CN108802216B - 一种使用区域敏感性蛋白组和/或多肽组鉴别刺参产地的方法 - Google Patents

Abstract

本发明涉及使用区域敏感性蛋白组和/或多肽组鉴别刺参产地的方法，所述区域敏感性蛋白组为，选自不同产地海参蛋白组中，在不同产地的刺参中含量差异显著且能在统计学上对海参产地进行判别区分的一组17种特征蛋白，其序列如SEQ ID No.76～92所示，所述的不同产地为：霞浦、大连、烟台、胶南、威海。所述的多肽组通过酶解所述的蛋白组获得，其序列如SEQ ID No.1～75所示。

Description

一种使用区域敏感性蛋白组和/或多肽组鉴别刺参产地的 方法

技术领域

本发明属于生物技术领域，具体而言，涉及一种使用区域敏感性蛋白组和/或多肽组鉴别刺参产地的方法。

背景技术

海参富含酸性黏多糖，对人体有免疫调节剂、抗凝血、控制血糖和降血脂等功效。故此，海参享有“营养宝库”之美誉，现代人把海参誉为“海味八珍之首”。市场上海参种类繁多，不同海参因其营养价值、口感等不同而价格相差巨大。美国肉参是海参的一种，自然生长在冷水海域中，其生长期较长，一般为7-10年，故营养价值极高，除具一般海参拥有的富含蛋白质和多种微量元素的优点外，还具有参体肥壮，发涨率高，参肉厚实，口感爽滑等特点。是烹饪和药用俱佳的极品。

同一品种的海参因产地不同会造成营养价值、口感的差异，类似“道地药材”，比如同一品种不同产地的海参有的粘多糖含量高，对心脑血管有好处；有的皂甙含量高，对抗癌有益；有的蛋白含量高，对蛋白质缺乏、营养不良的人群有好处。海参同人参一样，如果皂甙含量高，则药用价值高，但可能味道不好。

由于目前缺乏有效的识别海参产地的技术方法，靠传统的外观观察并无法鉴别海参的产地来源，导致不少不良商人以次充好、以假充真欺骗消费者，因此需要一种高效准确的鉴定海参产地的方法。

发明内容

由于目前缺乏有效的识别海参产地的技术方法，靠传统的外观观察并无法鉴别海参的产地来源，导致不少不良商人以次充好、以假充真欺骗消费者，因此需要一种高效准确的鉴定海参产地的方法。

本发明首先涉及一组可用来鉴别刺参(A.Japonicus)产地的区域敏感性蛋白组，所述区域敏感性蛋白组为，选自不同产地海参蛋白组中，在不同产地的刺参中含量差异显著且能在统计学上对海参产地进行判别区分的一组蛋白。

所述的不同产地优选为：霞浦、大连、烟台、胶南、威海。

所述的鉴别不同产地刺参的区域敏感性蛋白组中包含17种特征蛋白，其对应的GenBank编号分别为：

蛋白1：GenBank:APA22677.1collagen[Apostichopus japonicus]，GI:1095857067(Seq ID No.76)

蛋白2：GenBank:ADL27535.1tropomyosin[Apostichopus japonicus]，GI:302340969(Seq ID No.77)

蛋白3：GenBank:BAH79576.1major yolk protein 1[Apostichopus japonicus]，GI:240846031(Seq ID No.78)

蛋白4：GenBank:BAH79577.1major yolk protein 2[Apostichopus japonicus]，GI:240846033(Seq ID No.79)

蛋白5：GenBank:sp|Q9XY07.1|KARG_STIJA RecName:Full＝Arginine kinase；Short＝AK，GI:41017422(Seq ID No.80)

蛋白6：GenBank:ACJ70738.1actin isoform 2[Holothuria glaberrima]，GI:215882299(Seq ID No.81)

蛋白7：GenBank:AKN63087.1PDI[Apostichopus japonicus]，GI:852379636(SeqID No.82)

蛋白8：GenBank:AER60491.1heat shock protein gp96[Apostichopusjaponicus]，GI:355344590(Seq ID No.83)

蛋白9：GenBank:AEG90847.1glyceraldehyde-3-phosphate dehydrogenase,partial[Apostichopus japonicus]，GI:334821165(Seq ID No.84)

蛋白10：GenBank:sp|P48557.1|H2B_HOLTU RecName:Full＝Histone H2B，GI:1346252(Seq ID No.85)

蛋白11：GenBank:BAH79732.1actin[Apostichopus japonicus]，GI:241988732(Seq ID No.86)

蛋白12：GenBank:sp|P62776.2|H4_HOLTU RecName:Full＝Histone H4，GI:51317316(Seq ID No.87)

蛋白13：GenBank:BAV54301.1histone H3,partial[Holothuria sanctori]，GI:1060085899(Seq ID No.88)

蛋白14：GenBank:AFU36096.1profilin[Apostichopus japonicus]，GI:407894519(Seq ID No.89)

蛋白15：GenBank:AFG26286.1aldolase class-1protein[Apostichopusjaponicus]，GI:381356096(Seq ID No.90)

蛋白16：GenBank:ALI93846.1Piwi[Holothuria glaberrima]，GI:937655749(SeqID No.91)

蛋白17：GenBank:ACA05234.1vasa-like protein[Apostichopus japonicus]，GI:167860961(Seq ID No.92)

本发明还涉及一组可用来鉴别刺参产地的区域敏感性多肽组，所述的区域敏感性多肽组来源于所述区域敏感性蛋白组，通过对所述蛋白组酶解获得，可用来表征相关的区域敏感性蛋白组中的相关蛋白。所述的区域敏感性多肽组中的多肽序列、及其质谱检测特征质荷比离子(m/z)分别为：

SEQ ID NO.1：QEYDESGPSIVHR，368.9；

SEQ ID NO.2：ANDNEVLAK，402.7；

SEQ ID NO.3：SVLSGGTSMYPGIADR，952.9；

SEQ ID NO.4：QLLLAADDR，491.6；

SEQ ID NO.5：VIAGLNVMR，844.9；

SEQ ID NO.6：QPQGRPNGR，543.1；

SEQ ID NO.7：NSFVNDIFER，522.5；

SEQ ID NO.8：EIAQDFK，736.8；

SEQ ID NO.9：ASYDDIK，648.1；

SEQ ID NO.10：GEAGAPGATGPQGPAGQR，487.5；

SEQ ID NO.11：SFVNDIFER，409.4；

SEQ ID NO.12：YGIPIVSAGR，446.0；

SEQ ID NO.13：GYTFTTTAER，391.4；

SEQ ID NO.14：GEQGPPGGPGLGGQQGPR，772.8；

SEQ ID NO.15：DLYANSVLSGGTSMYPGIADR，517.1；

SEQ ID NO.16：DYDSEYTVPVISR，660.7；

SEQ ID NO.17：QLETELDDTSEK，738.8；

SEQ ID NO.18：IQPAEITALIAGFK，615.2；

SEQ ID NO.19：DNIQGITK，496.6；

SEQ ID NO.20：TVTAMDVVY，416.4；

SEQ ID NO.21：FEELNMELFR，653.2；

SEQ ID NO.22：VGLQVAAVT，667.7；

SEQ ID NO.23：IIEYFGLK，776.3；

SEQ ID NO.24：VTSFPTLK，531.1；

SEQ ID NO.25：VDQLVKGHFDVK，790.3；

SEQ ID NO.26：EGDILTKWEQR，823.8；

SEQ ID NO.27：DSTLIMQLLR，685.7；

SEQ ID NO.28：LLLPGELAK，384.4；

SEQ ID NO.29：DDFPNFEGHK，398.4；

SEQ ID NO.30：YLTADMYAK，495.5；

SEQ ID NO.31：AQQTAEEEMANLQR，704.7；

SEQ ID NO.32：LTGMAFR，590.6；

SEQ ID NO.33：GAAGMDGATGPR，444.9；

SEQ ID NO.34：VFLENVIR，810.3；

SEQ ID NO.35：GGDGQVGPPGPAGPQGEVGDR，647.2；

SEQ ID NO.36：GESIDDLVPK，591.1；

SEQ ID NO.37：MVSEALNSLAADLK，357.8；

SEQ ID NO.38：ISGLIYEETR，416.4；

SEQ ID NO.39：GAAQNIIPASTGAAK，425.9；

SEQ ID NO.40：DLAEVLFETAVLR，421.0；

SEQ ID NO.41：ETYSIYIYK，543.1；

SEQ ID NO.42：TVTAMDVVYALK，507.6；

SEQ ID NO.43：LTPEDIEK，506.5；

SEQ ID NO.44：ITITNDQNR，656.2；

SEQ ID NO.45：GILAADESTGSMAK，748.3；

SEQ ID NO.46：IIPASTGAAK，505.5；

SEQ ID NO.47：GGLLIPEK，492.0；

SEQ ID NO.48：TSLDQLEDELMIEK，447.0；

SEQ ID NO.49：VDATEETELAQK，455.5；

SEQ ID NO.50：DVATPSGYTLDR，554.6；

SEQ ID NO.51：ADIAEESLK，663.1；

SEQ ID NO.52：LVSIGTENTEENR，574.1；

SEQ ID NO.53：VLIEMEK，586.7；

SEQ ID NO.54：VLDATVVAVGSGLR，398.4；

SEQ ID NO.55：ADLVNNLGTIAK，643.7；

SEQ ID NO.56：EITALAPPTMK，567.1；

SEQ ID NO.57：SGSISITK，1094.7；

SEQ ID NO.58：LGPLTVLK，806.4；

SEQ ID NO.59：MLIPLLEGK，465.0；

SEQ ID NO.60：PSTQIMQLLR，731.8；

SEQ ID NO.61：SAAFESSLSQMK，431.5；

SEQ ID NO.62：IGSVFESVNR，537.0；

SEQ ID NO.63：VDEFTGIVGSLR，538.6；

SEQ ID NO.64：ELEEIVQPIVSK，443.9；

SEQ ID NO.65：ETLDLIK，508.0；

SEQ ID NO.66：NENLALIEK，500.1；

SEQ ID NO.67：ENANVLAR，676.2；

SEQ ID NO.68：VIAPPER，406.4；

SEQ ID NO.69：DEEGIISWVEK，488.1；

SEQ ID NO.70：VLLPEYGGTK，477.5；

SEQ ID NO.71：LLVPLLEGK，446.9；

SEQ ID NO.72：DSLVDIR，564.1；

SEQ ID NO.73：IFQIIFE，621.1；

SEQ ID NO.74：VADYLR，832.9；

SEQ ID NO.75：STELLIR，488.5；

本发明还涉及一种检测刺参产地的方法，所述方法包括如下步骤：

(1)对待测样本进行蛋白组测定前处理，获得待测多肽滤液：

(2)通过液相色谱-质谱法测定所述的区域敏感性蛋白组和/或区域敏感性多肽组成分，并以此构建正交偏最小二乘判别分析(OPLS-DA)模型，该模型以适当数量参考样本与液质测定获得的区域敏感蛋白组中相关蛋白的含量为数据矩阵，使用SIMCA软件(MKSUmetrics AB)构建OPLS-DA模型，使用模型输出的得分图(score plot)进行后续样品数据的判别分析。后续数据在得分图中被投射到的位置所代表的样品区域即预测为该测试样品的地理区域。

所述的对待测样本进行蛋白组测定前处理，获得待测多肽滤液步骤如下：

(1)称取1g海参样品均质成粉末状态，加入10mL蛋白提取液(8M尿素，50mMNH₄HCO₃)震荡提取蛋白，高速低温离心(20000r/min)，取上清液200μl转移到1ml EP管中；

(2)取2μL 1mol/L DTT(二硫苏糖醇)加入到100μL上述蛋白上清液液中，37℃反应1小时；

(3)取10μL现配的1mol/L的IAA(碘乙酰胺)配置过程需避光，现用现配，加入到已经冷却至室温的上述反应液中，室温避光反应1小时；

(4)采用10K滤膜20000g超滤30分钟，使用50mmol/L碳酸氢铵溶液反复冲洗滤膜上层，转移至新的EP管中；

(5)再加入碳酸氢铵溶液重复此步骤，合并溶液完成膜下蛋白提取；

(6)取5μl Trypsin酶溶液(1μg/μL的Trypsin酶溶液)加入到上述蛋白溶液中37℃酶解16-18小时；

(7)采用10K滤膜14000g超滤20分钟，收集下层的肽段滤液，待上机检测。

所述的通过液相色谱-质谱法测定所述的区域敏感性蛋白组和/或区域敏感性多肽组成分的方法为，

(1)采用液相色谱-四级杆/飞行时间质谱检测，

液相色谱条件：

流动相A：0.1％甲酸-乙腈，流动相B：0.1％甲酸-水，

流速：0.25mL/min，

质谱条件：

TOF扫描范围：350-1500Da，

正离子反应模式，GS1：35，GS2：45，Curtain Gas：35，ISVF：5500，TEM：500，DP：100，CE：10。

(2)采用液相色谱-三重四级杆检测，

流动相A：0.1％甲酸-乙腈，流动相B：0.1％甲酸-水，

流速：0.3mL/min，

电喷雾离子源，正离子反应模式，检测方式：MRM，喷雾电压：5500V，离子传输管温度：475℃；鞘气压力：40；辅助气压力：6。

本发明还涉及使用所述区域敏感性蛋白组和/或区域敏感性多肽组制备的用于鉴别刺参产地的检测试剂或检测试剂盒。

本发明还涉及所述的区域敏感性蛋白组和/或区域敏感性多肽组在制备用于鉴别刺参产地的试剂或试剂盒等中的应用。

本发明的有益效果是：

使用本发明所述不同地域的刺参按照多变量分析样本数量要求按上述方法进行分析，将所得蛋白组及(或)多肽组组分响应与不同地域刺参组成多变量分析矩阵，进行OPLS-DA分类判别后构建了标准品的分析模型。

