一种PKEK融合蛋白及制备方法与应用与流程

文档序号:26139612发布日期:2021-08-03 14:23阅读:168来源:国知局
一种PKEK融合蛋白及制备方法与应用与流程
本发明属于基因工程领域,特别涉及一种pkek融合蛋白及制备方法与应用。
背景技术
:专利申请w0/2008/08m94和w0/2009/068351中描述了一种新型的美白多肽pkek,体外细胞实验证实人角质细胞在紫外线uvb照射下,pkek可有效减少白介素-6、白介素-8和tnf-a的表达,并显著降低色素沉淀调控子(α-msh)的表达。在另外一组随机双盲人体皮肤功效测试试验中,10名健康受试者每天一次连续使用pkek四周后,与对照组相比,紫外线uvb照射下pkek受试组皮肤白介素-1α、白介素-6、白介素-8、肿瘤坏死因子-α以及酪氨酸酶表达显著下调;39名高加索地区女性将pkek与维他命c磷酸钠合用6周后,面部斑点明显褪去,而单独使用pkek或维他命c磷酸钠斑点褪去均不显著;由此可见,上述体内和体外实验均可证实pkek能够有效减少因户外紫外线导致的面部色素沉淀。目前pkek主要采用液相合成或固相合成方法(spps)生产,由于化学合成所用的原料赖氨酸和谷氨酸侧链均要进行保护,转肽反应结束后需进行脱帽工艺,再进行结晶,操作比较繁琐,而且化学合成过程中易于形成消旋手性杂质,导致高纯度pkek的制备成本居高不下。因此,通过基因工程技术制备多肽成为大势所趋。专利申请cn107881187a公开了利用eeeek、edk、ddddk作为连接肽,通过d或e的侧链羧基与k的侧链氨基形成盐键,以减少k位点的酰化修饰,然而在k前面引入d或e形成的连接肽,并没有影响蛋白酶的酶切效率,仍不能解决后续酶切工艺中pkek的错切问题。专利申请cn104725485a公开了一种重组活性肽的制备方法,具体地,利用类弹性蛋白纯化标签elps和sumo多肽融合一个或多个降血压肽单体和抗氧化肽单体表达,对包涵体表达的目标蛋白先进行变性与复性再进行标签技术纯化;通过elps标签纯化技术纯化融合蛋白以及采用sumo蛋白酶裂解及加盐离心去除二联体融合标签elps-sumo,获得重组活性肽,然后再采用胃蛋白酶、胰蛋白酶和胰凝乳蛋白酶酶解重组活性肽超滤得小肽混合体。该方法虽然可制备得到小肽,但是步骤工艺繁琐,需要昂贵且酶切效率低的sumo蛋白酶,而且酶切前需要对包涵体进行变性复性。另外,由于sumo蛋白酶不耐受变性液,故需要在酶切前利用层析技术去除变性复性液,导致通过生物合成法制备的小肽成本与化学合成法相比并无显著优势。专利申请cn106560475a公布了一种活性短肽的基因工程生物合成工艺,通过将活性短肽串联后与纯化标签在微生物细胞内以可溶性蛋白或包涵体蛋白表达,并采用高特异性的蛋白酶降解重组蛋白质最终形成单个活性短肽。虽然该方法在制备单个活性短肽方面具有一定的优势,但由于pkek含有多个碱性氨基酸,将多个pkek串联后等电点较高,如10个串联pkek多肽理论等电点为9.92,这类含有多个碱性氨基酸的多肽在微生物细胞中通常以可溶性蛋白形式表达,极难形成包涵体蛋白,这样不仅易于被降解,而且游离pkek串联多肽中的赖氨酸侧链氨基易于被乙酰化修饰,而且该方法仍无法解决pkek串联多肽在酶切工艺中的错切问题,导致最终得到美白多肽pkek的概率大大降低。因此,目前亟需一种高效方法生产美白多肽pkek。技术实现要素:本发明的首要目的在于克服现有技术的缺点与不足,提供一种pkek融合蛋白,所述融合蛋白在微生物细胞内以包涵体蛋白的形式表达,同时通过将美白多肽pkek与柔性连接肽依次串联构成融合蛋白,能够有效减少酶切过程中产生的错切杂质,进而高效获得高纯度美白多肽pkek,有利于工业化生产。本发明的另一目的在于提供一种上述pkek融合蛋白的编码基因。本发明的再一目的在于提供上述pkek融合蛋白的编码基因的应用。本发明的目的通过下述技术方案实现:一种pkek融合蛋白,包括将若干美白多肽pkek与柔性连接肽依次串联构成的蛋白,其结构式如下:(pkek-b)n;其中:b为柔性连接肽;n为1~30的整数。所述的pkek融合蛋白的结构式优选如下:a–dd-(pkek-b)n;其中:a为伴侣蛋白;d为蛋白酶酶切位点;b为柔性连接肽;n为1~30的整数;d为0~10的整数。所述的n优选为10~30;更优选为10~20。所述的d优选为1~5;进一步优选为1。所述的伴侣蛋白为ksi、pagp、pap3.30、taf12、gdf15、gygb、mksi、mpagp、mpap3.30、mgdf15、mgygb和mtaf12中的一种或两种以上形成的组合,m表示突变体。伴侣蛋白的作用是辅助融合蛋白在大肠杆菌中形成包涵体,促进包涵体的形成,提高包涵体产量和质量,降低被降解和侧链修饰的风险;且便于下游的分离纯化。现有野生型醛固酮异构酶ksi片段、野生型pagp蛋白片段、野生型taf12蛋白片段、野生型pap3.30蛋白片段、野生型gygb蛋白片段均具有高度疏水性,在大肠杆菌中易形成包涵体,并且能引导连接在其c端的小肽进行高效表达,因此选用ksi、pagp、pap3.30、gdf15、gygb或taf12蛋白作为伴侣蛋白;另外,由于上述野生型分子伴侣蛋白有多个赖氨酸残基及精氨酸残基,在后续的酶切工艺中会形成过多的错误酶切片段,若将上述野生型分子伴侣蛋白中的赖氨酸、精氨酸突变为其他氨基酸,能够有效减少杂质的形成,显著提高酶切纯度,故所述伴侣蛋白还可以是ksi、pagp、pap3.30、gdf15、gygb或taf12蛋白的突变体,即mksi、mpagp、mpap3.30、mgdf15、mgygb或mtaf12。