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蛋白的用途和筛选方法

文献发布时间:2023-06-19 16:09:34



技术领域

本发明属于生物技术领域,具体涉及一种蛋白的用途和筛选方法。

背景技术

发情鉴定是母猪繁殖生产中的重要内容,准确判断母猪是否发情,对于及时发现并解决母猪发情状况中的问题,适时开展配种,提高受胎率,增加经济效益具有指导意义。目前生产上常用的发情鉴定方法包括观察母猪发情表现、公猪试情法,这些方法对技术人员的经验要求很高,准确性和时机性把握较难,而基于体温变化进行数据化管理的方法需要消耗大量资金投入,管理维护困难,因而急需寻找更加简易和准确的发情鉴定方法。

血液、尿液、唾液等液体中存在种类多样的蛋白、类固醇、前列腺素等生物分子,这些分子在动物体内发挥重要的功能,其合成随动物所处的生理状态而发生明显的变化;此外,这些液体具有采集方便、分析样品量充足、成本低、稳定性强、且对动物应激小等特点,因而在家畜生理健康、疾病监控和早期诊断方面具有重要的生物学意义。

CN202110437164.0公开了一种用于母猪早期妊娠诊断的蛋白生物标志物,其特征在于:蛋白生物标志物为下列蛋白标志物之一:FLNC蛋白;ADIPOQ蛋白;HP蛋白;TERF2蛋白;母猪早期妊娠诊断方法,其特征在于:具体步骤如下:①、采集血样:采集配种后15天的母猪外周血;②、血样处理:对①步骤的血样进行分离血清或血浆处理:③、试剂盒检测:选用ELISA试剂盒对②步骤处理后的血样进行检测。

CN202010525872.5公开了一种用于母猪早期妊娠诊断的外泌体miRNA标志物及其应用,外泌体miRNA分子标志物包括ssc-miR-192、ssc-miR-146a-5p和ssc-miR-149,单独使用血清外泌体miRNA诊断区分早期妊娠与未妊娠母猪时,ROC曲线下面积(AUC)分别为0.863、0.885、0.857;ssc-miR-192、ssc-miR-146a-5p联合AUC为0.921;ssc-miR-192、ssc-miR-149联合AUC为0.902;ssc-miR-146a-5p、ssc-miR-149联合AUC为0.910,ssc-miR-192、ssc-miR-146a-5p、ssc-miR-149三种miRNA联合时,其AUC为0.959。

CN201911011796.X公开了一种血清外泌体ssc-miR-92b-3p作为母猪早期妊娠诊断分子标志物的应用。该发明确定了最早在怀孕的第9天血清中外泌体miRNA的水平变化并确定了在妊娠第9至15天期间ssc-miR-92b-3p表达的水平增加,该miRNA可以作为猪早期妊娠的新的生物标记物。

可见,本领域中技术人员已经针对猪血清、分泌物等物质中的蛋白进行分析,以尽早检测母猪妊娠。

需要说明的是,针对于母猪发情诊断还没有任何人提出过相应的鉴别指标。

CN201810106686 .0公开了一种新疆多浪羊不同生理时期繁殖相关蛋白的筛选与鉴定方法,属于生物技术领域,该方法包括以下步骤:步骤1、新疆多浪羊不同生理时期卵巢的获得:步骤2、蛋白提取;步骤3、Bradford蛋白定量和标准曲线的绘制;步骤4、样品标记和等量混合;步骤5、高PH条件下C18色谱柱的HPLC分级;步骤6、质谱分析步骤7、数据处理。本发明通过iTRAQ试剂标记,被标记的肽段进行高效液相色谱HPLC分离及质谱检测MS,共鉴定出4847个蛋白,其中表达显著差异的蛋白470个;但是该方案并未指出与发情相关的蛋白的相关情况和序列。

CN02113263.1 公开了一种可在现场快速诊断奶牛早孕与发情用的酶免疫分析测试盒,包括固相载体和检测用试剂两部分。检测试剂中有以抗孕酮抗体为第一抗体,还包括有抗第一抗体的第二抗体。检测试剂的组成为:使用前以磷酸盐缓冲液溶解的酶标孕酮,以磷酸盐缓冲液配制的孕酮标准品,使用前以磷酸盐缓冲液稀释的抗孕酮抗体,使用前以碳酸盐缓冲液稀释的抗第一抗体的第二抗体,过氧化氢和含有四甲基联苯胺缓冲液的第一和第二显色剂,pH7-7.4的磷酸盐缓冲液和pH9-9.8的碳酸盐缓冲液。用该发明测试盒可以在现场由奶牛的奶样品快速准确地判断奶牛是否已受孕或发情的分析检测结果。

但是该发明采用的是第一抗体的第二抗体来进行检测,其无法对猪的发情进行有效预测和判断。

为了解决母猪发情难于预测的问题,有申请人提出了信息化的判断方法,如CN201911083451.5、CN201810383668.7通过母猪行为结合计算机来判断。