实际应用中，将待测样品按同样方法进行前处理和液-质联用分析，将获得的蛋白组及(或)多肽组组分响应带入到分析模型中进行检测分析，通过模型自动计算分组，即可根据标准品模型判定相应刺参样本的产地信息。

附图说明

图1，5种刺参区域敏感性蛋白组指纹图1；

图2，5种刺参区域敏感性蛋白组指纹图2；

图3，5种刺参区域敏感性蛋白组指纹图3；

图4，霞浦刺参区域敏感性多肽组指纹色谱图；

图5，大连刺参区域敏感性多肽组指纹色谱图；

图6，烟台刺参区域敏感性多肽组指纹色谱图；

图7，胶南刺参区域敏感性多肽组指纹色谱图；

图8，威海刺参区域敏感性多肽组指纹色谱图；

图9，5种刺参区域敏感性多肽组指纹质谱图(横坐标1-75分别表示区域敏感性的多肽肽段)；

图10，5种刺参OPLS-DA分类判别分析模型图；

图11，待测样本的OPLS-DA分类判别分析模型预测分析。

具体实施方式

实施例1，区域敏感性蛋白组在5种刺参标准品中的指纹图谱分析

选取产自霞浦、大连、烟台、胶南、威海五个地区的海参样本，

首先，对所述的样本进行蛋白组测定前处理，步骤如下：

(1)称取1g海参样品均质成粉末状态，加入10mL蛋白提取液(8M尿素，50mMNH₄HCO₃)震荡提取蛋白，高速低温离心(20000r/min)，取上清液200μl转移到1ml EP管中；

(2)取2μL 1mol/L DTT(二硫苏糖醇)加入到100μL上述蛋白上清液液中，37℃反应1小时；

(3)取10μL现配的1mol/L的IAA(碘乙酰胺)，IAA溶液配置过程需避光，加入到已经冷却至室温的上述反应液中，室温避光反应1小时；

(4)采用10K滤膜20000g超滤30分钟，使用50mmol/L碳酸氢铵溶液反复冲洗滤膜上层，转移至新的EP管中；

(5)再加入碳酸氢铵溶液重复此步骤，合并溶液完成膜下蛋白提取获得蛋白溶液A(即为区域敏感性蛋白组)；

(6)取5μl Trypsin酶溶液(1μg/μL的Trypsin酶溶液)加入到上述蛋白溶液中，37℃酶解16-18小时；

(7)采用10K滤膜14000g超滤20分钟，收集下层的多肽段滤液B(即为区域敏感性多肽组)，待上机检测。

其次，通过液相色谱-质谱法联用测定所述多肽滤液的区域敏感性蛋白组成分进行分析，所述的液相色谱-质谱法联用的方法为，

(1)采用液相色谱-四级杆/飞行时间质谱检测，

液相色谱条件：

流动相A：0.1％甲酸-乙腈，流动相B：0.1％甲酸-水，

流速：0.25mL/min，

质谱条件：

TOF扫描范围：350-1500Da，

正离子反应模式，GS1：35，GS2：45，Curtain Gas：35，ISVF：5500，TEM：500，DP：100，CE：10。

(2)采用液相色谱-三重四级杆检测，

流动相A：0.1％甲酸-乙腈，流动相B：0.1％甲酸-水，

流速：0.3mL/min，

电喷雾离子源，正离子反应模式，检测方式：MRM，喷雾电压：5500V，离子传输管温度：475℃；鞘气压力：40；辅助气压力：6。

通过所述的区域敏感性多肽组的检测结果经过质谱定量及统计学软件分析并回归所述区域敏感性蛋白组，以所述区域敏感性蛋白组作图，可见，所述区域敏感性蛋白组在霞浦刺参、大连刺参、烟台刺参、胶南刺参、威海刺参中有显著性差异(见图1-图3)。

实施例2，区域敏感性多肽组在5种刺参中的色谱指纹图谱

对实施例1制备获得的和多肽滤液B进行色谱分析，分析方法同实施例1，具体为：

流动相A：0.1％甲酸-乙腈，流动相B：0.1％甲酸-水，

流速：0.25mL/min，

结果显示，所述的区域敏感性多肽组的色谱谱图在所述的霞浦刺参、大连刺参、烟台刺参、胶南刺参、威海刺参样品中有显著性差异(见图4-图8)。

实施例3，区域敏感性多肽组在5种刺参中的质谱指纹图谱

对实施例1制备获得的和多肽滤液B进行进行质谱分析，质谱分析的方法参数同实施例1，结果显示，所述的区域敏感性多肽组在霞浦刺参、大连刺参、烟台刺参、胶南刺参、威海刺参中有显著性差异(见图9)。

实施例4，5种刺参的OPLS-DA分类判别分析模型构建

使用实施例1～3所述的前处理及液相色谱质谱分析结果，将来自不同地域的刺参样品，按照多变量分析样本数量要求，将所得蛋白组和/或多肽组各组分响应与不同地域刺参组成多变量分析矩阵，使用SIMCA软件(MKS Umetrics AB)构建OPLS-DA模型，构建的5种刺参的OPLS-DA分类判别分析模型图见图10(图中的每个点表示一个标准样品)。

使用模型输出的得分图(score plot)进行后续样品数据的判别分析。后续数据在得分图中被投射到的位置所代表的样品区域即预测为该测试样品的地理区域。

实施例5，海参样本处理及检测步骤

取3例来源与大连的对待测海参样本，对其进行处理分析的步骤同实施例1所述的标准品处理步骤，具体包括：

(一)样本前处理步骤：

(1)称取1g海参样品均质成粉末状态，加入10mL蛋白提取液(8M尿素，50mMNH4HCO3)震荡提取蛋白，高速低温离心(20000r/min)，取上清液200μl转移到1ml EP管中；

(2)取2μL 1mol/L DTT加入到100μL上述蛋白溶液中37℃反应1小时；

(3)取10μL现配的1mol/L的IAA(配置过程需避光)加入到已经冷却至室温的上述反应液中，室温避光反应1小时；

(4)采用10K滤膜20000g超滤30分钟，使用200μL 50mmol/L碳酸氢铵溶液反复冲洗滤膜上层，转移至新的EP管中；

(5)再加入200μL碳酸氢铵溶液重复此步骤，合并溶液完成膜下蛋白提取；

(6)取5μl Trypsin酶溶液(1μg/μL的Trypsin酶溶液)加入到上述蛋白溶液中37℃酶解16-18小时；

(7)采用10K滤膜14000g超滤20分钟，收集下层的肽段滤液，待上机检测。

(二)上机检测，

采用液相色谱-四级杆/飞行时间质谱检测，

流动相A：0.1％甲酸-乙腈，流动相B：0.1％甲酸-水，

流速：0.25mL/min，

TOF扫描范围：350-1500Da，

正离子反应模式，GS1：35，GS2：45，Curtain Gas：35，ISVF：5500，TEM：500，DP：100，CE：10。

采用液相色谱-三重四级杆检测，

流动相A：0.1％甲酸-乙腈，流动相B：0.1％甲酸-水，

流速：0.3mL/min，

电喷雾离子源，正离子反应模式，检测方式：MRM，喷雾电压：5500V，离子传输管温度：475℃；鞘气压力：40；辅助气压力：6。

使用本发明构建的5种不同产地刺参的PCA分类判别分析模型，将获得的待测样品蛋白组及(或)多肽组组分响应带入模型中进行预测分析，通过模型自动计算得到待测样品产地。

鉴定结果如图11所示，所述待测样品通过模型归类分析，产于大连，分析结果准确无误。

最后需要说明的是，以上实施例仅用作帮助本领域技术人员理解本发明的实质，并不用作对本发明保护范围的限定。

SEQUENCE LISTING

<110> 山东出入境检验检疫局检验检疫技术中心

<120> 一种使用区域敏感性蛋白组和/或多肽组鉴别刺参产地的方法

<130> CP11802135M

<160> 92

<170> PatentIn version 3.5

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Ala Ser Tyr Asp Asp Ile Lys

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Ser Phe Val Asn Asp Ile Phe Glu Arg

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Tyr Gly Ile Pro Ile Val Ser Ala Gly Arg

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Gly Tyr Thr Phe Thr Thr Thr Ala Glu Arg

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Gly Glu Gln Gly Pro Pro Gly Gly Pro Gly Leu Gly Gly Gln Gln Gly

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Ile Gln Pro Ala Glu Ile Thr Ala Leu Ile Ala Gly Phe Lys

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Phe Glu Glu Leu Asn Met Glu Leu Phe Arg

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Val Gly Leu Gln Val Ala Ala Val Thr

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Ile Ile Glu Tyr Phe Gly Leu Lys

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Val Thr Ser Phe Pro Thr Leu Lys

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Val Asp Gln Leu Val Lys Gly His Phe Asp Val Lys

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Glu Gly Asp Ile Leu Thr Lys Trp Glu Gln Arg

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Asp Ser Thr Leu Ile Met Gln Leu Leu Arg

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Leu Leu Leu Pro Gly Glu Leu Ala Lys

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Asp Asp Phe Pro Asn Phe Glu Gly His Lys

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Tyr Leu Thr Ala Asp Met Tyr Ala Lys

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Ala Gln Gln Thr Ala Glu Glu Glu Met Ala Asn Leu Gln Arg

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Leu Thr Gly Met Ala Phe Arg

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Gly Ala Ala Gly Met Asp Gly Ala Thr Gly Pro Arg

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Val Phe Leu Glu Asn Val Ile Arg

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Gly Gly Asp Gly Gln Val Gly Pro Pro Gly Pro Ala Gly Pro Gln Gly

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Glu Val Gly Asp Arg

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Gly Glu Ser Ile Asp Asp Leu Val Pro Lys

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Met Val Ser Glu Ala Leu Asn Ser Leu Ala Ala Asp Leu Lys

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Ile Ser Gly Leu Ile Tyr Glu Glu Thr Arg

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Gly Ala Ala Gln Asn Ile Ile Pro Ala Ser Thr Gly Ala Ala Lys

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Asp Leu Ala Glu Val Leu Phe Glu Thr Ala Val Leu Arg

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Glu Thr Tyr Ser Ile Tyr Ile Tyr Lys

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Thr Val Thr Ala Met Asp Val Val Tyr Ala Leu Lys

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Leu Thr Pro Glu Asp Ile Glu Lys

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Gly Ile Leu Ala Ala Asp Glu Ser Thr Gly Ser Met Ala Lys

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Ile Ile Pro Ala Ser Thr Gly Ala Ala Lys

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Gly Gly Leu Leu Ile Pro Glu Lys

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Thr Ser Leu Asp Gln Leu Glu Asp Glu Leu Met Ile Glu Lys

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Val Asp Ala Thr Glu Glu Thr Glu Leu Ala Gln Lys

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Asp Val Ala Thr Pro Ser Gly Tyr Thr Leu Asp Arg

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Ala Asp Ile Ala Glu Glu Ser Leu Lys

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Leu Val Ser Ile Gly Thr Glu Asn Thr Glu Glu Asn Arg

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Val Leu Ile Glu Met Glu Lys

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Ala Asp Leu Val Asn Asn Leu Gly Thr Ile Ala Lys

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Glu Ile Thr Ala Leu Ala Pro Pro Thr Met Lys

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Ser Gly Ser Ile Ser Ile Thr Lys

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Leu Gly Pro Leu Thr Val Leu Lys

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Met Leu Ile Pro Leu Leu Glu Gly Lys

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Pro Ser Thr Gln Ile Met Gln Leu Leu Arg

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Ser Ala Ala Phe Glu Ser Ser Leu Ser Gln Met Lys

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Ile Gly Ser Val Phe Glu Ser Val Asn Arg

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Val Asp Glu Phe Thr Gly Ile Val Gly Ser Leu Arg

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Glu Leu Glu Glu Ile Val Gln Pro Ile Val Ser Lys

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Glu Thr Leu Asp Leu Ile Lys

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Asn Glu Asn Leu Ala Leu Ile Glu Lys

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Glu Asn Ala Asn Val Leu Ala Arg

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Val Ile Ala Pro Pro Glu Arg

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Asp Glu Glu Gly Ile Ile Ser Trp Val Glu Lys

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Val Leu Leu Pro Glu Tyr Gly Gly Thr Lys

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Leu Leu Val Pro Leu Leu Glu Gly Lys

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Asp Ser Leu Val Asp Ile Arg

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Ile Phe Gln Ile Ile Phe Glu

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Val Ala Asp Tyr Leu Arg

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Ser Thr Glu Leu Leu Ile Arg

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Met Tyr Asn Phe Val Ser Leu Cys Ser Arg His Arg Arg Thr Leu Leu

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Ala Leu Ala Val Phe Ile Thr Leu Tyr Ser Thr Val Ser Ser Gln Asp

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Val His Gly Leu Thr Gly Pro Lys Gly Pro Lys Gly Glu Pro Gly Gln

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Thr Pro Val Ile Gly Ser Gly Asp Leu Ile Pro Gly Pro Pro Gly Pro

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Gly Asn Ile Gly Ile Pro Gly Pro Arg Gly Gly Tyr Gly Ala Asp Gly

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Val Pro Gly Leu Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro

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Ser Gly Gln Leu Thr Ala Glu Ala Gln Val Ser Gln Gln Tyr Lys Gly

115 120 125

Pro Val Met Pro Ala Gly Leu Gly Tyr Pro Gln Ala Met Met Ala Gly

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Glu Pro Gly Gln Arg Gly Pro Pro Gly Gln Ala Gly Leu Arg Gly Pro

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Ala Gly Leu Thr Gly Ala Pro Gly Glu Pro Gly Glu Gln Gly Pro Thr

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Gly Pro Val Gly Leu Thr Gly Pro Ala Gly Ser Pro Gly Arg Pro Gly

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Ser Ala Gly Lys Asp Gly Ala Asn Gly Asn Pro Gly Pro Ala Gly Pro

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Ser Gly Pro Pro Gly Pro Ala Gly Gln Asn Gly Phe Pro Gly Pro Ser

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Gly Pro Lys Gly His Arg Gly Tyr Ser Gly Arg Ala Gly Gln Lys Gly

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Glu Arg Gly Asp Asp Gly Glu Arg Gly Asn Asp Gly Ala Ala Gly Ser

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Ile Gly Ala Pro Gly Pro Ala Gly Pro Met Gly Ser Pro Gly Glu Arg

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Gly Arg Thr Gly Pro Ser Gly Asp Thr Gly Pro Ala Gly Ser Asp Gly

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Ala Pro Gly Ala Gln Gly Pro Pro Gly Ser Ala Gly Ile Pro Gly Ala

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Pro Gly Val Pro Gly Ser Ser Gly Pro Lys Gly Asp Ala Gly Ser Pro

305 310 315 320

Gly Ala Thr Gly Pro Gln Gly Gln Gln Gly Ser Arg Gly Glu Arg Gly

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Pro Glu Gly Gln Gln Gly Gln Ala Gly Leu Gln Gly Ala Pro Gly Met

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Asn Gly Asn Pro Gly Ala Lys Gly Ala Ser Gly Ala Pro Gly Ala Gln

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Gly Pro Ile Gly Phe Ser Gly Leu Arg Gly Pro Asn Gly Ala Pro Gly

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Ser Gln Gly Pro Ala Gly Ser Lys Gly Asp Gln Gly Val Ala Gly Ala

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Pro Gly Glu Gln Gly Glu Glu Gly Pro Thr Gly Leu Pro Gly Asp Val

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Gly Pro Gln Gly Pro Pro Gly Ala Asn Gly Ala Ser Gly Glu Arg Gly

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Pro Arg Gly Ser Thr Gly Pro Ala Gly Pro Gln Gly Pro Ala Gly Asp

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Arg Gly Gln Leu Gly Ala Pro Gly Ser Ile Gly Pro Ala Gly Ala Ala

450 455 460

Gly Ala Lys Gly Glu Thr Gly Ala Arg Gly Gly Pro Gly Ser Pro Gly

465 470 475 480

Ser Lys Gly Ser Arg Gly Glu Pro Gly Arg Asn Gly Asp Pro Gly Leu

485 490 495

Pro Gly Thr Arg Gly Leu Ser Gly Asn Pro Gly Lys Leu Gly Lys Asp

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Gly Ser Pro Gly Pro Pro Gly Pro Pro Gly Lys Asn Gly Arg Gln Gly

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Ala Thr Gly Ala Thr Gly Gln Arg Gly Met Pro Gly Ala Gln Gly Leu

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Thr Gly Ala Pro Gly Ala Gln Gly Asp Arg Gly Gly Asp Gly Gln Val

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Gly Pro Pro Gly Pro Ala Gly Pro Gln Gly Glu Val Gly Asp Arg Gly

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Asn Pro Gly Pro Ala Gly Ala Pro Gly Ser Pro Gly Val Ala Gly Glu

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Arg Gly Ala Pro Gly Leu Gln Gly Pro Gln Gly Gln Gln Gly Pro Ala

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Gly Leu Asn Gly Pro Gln Gly Glu Ser Gly Ala Pro Gly Asp Ala Gly

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Pro Gln Gly Glu Thr Gly Ala Pro Gly Asn Pro Gly Asp Arg Gly Glu

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Arg Gly Gly Pro Gly Asp Arg Gly Pro Pro Gly Pro Ser Gly Arg Pro

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Gly Gly Arg Gly Val Ser Gly Arg Ala Gly Gln Asp Gly Ala Thr Gly

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Ala Pro Gly Pro Lys Gly Glu Gln Gly Pro Pro Gly Gly Pro Gly Leu

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Gly Gly Gln Gln Gly Pro Arg Gly Pro Pro Gly Pro Ser Gly Ser Arg

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Gly Pro Pro Gly Glu Asp Gly Asp Glu Gly Arg Pro Gly Pro Thr Gly

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Pro Ala Gly Pro Arg Gly Ser Ile Gly Pro Ile Gly Pro Thr Gly Ile

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Thr Gly Ser Pro Gly Glu Lys Gly Asp Gln Gly Ser Pro Gly Pro Ala

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Gly Ala Gln Gly Ala Arg Gly Asp Ala Gly Ala Arg Gly Ala Asn Gly

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Pro Ala Gly Pro Gln Gly Phe Pro Gly Pro Ala Gly Arg Pro Gly Ser

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Pro Gly Ala Arg Gly Glu Thr Gly Pro Ser Gly Ile Arg Gly Glu Thr

785 790 795 800

Gly Pro Ala Gly Ala Ala Gly Ala Thr Gly Asp Pro Gly Ala Gln Gly

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Pro Ala Gly Ala Val Gly Ala Gln Gly Glu Arg Gly Glu Ala Gly Asn

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Thr Gly Pro Gln Gly Pro Val Gly Asn Pro Gly Ile Gly Gly Pro Gln

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Gly Asn Gln Gly Pro Pro Gly Pro Ala Gly Gln Thr Gly Pro Ser Gly

850 855 860

Ala Pro Gly Ala Pro Gly Glu Arg Gly Asp Pro Gly Val Ala Gly Arg

865 870 875 880

Asp Gly Asp Gln Gly Pro Val Gly Ala Pro Gly Ala Ser Gly Glu Lys

885 890 895

Gly Asp Arg Gly Ser Asp Gly Asp Ile Gly Ser Ala Gly Pro Ala Gly

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Pro Pro Gly Pro Pro Gly Ala Ser Gly Pro Leu Gly Ile Ala Gly Ser

915 920 925

Met Gly Pro Arg Gly Pro Pro Gly Ala Pro Gly Gly Pro Gly Glu Gln

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Gly Ala Thr Gly Ser Pro Gly Ser Gln Gly Asn Arg Gly Ser Pro Gly

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Ala Ile Gly Ala Ser Gly Pro Ala Gly Ala Ala Gly Ala Asn Gly Gln

965 970 975

Asp Gly Glu Asp Gly Arg Asp Gly Ala Gln Gly Pro Ala Gly Gln Arg

980 985 990

Gly Glu Lys Gly Asp Thr Gly Ala Thr Gly Asn Pro Gly Ser Gln Gly

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Gly Pro Gly Pro Gln Gly Ala Ala Gly Gln Asn Gly Ala Pro Gly

1010 1015 1020

Glu Arg Gly Glu Ala Gly Ala Pro Gly Ala Thr Gly Pro Gln Gly

1025 1030 1035

Pro Ala Gly Gln Arg Gly Pro Ala Gly Pro Thr Gly Pro Thr Gly

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Pro Ala Gly Ala Thr Gly Glu Arg Gly Pro Ser Gly Pro Gln Gly

1055 1060 1065

Ala Lys Gly His Gln Gly Pro Thr Gly Leu Gln Gly Leu Thr Gly

1070 1075 1080

Pro Val Gly Ser Pro Gly Asp Thr Gly Val Pro Gly Pro Pro Gly

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Thr Ala Gly Glu Arg Gly Ser Pro Gly Asn Arg Gly Ala Ala Gly

1100 1105 1110

Met Asp Gly Ala Thr Gly Pro Arg Gly Phe Asp Gly Pro Glu Gly

1115 1120 1125

Pro Arg Gly Pro Pro Gly Ser Glu Gly Arg Gln Gly Ser Gln Gly

1130 1135 1140

Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Thr Val Gln

1145 1150 1155

Gly Phe Thr Tyr Ser Gln Tyr Pro Asn Gln Val Val Ser Gly Gln

1160 1165 1170

Asp Lys Gly Pro Ser Pro Tyr Tyr Tyr Gly Asp Asp Ala Asn Val

1175 1180 1185

Asp Ala Lys Asn Lys Glu Pro Glu Ile Leu Ala Thr Leu Lys Asp

1190 1195 1200

Ile Asn Asn Arg Phe Lys Ala Leu Lys Gln Pro Ser Gly Asp Ser

1205 1210 1215

Lys Leu His Ala Thr Arg Ser Cys Arg Asn Leu Phe Leu Asp His

1220 1225 1230

Pro Asp Lys Glu Ser Gly Ile Tyr Trp Val Asp Pro Asn Leu Gly

1235 1240 1245

Cys Thr Asp Asp Ala Phe Gln Ala Phe Cys Asp Lys Thr Asn Lys

1250 1255 1260

Ala Thr Cys Leu Thr Pro Glu Thr His Leu Val Thr Asn Ala Thr

1265 1270 1275

Trp Tyr Thr Gly Lys Pro Lys Arg Ile Tyr Tyr Ser Ser Met Arg

1280 1285 1290

Gly Gly Glu Lys Phe Thr Tyr Ala Glu Lys Ser Gln Leu Val Phe

1295 1300 1305

Leu Arg Leu Leu Ser Ile Ser Ala Lys Gln Ser Val Thr Phe Tyr

1310 1315 1320

Cys Arg Asn Val Ala Ala Asn Leu Glu Phe Leu Asn Ser Arg Glu

1325 1330 1335

Asp Val Met Arg Asp Tyr Gln Ile Leu Glu Asn Thr Cys Gln Ser

1340 1345 1350

Gln Ser Asp Ser Trp Gly Arg Val Val Leu Asp Tyr Glu Thr Asp

1355 1360 1365

Ile Thr Asp Asn Leu Pro Phe Glu Asp Phe Ser Val Gly Asp Leu

1370 1375 1380

Gly Ala Asp Asn Gln Glu Phe Gly Leu Asp Met Gly Lys Val Cys

1385 1390 1395

Phe Ala

1400

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Met Asp Thr Ile Lys Lys Lys Leu Ser Gln Leu Lys Ala Asp Lys Glu