所述伴侣蛋白是上述分子伴侣单个分子伴侣或分子伴侣的组合,优选为pagp+pagp、ksi+ksi、pagp+ksi、ksi+pagp、taf12+ksi、ksi+taf12、taf12+pagp、taf12+gygb或pagp+gygb;更优选为ksi+pagp、taf12+ksi、taf12+gygb或taf12+pagp。所述的柔性连接肽为如xm---x2-x1所示的肽序列;其中,x1为蛋白酶酶切位点,x2至xm为柔性片段;m为0~10的整数,m为0表示柔性连接肽缺失,m为1即所述的柔性连接肽仅为1个氨基酸,以此类推;优选地,m为0时,所述的d不能为0,n为1~30;或是m为0、d为0时,所述的n不能为1。所述的蛋白酶酶切位点为arg或lys;优选为lys。所述的柔性片段中的氨基酸不能为pro、thr或trp;优选为gly、ala或ser。所述的m优选为1~9的整数;更优选为2~6的整数。所述的柔性连接肽优选为ak、gk、aak、aagk、aaggk、ggaak、ggsask或aaggsgagk。所述的伴侣蛋白为taf12或是taf12与其他伴侣蛋白融合形成的伴侣蛋白时,所述的柔性连接肽优选为ak、gk、aak、aagk、aaggk或ggaak;更优选为gk、aak、aagk或ggaak;最优选为aak、aagk或ggaak;所述的n优选10~30;更优选为10~20。所述的其他伴侣蛋白优选为pagp、ksi或gygb。所述的伴侣蛋白为ksi或是ksi与其他伴侣蛋白融合形成的伴侣蛋白时,所述的柔性连接肽优选为ak、gk、aak、aagk、aaggk或ggaak;更优选为ggaak或ggsask;所述的n优选10~20。所述的其他伴侣蛋白优选为pagp或taf12。所述的伴侣蛋白为pagp或是pagp与其他伴侣蛋白融合形成的伴侣蛋白时,所述的柔性连接肽优选为ak、gk、aak、aagk、aaggk、ggaak或ggsask;更优选为aak;所述的n优选为10~20。所述的其他伴侣蛋白优选为ksi或taf12。一种编码上述pkek融合蛋白的基因,可通过密码子规则得到;更优选为依据宿主菌密码子使用偏好性设计得到的序列,如seqidno.4、seqidno.13、seqidno.15、seqidno.17、seqidno.19、seqidno.26、seqidno.28、seqidno.31、seqidno.33、seqidno.36、seqidno.38、seqidno.40、seqidno.42、seqidno.44或seqidno.52所示的序列;优选为如seqidno.4、seqidno.13、seqidno.15、seqidno.17、seqidno.19、seqidno.26、seqidno.28、seqidno.33、seqidno.36、seqidno.38、seqidno.40、seqidno.42或seqidno.52所示的序列。一种重组载体,含有上述编码pkek融合蛋白的基因;是将上述编码pkek融合蛋白的基因克隆入表达载体得到。所述的表达载体上的启动子包括t7启动子、t7lac启动子、tac启动子、lac启动子、trp启动子;优选为t7启动子和t7lac启动子。所述的表达载体优选为pet系列载体;更优选为pet-3a(+)、pet-29a(+)、pet-28a(+)、pet-22b(+)、pet-26b(+)或pet-31b(+);最优选为pet-31b(+)或pet29a(+)。一种重组表达菌株,含有上述重组载体;是将上述重组载体转入基因工程菌株获得。所述的基因工程菌株选自细菌、酵母和真菌;优选的是细菌;更优选的是大肠杆菌(escherichiacoli)。上述pkek融合蛋白可通过化学合成方法得到,也可通过基因工程方法表达得到;优选为通过基因工程方法表达得到,制备方法优选包括如下步骤:发酵培养上述重组表达菌株,收集融合蛋白的包涵体,得到pkek融合蛋白。上述pkek融合蛋白在制备美白多肽peke中的应用,优选的具体步骤如下:溶解、酶切融合蛋白的包涵体,分离纯化即得活性美白多肽pkek。一种利用基因工程技术生产美白多肽peke的制备方法,包括如下步骤:s1、获得包含上述pkek融合蛋白编码基因的重组载体,其编码的融合蛋白的结构如a-dd-(pkek-b)n所示;其中:a为伴侣蛋白;b为柔性连接肽,柔性连接肽为如xm---x2-x1所示的肽序列,x1为蛋白酶酶切位点,x2至xm为柔性片段,m为0~10的整数;d为蛋白酶切位点;n为1~30的整数;d为0~10的整数;s2、将步骤s1获得的重组载体转入基因工程菌株中,得到重组表达菌株;发酵培养重组表达菌株,收集融合蛋白的包涵体;s3、溶解、酶切融合蛋白的包涵体,分离纯化即得活性美白多肽pkek。步骤s2中所述的基因工程菌株为细菌、酵母或真菌,将含有美白多肽的重组表达载体导入基因工程菌株内,避免引入新杂质,方便后续工业化生产。所述的细菌优选为大肠杆菌。所述pkek融合蛋白可以在大肠杆菌中以包涵体形式进行表达,融合蛋白无需复性,只需在合适条件下溶解后、直接稀释酶切即可得到小分子蛋白或多肽,工艺简单且易于放大,适于工业化生产。步骤s2中所述的发酵中使用的发酵培养基由氮源、碳源和无机盐组成。所述的氮源为有机氮源和无机氮源中的一种或两种。所述的发酵培养基的组成优选如下:酵母粉2.5~3.5g/l、蛋白胨4~6g/l、nacl0.5~1.5g/l、kh2po42.5~3.5g/l、na2hpo43~3.5g/l、mgso40.5~1.5g/l、(nh4)2so45~7g/l、cacl2·2h2o0.01~0.015g/l、甘油4~4.2g/l、微量元素0.8~1ml/l,溶剂为水;灭菌后用氨水调ph至6.8~7.2;更优选如下:酵母粉3g/l、蛋白胨5g/l、nacl1g/l、kh2po43g/l、na2hpo43.25g/l、mgso41g/l、(nh4)2so46g/l、cacl2·2h2o0.01375g/l、甘油4.125g/l、微量元素0.875ml/l,溶剂为水;灭菌后用氨水调ph至7.0。