CN113136378A - 一种鼠李糖苷酶TpeRha突变体及其制备方法和应用 - Google Patents

一种鼠李糖苷酶TpeRha突变体及其制备方法和应用 Download PDF

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CN113136378A
CN113136378A CN202110693022.0A CN202110693022A CN113136378A CN 113136378 A CN113136378 A CN 113136378A CN 202110693022 A CN202110693022 A CN 202110693022A CN 113136378 A CN113136378 A CN 113136378A
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黄佳俊
李慧灵
胡浩轩
林育成
卢宇靖
周金林
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Abstract

一种鼠李糖苷酶TpeRha突变体及其制备方法和应用，本发明涉及生物医药技术领域，为突变体TpeRha‑R369A、突变体TpeRha‑W512A和突变体TpeRha‑K579A中的任一种；所述突变体TpeRha‑R369A，为将氨基酸序列为如SEQ ID NO：1所示的TpeRha酶的第369位的精氨酸突变为丙氨酸；所述突变体TpeRha‑W512A，为将氨基酸序列为所述TpeRha酶的第512位的色氨酸突变为丙氨酸。本发明通过定点突变技术构建α‑L‑鼠李糖苷酶TpeRha的组合突变体，催化转化淫羊藿总黄酮中的多组分黄酮糖苷制备淫羊藿次苷Ⅰ组合物，酶解时间短，转化率高。

Description

一种鼠李糖苷酶TpeRha突变体及其制备方法和应用
技术领域
本发明涉及生物医药技术领域，尤其涉及一种鼠李糖苷酶TpeRha突变体及其制备方法和应用。
背景技术
淫羊藿是小檗科淫羊藿属植物，是我国药典收载的传统中药，具有益精气、坚筋骨、补腰膝、强心力等功效，其主要活性成分是含有2-3个糖基的淫羊藿黄酮苷，淫羊藿黄酮苷具有抗癌、抗骨质疏松、抗衰老、免疫调节功能和补肾阳作用等功效。大量研究显示，朝藿定C、淫羊藿苷等淫羊藿黄酮原生苷在小肠吸收较差，其主要是以肠道代谢产物（次级苷或苷元）的形式被吸收而发挥疗效。因此，对淫羊藿黄酮苷进行稳定可控的水解，实现淫羊藿黄酮苷的有效生物转化，对于加强药物合成、增加生物利用度以提高药效发挥上具有重要意义。淫羊藿次苷Ⅰ是淫羊藿黄酮次级苷中的高活性物质，其通过菌群调节展示了潜在的增强宿主免疫功能和抗癌活性，具备很好的抗癌药物开发前景。但是，淫羊藿次苷Ⅰ在淫羊藿中含量极低，随着其活性功能不断被挖掘，其市场需求将越来越大。传统植物提取方法因其含量极低，后期纯化难度高，而淫羊藿提取物中含量相对较高是原生苷，如朝藿定C（占淫羊藿总黄酮的20.8%）和淫羊藿苷（占淫羊藿总黄酮的21.9%），以原生苷作为前体物质，利用α-L-鼠李糖苷酶进行淫羊藿次苷Ⅰ的制备是目前最有效的制备方法。
α-L-鼠李糖苷酶（α-L-rhamnosidase，EC 3.2.1.40）是一类具有广阔应用前景的糖苷水解酶，在植物、动物和微生物中广泛存在，能够将以醇羟基和半缩醛反应形成的糖苷键以外切或内切的方式断裂，在生物体中糖缀合物以及糖的合成和水解的过程中起到很重要的作用。α-L-鼠李糖苷酶在食品、医药等行业都具有广泛的应用价值，已有大量报道其可以有效地定向水解含有鼠李糖苷的天然活性物质或天然药物，如人参皂苷、柚皮苷、橙皮苷和芦丁等，提高原有物质的生物活性和生物利用度，是工业生产中常用的水解酶类。
然而，α-L-鼠李糖苷酶在进行规模化应用时仍然存在较大的技术壁垒，天然的α-L-鼠李糖苷酶活力不足，且其反应环境往往难以满足天然酶的最优条件。随着分子生物学、基因工程等相关技术逐渐成熟，对蛋白序列进行编辑与修改，可以加强酶催化机理的解读以及指导酶的设计。蛋白质工程理性设计是以定点突变技术为主要手段，根据对蛋白质结构与功能关系地了解，有目的地设计改造蛋白质，使其性能达到预期效果。目前，已有大量研究成功利用理性设计技术对天然酶的催化活性、底物特异性、热稳定性以及酶的别构效应等进行改造。然而，理性设计方法需要对酶的三维结构以及结构与功能的相互关系有较深入的了解。随着计算生物学的发展，计算机辅助设计指导酶改造的方法日趋成熟。利用计算机模拟的方法，建立酶的三维结构模型，分析酶与底物的构象关系，快速定位与催化反应相关的区域，缩小突变库容量，高效获得相关靶点，为蛋白质工程改造提供指导。
专利号为CN201710448315.6，名称为一种糖苷酶组合物及酶法制备淫羊藿苷元的方法的中国专利，其通过α-L-鼠李糖苷酶和β-葡萄糖苷酶组成制备淫羊藿苷元，具有较高淫羊藿苷元转化率，但是其中的淫羊藿次苷Ⅰ含量未知。专利号为CN201710332977.7，名称为一种酶法转化淫羊藿总黄酮制备淫羊藿苷元的方法，由耐热α-L-鼠李糖苷酶和耐热β-葡萄糖苷酶组成的双酶体系协同转化淫羊藿总黄酮中的多组分黄酮糖苷生成淫羊藿苷元，也无法高效催化转化得到淫羊藿次苷Ⅰ。因此，采用定点突变技术对α-L-鼠李糖苷酶TpeRha进行理性设计获得具备水解淫羊藿苷生成淫羊藿次苷Ⅰ的高效的鼠李糖苷酶TpeRha突变体是一个具备意义的研究方向。
发明内容
为了克服现有技术的不足，本发明的目的之一在于提供一种鼠李糖苷酶TpeRha突变体，用于制备淫羊藿次苷Ⅰ组合物，转化率高。
本发明的目的之二在于提供一种鼠李糖苷酶TpeRha突变体的制备方法。
本发明的目的之三在于提供一种鼠李糖苷酶TpeRha突变体在制备淫羊藿次苷Ⅰ组合物中的应用。
本发明的目的之一采用如下技术方案实现：
一种鼠李糖苷酶TpeRha突变体，为突变体TpeRha-R369A、突变体TpeRha-W512A 和突变体TpeRha-K579A中的任一种；
所述突变体TpeRha-R369A，为将氨基酸序列为如SEQ ID NO：1所示的TpeRha酶的第369位的精氨酸突变为丙氨酸；
所述突变体TpeRha-W512A，为将氨基酸序列为如SEQ ID NO：1所示的TpeRha酶的第512位的色氨酸突变为丙氨酸；
所述突变体TpeRha-K579A，为将氨基酸序列为如SEQ ID NO：1所示的TpeRha酶的第579位的赖氨酸突变为丙氨酸。
进一步地，所述突变体TpeRha-R369A的氨基酸序列为如SEQ ID NO：3所示；
所述突变体TpeRha-W512A的氨基酸序列为如SEQ ID NO：5所示；
所述突变体TpeRha-K579A的氨基酸序列为如SEQ ID NO：7所示。
进一步地，编码所述突变体TpeRha-R369A基因的核苷酸序列为如SEQ ID NO：4所示；
编码所述突变体TpeRha-W512A基因的核苷酸序列为如SEQ ID NO：6所示；
编码所述突变体TpeRha-K579A基因的核苷酸序列为如SEQ ID NO：8所示。
进一步地，所述TpeRha酶来源于石油热袍菌DSM 13995。
本发明的目的之二采用如下技术方案实现：
一种鼠李糖苷酶TpeRha突变体的制备方法，包括以下步骤：
S1，将TpeRha酶基因连接到质粒中，得到重组质粒；
S2，设计突变引物，采用突变引物并以所述重组质粒为模板进行PCR扩增，酶切消化去除模板DNA，得到突变产物；
S3，将突变产物转化至宿主细胞中，筛选得到鼠李糖苷酶TpeRha突变体表达菌株，诱导表达，得到鼠李糖苷酶TpeRha突变体。
进一步地，所述TpeRha酶基因的核苷酸序列如SEQ ID NO：2所示。
进一步地，突变引物序列如SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ IDNO：17、SEQ ID NO：22和SEQ ID NO：23所示。
进一步地，所述宿主细胞为大肠杆菌。
本发明的目的之三采用如下技术方案实现：
一种鼠李糖苷酶TpeRha突变体在制备淫羊藿次苷Ⅰ组合物中的应用。
进一步地，利用鼠李糖苷酶TpeRha突变体催化转化淫羊藿总黄酮中的多组分黄酮糖苷制备淫羊藿次苷Ⅰ组合物。