1 5 10 15

Lys Ala Leu Asp Glu Lys Asp Val Ala Glu Ala Ser Met Lys Glu Ala

20 25 30

Met Glu Arg Val Glu Gln Val Asn Asp Glu Asn Lys Glu Leu Gln Thr

35 40 45

Arg Ile Lys Gln Leu Glu Thr Glu Leu Asp Asp Thr Ser Glu Lys Leu

50 55 60

Asn Thr Thr Val Ile Lys Cys Glu Ala Ala Glu Lys Ala Gln Gln Thr

65 70 75 80

Ala Glu Glu Glu Met Ala Asn Leu Gln Arg Lys Leu Gln Leu Thr Glu

85 90 95

Glu Glu Leu Ser Arg Ser Glu Glu Arg Val Ala Asp Leu Gln Ser Lys

100 105 110

Tyr Thr Asp Ile Glu Gln Ser Ser Glu Glu Asn Glu Arg Gln Arg Lys

115 120 125

Val Leu Glu Ser Arg Ser Ala Ala Asp Asp Glu Arg Met Ser Glu Leu

130 135 140

Glu Thr Gln Val Met Ser Ser Lys Thr Ser Leu Glu Asp Ser Asp Arg

145 150 155 160

Lys Tyr Asp Glu Ala Ser Arg Lys Leu Thr Val Thr Glu Glu Glu Leu

165 170 175

Ala Arg Ser Glu Glu Arg Ser Ala Ala Phe Glu Ser Ser Leu Ser Gln

180 185 190

Met Lys Glu Glu Leu His Gln Leu His Asn Asn Val Lys Ser Leu Glu

195 200 205

Ala Gln Glu Glu Lys Phe Thr Glu Asn Glu Glu Met Tyr Glu Lys Lys

210 215 220

Val Arg Asp Leu Glu Asp Lys Leu Lys Val Ala Glu Asp Arg Ala Asp

225 230 235 240

Ile Ala Glu Glu Ser Leu Lys Ser Leu Lys Thr Ser Leu Asp Gln Leu

245 250 255

Glu Asp Glu Leu Met Ile Glu Lys Glu Lys Val Arg Glu Met Thr Glu

260 265 270

Glu Met Glu Arg Thr Ile Gln Glu Leu Asn Phe Glu Val

275 280 285

<210> 78

<211> 1365

<212> PRT

<213> Apostichopus japonicus

<400> 78

Met Lys Tyr Leu Leu Leu Leu Ser Val Ile Gly Leu Ala Phe Ala Gly

1 5 10 15

Pro Thr Pro Val Thr Lys Asp Val Val Thr Ala Gly Thr Cys Pro Val

20 25 30

Asp His Val Phe Pro Thr Arg Gly Met Cys Glu Pro Asp Cys Leu Asp

35 40 45

Asp Arg Tyr Cys Gln Glu Gly Leu Lys Cys Cys Met Thr Ala Asn Cys

50 55 60

Gly Leu Lys Cys Leu Ala Pro Val Val Gln Asn Ala Pro Glu Asn Ile

65 70 75 80

Thr Lys Thr Lys Gln Glu Ile Thr Glu Leu Leu Thr Thr Ile Leu Glu

85 90 95

Glu Arg Gln Asp Leu Ile Glu Lys Leu Glu Lys Phe Pro Pro Pro Met

100 105 110

Leu Arg Phe Ile Ala Met Lys Asn Arg Thr Asp Val Met Arg Met Cys

115 120 125

Val Thr Ser Pro Cys Glu Leu Arg Lys Cys Gln Arg Leu Ala Thr Glu

130 135 140

Leu Thr Tyr Asn Val Val Pro Arg Lys Glu His Phe Cys Gln Val Ala

145 150 155 160

Thr Ser Thr Lys Gln Cys Leu Phe Trp Val Glu Arg Gly Trp Ala Asp

165 170 175

Leu Val Val Ala Arg Asp Leu Asp Ile Tyr His Ala Lys Thr Lys Phe

180 185 190

Asn Val Thr Ala Phe Ala Gln Glu Thr Leu Lys Asn Glu Asn Leu Ala

195 200 205

Leu Ile Glu Lys Val Ser Gln Asn Ile Thr Leu Ala Val Thr Met Arg

210 215 220

Gln Ser His Ile His Thr Leu Asn Gln Leu Glu Gly Ala Pro Ile Cys

225 230 235 240

Ser Ala Gly Ile Asn Asn Thr Ala Ser Phe Ile Ser Pro Val Ala Thr

245 250 255

Leu Ile Ser Lys Gly Ile Ile Pro Ile Thr Gly Ser Val Leu Glu Ser

260 265 270

Ala Ala Asp Phe Phe Gly Asp Met Cys Val Pro Gly Ala Leu Asn Lys

275 280 285

Thr Thr Val Asn Thr Asn Glu Thr Leu Pro Glu Lys Phe Leu Thr Gly

290 295 300

Cys Asn Gln Arg Val Asp Glu Phe Thr Gly Ile Val Gly Ser Leu Arg

305 310 315 320

Cys Val Ser Ser Val Asn Gly Val Ala Phe Val Asp His Lys Ile Val

325 330 335

Lys Glu Met Met Glu Asn Thr Glu Tyr Asn Asp Asn Phe Arg Leu Val

340 345 350

Cys Glu Gly Glu Asn Lys Pro Leu Ser Ser Trp Val Glu Lys Pro Cys

355 360 365

Gln Ile Gly Tyr Thr Pro Arg Pro Ile Leu Phe Val Asn Pro Glu Arg

370 375 380

Asn Thr Thr Tyr Ile Gln Glu Leu Thr Thr Ile Met Thr Asn Val Val

385 390 395 400

Lys Lys Asn Thr Gly Lys Val Gly Ser Leu Asn Ile Phe Asn Ser Thr

405 410 415

Glu Ile Val Cys Gly Asn Glu Pro Asn Lys Asn Leu Ile Phe Met Asp

420 425 430

Glu Asn Asn Ala Phe Thr Ala Ile Asn Glu Ser Ile Ala Glu Gln Arg

435 440 445

Ile Glu Thr Lys Ser Tyr Phe Arg Thr Tyr Asn Ile Cys Glu Ser Leu

450 455 460

Val Pro Lys Pro Thr Val Arg Phe Cys Val Thr Lys Pro Val Glu Tyr

465 470 475 480

Asn Lys Cys Leu Thr Met Lys His Val Phe Glu Lys His Glu Lys Leu

485 490 495

Ser His Ile Ala Trp Gly Cys Val His Ala Pro Thr Ser Met Glu Cys

500 505 510

Met Arg Asn Val Leu Asn Gly Thr Ala Glu Ile Phe Ser Gly Asn Val

515 520 525

Glu Glu Ile Phe Ile Gly Gly Arg Asp Phe Leu Leu Gln Pro Ile Met

530 535 540

Met Gln Asp Pro Val Met Arg Glu Ser Ile Asn Asp Tyr Val Asn Lys

545 550 555 560

Val Phe Gly Thr His Ser Val Ser Val Met Lys Lys Ser Lys Leu Trp

565 570 575

Asn Lys Phe Gly Gln Asp Thr Lys Phe Leu Asn Ile Arg Asn Leu Thr

580 585 590

Val Cys Ser Ala Gly Ile Asn Lys Val Glu Ser Phe His Leu Pro Val

595 600 605

Gly Arg Met Leu Ser Thr Asn Val Met Pro Arg Ile Gly Ser Val Phe

610 615 620

Glu Ser Val Asn Arg Phe Phe Lys Ser Ala Cys Ile Pro Gly Ala Thr

625 630 635 640

Pro Val Ser Tyr Lys His Asp Leu Asp Leu Val Ile Gly His Glu Leu

645 650 655

Asn Trp Gly Ile Thr Gly Leu Ser Phe Tyr Asn Phe Thr Gly Phe Asp

660 665 670

Trp Phe Ile Trp Asn Ala Pro His Thr Trp Thr Phe Tyr Asn Tyr Asn

675 680 685

Arg Lys Thr Pro Gly Thr Leu Gln Arg Phe Met Lys Asn Arg Leu Leu

690 695 700

Val Pro Leu Leu Glu Gly Lys Leu Ser Leu Leu Met Ser Leu Thr Leu

705 710 715 720

Thr Pro Ser Thr Thr Ala Ser Leu Met Met Ser Ser Ala Leu Lys Thr

725 730 735

Arg Asp Ile Leu Ser Asp Ile Pro Asp Lys Ile Arg Asp Arg Leu Val

740 745 750

Asp Met Arg Val Ser Asn Lys Asp Lys Met Ser Thr Phe Lys Asp Leu

755 760 765

Tyr Glu Asp Arg Thr Gly Thr Glu Phe Val Arg Pro Lys Asp Lys Arg

770 775 780

Leu Ser Lys Gln Thr Leu Arg Ser Tyr Leu Gln Gln Ser Phe Thr Asn

785 790 795 800

Met Gln Asp Glu Trp Gly Ile Phe Glu Asn Val Asn Asn Pro Val Met

805 810 815

Thr Pro Asn Lys Pro Val Lys Ser Met Thr Gly Gly Lys Val Thr Asn

820 825 830

Val Arg Arg Phe Phe Asp Glu His Val Lys Glu Thr Asp Pro Asn Thr

835 840 845

Ile Ile Gly Asn Leu Leu Met Asp Tyr Asp Ser Glu Tyr Thr Val Pro

850 855 860

Leu Ile Thr Lys Ile Phe Ala Lys Val Leu Asn Gln Arg Phe Asp Lys

865 870 875 880

Leu Phe Gly Leu Ala Asp Ser Leu Ser Ile Leu His Gly Val Pro Thr

885 890 895

Met Ser Ser Val His Asp Glu Gln Tyr Glu Trp Ile Lys His Pro Ala

900 905 910

Val Gln Ala Phe Val Arg Ile Tyr Ala Pro Arg Leu Ala Thr Tyr Tyr

915 920 925

Ser Asp Leu Tyr Thr Asn Ser Glu Leu Asn Ser Met Val His Ser Arg

930 935 940

Tyr His Asn Pro Leu Trp Leu Thr Pro Thr Tyr Pro Glu Phe Leu Asp

945 950 955 960

Thr Met Lys Thr His Met Thr Asp Leu Ile His Ile Cys Gln Gly Phe

965 970 975

Gly Glu His Lys Gly Glu Leu Ser Asp Glu Glu Pro Phe Tyr Gly Leu

980 985 990

Ala Gly Ser Ile Glu Cys Ile Ala Asp Gly Ser Glu Gly Asp Ile Ala

995 1000 1005

Phe Val Glu Leu Glu Ser Leu Val Lys Glu Leu Pro Lys Met Gly

1010 1015 1020

Gln Ser Ile Leu Asp Tyr Val Ile Val Thr Pro Leu Gly Leu Val

1025 1030 1035

Lys Glu Val Thr Pro Glu Met Ile Thr Asn Ile Thr Phe Leu Arg

1040 1045 1050

Asn Val Thr Phe Gly Val Lys Ala Phe Pro Ala Leu Leu Thr Ser

1055 1060 1065

Phe Asn Lys Thr Gly Ser Trp Arg Trp Asn Val Thr Lys Ala Leu

1070 1075 1080

Leu Asp Ala Gln Lys Ile Phe Ser Asp Ala Gln Asn Asn Glu Asp

1085 1090 1095

Gln Tyr His Ile Phe Gly Ser Ala Ser Ile Phe Ala Pro Glu Thr

1100 1105 1110

Val Lys Leu Ala Pro Ile Pro Ile Gln Asp Gln Thr Tyr Ala Thr

1115 1120 1125

Tyr Leu Gly Pro Asn Leu Thr Arg Ser Phe Glu Ala Ile Ile Lys

1130 1135 1140

Pro Ser Thr Phe Asp Trp Trp Lys Glu Arg Arg His Ile Cys Thr

1145 1150 1155

Gly Glu Ser Tyr Thr Asn Val Ile Glu Gln Arg Asn Gly Thr Cys

1160 1165 1170

Lys Ala Ile Val Lys Asp Val Thr Cys Gly Gly Met Pro Arg Pro

1175 1180 1185

Lys Val Ile Ser Val Gly Thr Thr Glu Asn Lys Lys Pro Val Val

1190 1195 1200

Val Arg Met Cys Ser Arg Pro Thr Ser Phe Val Arg Glu Met Ala

1205 1210 1215

Glu Phe Val Cys Asp Asn Gly His Gly Tyr Val Thr Pro Val Met

1220 1225 1230

Val Pro Thr Thr Cys Ser Cys Val Pro Cys Glu Asp Ile Glu Tyr

1235 1240 1245

Ile Pro Asp Trp Thr Asn Asp Thr Val Trp Asn Ser Thr Thr Leu

1250 1255 1260

Asn His Thr Ile Thr Glu His Phe Glu Thr Leu Met Pro Leu Trp

1265 1270 1275

Gly Asn Lys Ala Phe Trp Met Asn His Thr Leu Asn Ser Asn Phe

1280 1285 1290

Met Ile Gly Val Pro Lys Asn Val Thr Arg Lys Asp Thr Thr Thr

1295 1300 1305

Leu Leu Gly Asp Val Asn Val Lys Pro Leu Asp Thr Cys Glu Ala

1310 1315 1320

Asn Trp Tyr Gly Phe Glu Trp His Lys Glu Trp Phe Pro Glu Thr

1325 1330 1335

Asn Arg Pro Val Cys Leu Gly Thr Val Asn Gly Leu Arg Arg Val

1340 1345 1350

Leu Thr Ser Glu Arg Val Gln Thr Lys Ile Met Pro

1355 1360 1365

<210> 79

<211> 1345

<212> PRT

<213> Apostichopus japonicus

<400> 79

Met Lys Tyr Leu Leu Leu Phe Cys Leu Ala Val Gly Ala Phe Ala Ser

1 5 10 15

Ile Asn Ile Glu Arg Glu Gly Glu Cys Pro Thr Asp Ile Thr Asn Val

20 25 30

Asp Ser Thr Leu Tyr Gly Val Cys Asn Asp Glu Cys Asp Val Asp Ser

35 40 45

Ser Cys Ser Asp Gln Leu His Lys Cys Cys Ala Thr Asn Cys Gly Arg

50 55 60

Arg Cys Val Asn Pro Met Arg Val Thr Pro Val Thr Glu Glu Glu Glu

65 70 75 80

Asn Lys Thr Arg Glu Glu Ile Thr Glu Met Leu Thr Thr Ile Leu Ser