所述的微量元素混合液(总体积100ml)的组成如下:fecl2·4h2o2~2.5g、zncl20.1~0.15g、cocl2·6h2o0.15~0.25g、na2mo4·2h2o0.15~0.25g、cacl2·2h2o0.05~0.15g、cucl2·2h2o0.1~0.15g、h3bo40.03~0.06g、mnso4·h2o0.2~0.23g、37wt%浓盐酸9.5~10.5ml,溶剂为水;优选如下:fecl2·4h2o2.287g、zncl20.131g、cocl2·6h2o0.2g、na2mo4·2h2o0.2g、cacl2·2h2o0.1g、cucl2·2h2o0.125g、h3bo40.05g、mnso4·h2o0.217g、37wt%浓盐酸10ml,溶剂为水。所述的发酵的方式为高密度发酵。所述的发酵的具体步骤优选如下:在36~38℃,ph控制为6.8~7.2,溶解氧(do)5~50%的最大氧饱和度之间发酵;当培养至碳源耗尽时开始补料,当培养至菌体浓度od600为75~85时,添加iptg诱导。所述的补料所用的补料培养基组成如下:450~550g/l甘油、30~40g/l酵母浸粉、60~70g/l酵母蛋白胨、3~4ml/l微量元素、余量为水;优选组成如下:500g/l甘油、33g/l酵母浸粉、66/lg酵母蛋白胨、3.5ml/l微量元素、余量为水。所述的补料的速率优选为0.4ml·min-1·l-1;更优选为0.3~0.5ml·min-1·l-1。所述的od600为80。所述的iptg在发酵体系中的终浓度为0.2~0.4mm;更优选为0.3mm。所述的诱导时间为至少3h;更优选为12~15h。步骤s2中所述的包涵体是通过高压匀浆法破碎菌体细胞得到。步骤s2中所述的包涵体的获得步骤优选如下:将发酵液进行固液分离,获得菌体;将菌体重悬于破碎液中,水浴下匀浆破碎菌体,离心收集沉淀,将沉淀溶于洗涤缓冲液进行洗涤,离心收集包涵体沉淀。所述的固液分离的方式优选为离心。所述的菌体和所述的破碎液优选按1g:5~10ml配比混合;更优选按1g:7ml配比混合。所述的破碎液的组成优选如下:6~6.2g/ltris和1.4~1.5g/ledta,余量为纯化水,调ph为7.8~8.2;更优选如下:6.05g/ltris和1.46g/ledta,余量为纯化水,调ph为8.0。所述的匀浆的条件优选为900bar。所述的沉淀和所述的洗涤缓冲液优选按1g:10~20ml配比混合;更优选按1g:15ml配比混合。所述的洗涤缓冲液的组成优选如下:6~6.1g/ltris、1.4~1.5g/ledta、2.8~3.0g/lnacl、9~11ml/ltritionx-100,余量为水;优选如下:6.05g/ltris、1.46g/ledta、2.9g/lnacl、10ml/ltritionx-100,余量为水。步骤s3中所述的酶切中使用的蛋白酶为牛胰蛋白酶、猪胰蛋白酶和赖氨酰特异性内切酶中的一种或至少两种。步骤s3中所述的溶解、酶切融合蛋白的包涵体的具体做法为:先将收集的融合蛋白的包涵体用尿素溶液溶解,稀释后用蛋白酶进行酶切。所述的尿素溶液的浓度为5m以上,更优选为8m。所述的尿素溶液的用量优选按1g包涵体配比5.5~6.5ml尿素溶液计算;更优选按1g包涵体配比6ml尿素溶液计算。上述利用基因工程技术生产美白多肽pkek的制备方法在工业化制备美白多肽peke中的应用。该利用基因工程技术生产美白多肽pkek的制备方法简单、高效,制备得到的美白多肽纯度高,适于工业化生产。本发明相对于现有技术具有如下优点及有益效果:本发明发明人通过将pkek与柔性连接肽依次串联,使pkek中的第二个赖氨酸的酶切效率显著高于第一个赖氨酸的酶切效率,最终得到大量的pkek完整多肽分子,错切杂质pk、ek显著降低;同时由于引入连接肽,使部分错切杂质以连接肽+pk、ek+连接肽形式存在,导致错切杂质与完整pkek美白多肽的理化性质有显著差别,这样通过层析工艺即可有效去除杂质,将错切杂质控制在较低水平。本发明提供了一种pkek融合蛋白,所述融合蛋白在微生物细胞内以包涵体蛋白的形式表达,同时通过将美白多肽pkek与柔性连接肽依次串联构成融合蛋白,能够有效减少酶切过程中产生的错切杂质,进而高效获得高纯度美白多肽pkek,且纯度可达95.4%;另外,利用本发明所述基因工程技术生产美白多肽peke的制备方法,在12h内即可大量获得pkek融合蛋白包涵体,且表达量最高可达45g/l,工艺简单高效且易于放大,有利于工业化生产。附图说明图1是实施例1所述重组质粒pet31b-taf12-k-(pkek-aak)10的质粒图谱图。图2是实施例1提供的基因工程菌株bl21(de)/pet31b-taf12-k-(pkek-aak)10摇瓶发酵诱导3h得到的样品sds-page图谱图;其中,泳道m为蛋白marker、泳道1为阴性对照、泳道2~7代表不同的单克隆。图3是实施例1提供的基因工程菌株bl21(de)/pet31b-taf12-k-(pkek-aak)10摇瓶发酵诱导15h得到的样品sds-page图谱图;其中,泳道m为蛋白marker、泳道1为阴性对照、泳道2~7代表不同单克隆。具体实施方式下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。