相比现有技术，本发明的有益效果在于：
本发明的一种鼠李糖苷酶TpeRha突变体，通过定点突变技术构建α-L-鼠李糖苷酶TpeRha的组合突变体，催化转化淫羊藿总黄酮中的多组分黄酮糖苷制备淫羊藿次苷Ⅰ组合物，酶解时间短，转化率高。
其中，突变体TpeRha-R369A在55℃，pH 4.6的反应体系中水解淫羊藿苷制备淫羊藿次苷Ⅰ的效率比野生型TpeRha提高了8.41倍；突变体TpeRha-W512A在55℃，pH 4.6的反应体系中水解淫羊藿苷制备淫羊藿次苷Ⅰ的效率比野生型TpeRha提高了4.73倍；突变体TpeRha-K579A在55℃，pH 4.6的反应体系中水解淫羊藿苷制备淫羊藿次苷Ⅰ的效率比野生型TpeRha提高了9.83倍。
本发明的一种鼠李糖苷酶TpeRha突变体的制备方法，通过计算机辅助蛋白质工程酶分子设计手段，分析来源于石油热袍菌（Thermotogapetrophila DSM 13995）的α-L-鼠李糖苷酶TpeRha的活性口袋区域，对其进行半理性设计新酶，使其在水解淫羊藿苷（Icarrin）生成淫羊藿次苷Ⅰ（IcarisideⅠ）的效率大大提高。
本发明的一种鼠李糖苷酶TpeRha突变体在制备淫羊藿次苷Ⅰ组合物中的应用，利用鼠李糖苷酶TpeRha突变体催化转化淫羊藿总黄酮中的多组分黄酮糖苷制备含淫羊藿次苷Ⅰ组合物。
附图说明
图1为TpeRha酶的三维结构模型图；
图2为TpeRha酶和DtRha酶结构比对图；
图3为pET-28a-TpeRha质粒载体图；
图4为淫羊藿次苷Ⅰ含量标准曲线图；
图5为TpeRha酶和各突变体反应后的HPLC结果图；
图6为各突变体反应后的HPLC结果图；
图7为TpeRha酶和各突变体对淫羊藿次苷Ⅰ的转化率对比图；
图8为生物炮制淫羊藿提取物前后各组分含量HPLC结果图。
具体实施方式
下面，结合附图和具体实施方式，对本发明做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。以下是本发明具体的实施例，在下述实施例中所采用的原材料、设备等除特殊限定外均可以通过购买方式获得。
下列实施例中为注明具体条件的实验方法，通常按照常规实验条件或制造厂商所建议的实验条件，如无特殊说明，实施例中涉及的各种反应试剂均可以通过商业渠道购买得到。本实施例中未作具体说明的分子生物学实验方法，可参照《分子克隆实验指南》。
实施例1
TpeRha酶三级结构模型的建立与突变位点的确定：
（一）建立TpeRha的三维结构模型
利用同源建模工具MODELLER对TpeRha进行同源建模，模型经评估后，根据评分标准评价获得可靠的三维结构模型，三维结构模型如图1所示。
（二）突变位点的确定
如图2所示，经同源建模获得的TpeRha三维结构模型是根据嗜热链球菌（Dictyoglomusthermophilum）α-L-鼠李糖苷酶DtRha（PDB ID：6i60）的三维结构模型建立的。研究显示，在DtRha酶与糖苷配体的晶体结构模型中，与鼠李糖基有相互作用的氨基酸残基涉及11个。将TpeRha酶与DtRha酶的三维结构模型进行构象比对，确定TpeRha酶的活性口袋，如图2a所示，定位对应的其中10个直接相关氨基酸。如图2b所示，优选地将位于活性口袋表面的6个非直接相关氨基酸H130、R369、W512、R514、Q569和K579作为候选突变位点，以丙氨酸扫描作为突变依据，这6个突变体组成一个小型突变文库。
实施例2
重组野生酶菌株BL21（DE3）/pET-28a-TpeRha的获得：
优化来自石油热袍菌DSM 13995的α-L-鼠李糖苷酶基因，其核苷酸序列如SEQ IDNO：2（GenBank：ABQ47687.1），将其连接到pET-28a质粒上，获得的重组质粒命名为pET-28a-TpeRha，如图3所示，将该质粒转化至大肠杆菌BL21（DE3）中，重组菌株命名为BL21（DE3）/pET-28a-TpeRha。该重组野生酶菌株表达的野生型TpeRha酶的氨基酸序列为如SEQ ID NO：1所示。
实施例3
TpeRha突变菌株的获得：
（一）全质粒PCR构建突变体载体
1、重组质粒pET-28a-TpeRha的质粒小量提取；
2、设计突变引物，引物有15 bp重叠区，15 bp延伸区，将突变位点设计于重叠区。以质粒pET-28a-TpeRha为模板进行PCR全质粒扩增，PCR体系如表1所示：
Figure 638867DEST_PATH_IMAGE001
所述引物Primer-F和Primer-R是根据不同突变位点进行相应设计的PCR上游引物和下游引物，具体引物信息如表2所示。
Figure 883903DEST_PATH_IMAGE002
引物对H130A-F和H130A-R用于获得突变体TpeRha-H130A，突变体TpeRha-H130A的氨基酸序列为SEQ ID NO：9。
引物对R369A-F和R369A-R用于获得突变体TpeRha-R369A，突变体TpeRha-R369A的氨基酸序列为SEQ ID NO：3；其编码基因的核苷酸序列为如SEQ ID NO：4所示。
引物对W512A-F和W512A-R用于获得突变体TpeRha-W512A，突变体TpeRha-W512A的氨基酸序列为SEQ ID NO：5；其编码基因的核苷酸序列为如SEQ ID NO：6所示。
引物对R514A-F和R514A-R用于获得突变体TpeRha-R514A，突变体TpeRha-R514A的氨基酸序列为SEQ ID NO：10。
引物对Q569A-F和Q569A-R用于获得突变体TpeRha-Q569A，突变体TpeRha-Q569A的氨基酸序列为SEQ ID NO：11。
引物对K579A-F和K579A-R用于获得突变体TpeRha-K579A，突变体TpeRha-K579A的氨基酸序列为SEQ ID NO：7，其编码基因的核苷酸序列为如SEQ ID NO：8所示。
PCR扩增程序：预变性98℃ 3 min；循环设定：变性98℃ 10 s，退火62℃ 15 s，延伸72℃ 3 min，30个循环；最后延伸72℃ 10 min；反应结束后，利用试剂盒进行PCR产物回收。
3、酶切消化去除模板DNA，将PCR回收产物进行酶切，酶切体系如表3所示：
Figure 929220DEST_PATH_IMAGE003
上述酶切体系置于37℃金属浴中消化1 h，反应结束后利用试剂盒进行酶切产物回收。
（二）测序验证突变体菌株构建成功
将上述酶切回收产物转化至大肠杆菌BL21（DE3）感受态细胞中，37℃倒置过夜培养，挑取拟阳性转化子进行测序验证，成功获得突变体表达菌株，命名规则同野生型重组菌株。
实施例4
TpeRha酶的诱导与全细胞催化反应：
（一）TpeRha野生酶重组菌株的诱导和全细胞酶液的准备
1、取BL21（DE3）/pET-28a-TpeRha野生酶重组菌株，在含Kan（100 μg/mL）的LB平板上划线活化。经37℃倒置培养过夜后，挑取单菌落接种至5 mL 含Kan的LB液体培养基中，37℃，200 r/min振荡培养12-16 h。按照1%接种量将过夜培养的种子液接种于20 mL新鲜的含Kan的TB液体培养基中，37℃，200 r/min振荡培养2-3 h至OD600为0.6-0.8时，加入终浓度为0.5 mM的IPTG，将培养液放至37℃，200 r/min振荡培养16 h进行蛋白的诱导表达。
2、诱导结束后，4℃，8000 rpm离心8 min收集菌体，菌体用柠檬酸-磷酸缓冲液（pH4.6）洗涤一次后，根据菌体湿重加入适量的柠檬酸-磷酸缓冲液（pH 4.6）重悬菌体，获得全细胞浓度为200 g/mL的细胞悬浊液，所有操作均在冰上或4℃进行。
（二）全细胞反应
1、以上述实验获得的野生型TpeRha酶的全细胞悬浊液作为酶液，配置如下反应体系如表4所示：
Figure 855587DEST_PATH_IMAGE004
2、反应体系在55℃下反应1 d。反应结束后，加入2倍体积的DMSO终止反应，充分涡旋后经12000 rpm，离心1 min去除细胞菌体，反应产物经0.45 μm有机滤头过滤后进行HPLC检测。
（三）淫羊藿次苷Ⅰ的检测方法