85 90 95

Glu Arg Arg Asp Leu Ile Lys Lys Leu Asp Met Tyr Pro Pro Pro Leu

100 105 110

Ala Arg Phe Ile Met Met Lys Asn Arg Thr Asp Val Val Arg Met Cys

115 120 125

Val Thr Thr Pro Cys Glu Leu Arg Lys Cys Gln Arg Ile Ala Gln Thr

130 135 140

Met Thr Tyr Lys Val Thr Pro Arg Lys Glu Trp Phe Cys Gln Leu Ala

145 150 155 160

Thr Thr Thr Glu Gln Cys Leu Phe Trp Ala Glu Arg Gly Trp Thr Asp

165 170 175

Thr Val Met Ala Arg Glu Asn Lys Ile Phe Val Ala Val Asp Lys Phe

180 185 190

Asn Val Thr Ser Leu Ala Tyr Glu Lys Asn Lys Asn Ala Pro Thr Glu

195 200 205

Ile Glu Lys Lys Val Gln Asn Ile Thr Leu Ala Leu Thr Leu Thr Thr

210 215 220

Ser Asn Ile Glu Thr Phe His His Leu Met Gly Arg Lys Val Cys Ser

225 230 235 240

Ala Gly Val Asn Ile Thr Ser Ala Phe Ile Thr Pro Met Cys Asn Leu

245 250 255

Ile Tyr Lys Asp Val Met Leu Ala Thr Gly Asp Val Val Glu Ser Ser

260 265 270

Ala Asp Phe Phe Gly Lys Met Cys Val Pro Gly Ile Leu Asn Lys Thr

275 280 285

Phe Asp Lys Asn Glu Thr Tyr Pro Glu Lys Leu Thr Thr Ala Cys His

290 295 300

Asn Leu Glu Thr Glu Tyr Thr Gly Ile Lys Gly Ser Leu Lys Cys Ile

305 310 315 320

Lys Ser Gly His Gly Glu Val Val Phe Val Asp Ser Lys Val Val Lys

325 330 335

Glu Leu Asp Glu Arg Phe Ala Gly Val Phe Lys Leu Val Cys Glu Asp

340 345 350

Gln Ser Leu Pro Leu Ser Gln Trp Glu Gln Thr Lys Cys His Leu Gly

355 360 365

Tyr Thr Pro Arg Pro Val Leu Phe Leu Asn Pro Glu Arg Asn Val Thr

370 375 380

Tyr Lys Asn Glu Leu Lys Glu Ile Ile Leu Glu Ala Gly Lys Met Lys

385 390 395 400

Thr Pro Thr Val Asp Leu Phe Asn Ser Ser Asp Tyr Val Cys Ile Lys

405 410 415

Glu Ala Pro Lys Asp Leu Ile Phe Met Asp Glu Asn Thr Asn Leu Glu

420 425 430

Phe Leu Glu Asp Pro Phe Ser Leu Pro Ala Val Gln Glu Tyr Met Lys

435 440 445

Val Phe Asn Thr Cys Gln Ala Leu Thr Pro Lys Pro Arg Ala Lys Ile

450 455 460

Cys Thr Thr Thr Pro Val Gln Tyr Gln Lys Cys Ile Thr Met Lys Lys

465 470 475 480

Val Phe Gln Thr Asp Ile Glu Leu Arg Asn Ile Ser Trp Gly Cys Val

485 490 495

Leu Ala Arg Thr Glu Met Asp Cys Met Arg Asn Val Leu Asn Gly Thr

500 505 510

Ala Asp Leu Tyr Ser Gly Asp Val Lys Glu Ile Phe Thr Ala Gly Asn

515 520 525

Asp Phe Gln Leu Gln Pro Met Met Ile Gln Asp Glu Asn Leu Arg Phe

530 535 540

Gln Lys Ser Leu Phe His Asn Ala Thr Val Lys Ser Tyr Thr Ile Ala

545 550 555 560

Val Met Lys Lys Ser Lys Phe Trp Lys Lys Phe Gly Glu Asp Thr Lys

565 570 575

Val Val Asn Ile Arg Asn Leu Thr Val Cys Ser Pro Asp Ile Lys Ser

580 585 590

Val Pro Asn Phe His Leu Pro Ile Gly His Leu Leu Ser Asn Gly Val

595 600 605

Ile Pro Arg Ile Gly Ser Val Phe Glu Ser Val Ser Arg Phe Tyr Lys

610 615 620

Ser Val Cys Leu Pro Gly Ala Ala Pro Val Asp Trp Thr Trp Asp Ser

625 630 635 640

Asp Leu Ile Leu Gly His Glu Val Asn Trp Gly Ile Pro Gly Leu Ser

645 650 655

Phe Tyr Asn Phe Thr Gly Phe Asp Trp Phe Ile Trp Asn Ala Pro His

660 665 670

Thr Trp Thr Phe Tyr Asn Met Asn Arg Lys Thr Pro Gly Thr Leu Lys

675 680 685

Glu Phe Met Lys Asn Arg Met Leu Ile Pro Leu Leu Glu Gly Lys Leu

690 695 700

Thr Val Pro Glu Gly Phe Asn Pro Asp Thr Phe Asp Tyr Ser Ile Leu

705 710 715 720

Asp Asp Val Leu Ser Val Glu Gly Leu Gly Asp Ile Leu Asn Asp Ile

725 730 735

Pro Asp Glu Ile Arg Asp Ser Leu Val Asp Ile Arg Thr Lys Arg Val

740 745 750

Asp Arg Lys Glu Lys Phe Arg Glu Leu Tyr Glu Glu Lys Thr Gly Asp

755 760 765

Ala Phe Val Ser Leu Arg Asp Arg Arg Leu Gly Lys Leu Thr Leu Arg

770 775 780

Gly Phe Leu Gln Gln Ser Phe Gln Asn Met Glu Asp Glu Trp Gly Ile

785 790 795 800

Phe Asp Asp Val Asn Ala Pro Val Leu Ser Ser Lys Ser Val Gly Lys

805 810 815

Gly Met Thr Gly Gly Lys Val Ser Asn Leu Arg Asp Ile Leu Arg Gly

820 825 830

Ala Ala Thr Thr Ala Asp Asp Asn Thr Val Ile Gly Asn Leu Leu Lys

835 840 845

Asp Tyr Asp Ser Glu Tyr Thr Val Pro Val Ile Ser Arg Ile Phe Ser

850 855 860

Lys Leu Leu Asn Glu Arg Phe Glu Thr Leu Asp Gly Leu Ala Lys Thr

865 870 875 880

Leu Glu Ile Leu His Arg Val Pro Thr Met Ser Ser Ile Arg Gly Thr

885 890 895

Asp Tyr Glu Trp Leu Lys His Pro Ala Val Gln Ser Tyr Leu Lys Ile

900 905 910

Tyr Ala Pro Arg Leu Ile Ser Phe His Ser Asp Leu Tyr Thr Ser Glu

915 920 925

Glu Leu Ala Lys Thr Gln Phe Ser Arg Tyr Leu Asn Pro Ile Trp Leu

930 935 940

Ser Pro Thr Phe Lys Asp Phe Leu Asp Val Thr Lys Thr His Met Thr

945 950 955 960

Lys Leu Thr Glu Met Cys Arg Gly Tyr Gly Asp Arg Gln Gly Val Gly

965 970 975

Asn Ser His Glu Pro Phe Tyr Gly Asn Glu Gly Ala Leu Arg Cys Ile

980 985 990

Glu Asp Thr Glu Glu Gly Asp Ile Ala Phe Ile Asp Thr Lys Asn Phe

995 1000 1005

Ala Thr Leu Ser Thr Thr Asp Tyr Val Met Val Thr Pro Leu Gly

1010 1015 1020

Ile Val Gln Pro Ile Asn Pro Glu Ser Ile Met Asn Gly Thr Phe

1025 1030 1035

Gly Thr Val Pro Phe Pro Ala Leu Met Thr Ala Phe Asn Lys Thr

1040 1045 1050

Gly Ser Trp Arg Trp Asn Val Thr Lys Ala Leu Leu Ile Ala Gln

1055 1060 1065

Lys Lys Tyr Pro Thr Asn Thr Ser Thr Asp Tyr Thr Met Tyr Gly

1070 1075 1080

Val Asp Ser Val Phe Met Pro Glu Thr Lys Lys Met Tyr Pro Val

1085 1090 1095

Pro Leu Asn Met Gln Thr His Pro Thr Tyr Leu Gly Ser Arg Leu

1100 1105 1110

Thr Arg Ala Phe Glu Ala Leu Ile Lys Pro Ser Thr His Asp Trp

1115 1120 1125

Trp Lys Glu Arg Arg His Ile Cys Ser Gly Glu Ser Tyr Thr Asn

1130 1135 1140

Val Ile Glu Gln Arg Asn Gly Thr Cys Lys Ala Ile Val Lys Asp

1145 1150 1155

Val Thr Cys Gly Gly Met Pro Arg Pro Lys Val Ile Ser Val Gly

1160 1165 1170

Thr Thr Glu Asn Lys Lys Pro Val Val Val Arg Met Cys Ser Arg

1175 1180 1185

Pro Thr Ser Phe Val Arg Glu Met Ala Glu Phe Arg Cys Asp Asn

1190 1195 1200

Gly Tyr Gly Tyr Leu Lys Pro Val Met Val Pro Thr Thr Cys Ser

1205 1210 1215

Cys Val Pro Cys Asp Glu Ile Glu Tyr Lys Pro Thr Trp Thr Thr

1220 1225 1230

Asp Ile Met Trp Asn Thr Thr Glu Lys Asn His Ile Ile Thr Glu

1235 1240 1245

Asn Ile Glu Thr Met Met Lys Leu Trp Gly Asn Glu Glu Phe Trp

1250 1255 1260

Thr Asn His Thr Leu Asn Ser Asn Phe Glu Ile Gly Val Val Asn

1265 1270 1275

Val Thr Ala Met Lys Asn Glu Thr Glu Lys Leu Gly Pro Leu Thr

1280 1285 1290

Val Leu Lys Ser Val Gly Ser Cys Glu Ala Asn Trp Tyr Gly Asn

1295 1300 1305

Gly Trp Asn Thr Glu Trp Phe Val Asn Thr Ser Arg Pro Val Cys

1310 1315 1320

Leu Gly Thr Ile Pro Gly Leu Arg Arg Thr Leu Thr Ala Gln Arg

1325 1330 1335

Ile Gln Thr Lys Leu Met Pro

1340 1345

<210> 80

<211> 370

<212> PRT

<213> Apostichopus japonicus

<400> 80

Met Ala Asn Leu Asn Gln Lys Lys Tyr Pro Ala Lys Asp Asp Phe Pro

1 5 10 15

Asn Phe Glu Gly His Lys Ser Leu Leu Ser Lys Tyr Leu Thr Ala Asp

20 25 30

Met Tyr Ala Lys Leu Arg Asp Val Ala Thr Pro Ser Gly Tyr Thr Leu

35 40 45

Asp Arg Ala Ile Gln Asn Gly Val Asp Asn Pro Asp Phe His Leu Gly

50 55 60

Leu Leu Ala Gly Asp Glu Glu Thr Tyr Thr Val Phe Ala Asp Leu Phe

65 70 75 80

Asp Pro Val Ile Glu Glu Tyr His Asn Gly Phe Lys Lys Thr Asp Asn

85 90 95

His Lys Thr Asp Leu Asp Ala Ser Lys Ile Leu Asp Asp Val Leu Asp

100 105 110

Pro Ala Tyr Val Ile Ser Ser Arg Val Arg Thr Gly Arg Asn Ile Arg

115 120 125

Gly Met Ala Leu Ser Pro His Val Cys Arg Ser Glu Arg Arg Ala Ile

130 135 140

Glu Lys Met Val Ser Glu Ala Leu Asn Ser Leu Ala Ala Asp Leu Lys

145 150 155 160

Gly Lys Tyr Tyr Ser Leu Met Lys Met Asp Glu Lys Thr Gln Gln Gln

165 170 175

Leu Ile Asp Asp His Phe Leu Phe Asp Arg Pro Val Ser Arg His Phe

180 185 190

Thr Ser Gly Gly Met Ala Arg Asp Phe Pro Asp Gly Arg Gly Ile Trp

195 200 205

His Asn Asp Lys Lys Asn Phe Leu Val Trp Ile Asn Glu Glu Asp His

210 215 220

Thr Arg Ile Ile Ser Met Gln Met Gly Gly Asn Met Lys Glu Val Phe

225 230 235 240

Glu Arg Phe Thr Arg Gly Leu Thr Glu Val Glu Lys His Ile Lys Asp

245 250 255

Lys Thr Gly Lys Glu Phe Met Lys Asn Asp His Leu Gly Phe Val Leu

260 265 270

Thr Cys Pro Ser Asn Leu Gly Thr Gly Val Arg Cys Ser Val His Ala

275 280 285

Lys Leu Pro His Met Ala Lys Asp Lys Arg Phe Glu Glu Ile Cys Thr

290 295 300

Lys Met Arg Leu Gln Lys Arg Gly Thr Ser Gly Glu Phe Thr Glu Ser

305 310 315 320

Val Gly Gly Val Tyr Asp Ile Ser Asn Leu Asp Arg Leu Gly Ser Ser

325 330 335

Glu Val Glu Gln Val Asn Cys Val Ile Lys Gly Val Lys Val Leu Ile

340 345 350

Glu Met Glu Lys Lys Leu Glu Lys Gly Glu Ser Ile Asp Asp Leu Val

355 360 365

Pro Lys

370

<210> 81

<211> 376

<212> PRT

<213> Apostichopus japonicus

<400> 81

Met Cys Glu Glu Glu Val Ala Ala Leu Val Val Asp Asn Gly Ser Gly

1 5 10 15

Met Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe

20 25 30

Pro Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly Met

35 40 45

Gly Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly

50 55 60

Ile Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp

65 70 75 80

Asp Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg

85 90 95