材料限制性内切酶ndei和xhoi购于neb公司;taf12-k-(pkek-aak)10融合基因序列(如seqidno.4所示),委托南京金斯瑞公司合成;琼脂糖凝胶回收试剂盒购于生工(生物工程)上海股份有限公司;dnaligationkit购于takara公司;大肠杆菌dh5α购于lifetechnologies公司;质粒提取试剂盒购于生工(生物工程)上海股份有限公司;鉴定含有taf12-k-(pkek-aak)10基因片段的克隆委托invitrogen公司测序;大肠杆菌bl21(de3)购于lifetechnologies公司;改良型lowry法蛋白浓度测定试剂盒购于生工(生物工程)上海股份有限公司;搅拌式发酵罐购于东方生工生物有限公司;重组牛胰蛋白酶购于上海雅心生物技术有限公司;重组猪胰蛋白酶购于roche罗氏试剂;重组赖氨酰内切酶购于日本和光纯药株式会社;unipmm30-500填料购于苏州纳微科技有限公司。实施例1一种利用基因工程技术生产美白多肽pkek的制备方法,包括如下步骤:1、构建重组质粒pet31b-taf12-k-(pkek-aak)101.1人工合成含分子伴侣、蛋白酶酶切位点、pkek和连接肽的基因序列taf12-k-(pkek-aak)10设计含分子伴侣、蛋白酶酶切位点、pkek和连接肽基因序列taf12-k-(pkek-aak)10的融合基因片段,依据密码子表将这些氨基酸序列转换为核苷酸序列,在转换过程中根据大肠杆菌的密码子使用偏好性,选用使用频率较高的密码子,调整其gc含量,去掉影响基因转录的顺式作用元件和重复序列而优化得到,同时在基因序列的3’端引入双终止密码子taatga,为了便于基因操作,在taf12-k-(pkek-aak)10融合基因序列的5’端引入ndei酶切位点序列catatg,在taf12-k-(pkek-aak)10融合基因3’端引入xhoi酶切位点ctcgag,优化后的taf12-k-(pkek-aak)10融合基因序列如seqidno.4所示。1.2pet31b-taf12-k-(pkek-aak)10重组表达载体的构建用限制性内切酶ndei和xhoi对质粒pet-31b(+)进行双酶切,取5μl酶切产物用质量体积比0.8%琼脂糖凝胶电泳进行分析,确认酶切完全后,将全部酶切产物进行质量体积比0.8%琼脂糖电泳,割下含约5.3kb的dna片段的凝胶,用琼脂糖凝胶回收试剂盒将凝胶中的dna片段纯化到30μl去离子水中,就得到带粘性末端的质粒片段。同理,用ndei和xhoi对taf12-k-(pkek-aak)10融合基因片段进行双酶切,将酶切后的dna片段纯化到20μl去离子水中,就得到带粘性末端的基因片段。用dna连接试剂盒(dnaligationkit)将基因片段和质粒片段进行连接,连接条件为16℃过夜,取10μl过夜连接的产物,加入到80μl用cacl2法(美国冷泉港实验室出版的《分子克隆实验指南》第三版)制备的大肠杆菌dh5α感受态细胞中,42℃处理90s,迅速加入300μl经过37℃预热的lb液体培养基(其中,蛋白胨10g/l、酵母粉5g/l、氯化钠5g/l,ph7.0~7.5),在37℃摇床中低速(100~150rpm)振荡培养45min,然后取100μl培养物涂布添加了氨苄青霉素(终浓度为100μg/ml)的lb固体培养基(其中,蛋白胨10g/l、酵母粉5g/l、氯化钠5g/l、琼脂粉15g/l,ph7.0~7.5)。在37℃培养箱中倒置培养约18h,直到长出单菌落,随机挑取部分单菌落进行菌液pcr鉴定,pcr反应的条件为:94℃5min;94℃30s、55℃30s、72℃30s,30个循环;72℃5min;引物为:t7启动子引物和t7终止子引物。t7启动子引物:5’-taatacgactcactataggg-3’(seqidno.5);t7终止子引物:5’-tgctagttattgctcagcgg-3’(seqidno.6)。将初步筛选得到的阳性克隆用质粒提取试剂盒,并按说明书进行抽提质粒,通过ndei和xhoi进行酶切鉴定,得到约465bp的基因片段和约5.3kb的载体片段的克隆,即为含有taf12-k-(pkek-aak)10基因片段的克隆。将鉴定含有taf12-k-(pkek-aak)10基因片段的克隆进行测序。将测序证明没有碱基突变和读码框移位的单克隆,接种至50ml添加了氨苄青霉素(终浓度为100μg/ml)的lb液体培养基,37℃、250rpm培养18h,获得的培养物用质粒提取剂盒抽提质粒,即为重组表达载体,命名为pet31b-taf12-k-(pkek-aak)10,如图1所示。2、bl21(de3)/pet31b-taf12-k-(pkek-aak)10表达菌株的筛选及产物鉴定按照美国冷泉港实验室出版的《分子克隆实验指南》第三版提供的氯化钙法,制备大肠杆菌bl21(de3)感受态细胞。取1μl实施例1中的重组表达载体pet31b-taf12-k-(pkek-aak)10转化上述大肠杆菌感受态细胞,转化方法同样按照1.2中的氯化钙法进行。将转化液分别涂布至添加了氨苄青霉素(终浓度为100μg/ml)的lb固体培养基,37℃倒置培养直到出现单菌落,即获得了pkek融合蛋白表达菌种库,命名为bl21(de3)/pet31b-taf12-k-(pkek-aak)10。同时,以pet-31b(+)转化大肠杆菌bl21(de3)感受态细胞,得到bl21(de3)/pet31b菌,作为阴性对照。