由此可见,现有技术中,对于母猪发情的预测和判断是非常具有难度的。

本案所要解决的技术问题是:如何对母猪的发情状况进行准确鉴别和量化。

发明内容

本发明的目的是提供一种蛋白的用途,该蛋白用于母猪发情阶段鉴别,可量化母猪发情周期内蛋白水平,本方案的鉴别准确性高、对母猪无损伤,检测指标量化,检测更直观。

本发明的另外一个目的在于,提出一种筛选此类蛋白的方法。

本发明的技术方案为:

蛋白的用途,所述蛋白为PHB domain-containing protein 蛋白、Growth factorreceptor bound protein 2蛋白、Elongation factor Tu蛋白、Carbonic anhydrase蛋白、Carboxypeptidase D 蛋白、Trefoil factor 3蛋白中任一种或多种组合:

PHB domain-containing protein(STOM, A0A4X1UP22)蛋白,其序列如氨基酸序列NO.1所示;

Growth factor receptor bound protein 2(GRB2, B6E241)蛋白,其序列如氨基酸序列NO.2所示;

Elongation factor Tu(A0A4X1UAP2)蛋白,其序列如氨基酸序列NO.3所示;

Carboxypeptidase D (CPD, F1RN68)蛋白,其序列如氨基酸序列NO.4所示;

Carbonic anhydrase(CA6, A0A4X1W9S1)蛋白,其序列如氨基酸序列NO.5所示;

Trefoil factor 3(TFF3, A0A4X1TI96)蛋白,其序列如氨基酸序列NO.6所示;

所述蛋白用作母猪发情鉴定的标志物。

氨基酸序列NO.1如下:

MRAPFSSKSSAQKGAFFFFVAFSQAITQLPLPSLFSSRPPAPRLLPLPSGAFSPFRCCQSPDPLFRGASPLRPRPPARGLEGSAPEAAAPEGRAVEGGGVSSLVLGVDWPGLREGAGSLPGSRDPESSRGIPLSSFSSSGPGDCDARVATLVRAACLTSGQQRTPKLGGSPTPSRIIKEYERAIIFRLGRILQGGAKGPGLFFILPCTDSFIKVDMRTISFDIPPQEILTKDSVTISVDGVVYYRVQNATLAVANITNADSATRLLAQTTLRNVLGTKNLSQILSDREEIAHNMQCTLDDATDDWGIKVERVEIKDVKLPVQLQRAMAAEAEASREARAKVIAAEGEMNASRALKEASMVITESPAALQLRYLQTLTTIAAEKNSTIVFPLPIDMLQGIMGARK

氨基酸序列NO.2如下:

MEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPKNYIEMKPHPWFFGKIPRAKAEEMLSKQRHDGAFLIRESESAPGDFSLSVKFGNDVQHFKVLRDGAGKYFLWVVKFNSLNELVDYHRSTSVSRNQQIFLRDIEQVPQQPTYVQALFDFDPQEDGELGFRRGDFIHVMDNSDPNWWKGACHGQTGMFPRNYVTPVNRNV

氨基酸序列NO.3如下:

MAPRIQEPPWTGSLKWPMLMSQTATQMREMTLDSCSPNSSSFCCRGVLSCSVAAIWSRIFPISVPSPEVDREGHRDSGLEVPKLALSALTSAGSRFQPQLPFAWCTRRGPCAVPTVPGARKTTMAAATLLRATPLFSGLGAGPTSLLQGLLRPLKAPALPLLCRGLAVEAKKTYVRDKPHVNVGTIGHVDHGKTTLTAAITKILAEGGGAKFKKYEEIDNAPEERARGITINAAHVEYSTANRHYAHTDCPGHADYVKNMITGTAPLDGCILVVAANDGPMPQTREHLLLAKQIGVEHVVVYVNKADAVQDSEMVELVELEIRELLTEFGYKGEETPIIVGSALCALEQRDPELGLKSVQKLLDAVDTYIPVPTRDLEKPFLLPVESVYSIPGRGTVVTGTLERGILKKGDECEFLGHSKNIRTVVTGIEMFHKSLERAEAGDNLGALVRGLKREDLRRGLVMAKPGSIQTHQKVEAQVYILSKEEGGRHKPFVSHFMPVMFSLTWDMACRIILPQGKELAMPGEDLKLTLILRQPMILEKGQRFTLRDGNRTIGTGLVTDTPAMTEEDKNIKWS

氨基酸序列NO.4如下:

MASGWDARPPWRRGRLPLLMCLLLLLRSPVQAAHIKKAEATTTTTSAGAEADEGQFDRYYHEEELGSVLREAAAAGPPGLARLFSIGSSVEGRPLWVLRLTAGLGPPPPDGDTGLDAAGPLLPGRPQVKLVGNMHGDETVSRQVLIYLARELAAGYRRGDPRLVRLLNTTDVYVLPSLNPDGFERAREGDCGLSDSGPPGASGRDNSRGRDLNRSFPDQFSTGEPPSLDDVPEVRALIEWIRRNKFVLSGNLHGGSVVASYPFDDSPEHKATGIYSKTSDDEVFKYLAKAYASNHPIMKTGAPHCPGDEDETFKDGITNGAHWYDVEGGMQDYNYVWANCFEITLELSCCKYPPASQLKQEWENNRESLITLIEKVHIGVKGFVKDSVTGAGLENATISVAGISHNITTGRFGDFHRLLVPGTYNITVVSTGYMPLTINNIIVKEGPAAIVNFSLRPTVASLIPAATEVVVTASTVAILNSALGTQSSHQPVQPQDFHHHHFPDMEIFLRRFANEYPNITRLYSLGKSVESRELYVMEISDNPGVHEPGEPEFKYIGNMHGNEVVGRELLLNLIEYLCKNFGTDPEVTDLVLNTRIHLMPSMNPDGYEKAQEGDSISVIGRNNSNNFDLNRNFPDQFIQITDPTQPETIAVMSWMKSYPFVLSANLHGGSLVVNYPFDDDEQGLATYSKSPDDAVFQQIALSYSKENSQMFQGRPCKNMYPNEYFPHGITNGASWYNVPGGMQDWNYLQTNCFEVTIELGCIKYPFEKDLPKFWEQNRRSLIQFMKQVHQGVKGFVLDATDGRGILNATISVAEINHPVTTYKTGDYWRLLVPGTYKITASARGYNPVTKNVTVKSEGAVQVNFTLVRSSTDSNNESKKGKGDSTNADDASDPTTKEFETLIKDLSAENGLEGFILRTSSNLALYRYHSYKDLSEFLRGLVMNYPHITNLTNLGQSAEYRHIWSLEISNKPNVSEPEEPKIRFVAGIHGNAPVGTELLLALAEFLCLNYKKNPAVTQLVDRTRIVIVPSLNPDGRERAQEKDCTSKIGQTNARGKDLDTDFTNNASQPETKAIIENLIQKQDFSLSVALDGGSVLVTYPYDKPVQTVENKETLKHLASLYANNHPSMHMGQPSCPNKSDENIPGGVMRGAEWHSHLGSMKDYSVTYGHCPEITVYTSCCYFPSAAQLPSLWAENKKSLLSMLVEVHKGVHGFVKDKTGKPISKAVIVLNEGIKVHTKEGGYFHVLLAPGVHNINAIADGYQQQHSQVFVHHDAASSVVIVFDTDNRIFGLPRELVVTVSGATMSALILTACIIWCICSIKSNRHKDGFHRLRQHHDEYEDEIRMMSTGSKKSLLSHEFQDETDTEEETLYSSKH

氨基酸序列NO.5如下:

MTALVTLLFLLLLGAQTQHGAEWTYSDGELDEAHWSREYPDCDGRRQSPIDVQRKSVQYNPALRALSLTGYEAQEGEFSMINNGHTVQISLPSTMRLTAPDGTQYIAKQMHFHWGGAFSEISGSEHTIDGIRYVTEVHVVHYNSKYKSYDEAQTAPDGLAVLAALFEIKDYAENTYYSDFISHLKNIRIPGQTTVLSSLNLQDMLPENLHSYYSYQGSLTTPPCTENVHWFVLADTVKLSRTQVWKLQNSLLNDQNKTIHNDYRRTQPLNHRVVEANFKSLPNPRSELHFYLNNIDNNLEYLRRVIEQKKAKGKGPW

氨基酸序列NO.6如下:

MEARMFWLLVVLLALASSSSAGEYVGLSANQCAVPAKDRVDCGYPQVTPEQCNNRGCCFDSSIPGVPWCFKPLQETECTF。

在上述的蛋白的用途中,所述蛋白由第一蛋白组中至少一种蛋白、第二蛋白组中至少一种蛋白组成;

所述第一蛋白组由PHB domain-containing protein蛋白、Growth factorreceptor bound protein 2蛋白、Elongation factor Tu蛋白组成;

所述第二蛋白组由Carboxypeptidase D蛋白、Carbonic anhydrase蛋白、Trefoilfactor 3蛋白组成。

在上述的蛋白的用途中,所述蛋白由Carbonic anhydrase蛋白和第一蛋白组中的至少一种蛋白组成。

同时,本发明还公开了一种母猪发情鉴别的标志物蛋白的筛选方法,步骤1:获取母猪发情周期中不同时间点的唾液;

步骤2:对步骤1获得的唾液蛋白进行提取;

步骤3:对提取的唾液蛋白在Label free模式下进行液质检测,获得各类蛋白的质谱检测原始数据;

步骤4:将质谱检测原始数据导入到Proteome Discoverer2.2软件,在sus scrofaUniProt数据库检索,谱肽、蛋白定量;

在进行蛋白相对定量分析时,以发情期的蛋白丰度作为参考,对不同时间点的蛋白丰度进行定量比较分析;

获得在不同时间点的蛋白丰度有表达差异的蛋白;

步骤5:对步骤4所获得的蛋白进行PRM靶向验证;验证通过,则判断该蛋白对母猪发情鉴别具有标志物的作用;

通过步骤5筛选得到的蛋白为PHB domain-containing protein 蛋白、Growthfactor receptor bound protein 2蛋白、Elongation factor Tu蛋白、Carbonicanhydrase蛋白、Carboxypeptidase D 蛋白、Trefoil factor 3蛋白:

PHB domain-containing protein 蛋白,其序列如氨基酸序列NO.1所示;

Growth factor receptor bound protein 2蛋白,其序列如氨基酸序列NO.2所示;