Val Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn

100 105 110

Pro Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe

115 120 125

Asn Ala Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr

130 135 140

Ala Ser Gly Arg Thr Thr Gly Ile Val Leu Asp Ser Gly Asp Gly Val

145 150 155 160

Thr His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile

165 170 175

Leu Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys

180 185 190

Ile Leu Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu

195 200 205

Ile Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe

210 215 220

Glu Gln Glu Met Ala Thr Ala Ala Ser Ser Ser Ser Leu Glu Lys Ser

225 230 235 240

Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe

245 250 255

Arg Cys Pro Glu Thr Leu Phe Gln Pro Ser Phe Ile Gly Met Glu Ser

260 265 270

Ala Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp Ile

275 280 285

Asp Ile Arg Lys Asp Leu Tyr Ala Asn Ser Val Leu Ser Gly Gly Thr

290 295 300

Ser Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr Ala

305 310 315 320

Leu Ala Pro Pro Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg

325 330 335

Lys Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr

340 345 350

Phe Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro

355 360 365

Ser Ile Val His Arg Lys Cys Phe

370 375

<210> 82

<211> 503

<212> PRT

<213> Apostichopus japonicus

<400> 82

Met Lys Phe Phe Phe Ser Val Leu Ala Ile Leu Val Ala Val Ser Tyr

1 5 10 15

Ala Asp Glu Val Ala Glu Glu Asp Asn Val Ala Ile Leu Thr Asp Lys

20 25 30

Asn Phe Asp Gly Phe Val Ser Glu Asn Thr Tyr Val Leu Val Glu Phe

35 40 45

Tyr Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala Glu Pro Tyr Gly

50 55 60

Lys Ala Ala Thr Ala Leu Lys Glu Lys Gly Ser Glu Ile Arg Leu Gly

65 70 75 80

Lys Val Asp Ala Thr Glu Glu Thr Glu Leu Ala Gln Lys Phe Glu Val

85 90 95

Arg Gly Tyr Pro Thr Leu Lys Phe Phe Lys His Gly Val Pro Lys Asp

100 105 110

Tyr Gly Gly Gly Arg Asp Glu Glu Gly Ile Ile Ser Trp Val Glu Lys

115 120 125

Asn Thr Gly Pro Pro Ala Gln Thr Leu Glu Asn Ser Asp Asp Tyr Glu

130 135 140

Lys Phe Lys Asp Gly Lys Glu Val His Val Val Gly Phe Phe Lys Asn

145 150 155 160

Val Glu Ser Glu Asn Ala Val Ala Phe Leu Thr Ala Ala Asp Met Asp

165 170 175

Asp Ser Val Asp Tyr Ala Ile Thr Ser Ser Asp Asp Leu Ile Lys Glu

180 185 190

Leu Ser Ser Lys Asp Asp Gly Ile Val Val Phe Lys Glu Phe Asp Asn

195 200 205

Lys Arg Glu Asp Tyr Asp Gly Glu Phe Ser Ala Glu Ala Ile Ser Lys

210 215 220

Phe Val Gly Ala Ala Ser Met Pro Leu Val Ile Glu Phe Ser Asp Lys

225 230 235 240

Ser Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Val His Asn Leu Leu

245 250 255

Phe Val Lys Ala Glu Asp Glu Lys Phe Asn Val Ile His Gly Glu Phe

260 265 270

Gln Lys Ala Ala Ser Ser Tyr Lys Gly Lys Cys Leu Phe Val Tyr Ile

275 280 285

Asn Ala Glu Lys Glu Glu Asn Lys Arg Ile Ile Glu Tyr Phe Gly Leu

290 295 300

Lys Asp Glu Asp Leu Pro Thr Met Arg Val Ile Thr Leu Asp Gly Glu

305 310 315 320

Met Lys Lys Phe Lys Pro Pro Thr Asp Glu Ile Thr Glu Ser Ser Val

325 330 335

Lys Thr Phe Met Asp Gly Phe Phe Asp Gly Ser Leu Lys Pro His Leu

340 345 350

Met Ser Glu Glu Val Pro Glu Asp Trp Asp Ala Thr Glu Val Arg Val

355 360 365

Ile Val Gly Lys Asn Phe Lys Glu Val Val Leu Asp Lys Ser Lys Asn

370 375 380

Val Leu Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Gln Leu

385 390 395 400

Ala Pro Ile Tyr Asp Glu Leu Gly Glu Lys Tyr Lys Asp Asn Glu Asp

405 410 415

Ile Val Ile Ala Lys Met Asp Ala Thr Lys Asn Glu Ile Glu Glu Val

420 425 430

Lys Val Thr Ser Phe Pro Thr Leu Lys Tyr Phe Arg Lys Glu Thr Asn

435 440 445

Glu Ile Val Asp Tyr Asn Gly Gly Arg Thr Leu Glu Asp Leu Ala Ala

450 455 460

Phe Ile Asp Ser Asp Gly Lys Thr Gly Ala Gly Pro Ser Asp Glu Asp

465 470 475 480

Leu Glu Glu Asp Leu Glu Glu Glu Glu Glu Glu Asp Glu Glu Glu Gly

485 490 495

Gly Glu Val Lys Asp Glu Leu

500

<210> 83

<211> 815

<212> PRT

<213> Apostichopus japonicus

<400> 83

Met Thr Lys Trp Leu Phe Phe Val Gly Leu Leu Gly Ile Leu Leu Cys

1 5 10 15

Thr Gly Leu Val Pro Ala Glu Thr Asp Val Asp Gly Asp Ala Pro Val

20 25 30

Val Asp Glu Asp Ile Gly Lys Ser Arg Asp Gly Ser Lys Thr Asp Asp

35 40 45

Glu Val Val Glu Arg Glu Glu Glu Ala Ile Lys Leu Asp Gly Leu Asn

50 55 60

Val Ser Gln Met Lys Glu Leu Arg Glu Gln Ser Glu Lys His Val Phe

65 70 75 80

Gln Ala Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr

85 90 95

Arg Asn Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp

100 105 110

Ala Leu Asp Lys Ile Arg Leu Leu Ser Leu Thr Asp Lys Thr Ala Leu

115 120 125

Asp Ala Thr Glu Glu Leu Ser Ile Lys Ile Lys Ala Asp Lys Glu Asn

130 135 140

His Met Leu His Ile Thr Asp Thr Gly Ile Gly Met Thr Lys Ala Asp

145 150 155 160

Leu Val Asn Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe

165 170 175

Phe Glu Lys Leu Ala Ser Val Asp Ala Gly Glu Ala Asn Asp Leu Ile

180 185 190

Gly Gln Phe Gly Val Gly Phe Tyr Ser Ser Phe Leu Val Ser Glu Thr

195 200 205

Val Ile Val Thr Ser Lys His Asn Ser Asp Lys Gln Tyr Ile Trp Glu

210 215 220

Ser Asn Ser Ala Glu Tyr Ser Ile Ala Asp Asp Pro Arg Gly Asp Thr

225 230 235 240

Leu Leu Arg Gly Thr Thr Val Ser Leu Leu Leu Lys Glu Glu Ala Tyr

245 250 255

Asp Tyr Leu Glu Ala Asp Thr Ile Glu Gln Leu Val Lys Lys Tyr Ser

260 265 270

Gln Phe Ile Asn Phe Pro Ile Tyr Leu Trp Ser Ser Lys Thr Glu Thr

275 280 285

Val Glu Glu Pro Ile Glu Asp Asp Glu Glu Glu Ala Glu Ala Val Glu

290 295 300

Asp Thr Lys Glu Glu Asp Thr Lys Glu Glu Gly Glu Glu Asp Glu Asp

305 310 315 320

Val Glu Val Glu Glu Glu Glu Glu Glu Glu Asp Lys Pro Lys Thr Lys

325 330 335

Ser Val Glu Lys Thr Thr Trp Asp Trp Ala Leu Met Asn Gly Asn Lys

340 345 350

Pro Ile Trp Thr Arg Asn Ala Arg Glu Ile Glu Asn Glu Glu Tyr Ser

355 360 365

Glu Phe Tyr Lys Ser Phe Thr Lys Asp Ser Asp Asp Pro Leu Thr Lys

370 375 380

Thr His Phe Asn Val Glu Gly Glu Val Thr Phe Lys Ser Ile Leu Phe

385 390 395 400

Val Pro Gly Lys Ala Pro Ser Glu Met Phe Lys Asp Tyr Gly Lys His

405 410 415

Phe Asp Ser Ile Lys Leu Tyr Val Arg Arg Val Phe Ile Thr Glu Asp

420 425 430

Phe Glu Asp Met Met Pro Lys Tyr Leu Ser Phe Ile Lys Gly Val Val

435 440 445

Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg Glu Thr Leu Gln Gln

450 455 460

His Lys Leu Leu Lys Val Ile Lys Lys Lys Leu Val Arg Lys Thr Leu

465 470 475 480

Asp Met Ile Lys Lys Ile Asp Ala Asp Asp Tyr Ile Glu Lys Phe Trp

485 490 495

Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val Ile Glu Asp His Ser

500 505 510

Asn Arg Thr Arg Leu Ala Lys Leu Val Arg Phe Gln Ser Ser Asn Ser

515 520 525

Asp Thr Glu Tyr Thr Ser Leu Glu Glu Tyr Val Glu Arg Met Lys Glu

530 535 540

Lys Gln Glu His Ile Tyr Phe Met Ala Gly Ser Ser Arg Gln Glu Val

545 550 555 560

Glu Ser Ser Pro Phe Ser Glu Lys Leu Leu Lys Lys Gly Tyr Glu Val

565 570 575

Leu Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys Ile Gln Ser Leu Pro

580 585 590

Glu Phe Glu Gly Lys Lys Phe Gln Asn Ile Ala Lys Glu Gly Leu Val

595 600 605

Leu Asp Glu Ser Glu Lys Ala Lys Glu Arg Lys Glu Glu Leu Glu Lys

610 615 620

Glu Tyr Glu Gly Leu Thr Glu Trp Leu Lys Glu Thr Ala Leu Lys Asp

625 630 635 640

Lys Ile Lys Asp Ala Lys Ile Ser Ser Arg Leu Thr Glu Thr Pro Cys

645 650 655

Ala Leu Val Ala Gly Thr Tyr Gly Trp Ser Gly Asn Met Glu Arg Ile

660 665 670

Met Leu Ser Gln Ala Tyr Ser Gln Pro Gly Ser Asp Ser Ser Ser Phe

675 680 685

Tyr Lys Gly Gln Lys Lys Thr Leu Glu Ile Asn Val Arg His Pro Leu

690 695 700

Ile Lys Lys Leu Ala Ala Gln Val Glu Glu Asp Gly Glu Ser Gln Thr

705 710 715 720

Ala Lys Asp Leu Ala Glu Val Leu Phe Glu Thr Ala Val Leu Arg Ser

725 730 735

Gly Phe Met Leu Glu Asp Ser Ala Ala Phe Ala Gly Arg Ile Glu Arg

740 745 750

Met Leu Arg Leu Ser Met Asp Ile Asp Leu Asp Glu Lys Ile Glu Glu

755 760 765

Glu Pro Glu Glu Glu Asp Asp Glu Asp Glu Asp Glu Glu Glu Glu Glu

770 775 780

Glu Glu Glu Asp Glu Glu Glu Glu Asp Asp Asp Glu Glu Glu Glu Gly

785 790 795 800

Asp Gly Glu Val Glu Gly Glu Glu Asp Ala Asp His Asp Glu Leu

805 810 815

<210> 84

<211> 153

<212> PRT

<213> Apostichopus japonicus

<400> 84

Ala Thr Gln Lys Thr Val Asp Gly Pro Ser Ala Lys Lys Trp Arg Asp

1 5 10 15

Gly Arg Gly Ala Ala Gln Asn Ile Ile Pro Ala Ser Thr Gly Ala Ala

20 25 30

Lys Ala Val Gly Lys Val Ile Pro Asp Leu Asn Gly Lys Leu Thr Gly

35 40 45

Met Ala Phe Arg Val Pro Val Pro Asp Val Ser Val Val Asp Leu Thr

50 55 60

Val Arg Leu Lys Lys Lys Ala Ser Tyr Asp Asp Ile Lys Ala Val Ile

65 70 75 80

Lys Lys Ala Ala Ser Ser Thr Ser Leu Gln Ser Ile Leu Gly Tyr Thr

85 90 95

Glu Asp Gln Val Val Ser Gln Asp Phe Arg Gly Asp Thr Arg Ser Ser

100 105 110

Ile Phe Asp Ala Lys Ala Gly Ile Ala Leu Asn Glu Asn Phe Val Lys

115 120 125