用无菌牙签分别挑取上述工程菌在lb固体培养基形成的单菌落,挑取6个单克隆接种至50mllb液体培养基,37℃、250rpm振荡培养,当菌液od600=0.5~1.0时,取3~6ml菌液接种至100mllb液体培养基,并取3~6mlbl21(de3)/pet31b菌液接种至100mllb液体培养基作为阴性对照,37℃、250rpm振荡培养至od600=0.6~0.8时添加iptg(异丙基-β-d-硫代吡喃半乳糖苷,终浓度为1mmol/l)开始诱导,并分别取诱导3h、15h菌液(4ml),12000rpm离心1分钟,去掉上清液,将菌体保存在-20℃备用。取出-20℃冻存的菌体,加入5ml浓度为8mol/l的尿素溶液重悬菌体,在冰水混合物中超声波破碎10分钟(超声3秒、停5秒,如此循环)。取15μl破碎液,加入15μl上样缓冲液,充分混匀后取10μl进行sds-page(其中浓缩胶含体积百分比5%的丙烯酰胺-甲叉双丙烯酰胺(按照体积比29:1配制)、分离胶含体积百分比15%的丙烯酰胺-甲叉双丙烯酰胺。电泳条件为:浓缩胶设定电流为11ma,分离胶设定电流为22ma。电泳结束后,取出凝胶,用考马斯亮蓝染色液(每升含0.6g考马斯亮蓝r-250、450ml乙醇、100ml冰醋酸,余量为纯化水)染色过夜,用脱色液(每升含250ml乙醇、80ml冰醋酸,余量为纯化水)脱色直到背景透明,随后将凝胶在透明背景下拍照提取图像,如图2和图3所示,用生物图像处理软件gel-proanalyzer4.0进行灰度扫描分析目的条带占总条带的比例,即为pkek融合蛋白在总蛋白质中的纯度。使用改良型lowry法蛋白浓度测定试剂盒按说明书进行操作,测定菌体破碎液的总蛋白质浓度,再通过稀释倍数换算得到菌液的总蛋白浓度。根据pkek融合蛋白在总蛋白质中的纯度和菌液的总蛋白浓度,换算得到pkek融合蛋白的表达量。根据电泳结果发现,基因工程菌bl21(de3)/pet31b-taf12-k-(pkek-aak)10在诱导前不会表达目的蛋白质,这可以有效降低目的蛋白质对宿主细胞的毒性,有利于延长蛋白的表达时间。不含有pkek基因的对照菌bl21(de3)/pet-31b(+)经iptg诱导后可表达分子量大小约为14kda的醛固酮异构酶,而基因工程菌bl21(de3)/pet31b-taf12-k-(pkek-aak)10经iptg诱导后可表达大小约为17kda的蛋白质,这与pkek融合蛋白的理论分子量16.947kda(使用在线软件http://web.expasy.org/compute_pi/预测得到)相符,说明基因工程菌中已成功导入pkek基因。将上述基因工程菌bl21(de3)/pet31b-taf12-k-(pkek-aak)10,在加有终浓度为100μg/ml的氨苄青霉素新鲜lb固体培养基上划线进行纯培养,挑取单菌落用lb液体培养基培养至od600=0.8~1.0,制成含体积百分比15%甘油的菌液,存放至-70℃冰箱备用。3、pkek融合蛋白基因工程菌bl21(de3)/pet31b-taf12-k-(pkek-aak)10发酵试验3.1制备种子培养物取20μl在-70℃冷冻保存的bl21(de3)/pet31b-taf12-k-(pkek-aak)10菌种,接种至50ml添加了氨苄青霉素(终浓度为100μg/ml)的lb液体培养基,在28℃、250rpm的摇床中培养16小时,从而活化菌种,再将50ml活化过的菌种接种到400ml的lb液体培养基中,在28℃、250rpm的条件下继续培养3h,获得种子培养物,控制菌体浓度od600在2.0~3.0之间。3.220l发酵罐中的发酵培养使用20l搅拌式发酵罐,按照表1所示的发酵培养基的配方经灭菌后用氨水调ph至7.0后进行投料,投料体积为8l,严格控制发酵条件:温度控制为37℃,ph控制为7.0,溶解氧(do)控制在5~50%的最大氧饱和度之间(氧饱和度“0”的校正条件是:饱和亚硫酸钠溶液;最大氧饱和度的校正条件是:转速为150rpm,空气流速4l/min),发酵转速控制在150~700rpm之间,具体发酵转速根据do的变化进行调控,空气流速控制在4~20l/min之间,当培养至碳源耗尽时开始补料,其中每千克补料液含有:500g甘油、33g酵母浸粉、66g酵母蛋白胨、3.5ml微量元素、余量为水,全程以0.4ml·min-1·l-1的补料速率进行匀速补料。当培养至菌体浓度od600约等于80时,开始添加iptg至终浓度0.3mm,开始诱导。表1:发酵培养基配方组分用量(g)酵母粉(yeastextract)24.0蛋白胨(tryptone)40.0nacl8.0kh2po424.0na2hpo426.0mgso48.0(nh4)2so448.0cacl2·2h2o0.11甘油33.0微量元素混合液(见表2)7ml表2:微量元素混合液(100ml)配方表组分用量(g)fecl2·4h2o2.287zncl20.131cocl2·6h2o0.2na2mo4·2h2o0.2cacl2·2h2o0.1cucl2·2h2o0.125h3bo40.05mnso4·h2o0.21737wt%浓盐酸10ml诱导12h停止发酵,分别在诱导0h和12h取1ml发酵液,用纯化水稀释10倍后,测定taf12-k-(pkek-aak)10融合蛋白的表达量(两个平行批次)。基因工程菌株bl21(de)/pet31b-taf12-k-(pkek-aak)10发酵试验换算后含有融合蛋白包涵体的平均产量为45g/l(按下面步骤4.1分离得到包涵体测定得到),其中融合蛋白包涵体中的融合蛋白含量为44%,即19.8g/l。