Elongation factor Tu蛋白,其序列如氨基酸序列NO.3所示;

Carboxypeptidase D蛋白,其序列如氨基酸序列NO.4所示;

Carbonic anhydrase蛋白,其序列如氨基酸序列NO.5所示;

Trefoil factor 3蛋白,其序列如氨基酸序列NO.6所示。

本发明的有益效果如下:

本方案利用Label free蛋白质组学方法,将母猪发情周期分为不同阶段(发情前期、发情期、发情后期、间情期)唾液提取的蛋白进行酶解之后,肽段进行高效液相色谱分离和质谱检测,共鉴定出个1155种蛋白;筛选差异表达蛋白进行PRM靶向定量验证。

结果显示,相比发情前期(Day-3),3种蛋白在发情期(Day0)的表达量上升,且在发情期的表达量最高,这3种蛋白分别是A0A4X1UP22(PHB domain-containing protein,STOM)、B6E241(Growth factor receptor bound protein 2,GRB2)、A0A4X1UAP2(Elongation factor Tu,N/A),且3种蛋白在发情周期4个时间点的相对表达趋势与labelfree中的表达趋势一致; 3种蛋白在发情期(Day0)的表达量下降,在发情期的表达量最低,这3种蛋白分别是F1RN68(Carboxypeptidase D,CPD)、A0A4X1W9S1(Carbonic anhydrase,CA6)、A0A4X1TI96(Trefoil factor 3,TFF3),且3种蛋白在发情周期4个时间点的相对表达趋势与label free中的表达趋势一致。因此这6种蛋白可以作为母猪发情诊断的候选标志蛋白。

附图说明

图1 A0A4X1UP22蛋白在母猪发情周期不同时间唾液蛋白质组(Label free,标号A)和PRM靶向定量验证(标号B)中的相对表达水平;

图2 B6E241蛋白在母猪发情周期不同时间唾液蛋白质组(Label free,标号A)和PRM靶向定量验证(标号B)中的相对表达水平;

图3 A0A4X1UAP2蛋白在母猪发情周期不同时间唾液蛋白质组(Label free,标号A)和PRM靶向定量验证(标号B)中的相对表达水平;

图4 F1RN68蛋白在母猪发情周期不同时间唾液蛋白质组(Label free,标号A)和PRM靶向定量验证(标号B)中的相对表达水平;

图5 A0A4X1W9S1蛋白在母猪发情周期不同时间唾液蛋白质组(Label free,标号A)和PRM靶向定量验证(标号B)中的相对表达水平;

图6 A0A4X1TI96蛋白在母猪发情周期不同时间唾液蛋白质组(Label free,标号A)和PRM靶向定量验证(标号B)中的相对表达水平。

具体实施方式

下面结合附图和具体实施方式,对本发明的技术方案作进一步的详细说明,但不构成对本发明的任何限制。

(1)母猪发情周期不同时间点唾液的获得

选择杜洛克公猪与藏母猪杂交生产的杜藏二元杂母猪进行试验。根据母猪行为表现及发情规律,确定母猪发情阶段,将其划分为不同的阶段进行唾液采集。采用传统的发情鉴定方法进行鉴定:母猪发情行为表现静立反射、食欲不振、外阴红肿、流黏液,联合公猪试情法。以母猪开始接受公猪爬跨当天计为发情期(Day0),发情期前三天为发情前期(Day-3),发情后第3天和第8天分别计为发情后期(Day3)和发情末期(Day8)。使用唾液采集袋采集母猪唾液,具体方法为:母猪自然咬住唾液采集袋,咀嚼2-3 min后将采集袋放置小样品袋中挤压,挤出唾液,将盛有唾液的样品袋放置冰上保存,再让母猪咀嚼唾液采集袋2-3min后挤出,如此反复,直至唾液量满足需求;唾液4800rpm离心5min后分装冷冻保存在液氮罐,直至送样。采集时间统一设在早上喂食前,此时母猪为空腹状态。

(2)唾液蛋白提取及质检

对总蛋白进行提取,使用 Bradford 蛋白质定量试剂盒进行定量,SDS-PAGE凝胶电泳检测蛋白提取质量。取等量蛋白进行蛋白酶解、C18 除盐柱脱盐、冻干。

(3)Label free模式液质检测

使用 EASY-nLCTM 1200 纳升级 UHPLC 系统,使用 Q Exactive

(4)数据处理

质谱下机数据导入到Proteome Discoverer2.2软件进行sus scrofa UniProt数据库检索,谱肽、蛋白定量。分析参数中前体离子搜库时和碎片离子搜库时的质量偏差容忍范围分别设为10ppm和0.02Da。保留可信度在99%以上的谱肽(Peptide Spectrum Matches,简称PSMs)为可信PSMs,至少包含一个unique肽段(特有肽段)的蛋白为可信蛋白,FDR(false discovery rate)≤1%。用T-test检验对蛋白质定量结果进行统计分析。在进行蛋白相对定量分析时,以发情期的蛋白丰度作为参考,与其他3个阶段的蛋白丰度进行比较,同时对不同时间点进行定量比较分析。p < 0.05 , |log2FC| > * (FC > * or FC < *[fold change, FC] 时差异显著。