采用HPLC进行淫羊藿次苷Ⅰ的定量分析，色谱条件如下所述：
高效液相色谱仪：Agilent 1100 Series
色谱柱：Diamonsil® C18（250 mm×4.6 mm×5 μm）
检测器：VWD检测器，检测波长270 nm
流动相比例与洗脱条件：流速1 mL/min；柱温30℃；进样量10μL；梯度洗脱体系如表5所示：
Figure 222022DEST_PATH_IMAGE005
（四）淫羊藿次苷Ⅰ标准曲线的制备
以DMSO作为溶剂，称量淫羊藿次苷Ⅰ对照品（98.6% HPLC）分别配置0、0.1 mg/mL、0.2 mg/mL、0.3 mg/mL、0.4 mg/mL的标准溶液，利用上述HPLC检测条件进行检测，标准曲线如图4所示，HPLC结果如图5所示。
（五）结果分析
参照图5和图7，全细胞催化反应结果显示，55℃，pH 4.6的反应条件下，反应1 d，利用野生型TpeRha酶反应能水解转化淫羊藿苷生成淫羊藿次苷Ⅰ，但是产物转化率很低，只有3.73±0.09%。
实施例5
突变文库的全细胞催化反应筛选：
以实施例4的全细胞催化反应方法进行突变体的检验，所加入全细胞酶液浓度均为40 mg/mL，55℃，pH 4.6条件下反应1 d后，将各突变体水解淫羊藿苷生成淫羊藿次苷Ⅰ的HPLC结果如图5、图6所示，转化率比较结果如图7所示。
参照图5至图7，野生型TpeRha酶转化率3.73±0.09%，淫羊藿次苷Ⅰ转化率提高的突变体一共四个，分别是TpeRha-R369A（转化率31.34±0.03%），TpeRha-W512A（转化率17.64±0.36%），TpeRha-Q569A（转化率5.24±0.05%）和TpeRha-K579A（转化率36.64±0.37%）；转化率降低的突变体一共两个，分别是TpeRha-H130A（转化率1.09±0.14%）和TpeRha-R514A（转化率3.65±0.24%）。其中，TpeRha-R369A、TpeRha-W512A和TpeRha-K579A对淫羊藿次苷Ⅰ的转化率都得到了大幅提升（分别提高8.41倍、4.73倍和9.83倍）。
实施例6
突变菌BL21（DE3）/pET-28a-TpeRha-K579A在淫羊藿提取物中的生物炮制应用：
以100 mL pH为5的TB培养基作为发酵培养基，培养基配方为1.2%（w/v）胰蛋白胨，2.4%（w/v）酵母提取粉，0.4%（v/v）甘油，17 mM KH2PO4，72 mM KH2PO4，加入淫羊藿提取物（含20%朝藿定C和淫羊藿苷）1g。接种2%突变菌BL21（DE3）/pET-28a-TpeRha-K579A，并在37℃，200rpm条件下发酵1d，随后调整发酵温度为55℃，继续发酵1 d，取发酵液加入5倍体积的DMSO充分混合并离心取上清进行HPLC检测，结果如图8所示。
参照图8，发酵前后淫羊藿提取物组分发生明显变化，证明突变体TpeRha-K579A具有优异的催化效果，发酵后朝藿定C（保留时间在6.382 min处）含量明显降低，淫羊藿苷（保留时间在7.209min处）含量和高生物活性的淫羊藿次苷Ⅰ（保留时间在14.145 min处）含量明显提高，淫羊藿次苷Ⅱ（保留时间在15.683 min处）也有所生成，抗肿瘤新药主要成分淫羊藿素（保留时间在23.128 min处）含量也有所提高。
以上所述仅是本发明的优选实施方式，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的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 广东金骏康生物技术有限公司
<120> 一种鼠李糖苷酶TpeRha突变体及其制备方法和应用
<130> SEQ ID No.1-23
<160> 23
<170> PatentIn version 3.3
<210> 1
<211> 876
<212> PRT
<213> Thermotoga petrophila DSM 13995
<400> 1
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<210> 2
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gatgaacgta tgggctggct gggtgacgcc cagctgaccg tggaagaagc catgtataat 1440
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755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 4
<211> 2628
<212> DNA
<213> 人工合成
<400> 4
atgatccagg catgtgatct gcgttgtgaa tatctgacca gcccggttct gggtctggat 60
gttattccgc gttttagctg gcgtctgaaa ggtaatggca aaaaacagac ccgctataaa 120
attattgtga gcgataattt cgacgatatt gaacgcggca ttggtaatgt gtgggaaagc 180
gaaaaagata gcagtaaaaa tctgaatatc gagtatgaag gcccgaaact gaaagcatat 240
aaaggctatt attggcgtgt gaaactgtgg gatgaaaaag aaaatggtcc gtggagcgaa 300
accgcatatt ttgaaatggg cccgctggaa gattggcgtg gtaaatggat taccatgccg 360
agtccgctga gctttaaaga tccggcccat cgtcatgaac tgttttatgc catgtatttt 420
cgtaaagaat ttctgctgaa caaggaagtg gaaaaagccc gtgtttatgt tagtggtctg 480
ggtgtttatg aactgcatct gaatggtaaa cgcgtgggca ataatgtgct ggaccctgca 540
ccgaccgatt ataataaggt tgccctgtat agcacctatg atgtgaccca gtatctgacc 600
accggtaaaa ataccattgg cgttattctg ggcaatggtc gtcatattcg tgattatggt 660
tatagtaaac cgaaactgta tctgcagctg ctggttttct ataaagatgg tagccgcgag 720
tttatttgta gcgatgaaac ctggaaagtg agtcatggtc cgctgaaaga aaatggcatc 780
tattttggtg aagtttatga tgcccgtgat gaaattagcg gttgggatag cccgggtttt 840
gatgatcgca attggagtga agtggaaatt gttgaaggtc cgagcctgaa agcccagctg 900
attccggtta ttcgtgtgtg tgaagttatt aagccgaaac gtctgtggct gagcagccgc 960
ggcaccttta ttgttgattt tggcaaaaat atcagcggtt gggttaaact gcgcgtgaat 1020
aatggtaaac gtggtgaaaa aattatcatc cgttatgcag aagtgctgga ccctagtatg 1080
gatcgtctgg atacccgtaa tctggcactg gcccgcgcaa ccgatgaata tattctgaaa 1140
ggtcagggtg tggaaatcta tgaaccgcgt tttacctatc atggttttcg ctatgttgaa 1200
gttgaagatt atccgggtac cctgaccagc gataatattg aagcaatgtt tgttcatacc 1260