4、pkek融合蛋白包涵体的提取和酶切4.1包涵体的分离提取取上述步骤3中的发酵液以4500rpm离心30分钟收集菌体,以质量体积比为1:7的比例将菌体重悬于破碎液(每升含有6.05gtris和1.46gedta,余量为纯化水,调ph为8.0)中,水浴下高压(900bar)匀浆破碎菌体,离心收集沉淀,再以质量体积比为1:15的比例将收集沉淀溶于洗涤缓冲液(每升含有6.05gtris、1.46gedta、2.9gnacl、10mltritionx-100,余量为纯化水)进行洗涤,离心收集包涵体沉淀。4.2包涵体变性液酶切将上述收集的包涵体2.5g用15ml变性液(每升含有8mol尿素,余量为纯化水)溶解后,再用5mmtris溶液稀释1倍,调ph为9.0,室温下搅拌2~5h。采用重组牛胰蛋白酶按照说明书进行酶切,经酶切即可得到pkek粗品。pkek粗品液相检测条件:beh-c8(填料规格为1.7μm粒径,柱子规格为2.1×100mm)分析柱,柱温为35℃,0.2ml/min流速。流动相a:含0.1%(v/v)tfa的水溶液;流动相b:含0.09%(v/v)tfa的乙腈;梯度:0~25min,b相从30%升至80%;25~38min:b相从80%升至100%。pkek出峰时间为21.5min,统计峰面积占比为13.2%,即pkek粗品纯度为13.2%。5、酶切后的pkek粗品纯化将100ml按照上述步骤4中制备的经酶切后的pkek粗品上样到装有10mlunipmm30-500填料的层析柱上(其预先用含0.1%(v/v)tfa的20%异丙醇的缓冲液平衡),上样结束后用平衡缓冲液(含0.1%(v/v)tfa的20%异丙醇的缓冲液)冲洗至基线平稳。再以100ml浓度为20%(v/v)异丙醇a液(含0.1%(v/v)tfa缓冲液)和100ml浓度为40%(v/v)异丙醇b液(含0.1%(v/v)tfa缓冲液)线性梯度洗脱,洗脱流速≤2倍柱体积每小时。主峰分段收集,约20%峰高开始收集,检测波长为280nm,即得到pkek。纯化工艺制备的pkek纯度达到95.4%。pkek液相检测条件:beh-c8(填料规格为1.7μm粒径,柱子规格为2.1×100mm)分析柱,柱温为35℃,0.2ml/min流速。流动相a:含0.1%(v/v)tfa的水溶液;流动相b:含0.09%(v/v)tfa的乙腈;梯度:0~25min,b相从30%升至80%;25~38min:b相从80%升至100%。质谱鉴定:赛默飞高分辨液质联用仪取peke主峰进行高分辨质谱分析,使用thermobiopharmafinder2.0解卷积,采集范围:ms100~1000。结果显示本发明制备得到的pkek分子量大小为500.3da,与理论分子量完全一致,说明通过上述方法制备的多肽即为高纯度美白多肽pkek。实施例2本实施例与实施例1所述方法基本相同,不同之处在于,本实施例用于酶切的蛋白酶为重组猪胰蛋白酶,16℃反应2h后调酸终止反应,经酶切即可得到pkek粗品,pkek粗品纯度为13.2%。实施例3本实施例与实施例1所述方法基本相同,不同之处在于,本实施例用于酶切的蛋白酶为重组赖氨酰内切酶,25℃反应2h后调酸终止反应,经酶切即可得到pkek粗品,pkek粗品纯度为13.5%。实施例4本实施例与实施例1所述方法基本相同,不同之处在于,本实施例选用的重组表达载体中含有的分子伴侣、连接肽和pkek串联数量不同。不同分子伴侣和连接肽融合蛋白的相对产量如表3所示,酶切后的pkek相对纯度如表4所示。表3:不同分子伴侣和连接肽融合蛋白发酵产量试验说明:上表中的相对产量仅指融合蛋白分子中pkek部分的相对产量,计算时已扣除分子伴侣,蛋白酶切位点和连接肽。上表中,蛋白分子核苷酸酶切位点均为ndei、xhoi。ksi分子伴侣的氨基酸序列如seqidno.29所示;pagp分子伴侣的氨基酸序列如seqidno.24所示;taf12分子伴侣的氨基酸序列如seqidno.1所示;pap3.30分子伴侣的氨基酸序列如seqidno.45所示;gdf15分子伴侣的氨基酸序列如seqidno.48所示;gygb分子伴侣的氨基酸序列如seqidno.34所示。seqidno.4所用的重组表达载体通过实施例1制备得到。本发明中所有的核酸序列5’端均为酶切位点ndei,3’端为终止密码子(taatga)及酶切位点xhoi,氨基酸序列本身起始的第一个氨基酸为m,其来自于ndei酶切位点。表3的结果说明,加入分子伴侣后,pkek的产量明显增加。某些具有疏水性的分子伴侣共同表达可进一步提高pkek产量。按实施例1将不同的包涵体进行酶切,酶切得到的pkek单体相对纯度如表4所示。表4:不同分子伴侣和连接肽融合蛋白酶切后的pkek相对纯度分子伴侣蛋白酶酶切位点连接肽pkek-连接肽串联数pkek单体相对纯度taf12k无1038%taf12k无2040%taf12kaak10100%taf12kak1099%taf12kgk10106%taf12kaagk10110%taf12kggaak10155%taf12kwtk1039%taf12kppk1035%taf12+pagpkaak10100%taf12+pagpkaak20106%taf12+ksikaak1088%taf12+ksikaak2092%taf12+gygbkaak30109%ksikggaak1096%ksikggsask20104%pagpkaak10103%ksi+pagpkaak1091%pap3.30kaak1087%gdf15kaak1095%gygbkaak10108%说明:上表中所有组酶切时包涵体中pkek含量相同,已扣除分子伴侣和连接肽。百分比表示酶切后的相对纯度,纯度越高,表示错切的比例越少。表4的结果说明:无连接肽时,酶切获得的单体pkek远低于有连接肽的分子。