(5)蛋白表达差异验证

筛选差异表达蛋白进行蛋白PRM靶向验证。预实验中质谱下机数据使用 Skyline软件进行分析,根据重现性与稳定性来确定选择的肽段是否可用。正式试验中质谱下机数据使用 Skyline 软件进行分析,使用内标肽段进行峰面积的矫正。

本实施例利用Label free蛋白质组学方法,将母猪发情周期内不同时间点唾液提取的蛋白进行酶解之后,肽段进行高效液相色谱分离和质谱检测,共鉴定出1155种蛋白;筛选差异表达蛋白进行PRM靶向定量验证。结果显示,相比发情前期(Day-3),3种蛋白在发情期(Day0)的表达量上升,且在发情期的表达量最高,这3种蛋白分别是A0A4X1UP22(PHBdomain-containing protein,STOM)、B6E241(Growth factor receptor bound protein2,GRB2)、A0A4X1UAP2(Elongation factor Tu,N/A),且3种蛋白在发情周期4个时间点的相对表达趋势与label free中的表达趋势一致;3种蛋白在发情期(Day0)的表达量下降,在发情期的表达量最低,这3种蛋白分别是F1RN68(Carboxypeptidase D,CPD)、A0A4X1W9S1(Carbonic anhydrase,CA6)、A0A4X1TI96(Trefoil factor 3,TFF3),且3种蛋白在发情周期4个时间点的相对表达趋势与label free中的表达趋势一致。因此这6种蛋白可以作为母猪发情诊断的候选标志蛋白。

优选地,所述蛋白由第一蛋白组中至少一种蛋白、第二蛋白组中至少一种蛋白组成;

所述第一蛋白组由PHB domain-containing protein蛋白、Growth factorreceptor bound protein 2蛋白、Elongation factor Tu蛋白组成;第一蛋白组符合在发情期(Day0)的表达量上升的规律;

所述第二蛋白组由Carboxypeptidase D蛋白、Carbonic anhydrase蛋白、Trefoilfactor 3蛋白组成。第二蛋白组符合在发情期(Day0)的表达量下降的规律。

更为优选地,所述蛋白由Carbonic anhydrase蛋白和第一蛋白组中的至少一种蛋白组成,由于A0A287AI92(Carbonic anhydrase,CA1)蛋白表达峰值和其他三种蛋白具有明显的区别,因此本实施例中,更为优选的是,将A0A287AI92(Carbonic anhydrase,CA1)蛋白与其他三种蛋白中的一种或两种或三种进行组合,用于鉴定藏猪在12天、15-16天的相关蛋白含量,可以更为准确的确定藏猪的早期妊娠。

本发明通过Label free蛋白质组学技术和PRM靶向定量验证技术筛选和鉴定母猪发情诊断标志蛋白,从而达到量化发情诊断指标,提高发情鉴定的准确率,丰富诊断方法,提高畜牧繁殖生产效益的目的。

上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其它的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。

序列表

<110> 广东省农业科学院动物科学研究所

岭南现代农业科学与技术广东省实验室茂名分中心

<120> 蛋白的用途和筛选方法

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 404

<212> PRT

<213> STOM蛋白(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

Met Arg Ala Pro Phe Ser Ser Lys Ser Ser Ala Gln Lys Gly Ala Phe

1 5 10 15

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

20 25 30

Ser Leu Phe Ser Ser Arg Pro Pro Ala Pro Arg Leu Leu Pro Leu Pro

35 40 45

Ser Gly Ala Phe Ser Pro Phe Arg Cys Cys Gln Ser Pro Asp Pro Leu

50 55 60

Phe Arg Gly Ala Ser Pro Leu Arg Pro Arg Pro Pro Ala Arg Gly Leu

65 70 75 80

Glu Gly Ser Ala Pro Glu Ala Ala Ala Pro Glu Gly Arg Ala Val Glu

85 90 95

Gly Gly Gly Val Ser Ser Leu Val Leu Gly Val Asp Trp Pro Gly Leu

100 105 110

Arg Glu Gly Ala Gly Ser Leu Pro Gly Ser Arg Asp Pro Glu Ser Ser

115 120 125

Arg Gly Ile Pro Leu Ser Ser Phe Ser Ser Ser Gly Pro Gly Asp Cys

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Val Val Tyr Tyr Arg Val Gln Asn Ala Thr Leu Ala Val Ala Asn Ile