gatgttgaaa aagtgggtga ctttgcatgc agcagcgaac tgctgaataa gattcatagt 1320
tgcgtggtga atagccagct ggcaaatctg atgggtattc cgaccgattg cccgcagcgt 1380
gatgaacgta tgggctggct gggtgacgcc cagctgaccg tggaagaagc catgtataat 1440
tttgatatgg ccgcctttta taccaaatat ctgatggata ttaagctgag tcagaaagaa 1500
gatggtagta ttagtgatgt ggcaccgccg tattggaaac gctatccgag cgatccggcc 1560
tggggtaccg cctatgcaac cattctgtgg tatctgtatt tcttttatga ggatcgccgc 1620
gttctggaag aacattatga tagtctgaaa cgctatgtgg aatttctgcg taaaaatagc 1680
ccgaatcatc tgaccaaact gggtcagcat ggtgactggt gtccgccggg tgacaaattt 1740
ccgaaacgta ccccgctgat tctgaccagt acctggtatt attatcatga taccctgatt 1800
ctgagtgaaa ttgccaaaat tctgggtaaa aaagaagatg aacacgaata tcgtaagctg 1860
gccggtgaaa ttaaggaagc ctttaatcgt cattttctgc gcaaagtgga agatcatacc 1920
ggtcgtattg tttgttttta tcgcggcatt aagctgagcc cgaaagatcg cattccgacc 1980
acccagacct gtaatgtgct gccgctgtgg aataagatgg ttccggaaga atgtcgcgaa 2040
gatgttttta aagttctgga acgcctgatt gaagttgata atgataccca ttttgacacc 2100
ggcattgtgg gcacccgcta tattctggaa gttctgagcg aaaatggtcg caaagatctg 2160
gccctgaaac tgctgctgaa agaagattat cctagctttg gttatatgat taagaacggt 2220
gccaccaccc tgtgggaacg ctgggaaaaa ctggaaggca ccggtatgaa tagccataat 2280
catgttatgc tgggtagcgt tgatacctgg ttttataaat atctgagcgg cattaagccg 2340
gttgcaccgg gctggaaaaa gattcgtatt gaaccgtatt ttgcagatca gattgatttt 2400
gtgagtgcaa aaattaagac cccgaatggc agcctggaag tgagttggaa aaaacagaat 2460
aaggaatatg agatccagat tattatcccg gtgaataccg ttggtatttt tgcagtgccg 2520
gaaagtttta aagttagtgc aattaatagc aagcaggtta gttatccgag tgaatttgaa 2580
ctggaaccgg gtgcctataa tattgtgctg gaacgcgttc gcgaatgt 2628
<210> 5
<211> 876
<212> PRT
<213> 人工合成
<400> 5
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Ala
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln His Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Lys Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 6
<211> 2628
<212> DNA
<213> 人工合成
<400> 6
atgatccagg catgtgatct gcgttgtgaa tatctgacca gcccggttct gggtctggat 60
gttattccgc gttttagctg gcgtctgaaa ggtaatggca aaaaacagac ccgctataaa 120
attattgtga gcgataattt cgacgatatt gaacgcggca ttggtaatgt gtgggaaagc 180
gaaaaagata gcagtaaaaa tctgaatatc gagtatgaag gcccgaaact gaaagcatat 240
aaaggctatt attggcgtgt gaaactgtgg gatgaaaaag aaaatggtcc gtggagcgaa 300
accgcatatt ttgaaatggg cccgctggaa gattggcgtg gtaaatggat taccatgccg 360
agtccgctga gctttaaaga tccggcccat cgtcatgaac tgttttatgc catgtatttt 420
cgtaaagaat ttctgctgaa caaggaagtg gaaaaagccc gtgtttatgt tagtggtctg 480
ggtgtttatg aactgcatct gaatggtaaa cgcgtgggca ataatgtgct ggaccctgca 540
ccgaccgatt ataataaggt tgccctgtat agcacctatg atgtgaccca gtatctgacc 600
accggtaaaa ataccattgg cgttattctg ggcaatggtc gtcatattcg tgattatggt 660
tatagtaaac cgaaactgta tctgcagctg ctggttttct ataaagatgg tagccgcgag 720
tttatttgta gcgatgaaac ctggaaagtg agtcatggtc cgctgaaaga aaatggcatc 780
tattttggtg aagtttatga tgcccgtgat gaaattagcg gttgggatag cccgggtttt 840
gatgatcgca attggagtga agtggaaatt gttgaaggtc cgagcctgaa agcccagctg 900
attccggtta ttcgtgtgtg tgaagttatt aagccgaaac gtctgtggct gagcagccgc 960
ggcaccttta ttgttgattt tggcaaaaat atcagcggtt gggttaaact gcgcgtgaat 1020
aatggtaaac gtggtgaaaa aattatcatc cgttatgcag aagtgctgga ccctagtatg 1080
gatcgtctgg atacccgtaa tctgcgcctg gcccgcgcaa ccgatgaata tattctgaaa 1140
ggtcagggtg tggaaatcta tgaaccgcgt tttacctatc atggttttcg ctatgttgaa 1200
gttgaagatt atccgggtac cctgaccagc gataatattg aagcaatgtt tgttcatacc 1260
gatgttgaaa aagtgggtga ctttgcatgc agcagcgaac tgctgaataa gattcatagt 1320