Leu Val Ser Trp Tyr Asp Asn Glu Tyr Gly Tyr Ser His Arg Val Ile

130 135 140

Asp Leu Ile Leu Tyr Met Ala Lys Lys

145 150

<210> 85

<211> 123

<212> PRT

<213> Apostichopus japonicus

<400> 85

Met Ala Pro Lys Ala Pro Gly Lys Gly Ala Lys Lys Ala Ala Lys Ser

1 5 10 15

Lys Ala Pro Arg Ala Pro Gly Asp Arg Lys Arg Lys Arg Thr Arg Arg

20 25 30

Glu Ser Tyr Ser Ile Tyr Ile Tyr Lys Val Met Lys Gln Val His Pro

35 40 45

Asp Thr Gly Ile Ser Ser Arg Ala Met Ser Ile Met Asn Ser Phe Val

50 55 60

Asn Asp Ile Phe Glu Arg Ile Ala Ala Glu Ala Ser Arg Leu Ala His

65 70 75 80

Tyr Asn Arg Lys Ser Thr Ile Thr Ser Arg Glu Val Gln Thr Ala Val

85 90 95

Arg Leu Leu Leu Pro Gly Glu Leu Ala Lys His Ala Val Ser Glu Gly

100 105 110

Thr Lys Ala Val Thr Lys Tyr Thr Thr Ser Lys

115 120

<210> 86

<211> 375

<212> PRT

<213> Apostichopus japonicus

<400> 86

Met Glu Asp Glu Val Gln Ala Leu Val Ile Asp Asn Gly Ser Gly Met

1 5 10 15

Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe Pro

20 25 30

Ser Leu Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly Met Gly

35 40 45

Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile

50 55 60

Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp

65 70 75 80

Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val

85 90 95

Ala Pro Glu Glu His Pro Val Leu Cys Thr Glu Ala Pro Leu Asn Pro

100 105 110

Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe Asn

115 120 125

Thr Pro Ala Phe Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr Ala

130 135 140

Ser Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Ser

145 150 155 160

His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu

165 170 175

Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile

180 185 190

Leu Thr Glu Arg Gly Tyr Thr Phe Thr Thr Thr Ala Glu Arg Glu Ile

195 200 205

Val Arg Asp Ile Lys Glu Lys Leu Asn Tyr Val Ala Leu Asp Phe Glu

210 215 220

Gln Glu Met Gln Thr Ala Ala Ser Ser Ser Ser Leu Glu Lys Ser Tyr

225 230 235 240

Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe Arg

245 250 255

Cys Pro Glu Ala Met Phe Gln Pro Ser Phe Leu Gly Met Glu Ser Ala

260 265 270

Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp Val Asp

275 280 285

Ile Arg Lys Asp Leu Tyr Ala Asn Ile Val Leu Ser Gly Gly Thr Thr

290 295 300

Met Phe Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Ser Ala Leu

305 310 315 320

Ala Pro Pro Thr Met Lys Ile Lys Val Ile Ala Pro Pro Glu Arg Lys

325 330 335

Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe

340 345 350

Gln Gln Met Trp Ile Ser Lys Glu Glu Tyr Asp Glu Ser Gly Pro Ser

355 360 365

Ile Val His Arg Lys Cys Phe

370 375

<210> 87

<211> 103

<212> PRT

<213> Apostichopus japonicus

<400> 87

Met Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala

1 5 10 15

Lys Arg His Arg Lys Val Leu Arg Asp Asn Ile Gln Gly Ile Thr Lys

20 25 30

Pro Ala Ile Arg Arg Leu Ala Arg Arg Gly Gly Val Lys Arg Ile Ser

35 40 45

Gly Leu Ile Tyr Glu Glu Thr Arg Gly Val Leu Lys Val Phe Leu Glu

50 55 60

Asn Val Ile Arg Asp Ala Val Thr Tyr Cys Glu His Ala Lys Arg Lys

65 70 75 80

Thr Val Thr Ala Met Asp Val Val Tyr Ala Leu Lys Arg Gln Gly Arg

85 90 95

Thr Leu Tyr Gly Phe Gly Gly

100

<210> 88

<211> 111

<212> PRT

<213> Apostichopus japonicus

<400> 88

Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala Pro Arg Lys Gln Leu Ala

1 5 10 15

Thr Lys Ala Ala Arg Lys Ser Ala Pro Ala Thr Gly Gly Val Lys Lys

20 25 30

Pro His Arg Tyr Arg Pro Gly Thr Val Ala Leu Arg Glu Ile Arg Arg

35 40 45

Tyr Gln Lys Ser Thr Glu Leu Leu Ile Arg Lys Leu Pro Phe Gln Arg

50 55 60

Leu Val Arg Glu Ile Ala Gln Asp Phe Lys Thr Glu Leu Arg Phe Gln

65 70 75 80

Ser Ser Ala Val Met Ala Leu Gln Glu Ala Ser Glu Ala Tyr Leu Val

85 90 95

Gly Leu Phe Glu Asp Thr Asn Leu Cys Ala Ile His Ala Lys Arg

100 105 110

<210> 89

<211> 125

<212> PRT

<213> Apostichopus japonicus

<400> 89

Met Ser Trp Asp Ala Tyr Ile Thr Asn Leu Leu Ala Thr Asn Tyr Val

1 5 10 15

Thr Glu Gly Val Ile Leu Gly His Asn Gly Ser Val Trp Ala Ala Ser

20 25 30

Pro Gly Leu Lys Ile Gln Pro Ala Glu Ile Thr Ala Leu Ile Ala Gly

35 40 45

Phe Lys Glu Asn Ser Pro Leu His Ala Thr Gly Val His Ile Asn Gly

50 55 60

Val Lys Tyr Phe Thr Leu Arg Ala Asn Asp Asn Glu Val Leu Ala Lys

65 70 75 80

Lys Gly Pro Thr Gly Ile Ala Cys Tyr Lys Thr Thr Gln Ala Ile Val

85 90 95

Ile Gly Phe His Pro Glu Ser Val Gln Pro Gly Gln Cys Thr Thr Glu

100 105 110

Val Ala Lys Val Ala Asp Tyr Leu Arg Glu Gln Gly Tyr

115 120 125

<210> 90

<211> 364

<212> PRT

<213> Apostichopus japonicus

<400> 90

Met Pro Tyr Gln Tyr Pro Ala Leu Thr Pro Glu Gln Lys Lys Glu Leu

1 5 10 15

Ser Asp Ile Ala His Arg Ile Val Ala Pro Gly Lys Gly Ile Leu Ala

20 25 30

Ala Asp Glu Ser Thr Gly Ser Ile Ala Lys Arg Leu Gln Ser Ile Gly

35 40 45

Thr Glu Asn Thr Glu Glu Asn Arg Arg Phe Tyr Arg Gln Leu Leu Leu

50 55 60

Thr Ala Asp Asp Arg Val Asn Pro Cys Ile Gly Gly Val Ile Leu Phe

65 70 75 80

His Glu Thr Leu Tyr Gln Lys Ala Asp Asp Gly Arg Pro Phe Pro Gln

85 90 95

Val Ile Lys Ser Lys Gly Gly Val Val Gly Ile Lys Val Asp Lys Gly

100 105 110

Val Val Pro Leu Ala Gly Thr Asn Gly Glu Thr Thr Thr Gln Gly Leu

115 120 125

Asp Gly Leu Ser Glu Arg Cys Ala Gln Tyr Lys Lys Asp Gly Ala Asp

130 135 140

Phe Ala Lys Trp Arg Cys Val Leu Lys Ile Gly Glu His Thr Pro Ser

145 150 155 160

Ala Leu Ala Ile Met Glu Asn Ala Asn Val Leu Ala Arg Tyr Ala Ser

165 170 175

Ile Cys Gln Gln Asn Gly Ile Val Pro Ile Val Glu Pro Glu Ile Leu

180 185 190

Pro Asp Gly Asp His Asp Leu Lys Arg Cys Gln Tyr Val Thr Glu Lys

195 200 205

Val Leu Ala Ala Val Tyr Lys Ala Leu Ser Asp His His Ile Tyr Leu

210 215 220

Glu Gly Thr Leu Leu Lys Pro Asn Met Val Thr Pro Gly His Ala Cys

225 230 235 240

Thr Gln Lys Phe Ser His Glu Glu Ile Ala Met Ala Thr Val Thr Ala

245 250 255

Leu Arg Arg Thr Val Pro Pro Ala Val Thr Gly Ile Thr Phe Leu Ser

260 265 270

Gly Gly Gln Ser Glu Glu Glu Ala Ser Ile Asn Leu Asn Ala Ile Asn

275 280 285

Lys Cys Pro Leu Leu Lys Pro Trp Ala Leu Thr Phe Ser Tyr Gly Arg

290 295 300

Ala Leu Gln Ala Xaa Ala Leu Lys Ala Trp Gly Gly Lys Lys Glu Asn

305 310 315 320

Leu Lys Ala Ala Gln Glu Glu Tyr Val Lys Arg Ala Leu Ala Asn Ser

325 330 335

Leu Ala Cys Gln Gly Lys Tyr Thr Pro Ser Gly Gln Ala Gly Ala Ala

340 345 350

Ala Ser Glu Ser Leu Phe Val Ser Asn His Ala Tyr

355 360

<210> 91

<211> 917

<212> PRT

<213> Apostichopus japonicus

<400> 91

Met Ser Gln Pro Gln Gly Arg Pro Arg Gly Arg Ala Arg Gly Arg Gly

1 5 10 15

Arg Pro Ser Gly Thr Ala Ala Pro Pro Gln Gln Glu Ala Gln Arg Pro

20 25 30

Gly Glu Pro Pro Gln Pro Arg Ala Pro Pro Pro Ser Asp Val Gln Val

35 40 45

Pro Val Pro Gly Arg Gly Arg Ser Arg Gly Ala Met Ala Gln Val Ala

50 55 60

Pro Gly Gly Ala Pro Ala Gln Ala Pro Ala Pro Pro Thr Ala Gln Met

65 70 75 80

Ala Ala Met Gln Val Arg Ala Gln Ala Gly Gly Glu Pro Met Gly Gly

85 90 95

Ala Thr Arg Arg Arg Arg Asp Glu Val Phe Ile Glu Pro Pro Thr Arg

100 105 110

Gly Asp Leu Pro Asp Lys Arg Gly Thr Ser Gly Thr Pro Val Lys Leu

115 120 125

Leu Thr Asn Cys Phe Lys Met Lys Thr Pro Gln Asn Trp Leu Ile Tyr

130 135 140

Gln Tyr Lys Val Ser Phe Glu Pro Pro Ile Asp Tyr Lys Lys Val Arg

145 150 155 160

Leu Ala Val Met Gly Ser Leu Arg His Ile Phe Ile Asn Glu Gln Tyr

165 170 175

Val Phe Asp Gly Glu Thr Met Phe Thr Ile Thr Arg Phe Asn Glu Lys

180 185 190

Ile Thr Arg His Val Val Gln Arg Arg Pro Met Arg Asp Gly Thr Pro

195 200 205

Asn Pro Asp Val Glu Ile Thr Leu Glu Leu Thr Asn Glu Cys Pro Pro

210 215 220

Gly Ser Ala Glu His Leu Arg Ile Phe Asn Ile Ile Phe Lys Arg Ile

225 230 235 240

Leu Lys Met Met Lys Leu Glu Gln Ile Gly Arg Asn Tyr Phe Asn Pro

245 250 255

His Asp Ala Pro Gln Ile Pro Gln His Asn Leu Thr Leu Trp Pro Gly

260 265 270

Tyr Val Thr Ser Ile Ser Gln Phe Glu Glu Asn Val Met Leu Met Ala

275 280 285

Asp Leu Thr Phe Lys Ile Leu His Thr Asp Ser Val Leu Asn Val Phe

290 295 300

Arg Asp Leu His Asp Lys Val Arg Gly Asn Ala Ser Gln Phe Arg Asp

305 310 315 320

Leu Ala Thr Lys Lys Leu Val Gly Glu Ile Val Met Thr Arg Tyr Asn

325 330 335

Asn Lys Thr Tyr Arg Val Asp Glu Ile Val Trp Asp Gln Asn Pro Gln

340 345 350

Ser Lys Phe Ser Thr Pro Arg Gly Asp Ile Ser Phe Val Glu Tyr Tyr

355 360 365

Glu Lys Gln Tyr Glu Arg Lys Ile Thr Asp Pro Thr Gln Pro Leu Leu

370 375 380

Met Ser Leu Pro Lys Ala Lys Gln Thr Ala Glu Ala Arg Gly Pro Ile

385 390 395 400

Leu Leu Val Pro Glu Leu Cys Leu Met Thr Gly Leu Ser Glu Glu Met

405 410 415

Arg Ser Asn Phe Thr Ile Met Lys Asp Val Gly Val His Thr Arg Val

420 425 430

Val Pro Gly Gln Arg Thr Arg Glu Leu Gln Lys Phe Leu Asn Gln Ile

435 440 445

Asn Asn Asn Thr Glu Ile Val Glu Tyr Thr Lys Lys Trp Gln Val Glu

450 455 460