245 250 255

Thr Asn Ala Asp Ser Ala Thr Arg Leu Leu Ala Gln Thr Thr Leu Arg

260 265 270

Asn Val Leu Gly Thr Lys Asn Leu Ser Gln Ile Leu Ser Asp Arg Glu

275 280 285

Glu Ile Ala His Asn Met Gln Cys Thr Leu Asp Asp Ala Thr Asp Asp

290 295 300

Trp Gly Ile Lys Val Glu Arg Val Glu Ile Lys Asp Val Lys Leu Pro

305 310 315 320

Val Gln Leu Gln Arg Ala Met Ala Ala Glu Ala Glu Ala Ser Arg Glu

325 330 335

Ala Arg Ala Lys Val Ile Ala Ala Glu Gly Glu Met Asn Ala Ser Arg

340 345 350

Ala Leu Lys Glu Ala Ser Met Val Ile Thr Glu Ser Pro Ala Ala Leu

355 360 365

Gln Leu Arg Tyr Leu Gln Thr Leu Thr Thr Ile Ala Ala Glu Lys Asn

370 375 380

Ser Thr Ile Val Phe Pro Leu Pro Ile Asp Met Leu Gln Gly Ile Met

385 390 395 400

Gly Ala Arg Lys

<210> 2

<211> 217

<212> PRT

<213> GRB2蛋白(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 2

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

1 5 10 15

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

20 25 30

Asp Gln Asn Trp Tyr Lys Ala Glu Leu Asn Gly Lys Asp Gly Phe Ile

35 40 45

Pro Lys Asn Tyr Ile Glu Met Lys Pro His Pro Trp Phe Phe Gly Lys

50 55 60

Ile Pro Arg Ala Lys Ala Glu Glu Met Leu Ser Lys Gln Arg His Asp

65 70 75 80

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

85 90 95

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

100 105 110

Asp Gly Ala Gly Lys Tyr Phe Leu Trp Val Val Lys Phe Asn Ser Leu

115 120 125

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

130 135 140

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

145 150 155 160

Val Gln Ala Leu Phe Asp Phe Asp Pro Gln Glu Asp Gly Glu Leu Gly

165 170 175

Phe Arg Arg Gly Asp Phe Ile His Val Met Asp Asn Ser Asp Pro Asn

180 185 190

Trp Trp Lys Gly Ala Cys His Gly Gln Thr Gly Met Phe Pro Arg Asn

195 200 205

Tyr Val Thr Pro Val Asn Arg Asn Val

210 215

<210> 3

<211> 575

<212> PRT

<213> A0A4X1UAP2蛋白(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 3

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

1 5 10 15

Pro Met Leu Met Ser Gln Thr Ala Thr Gln Met Arg Glu Met Thr Leu

20 25 30

Asp Ser Cys Ser Pro Asn Ser Ser Ser Phe Cys Cys Arg Gly Val Leu

35 40 45

Ser Cys Ser Val Ala Ala Ile Trp Ser Arg Ile Phe Pro Ile Ser Val

50 55 60

Pro Ser Pro Glu Val Asp Arg Glu Gly His Arg Asp Ser Gly Leu Glu

65 70 75 80

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

85 90 95

Gln Pro Gln Leu Pro Phe Ala Trp Cys Thr Arg Arg Gly Pro Cys Ala

100 105 110

Val Pro Thr Val Pro Gly Ala Arg Lys Thr Thr Met Ala Ala Ala Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Asp Lys Pro His Val Asn Val Gly Thr Ile Gly His Val Asp His Gly