tgcgtggtga atagccagct ggcaaatctg atgggtattc cgaccgattg cccgcagcgt 1380
gatgaacgta tgggctggct gggtgacgcc cagctgaccg tggaagaagc catgtataat 1440
tttgatatgg ccgcctttta taccaaatat ctgatggata ttaagctgag tcagaaagaa 1500
gatggtagta ttagtgatgt ggcaccgccg tatgcaaaac gctatccgag cgatccggcc 1560
tggggtaccg cctatgcaac cattctgtgg tatctgtatt tcttttatga ggatcgccgc 1620
gttctggaag aacattatga tagtctgaaa cgctatgtgg aatttctgcg taaaaatagc 1680
ccgaatcatc tgaccaaact gggtcagcat ggtgactggt gtccgccggg tgacaaattt 1740
ccgaaacgta ccccgctgat tctgaccagt acctggtatt attatcatga taccctgatt 1800
ctgagtgaaa ttgccaaaat tctgggtaaa aaagaagatg aacacgaata tcgtaagctg 1860
gccggtgaaa ttaaggaagc ctttaatcgt cattttctgc gcaaagtgga agatcatacc 1920
ggtcgtattg tttgttttta tcgcggcatt aagctgagcc cgaaagatcg cattccgacc 1980
acccagacct gtaatgtgct gccgctgtgg aataagatgg ttccggaaga atgtcgcgaa 2040
gatgttttta aagttctgga acgcctgatt gaagttgata atgataccca ttttgacacc 2100
ggcattgtgg gcacccgcta tattctggaa gttctgagcg aaaatggtcg caaagatctg 2160
gccctgaaac tgctgctgaa agaagattat cctagctttg gttatatgat taagaacggt 2220
gccaccaccc tgtgggaacg ctgggaaaaa ctggaaggca ccggtatgaa tagccataat 2280
catgttatgc tgggtagcgt tgatacctgg ttttataaat atctgagcgg cattaagccg 2340
gttgcaccgg gctggaaaaa gattcgtatt gaaccgtatt ttgcagatca gattgatttt 2400
gtgagtgcaa aaattaagac cccgaatggc agcctggaag tgagttggaa aaaacagaat 2460
aaggaatatg agatccagat tattatcccg gtgaataccg ttggtatttt tgcagtgccg 2520
gaaagtttta aagttagtgc aattaatagc aagcaggtta gttatccgag tgaatttgaa 2580
ctggaaccgg gtgcctataa tattgtgctg gaacgcgttc gcgaatgt 2628
<210> 7
<211> 876
<212> PRT
<213> 人工合成
<400> 7
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Trp
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln His Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Ala Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 8
<211> 2628
<212> DNA
<213> 人工合成
<400> 8
atgatccagg catgtgatct gcgttgtgaa tatctgacca gcccggttct gggtctggat 60
gttattccgc gttttagctg gcgtctgaaa ggtaatggca aaaaacagac ccgctataaa 120
attattgtga gcgataattt cgacgatatt gaacgcggca ttggtaatgt gtgggaaagc 180
gaaaaagata gcagtaaaaa tctgaatatc gagtatgaag gcccgaaact gaaagcatat 240
aaaggctatt attggcgtgt gaaactgtgg gatgaaaaag aaaatggtcc gtggagcgaa 300
accgcatatt ttgaaatggg cccgctggaa gattggcgtg gtaaatggat taccatgccg 360
agtccgctga gctttaaaga tccggcccat cgtcatgaac tgttttatgc catgtatttt 420
cgtaaagaat ttctgctgaa caaggaagtg gaaaaagccc gtgtttatgt tagtggtctg 480
ggtgtttatg aactgcatct gaatggtaaa cgcgtgggca ataatgtgct ggaccctgca 540
ccgaccgatt ataataaggt tgccctgtat agcacctatg atgtgaccca gtatctgacc 600
accggtaaaa ataccattgg cgttattctg ggcaatggtc gtcatattcg tgattatggt 660
tatagtaaac cgaaactgta tctgcagctg ctggttttct ataaagatgg tagccgcgag 720
tttatttgta gcgatgaaac ctggaaagtg agtcatggtc cgctgaaaga aaatggcatc 780
tattttggtg aagtttatga tgcccgtgat gaaattagcg gttgggatag cccgggtttt 840
gatgatcgca attggagtga agtggaaatt gttgaaggtc cgagcctgaa agcccagctg 900
attccggtta ttcgtgtgtg tgaagttatt aagccgaaac gtctgtggct gagcagccgc 960
ggcaccttta ttgttgattt tggcaaaaat atcagcggtt gggttaaact gcgcgtgaat 1020
aatggtaaac gtggtgaaaa aattatcatc cgttatgcag aagtgctgga ccctagtatg 1080
gatcgtctgg atacccgtaa tctgcgcctg gcccgcgcaa ccgatgaata tattctgaaa 1140
ggtcagggtg tggaaatcta tgaaccgcgt tttacctatc atggttttcg ctatgttgaa 1200
gttgaagatt atccgggtac cctgaccagc gataatattg aagcaatgtt tgttcatacc 1260
gatgttgaaa aagtgggtga ctttgcatgc agcagcgaac tgctgaataa gattcatagt 1320
tgcgtggtga atagccagct ggcaaatctg atgggtattc cgaccgattg cccgcagcgt 1380