Phe Ser Pro Gln Leu Glu Glu Ile Gln Ala Arg Ile Leu Ala Pro Glu

465 470 475 480

Asp Leu Tyr Gln Leu Gln Ser Ser Arg Gly Gln Met Gly Glu Thr Cys

485 490 495

Tyr Ser Tyr Gln Gln His Glu Ala Asp Trp Ser Arg Ser Leu Arg Gly

500 505 510

Asn Ile Leu Arg Ser Ala Val Asn Leu Asp Gln Trp Leu Phe Ile Cys

515 520 525

Pro Arg Arg Glu Met Asn Asn Gly Ser Ser Phe Val Asp Asn Leu Lys

530 535 540

Lys Val Gly Pro Pro Met Gly Met Gln Ile Ala Asp Pro Glu Lys Val

545 550 555 560

Ile Ile Asn Asp Asp Arg Ile Asp Ser Tyr Val Ser Thr Ile Arg Gly

565 570 575

Ser Tyr Gly Lys His Gln Leu Val Met Cys Ile Leu Thr Ser Ala Arg

580 585 590

Lys Glu Arg Tyr Asp Ser Ile Lys Lys Val Cys Val Asn Glu Cys Pro

595 600 605

Ile Pro Ser Gln Met Ile Leu Ser Lys Thr Ile Phe Lys Lys Asn Thr

610 615 620

Leu Met Ser Val Ala Thr Lys Val Ala Met Gln Leu Asn Cys Lys Leu

625 630 635 640

Gly Gly Asp Leu Trp Arg Leu Asp Ile Pro Met Ser Asp Leu Met Val

645 650 655

Val Gly Ile Asp Cys Tyr His Asp Ser Ser Thr Arg Gly Arg Ser Ala

660 665 670

Val Gly Phe Val Ala Ser Met Asn Lys Thr Leu Thr Lys Tyr His Ser

675 680 685

Arg Cys Thr Phe Gln Thr Thr His Gln Glu Ile Ile Asp Asn Leu Met

690 695 700

Tyr Cys Met Thr Ala Cys Leu Lys Arg Tyr His Glu Ile Asn Lys Ser

705 710 715 720

Tyr Pro Gln Lys Ile Val Val Tyr Arg Asp Gly Val Gly Asp Gly Gln

725 730 735

Leu Pro Ala Val Val Gln His Glu Val Pro Gln Leu Glu Asp Cys Phe

740 745 750

Ser Arg Val Ile Pro Asn Tyr Lys Pro Lys Leu Ser Val Val Ile Val

755 760 765

Lys Lys Arg Ile Asn Asn Arg Phe Phe Leu Pro Ser Asn Gly Gly Gly

770 775 780

Gly Gly Tyr Arg Gly Gly Gly Arg Gly Asp Arg Gly Gly Arg Gly Gly

785 790 795 800

Arg Gly Arg Gly Gly Gly Gly Asp Asp Gly Gly Met Val Asn Pro Pro

805 810 815

Pro Gly Thr Val Val Asp Ser Val Val Thr Lys Ser Gln Leu Phe Asp

820 825 830

Phe Phe Leu Val Ser Gln Ser Val Arg Gln Gly Thr Val Ser Pro Thr

835 840 845

Ser Tyr Asn Val Val Lys Asn Gly Val Gly Leu Lys Pro Asp His Leu

850 855 860

Gln Arg Leu Thr Tyr Lys Leu Thr His Leu Tyr Phe Asn Trp Pro Gly

865 870 875 880

Thr Ile Arg Val Pro Ala Pro Cys Leu Tyr Ala His Lys Leu Ala Phe

885 890 895

Leu Val Gly Gln Ser Leu His Gln Leu Pro Pro Pro Asp Leu Ala Asp

900 905 910

Thr Leu Phe Tyr Leu

915

<210> 92

<211> 530

<212> PRT

<213> Apostichopus japonicus

<400> 92

Met Gly His Phe Ala Arg Glu Cys Pro Asn Ala Glu Arg Val Asp Ala

1 5 10 15

Glu Asn Gly Asp Asp Arg Pro Ala Ala Ala Asp Tyr Val Pro Pro Glu

20 25 30

Pro Ser Gln Asp Glu Glu Val Ile Phe Ser Thr Ile Gln Lys Gly Ile

35 40 45

Asn Phe Asp Lys Tyr Asp Asp Ile Pro Val Glu Val Ser Gly Asn Asp

50 55 60

Pro Cys Arg Cys Ile Asn Ser Phe Gly Glu Ala Asp Leu Tyr Asp Ser

65 70 75 80

Ile Gly Glu Asn Val Arg Arg Ala Lys Tyr Asp Lys Pro Thr Pro Val

85 90 95

Gln Lys Tyr Gly Ile Pro Ile Val Ser Ala Gly Arg Asp Leu Met Ala

100 105 110

Cys Ala Gln Thr Gly Ser Gly Lys Thr Ala Ala Phe Leu Leu Pro Ile

115 120 125

Leu Ser Gly Leu Leu Arg Asp Gly Leu Gln Ser Ser Ala Leu Ser Gly

130 135 140

Gln Gln Cys Pro Gln Cys Ile Val Val Ser Pro Thr Arg Glu Leu Ala

145 150 155 160

Ile Gln Ile Phe Asp Glu Ala Arg Lys Phe Ser Tyr Lys Thr Met Ile

165 170 175

Lys Cys Val Val Ile Tyr Gly Gly Thr Lys Val Gln His Gln Thr Ser

180 185 190

Met Val Asp Arg Gly Cys Asn Val Leu Val Ala Thr Pro Gly Arg Leu

195 200 205

Leu His Phe Ile Asn Ser Gly Met Ile Ser Val Glu Lys Val Lys Tyr

210 215 220

Leu Val Leu Asp Glu Ala Asp Arg Met Leu Asp Met Gly Phe Gly Pro

225 230 235 240

Asp Met Glu Lys Leu Val Asn Asn Pro Ala Met Pro Lys Lys Gly Glu

245 250 255

Arg His Thr Leu Met Phe Ser Ala Thr Phe Pro Asn Glu Val Gln Glu

260 265 270

Arg Ala Ala Glu Tyr Leu Asp Asn Tyr Leu Phe Leu Thr Val Gly Arg

275 280 285

Val Gly Gly Ala Asn Ser Asp Val Arg Gln Val Val Val Glu Val Pro

290 295 300

Gln Ala Ala Lys Lys Asp Lys Leu Ile Glu Ile Leu Gln Ser Gln Pro

305 310 315 320

Glu Asn Asp Arg Thr Leu Val Phe Thr Glu Thr Lys Arg Gly Ala Asp

325 330 335

Phe Leu Ala Ser Tyr Leu Ser Gln Ser Gly Phe Pro Thr Thr Ser Ile

340 345 350

His Gly Asp Arg Glu Gln Arg Glu Arg Glu Glu Ala Leu Asn Asp Phe

355 360 365

Lys Arg Asn Arg Ala Ser Val Leu Val Ala Thr Ser Val Ala Ser Arg

370 375 380

Gly Leu Asp Ile Pro Ala Val Lys His Val Val Asn Phe Asp Leu Pro

385 390 395 400

Ser Glu Ile Asp Asp Tyr Val His Arg Ile Gly Arg Thr Gly Arg Cys

405 410 415

Gly Asn Thr Gly Leu Ser Thr Ser Phe Tyr Asn Pro Glu Lys Asp Ala

420 425 430

Thr Ile Ser Arg Ala Leu Ile Lys Ile Leu Ala Asp Ala His Gln Asp

435 440 445

Val Pro Glu Phe Leu Glu Asn Val Ala Asp Ser Ala Ile Gly Thr Tyr

450 455 460

His Gly Asn Thr Gly Gly Gly Phe Gly Gly Arg Asp Thr Arg Ser Gly

465 470 475 480

Pro Arg Arg Gly Gly Gly Gly Gly Gly Asn Arg Gly Asn Arg Asp Trp

485 490 495

Asn Ser Gly Gly Gln Gly Gly Gly Gln Glu Ser Trp Asn Ser Gly Gly

500 505 510

Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala Glu Asp Glu Glu Ala

515 520 525

Trp Asp

530

Claims (7)

1.一组用来鉴别刺参(A.Japonicus)产地的区域敏感性蛋白组，所述区域敏感性蛋白组为，选自不同产地海参蛋白组中，在不同产地的刺参中含量差异显著且能在统计学上对海参产地进行判别区分的一组蛋白，其特征在于，所述的蛋白组中包含序列如SEQ IDNo.76～92所示的17种特征蛋白，所述的不同产地为：霞浦、大连、烟台、胶南、威海。

2.一组用来鉴别刺参产地的区域敏感性多肽组，其特征在于，所述的区域敏感性多肽组通过酶解权利要求1所述的蛋白组获得，所述的不同产地为：霞浦、大连、烟台、胶南、威海；所述的区域敏感性多肽组中的多肽序列及其质谱检测特征质荷比离子(m/z)分别为：

SEQ ID NO.1：QEYDESGPSIVHR，368.9；

SEQ ID NO.2：ANDNEVLAK，402.7；

SEQ ID NO.3：SVLSGGTSMYPGIADR，952.9；

SEQ ID NO.4：QLLLAADDR，491.6；

SEQ ID NO.5：VIAGLNVMR，844.9；

SEQ ID NO.6：QPQGRPNGR，543.1；

SEQ ID NO.7：NSFVNDIFER，522.5；

SEQ ID NO.8：EIAQDFK，736.8；

SEQ ID NO.9：ASYDDIK，648.1；

SEQ ID NO.10：GEAGAPGATGPQGPAGQR，487.5；

SEQ ID NO.11：SFVNDIFER，409.4；

SEQ ID NO.12：YGIPIVSAGR，446.0；

SEQ ID NO.13：GYTFTTTAER，391.4；

SEQ ID NO.14：GEQGPPGGPGLGGQQGPR，772.8；

SEQ ID NO.15：DLYANSVLSGGTSMYPGIADR，517.1；

SEQ ID NO.16：DYDSEYTVPVISR，660.7；

SEQ ID NO.17：QLETELDDTSEK，738.8；

SEQ ID NO.18：IQPAEITALIAGFK，615.2；

SEQ ID NO.19：DNIQGITK，496.6；

SEQ ID NO.20：TVTAMDVVY，416.4；

SEQ ID NO.21：FEELNMELFR，653.2；

SEQ ID NO.22：VGLQVAAVT，667.7；

SEQ ID NO.23：IIEYFGLK，776.3；

SEQ ID NO.24：VTSFPTLK，531.1；

SEQ ID NO.25：VDQLVKGHFDVK，790.3；

SEQ ID NO.26：EGDILTKWEQR，823.8；

SEQ ID NO.27：DSTLIMQLLR，685.7；

SEQ ID NO.28：LLLPGELAK，384.4；

SEQ ID NO.29：DDFPNFEGHK，398.4；

SEQ ID NO.30：YLTADMYAK，495.5；

SEQ ID NO.31：AQQTAEEEMANLQR，704.7；

SEQ ID NO.32：LTGMAFR，590.6；

SEQ ID NO.33：GAAGMDGATGPR，444.9；

SEQ ID NO.34：VFLENVIR，810.3；

SEQ ID NO.35：GGDGQVGPPGPAGPQGEVGDR，647.2；

SEQ ID NO.36：GESIDDLVPK，591.1；

SEQ ID NO.37：MVSEALNSLAADLK，357.8；

SEQ ID NO.38：ISGLIYEETR，416.4；

SEQ ID NO.39：GAAQNIIPASTGAAK，425.9；

SEQ ID NO.40：DLAEVLFETAVLR，421.0；

SEQ ID NO.41：ETYSIYIYK，543.1；

SEQ ID NO.42：TVTAMDVVYALK，507.6；

SEQ ID NO.43：LTPEDIEK，506.5；

SEQ ID NO.44：ITITNDQNR，656.2；

SEQ ID NO.45：GILAADESTGSMAK，748.3；

SEQ ID NO.46：IIPASTGAAK，505.5；

SEQ ID NO.47：GGLLIPEK，492.0；

SEQ ID NO.48：TSLDQLEDELMIEK，447.0；

SEQ ID NO.49：VDATEETELAQK，455.5；

SEQ ID NO.50：DVATPSGYTLDR，554.6；

SEQ ID NO.51：ADIAEESLK，663.1；

SEQ ID NO.52：LVSIGTENTEENR，574.1；

SEQ ID NO.53：VLIEMEK，586.7；

SEQ ID NO.54：VLDATVVAVGSGLR，398.4；

SEQ ID NO.55：ADLVNNLGTIAK，643.7；

SEQ ID NO.56：EITALAPPTMK，567.1；

SEQ ID NO.57：SGSISITK，1094.7；

SEQ ID NO.58：LGPLTVLK，806.4；

SEQ ID NO.59：MLIPLLEGK，465.0；

SEQ ID NO.60：PSTQIMQLLR，731.8；

SEQ ID NO.61：SAAFESSLSQMK，431.5；

SEQ ID NO.62：IGSVFESVNR，537.0；

SEQ ID NO.63：VDEFTGIVGSLR，538.6；

SEQ ID NO.64：ELEEIVQPIVSK，443.9；

SEQ ID NO.65：ETLDLIK，508.0；

SEQ ID NO.66：NENLALIEK，500.1；

SEQ ID NO.67：ENANVLAR，676.2；

SEQ ID NO.68：VIAPPER，406.4；

SEQ ID NO.69：DEEGIISWVEK，488.1；

SEQ ID NO.70：VLLPEYGGTK，477.5；

SEQ ID NO.71：LLVPLLEGK，446.9；

SEQ ID NO.72：DSLVDIR，564.1；

SEQ ID NO.73：IFQIIFE，621.1；

SEQ ID NO.74：VADYLR，832.9；

SEQ ID NO.75：STELLIR，488.5。

3.一种检测刺参产地的方法，其特征在于，所述方法包括如下步骤：

(1)对待测样本进行蛋白组测定前处理，获得权利要求1所述蛋白组溶液或权利要求2所述多肽组溶液：

(2)通过液相色谱-质谱法测定所述的区域敏感性蛋白组和/或区域敏感性多肽组成分，对照标准品的信息，判断刺参产地来源。

4.根据权利要求3所述的方法，其特征在于，所述的对待测样本进行蛋白组测定前处理，获得待测蛋白组溶液或多肽组溶液步骤如下：

(1)称取一定量海参样品均质成粉末状态，加入蛋白提取液震荡提取蛋白，高速低温离心，取上清液；

(2)将DTT加入到步骤(1)所获得的上清液中，反应一定时间得反应液1；

(3)将现配的碘乙酰胺加入到已经冷却至室温的步骤(2)所获得反应液1中，室温避光反应的反应液2；

(4)采用10K滤膜超滤30分钟步骤(3)所获得的反应液2，使用碳酸氢铵溶液反复冲洗滤膜获得蛋白组溶液；

(5)再加入碳酸氢铵溶液重复此步骤，合并两次溶液即得蛋白组溶液；

(6)使用胰酶溶液消化步骤(5)所获得的蛋白组溶液中16-18小时；

(7)采用10K滤膜超滤，收集下层的滤液即为多肽组溶液。

5.根据权利要求3所述的方法，其特征在于，所述的通过液相色谱-质谱法测定所述的区域敏感性蛋白组和/或区域敏感性多肽组成分的方法为，

方法(1)：采用液相色谱-四级杆/飞行时间质谱检测，

液相色谱条件：

流动相A：0.1％甲酸-乙腈，流动相B：0.1％甲酸-水，

流速：0.25mL/min，

质谱条件：

TOF扫描范围：350-1500Da，

正离子反应模式，GS1：35，GS2：45，Curtain Gas：35，ISVF：5500，TEM：500，DP：100，CE：10；

方法(2)：采用液相色谱-三重四级杆检测，

流动相A：0.1％甲酸-乙腈，流动相B：0.1％甲酸-水，

流速：0.3mL/min，

电喷雾离子源，正离子反应模式，检测方式：MRM，喷雾电压：5500V，离子传输管温度：475℃；鞘气压力：40；辅助气压力：6。

6.使用权利要求1所述蛋白组和/或权利要求2所述多肽组制备的用于鉴别刺参产地的检测试剂或检测试剂盒。

7.权利要求1所述蛋白组和/或权利要求2所述多肽组在制备用于鉴别刺参产地的试剂或试剂盒中的应用。

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