180 185 190

Lys Thr Thr Leu Thr Ala Ala Ile Thr Lys Ile Leu Ala Glu Gly Gly

195 200 205

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

210 215 220

Arg Ala Arg Gly Ile Thr Ile Asn Ala Ala His Val Glu Tyr Ser Thr

225 230 235 240

Ala Asn Arg His Tyr Ala His Thr Asp Cys Pro Gly His Ala Asp Tyr

245 250 255

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

260 265 270

Val Val Ala Ala Asn Asp Gly Pro Met Pro Gln Thr Arg Glu His Leu

275 280 285

Leu Leu Ala Lys Gln Ile Gly Val Glu His Val Val Val Tyr Val Asn

290 295 300

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

305 310 315 320

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

325 330 335

Pro Ile Ile Val Gly Ser Ala Leu Cys Ala Leu Glu Gln Arg Asp Pro

340 345 350

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

355 360 365

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

370 375 380

Val Glu Ser Val Tyr Ser Ile Pro Gly Arg Gly Thr Val Val Thr Gly

385 390 395 400

Thr Leu Glu Arg Gly Ile Leu Lys Lys Gly Asp Glu Cys Glu Phe Leu

405 410 415

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

420 425 430

His Lys Ser Leu Glu Arg Ala Glu Ala Gly Asp Asn Leu Gly Ala Leu

435 440 445

Val Arg Gly Leu Lys Arg Glu Asp Leu Arg Arg Gly Leu Val Met Ala

450 455 460

Lys Pro Gly Ser Ile Gln Thr His Gln Lys Val Glu Ala Gln Val Tyr

465 470 475 480

Ile Leu Ser Lys Glu Glu Gly Gly Arg His Lys Pro Phe Val Ser His

485 490 495

Phe Met Pro Val Met Phe Ser Leu Thr Trp Asp Met Ala Cys Arg Ile

500 505 510

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

515 520 525

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

530 535 540

Phe Thr Leu Arg Asp Gly Asn Arg Thr Ile Gly Thr Gly Leu Val Thr

545 550 555 560

Asp Thr Pro Ala Met Thr Glu Glu Asp Lys Asn Ile Lys Trp Ser

565 570 575

<210> 4

<211> 1374

<212> PRT

<213> CPD蛋白(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 4

Met Ala Ser Gly Trp Asp Ala Arg Pro Pro Trp Arg Arg Gly Arg Leu

1 5 10 15

Pro Leu Leu Met Cys Leu Leu Leu Leu Leu Arg Ser Pro Val Gln Ala

20 25 30

Ala His Ile Lys Lys Ala Glu Ala Thr Thr Thr Thr Thr Ser Ala Gly

35 40 45

Ala Glu Ala Asp Glu Gly Gln Phe Asp Arg Tyr Tyr His Glu Glu Glu

50 55 60

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

65 70 75 80

Ala Arg Leu Phe Ser Ile Gly Ser Ser Val Glu Gly Arg Pro Leu Trp

85 90 95

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

100 105 110

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

115 120 125

Lys Leu Val Gly Asn Met His Gly Asp Glu Thr Val Ser Arg Gln Val

130 135 140

Leu Ile Tyr Leu Ala Arg Glu Leu Ala Ala Gly Tyr Arg Arg Gly Asp

145 150 155 160

Pro Arg Leu Val Arg Leu Leu Asn Thr Thr Asp Val Tyr Val Leu Pro

165 170 175

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

180 185 190

Leu Ser Asp Ser Gly Pro Pro Gly Ala Ser Gly Arg Asp Asn Ser Arg

195 200 205

Gly Arg Asp Leu Asn Arg Ser Phe Pro Asp Gln Phe Ser Thr Gly Glu

210 215 220

Pro Pro Ser Leu Asp Asp Val Pro Glu Val Arg Ala Leu Ile Glu Trp

225 230 235 240

Ile Arg Arg Asn Lys Phe Val Leu Ser Gly Asn Leu His Gly Gly Ser

245 250 255

Val Val Ala Ser Tyr Pro Phe Asp Asp Ser Pro Glu His Lys Ala Thr

260 265 270

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

275 280 285

Lys Ala Tyr Ala Ser Asn His Pro Ile Met Lys Thr Gly Ala Pro His

290 295 300

Cys Pro Gly Asp Glu Asp Glu Thr Phe Lys Asp Gly Ile Thr Asn Gly

305 310 315 320

Ala His Trp Tyr Asp Val Glu Gly Gly Met Gln Asp Tyr Asn Tyr Val

325 330 335

Trp Ala Asn Cys Phe Glu Ile Thr Leu Glu Leu Ser Cys Cys Lys Tyr

340 345 350

Pro Pro Ala Ser Gln Leu Lys Gln Glu Trp Glu Asn Asn Arg Glu Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ala Ala Thr Glu Val Val Val Thr Ala Ser Thr Val Ala Ile Leu Asn