gatgaacgta tgggctggct gggtgacgcc cagctgaccg tggaagaagc catgtataat 1440
tttgatatgg ccgcctttta taccaaatat ctgatggata ttaagctgag tcagaaagaa 1500
gatggtagta ttagtgatgt ggcaccgccg tattggaaac gctatccgag cgatccggcc 1560
tggggtaccg cctatgcaac cattctgtgg tatctgtatt tcttttatga ggatcgccgc 1620
gttctggaag aacattatga tagtctgaaa cgctatgtgg aatttctgcg taaaaatagc 1680
ccgaatcatc tgaccaaact gggtcagcat ggtgactggt gtccgccggg tgacgcattt 1740
ccgaaacgta ccccgctgat tctgaccagt acctggtatt attatcatga taccctgatt 1800
ctgagtgaaa ttgccaaaat tctgggtaaa aaagaagatg aacacgaata tcgtaagctg 1860
gccggtgaaa ttaaggaagc ctttaatcgt cattttctgc gcaaagtgga agatcatacc 1920
ggtcgtattg tttgttttta tcgcggcatt aagctgagcc cgaaagatcg cattccgacc 1980
acccagacct gtaatgtgct gccgctgtgg aataagatgg ttccggaaga atgtcgcgaa 2040
gatgttttta aagttctgga acgcctgatt gaagttgata atgataccca ttttgacacc 2100
ggcattgtgg gcacccgcta tattctggaa gttctgagcg aaaatggtcg caaagatctg 2160
gccctgaaac tgctgctgaa agaagattat cctagctttg gttatatgat taagaacggt 2220
gccaccaccc tgtgggaacg ctgggaaaaa ctggaaggca ccggtatgaa tagccataat 2280
catgttatgc tgggtagcgt tgatacctgg ttttataaat atctgagcgg cattaagccg 2340
gttgcaccgg gctggaaaaa gattcgtatt gaaccgtatt ttgcagatca gattgatttt 2400
gtgagtgcaa aaattaagac cccgaatggc agcctggaag tgagttggaa aaaacagaat 2460
aaggaatatg agatccagat tattatcccg gtgaataccg ttggtatttt tgcagtgccg 2520
gaaagtttta aagttagtgc aattaatagc aagcaggtta gttatccgag tgaatttgaa 2580
ctggaaccgg gtgcctataa tattgtgctg gaacgcgttc gcgaatgt 2628
<210> 9
<211> 876
<212> PRT
<213> 人工合成
<400> 9
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
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Ala Ala Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
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Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
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Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
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Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
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Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
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Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
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Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
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Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
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Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
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Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
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Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Trp
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
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Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln His Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Lys Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 10
<211> 876
<212> PRT
<213> 人工合成
<400> 10
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
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Lys Gly Tyr Tyr Trp Arg Val Lys Leu Trp Asp Glu Lys Glu Asn Gly
85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Trp
500 505 510
Lys Ala Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln His Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Lys Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
610 615 620
