe
275 280 285
Asp Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Pro Asn Ala
290 295 300
Tyr Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys
305 310 315 320
Ala Tyr Phe Tyr Gln Asp Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe
325 330 335
Lys Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asn Asn Pro Phe Ile
340 345 350
Phe Tyr Arg Asp Leu His Glu Pro Leu Ile Ser Ile Asp Asp Leu Arg
355 360 365
Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn
370 375 380
Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp
385 390 395 400
Asp Leu Arg Val Asn Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly
405 410 415
Lys
<210> 14
<211> 1251
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgttccagc cgctcctgga cgctttcatc gaatccgctt ctatcgagaa aatggtgtct 60
aaaagcccgc ctccgcctct gaaaatcgcc gtagcgaact ggtggggtga cgaggaaatc 120
aaggaattca agaaatccgt tctgtacttc atcctgtccc agcgctacgc gatcactctg 180
catcagaacc ctaatgaaag ctctgatctg gttttcagca acccgctggg cgcggctcgt 240
aaaattctgt cctatcagaa cacgaaacgt gtgttttaca ccggcgaaaa cgagagcccg 300
aacttcaatc tgtttgacta cgcgatcggt ttcgacgaac tggacttcaa cgaccgttac 360
ctccgcatgc cgctgtacta tgcgcacctg cactatgagg ccgaactggt caatgatacc 420
actgctccgt acaaactgaa agacaacagc ctgtacgcgc tgaagaaacc gtctcaccat 480
ttcaaagaaa accatccgaa cctgtgcgcg gttgtcaatg acgaaagcga cctcctgaag 540
cgcggcttcg caagcttcgt agcctccaac gcaaacgcac caatgcgtaa cgctttctac 600
gacgcgctga actccatcga accggtcacc ggcggtggca gcgttcgcaa tactctgggt 660
tacaaagtgg gcaacaaatc tgagttcctc tcccaataca aattcaacct gtgcttcgaa 720
aacagccagg gttacggtta tgttaccgaa aaaatcctgg acgcatactt ctcccacacc 780
atcccgatct actggggtag cccgagcgtt gcaaaggact tcaacccaaa gtcttttgta 840
aacgttcacg actttaataa cttcgatgaa gcgatcgatt acatcaaata cctccatact 900
catccgaacg cgtacctgga tatgctgtac gaaaacccgc tgaacactct ggacggtaaa 960
gcatacttct accaggacct gtccttcaag aaaatcctgg attttttcaa aaccatcctg 1020
gaaaacgata ctatttacca caacaatccg ttcatcttct atcgcgatct ccatgaaccg 1080
ctgatctcta ttgatgacct gcgtgttaac tatgacgatc tgcgtgtcaa ctacgacgat 1140
ctgcgtgtaa actatgatga cctgcgtgta aactacgatg acctgcgtgt aaactatgat 1200
gacctccgtg ttaacgaaga tcaggtagat cctcgtctga tcgatggcaa a 1251
<210> 15
<211> 476
<212> PRT
<213> 幽门螺杆菌(Helicobacter pylori)
<400> 15
Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Glu
1 5 10 15
Lys Met Val Ser Lys Ser Pro Pro Pro Pro Leu Lys Ile Ala Val Ala
20 25 30
Asn Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Lys Ser Val Leu
35 40 45
Tyr Phe Ile Leu Ser Gln Arg Tyr Ala Ile Thr Leu His Gln Asn Pro
50 55 60
Asn Glu Ser Ser Asp Leu Val Phe Ser Asn Pro Leu Gly Ala Ala Arg
65 70 75 80
Lys Ile Leu Ser Tyr Gln Asn Thr Lys Arg Val Phe Tyr Thr Gly Glu
85 90 95
Asn Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp
100 105 110
Glu Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Ala
115 120 125
His Leu His Tyr Glu Ala Glu Leu Val Asn Asp Thr Thr Ala Pro Tyr
130 135 140
Lys Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His His
145 150 155 160
Phe Lys Glu Asn His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser
165 170 175
Asp Leu Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Ala Asn
180 185 190
Ala Pro Met Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro
195 200 205
Val Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Lys Val Gly
210 215 220
Asn Lys Ser Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu
225 230 235 240
Asn Ser Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Leu Asp Ala Tyr
245 250 255
Phe Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys
260 265 270
Asp Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Asn Asn Phe
275 280 285
Asp Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Pro Asn Ala
290 295 300
Tyr Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys
305 310 315 320
Ala Tyr Phe Tyr Gln Asp Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe
325 330 335
Lys Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asn Asn Pro Phe Ile
340 345 350
Phe Tyr Arg Asp Leu His Glu Pro Leu Ile Ser Ile Asp Asp Leu Arg
355 360 365
Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn
370 375 380
Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp
385 390 395 400
Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu
405 410 415
Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val
420 425 430
Asn Tyr Asp Arg Leu Leu Gln Asn Ala Ser Pro Leu Leu Glu Leu Ser
435 440 445
Gln Asn Thr Thr Phe Lys Ile Tyr Arg Lys Ala Tyr Gln Lys Ser Leu
450 455 460
Pro Leu Leu Arg Thr Ile Arg Arg Trp Val Lys Lys
465 470 475

Claims (8)

1. 一种幽门螺杆菌的岩藻糖基转移酶的突变体，其特征在于，所述突变体的氨基酸序列如SEQ ID NO. 13所示。
2. 一种编码权利要求1所述幽门螺杆菌的岩藻糖基转移酶的突变体的多核苷酸，其特征在于，所述多核苷酸的序列如SEQ ID NO. 14所示。
3.一种含有权利要求2所述多核苷酸的表达载体。
4.一种含有权利要求3所述表达载体的宿主细胞。
5.根据权利要求4所述的宿主细胞，其特征在于，所述宿主细胞为大肠杆菌。
6.一种应用权利要求1所述的重组岩藻糖基转移酶突变体将靶分子标记于目的细胞或者目的蛋白质的非诊断或治疗的方法，其特征在于，所述方法包括：
（1）将供体底物与靶分子相缀合获得连接复合物；
（2）在所述的重组岩藻糖基转移酶突变体存在的条件下，使步骤（1）获得的连接复合物与含有GlcNac受体分子的目的细胞或者目的蛋白质一起孵育，获得被标记了靶分子的目的细胞。
7. 根据权利要求6所述的方法，其特征在于，所述供体底物的分子量为500D 至150KD的GDP-Fucose-(PEG4)n，所述n为1-10的整数。
8.根据权利要求7所述的方法，其特征在于，所述靶分子为IgG，所述n=2。
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