465 470 475 480

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

485 490 495

Phe His His His His Phe Pro Asp Met Glu Ile Phe Leu Arg Arg Phe

500 505 510

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

515 520 525

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

530 535 540

Val His Glu Pro Gly Glu Pro Glu Phe Lys Tyr Ile Gly Asn Met His

545 550 555 560

Gly Asn Glu Val Val Gly Arg Glu Leu Leu Leu Asn Leu Ile Glu Tyr

565 570 575

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

580 585 590

Asn Thr Arg Ile His Leu Met Pro Ser Met Asn Pro Asp Gly Tyr Glu

595 600 605

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

610 615 620

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

625 630 635 640

Thr Asp Pro Thr Gln Pro Glu Thr Ile Ala Val Met Ser Trp Met Lys

645 650 655

Ser Tyr Pro Phe Val Leu Ser Ala Asn Leu His Gly Gly Ser Leu Val

660 665 670

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

675 680 685

Lys Ser Pro Asp Asp Ala Val Phe Gln Gln Ile Ala Leu Ser Tyr Ser

690 695 700

Lys Glu Asn Ser Gln Met Phe Gln Gly Arg Pro Cys Lys Asn Met Tyr

705 710 715 720

Pro Asn Glu Tyr Phe Pro His Gly Ile Thr Asn Gly Ala Ser Trp Tyr

725 730 735

Asn Val Pro Gly Gly Met Gln Asp Trp Asn Tyr Leu Gln Thr Asn Cys

740 745 750

Phe Glu Val Thr Ile Glu Leu Gly Cys Ile Lys Tyr Pro Phe Glu Lys

755 760 765

Asp Leu Pro Lys Phe Trp Glu Gln Asn Arg Arg Ser Leu Ile Gln Phe

770 775 780

Met Lys Gln Val His Gln Gly Val Lys Gly Phe Val Leu Asp Ala Thr

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

Thr Lys Asn Val Thr Val Lys Ser Glu Gly Ala Val Gln Val Asn Phe

850 855 860

Thr Leu Val Arg Ser Ser Thr Asp Ser Asn Asn Glu Ser Lys Lys Gly

865 870 875 880

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

885 890 895

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

900 905 910

Gly Phe Ile Leu Arg Thr Ser Ser Asn Leu Ala Leu Tyr Arg Tyr His

915 920 925

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

930 935 940

Pro His Ile Thr Asn Leu Thr Asn Leu Gly Gln Ser Ala Glu Tyr Arg

945 950 955 960

His Ile Trp Ser Leu Glu Ile Ser Asn Lys Pro Asn Val Ser Glu Pro

965 970 975

Glu Glu Pro Lys Ile Arg Phe Val Ala Gly Ile His Gly Asn Ala Pro

980 985 990

Val Gly Thr Glu Leu Leu Leu Ala Leu Ala Glu Phe Leu Cys Leu Asn

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

Lys Asp Cys Thr Ser Lys Ile Gly Gln Thr Asn Ala Arg Gly Lys Asp

1045 1050 1055

Leu Asp Thr Asp Phe Thr Asn Asn Ala Ser Gln Pro Glu Thr Lys Ala

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

Gln Thr Val Glu Asn Lys Glu Thr Leu Lys His Leu Ala Ser Leu Tyr

1105 1110 1115 1120

Ala Asn Asn His Pro Ser Met His Met Gly Gln Pro Ser Cys Pro Asn

1125 1130 1135

Lys Ser Asp Glu Asn Ile Pro Gly Gly Val Met Arg Gly Ala Glu Trp

1140 1145 1150

His Ser His Leu Gly Ser Met Lys Asp Tyr Ser Val Thr Tyr Gly His

1155 1160 1165

Cys Pro Glu Ile Thr Val Tyr Thr Ser Cys Cys Tyr Phe Pro Ser Ala

1170 1175 1180

Ala Gln Leu Pro Ser Leu Trp Ala Glu Asn Lys Lys Ser Leu Leu Ser

1185 1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Lys Val His Thr Lys Glu Gly Gly Tyr Phe His Val Leu Leu Ala Pro

1235 1240 1245

Gly Val His Asn Ile Asn Ala Ile Ala Asp Gly Tyr Gln Gln Gln His

1250 1255 1260

Ser Gln Val Phe Val His His Asp Ala Ala Ser Ser Val Val Ile Val

1265 1270 1275 1280

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

1285 1290 1295

Thr Val Ser Gly Ala Thr Met Ser Ala Leu Ile Leu Thr Ala Cys Ile

1300 1305 1310

Ile Trp Cys Ile Cys Ser Ile Lys Ser Asn Arg His Lys Asp Gly Phe

1315 1320 1325

His Arg Leu Arg Gln His His Asp Glu Tyr Glu Asp Glu Ile Arg Met

1330 1335 1340

Met Ser Thr Gly Ser Lys Lys Ser Leu Leu Ser His Glu Phe Gln Asp

1345 1350 1355 1360

Glu Thr Asp Thr Glu Glu Glu Thr Leu Tyr Ser Ser Lys His

1365 1370

<210> 5

<211> 317

<212> PRT

<213> CA6蛋白(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 5

Met Thr Ala Leu Val Thr Leu Leu Phe Leu Leu Leu Leu Gly Ala Gln

1 5 10 15

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

20 25 30

Ala His Trp Ser Arg Glu Tyr Pro Asp Cys Asp Gly Arg Arg Gln Ser

35 40 45

Pro Ile Asp Val Gln Arg Lys Ser Val Gln Tyr Asn Pro Ala Leu Arg

50 55 60

Ala Leu Ser Leu Thr Gly Tyr Glu Ala Gln Glu Gly Glu Phe Ser Met

65 70 75 80

Ile Asn Asn Gly His Thr Val Gln Ile Ser Leu Pro Ser Thr Met Arg

85 90 95

Leu Thr Ala Pro Asp Gly Thr Gln Tyr Ile Ala Lys Gln Met His Phe

100 105 110

His Trp Gly Gly Ala Phe Ser Glu Ile Ser Gly Ser Glu His Thr Ile

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Tyr Ser Asp Phe Ile Ser His Leu Lys Asn Ile Arg Ile Pro Gly Gln

180 185 190

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

195 200 205

Leu His Ser Tyr Tyr Ser Tyr Gln Gly Ser Leu Thr Thr Pro Pro Cys

210 215 220

Thr Glu Asn Val His Trp Phe Val Leu Ala Asp Thr Val Lys Leu Ser

225 230 235 240

Arg Thr Gln Val Trp Lys Leu Gln Asn Ser Leu Leu Asn Asp Gln Asn

245 250 255

Lys Thr Ile His Asn Asp Tyr Arg Arg Thr Gln Pro Leu Asn His Arg

260 265 270

Val Val Glu Ala Asn Phe Lys Ser Leu Pro Asn Pro Arg Ser Glu Leu

275 280 285

His Phe Tyr Leu Asn Asn Ile Asp Asn Asn Leu Glu Tyr Leu Arg Arg

290 295 300

Val Ile Glu Gln Lys Lys Ala Lys Gly Lys Gly Pro Trp

305 310 315

<210> 6

<211> 80

<212> PRT

<213> TFF3蛋白(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 6

Met Glu Ala Arg Met Phe Trp Leu Leu Val Val Leu Leu Ala Leu Ala

1 5 10 15

Ser Ser Ser Ser Ala Gly Glu Tyr Val Gly Leu Ser Ala Asn Gln Cys

20 25 30

Ala Val Pro Ala Lys Asp Arg Val Asp Cys Gly Tyr Pro Gln Val Thr

35 40 45

Pro Glu Gln Cys Asn Asn Arg Gly Cys Cys Phe Asp Ser Ser Ile Pro

50 55 60

Gly Val Pro Trp Cys Phe Lys Pro Leu Gln Glu Thr Glu Cys Thr Phe

65 70 75 80

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