Lys Glu Ala Phe Asn Arg His Phe Leu Arg Lys Val Glu Asp His Thr
625 630 635 640
Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
660 665 670
Met Val Pro Glu Glu Cys Arg Glu Asp Val Phe Lys Val Leu Glu Arg
675 680 685
Leu Ile Glu Val Asp Asn Asp Thr His Phe Asp Thr Gly Ile Val Gly
690 695 700
Thr Arg Tyr Ile Leu Glu Val Leu Ser Glu Asn Gly Arg Lys Asp Leu
705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
Val Ser Ala Lys Ile Lys Thr Pro Asn Gly Ser Leu Glu Val Ser Trp
805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
850 855 860
Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 11
<211> 876
<212> PRT
<213> 人工合成
<400> 11
Met Ile Gln Ala Cys Asp Leu Arg Cys Glu Tyr Leu Thr Ser Pro Val
1 5 10 15
Leu Gly Leu Asp Val Ile Pro Arg Phe Ser Trp Arg Leu Lys Gly Asn
20 25 30
Gly Lys Lys Gln Thr Arg Tyr Lys Ile Ile Val Ser Asp Asn Phe Asp
35 40 45
Asp Ile Glu Arg Gly Ile Gly Asn Val Trp Glu Ser Glu Lys Asp Ser
50 55 60
Ser Lys Asn Leu Asn Ile Glu Tyr Glu Gly Pro Lys Leu Lys Ala Tyr
65 70 75 80
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85 90 95
Pro Trp Ser Glu Thr Ala Tyr Phe Glu Met Gly Pro Leu Glu Asp Trp
100 105 110
Arg Gly Lys Trp Ile Thr Met Pro Ser Pro Leu Ser Phe Lys Asp Pro
115 120 125
Ala His Arg His Glu Leu Phe Tyr Ala Met Tyr Phe Arg Lys Glu Phe
130 135 140
Leu Leu Asn Lys Glu Val Glu Lys Ala Arg Val Tyr Val Ser Gly Leu
145 150 155 160
Gly Val Tyr Glu Leu His Leu Asn Gly Lys Arg Val Gly Asn Asn Val
165 170 175
Leu Asp Pro Ala Pro Thr Asp Tyr Asn Lys Val Ala Leu Tyr Ser Thr
180 185 190
Tyr Asp Val Thr Gln Tyr Leu Thr Thr Gly Lys Asn Thr Ile Gly Val
195 200 205
Ile Leu Gly Asn Gly Arg His Ile Arg Asp Tyr Gly Tyr Ser Lys Pro
210 215 220
Lys Leu Tyr Leu Gln Leu Leu Val Phe Tyr Lys Asp Gly Ser Arg Glu
225 230 235 240
Phe Ile Cys Ser Asp Glu Thr Trp Lys Val Ser His Gly Pro Leu Lys
245 250 255
Glu Asn Gly Ile Tyr Phe Gly Glu Val Tyr Asp Ala Arg Asp Glu Ile
260 265 270
Ser Gly Trp Asp Ser Pro Gly Phe Asp Asp Arg Asn Trp Ser Glu Val
275 280 285
Glu Ile Val Glu Gly Pro Ser Leu Lys Ala Gln Leu Ile Pro Val Ile
290 295 300
Arg Val Cys Glu Val Ile Lys Pro Lys Arg Leu Trp Leu Ser Ser Arg
305 310 315 320
Gly Thr Phe Ile Val Asp Phe Gly Lys Asn Ile Ser Gly Trp Val Lys
325 330 335
Leu Arg Val Asn Asn Gly Lys Arg Gly Glu Lys Ile Ile Ile Arg Tyr
340 345 350
Ala Glu Val Leu Asp Pro Ser Met Asp Arg Leu Asp Thr Arg Asn Leu
355 360 365
Arg Leu Ala Arg Ala Thr Asp Glu Tyr Ile Leu Lys Gly Gln Gly Val
370 375 380
Glu Ile Tyr Glu Pro Arg Phe Thr Tyr His Gly Phe Arg Tyr Val Glu
385 390 395 400
Val Glu Asp Tyr Pro Gly Thr Leu Thr Ser Asp Asn Ile Glu Ala Met
405 410 415
Phe Val His Thr Asp Val Glu Lys Val Gly Asp Phe Ala Cys Ser Ser
420 425 430
Glu Leu Leu Asn Lys Ile His Ser Cys Val Val Asn Ser Gln Leu Ala
435 440 445
Asn Leu Met Gly Ile Pro Thr Asp Cys Pro Gln Arg Asp Glu Arg Met
450 455 460
Gly Trp Leu Gly Asp Ala Gln Leu Thr Val Glu Glu Ala Met Tyr Asn
465 470 475 480
Phe Asp Met Ala Ala Phe Tyr Thr Lys Tyr Leu Met Asp Ile Lys Leu
485 490 495
Ser Gln Lys Glu Asp Gly Ser Ile Ser Asp Val Ala Pro Pro Tyr Trp
500 505 510
Lys Arg Tyr Pro Ser Asp Pro Ala Trp Gly Thr Ala Tyr Ala Thr Ile
515 520 525
Leu Trp Tyr Leu Tyr Phe Phe Tyr Glu Asp Arg Arg Val Leu Glu Glu
530 535 540
His Tyr Asp Ser Leu Lys Arg Tyr Val Glu Phe Leu Arg Lys Asn Ser
545 550 555 560
Pro Asn His Leu Thr Lys Leu Gly Gln His Gly Asp Trp Cys Pro Pro
565 570 575
Gly Asp Ala Phe Pro Lys Arg Thr Pro Leu Ile Leu Thr Ser Thr Trp
580 585 590
Tyr Tyr Tyr His Asp Thr Leu Ile Leu Ser Glu Ile Ala Lys Ile Leu
595 600 605
Gly Lys Lys Glu Asp Glu His Glu Tyr Arg Lys Leu Ala Gly Glu Ile
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Gly Arg Ile Val Cys Phe Tyr Arg Gly Ile Lys Leu Ser Pro Lys Asp
645 650 655
Arg Ile Pro Thr Thr Gln Thr Cys Asn Val Leu Pro Leu Trp Asn Lys
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690 695 700
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705 710 715 720
Ala Leu Lys Leu Leu Leu Lys Glu Asp Tyr Pro Ser Phe Gly Tyr Met
725 730 735
Ile Lys Asn Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Glu
740 745 750
Gly Thr Gly Met Asn Ser His Asn His Val Met Leu Gly Ser Val Asp
755 760 765
Thr Trp Phe Tyr Lys Tyr Leu Ser Gly Ile Lys Pro Val Ala Pro Gly
770 775 780
Trp Lys Lys Ile Arg Ile Glu Pro Tyr Phe Ala Asp Gln Ile Asp Phe
785 790 795 800
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805 810 815
Lys Lys Gln Asn Lys Glu Tyr Glu Ile Gln Ile Ile Ile Pro Val Asn
820 825 830
Thr Val Gly Ile Phe Ala Val Pro Glu Ser Phe Lys Val Ser Ala Ile
835 840 845
Asn Ser Lys Gln Val Ser Tyr Pro Ser Glu Phe Glu Leu Glu Pro Gly
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Ala Tyr Asn Ile Val Leu Glu Arg Val Arg Glu Cys
865 870 875
<210> 12
<211> 30
<212> DNA
<213> 人工合成
<400> 12
ccggccgcac gtcatgaact gttttatgcc 30
<210> 13
<211> 30
<212> DNA
<213> 人工合成
<400> 13
atgacgtgcg gccggatctt taaagctcag 30
<210> 14
<211> 30
<212> DNA
<213> 人工合成
<400> 14
aatctggcac tggcccgcgc aaccgatgaa 30
<210> 15
<211> 30
<212> DNA
<213> 人工合成
<400> 15
ggccagtgcc agattacggg tatccagacg 30
<210> 16
<211> 30
<212> DNA
<213> 人工合成
<400> 16
ccgtatgcaa aacgctatcc gagcgatccg 30
<210> 17
<211> 30
<212> DNA
<213> 人工合成
<400> 17
gcgttttgca tacggcggtg ccacatcact 30
<210> 18
<211> 30
<212> DNA
<213> 人工合成
<400> 18
tggaaagcat atccgagcga tccggcctgg 30
<210> 19
<211> 30
<212> DNA
<213> 人工合成
<400> 19
cggatatgct ttccaatacg gcggtgccac 30
<210> 20
<211> 30
<212> DNA
<213> 人工合成
<400> 20
ctgggtgcac atggtgactg gtgtccgccg 30
<210> 21
<211> 30
<212> DNA
<213> 人工合成
<400> 21
accatgtgca cccagtttgg tcagatgatt 30
<210> 22
<211> 30
<212> DNA
<213> 人工合成
<400> 22
ggtgacgcat ttccgaaacg taccccgctg 30
<210> 23
<211> 30
<212> DNA
<213> 人工合成
<400> 23
cggaaatgcg tcacccggcg gacaccagtc 30

Claims (10)

1.一种鼠李糖苷酶TpeRha突变体，其特征在于，为突变体TpeRha-R369A、突变体TpeRha-W512A 和突变体TpeRha-K579A中的任一种；
所述突变体TpeRha-R369A，为将氨基酸序列为如SEQ ID NO：1所示的TpeRha酶的第369位的精氨酸突变为丙氨酸；
所述突变体TpeRha-W512A，为将氨基酸序列为如SEQ ID NO：1所示的TpeRha酶的第512位的色氨酸突变为丙氨酸；
所述突变体TpeRha-K579A，为将氨基酸序列为如SEQ ID NO：1所示的TpeRha酶的第579位的赖氨酸突变为丙氨酸。
2.根据权利要求1所述的一种鼠李糖苷酶TpeRha突变体，其特征在于，所述突变体TpeRha-R369A的氨基酸序列为如SEQ ID NO：3所示；
所述突变体TpeRha-W512A的氨基酸序列为如SEQ ID NO：5所示；
所述突变体TpeRha-K579A的氨基酸序列为如SEQ ID NO：7所示。
3.根据权利要求2所述的一种鼠李糖苷酶TpeRha突变体，其特征在于，编码所述突变体TpeRha-R369A基因的核苷酸序列为如SEQ ID NO：4所示；
编码所述突变体TpeRha-W512A基因的核苷酸序列为如SEQ ID NO：6所示；
编码所述突变体TpeRha-K579A基因的核苷酸序列为如SEQ ID NO：8所示。
4.根据权利要求1所述的一种鼠李糖苷酶TpeRha突变体，其特征在于，所述TpeRha酶来源于石油热袍菌DSM 13995。
5.一种权利要求1-4任一项所述的鼠李糖苷酶TpeRha突变体的制备方法，其特征在于，包括以下步骤：
S1，将TpeRha酶基因连接到质粒中，得到重组质粒；
S2，设计突变引物，采用突变引物并以所述重组质粒为模板进行PCR扩增，酶切消化去除模板DNA，得到突变产物；
S3，将突变产物转化至宿主细胞中，筛选得到鼠李糖苷酶TpeRha突变体表达菌株，诱导表达，得到鼠李糖苷酶TpeRha突变体。
6.根据权利要求5所述的一种鼠李糖苷酶TpeRha突变体的制备方法，其特征在于，所述TpeRha酶基因的核苷酸序列如SEQ ID NO：2所示。
7.根据权利要求5所述的一种鼠李糖苷酶TpeRha突变体的制备方法，其特征在于，突变引物序列如SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：22和SEQ ID NO：23所示。
8.根据权利要求5所述的一种鼠李糖苷酶TpeRha突变体的制备方法，其特征在于，所述宿主细胞为大肠杆菌。
9.权利要求1-4任一项所述的一种鼠李糖苷酶TpeRha突变体在制备淫羊藿次苷Ⅰ组合物中的应用。
10.根据权利要求9所述的应用，其特征在于，利用鼠李糖苷酶TpeRha突变体催化转化淫羊藿总黄酮中的多组分黄酮糖苷制备淫羊藿次苷Ⅰ组合物。
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