同时,当连接肽中的xm---x2为a、g、aa、aag、ggaa、ggsas等小侧链疏水柔性多肽时,其酶切后获得的pkek单体远高于wt、pp等大侧链疏水性多肽。wt、pp等大侧链疏水性多肽为连接肽,酶切后的pkek单体产量仅仅与无连接肽组相当。可见,本发明提供的连接肽可大幅提高pkek单体酶切收率。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,其他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>珠海联邦制药股份有限公司<120>一种pkek融合蛋白及制备方法与应用<160>56<210>1<223>taf12metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpile657075<210>2<223>(pkek-aak)10prolysglulysalaalalysprolysglulysalaalalysprolys151015glulysalaalalysprolysglulysalaalalysprolysglulys202530alaalalysprolysglulysalaalalysprolysglulysalaala354045lysprolysglulysalaalalysprolysglulysalaalalyspro505560lysglulysalaalalys6570<210>3<223>taf12-k-(pkek-aak)10metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilelys65707580prolysglulysalaalalysprolysglulysalaalalysprolys859095glulysalaalalysprolysglulysalaalalysprolysglulys100105110alaalalysprolysglulysalaalalysprolysglulysalaala115120125lysprolysglulysalaalalysprolysglulysalaalalyspro130135140lysglulysalaalalys145150<210>4<223>taf12-k-(pkek-aak)10catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240aaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagct300gctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaa360gctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaa420aaagctgctaaaccgaaagaaaaagctgctaaataatgactcgag465<210>5<223>t7启动子引物taatacgactcactataggg20<210>6<223>t7终止子引物tgctagttattgctcagcgg20<210>7<223>(pkek)10prolysglulysprolysglulysprolysglulysprolysglulys151015prolysglulysprolysglulysprolysglulysprolysglulys202530prolysglulysprolysglulys3540<210>8<223>(pkek)10catatgccgaaagaaaaaccgaaagaaaaaccgaaagaaaaaccgaaagaaaaaccgaaa60gaaaaaccgaaagaaaaaccgaaagaaaaaccgaaagaaaaaccgaaagaaaaaccgaaa120gaaaaataatgactcgag138<210>9<223>(pkek-aak)10catatgccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaa60gctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaa120aaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaa180gaaaaagctgctaaaccgaaagaaaaagctgctaaataatgactcgag228<210>10<223>(pkek-aak)20prolysglulysalaalalysprolysglulysalaalalysprolys151015glulysalaalalysprolysglulysalaalalysprolysglulys202530alaalalysprolysglulysalaalalysprolysglulysalaala354045lysprolysglulysalaalalysprolysglulysalaalalyspro505560lysglulysalaalalysprolysglulysalaalalysprolysglu65707580lysalaalalysprolysglulysalaalalysprolysglulysala859095alalysprolysglulysalaalalysprolysglulysalaalalys100105110prolysglulysalaalalysprolysglulysalaalalysprolys115120125glulysalaalalysprolysglulysalaalalys130135140<210>11<223>(pkek-aak)20catatgccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaa60gctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaa120aaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaa180gaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccg240aaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaa300ccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgct360aaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagct420gctaaataatgactcgag438<210>12<223>taf12-k-(pkek-ak)10metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilelys65707580prolysglulysalalysprolysglulysalalysprolysglulys859095alalysprolysglulysalalysprolysglulysalalysprolys100105110glulysalalysprolysglulysalalysprolysglulysalalys115120125prolysglulysalalysprolysglulysalalys130135140<210>13<223>taf12-k-(pkek-ak)10catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240aaaccgaaagaaaaagctaaaccgaaagaaaaagctaaaccgaaagaaaaagctaaaccg300aaagaaaaagctaaaccgaaagaaaaagctaaaccgaaagaaaaagctaaaccgaaagaa360aaagctaaaccgaaagaaaaagctaaaccgaaagaaaaagctaaaccgaaagaaaaagct420aaataatgactcgag435<210>14<223>taf12-k-(pkek-gk)10metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilelys65707580prolysglulysglylysprolysglulysglylysprolysglulys859095glylysprolysglulysglylysprolysglulysglylysprolys100105110glulysglylysprolysglulysglylysprolysglulysglylys115120125prolysglulysglylysprolysglulysglylys130135140<210>15<223>taf12-k-(pkek-gk)10catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240aaaccgaaagaaaaaggtaaaccgaaagaaaaaggtaaaccgaaagaaaaaggtaaaccg300aaagaaaaaggtaaaccgaaagaaaaaggtaaaccgaaagaaaaaggtaaaccgaaagaa360aaaggtaaaccgaaagaaaaaggtaaaccgaaagaaaaaggtaaaccgaaagaaaaaggt420aaataatgactcgag435<210>16<223>taf12-k-(pkek-aagk)10metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilelys65707580prolysglulysalaalaglylysprolysglulysalaalaglylys859095prolysglulysalaalaglylysprolysglulysalaalaglylys100105110prolysglulysalaalaglylysprolysglulysalaalaglylys115120125prolysglulysalaalaglylysprolysglulysalaalaglylys130135140prolysglulysalaalaglylysprolysglulysalaalaglylys145150155160<210>17<223>taf12-k-(pkek-aagk)10catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240aaaccgaaagaaaaagctgctggtaaaccgaaagaaaaagctgctggtaaaccgaaagaa300aaagctgctggtaaaccgaaagaaaaagctgctggtaaaccgaaagaaaaagctgctggt360aaaccgaaagaaaaagctgctggtaaaccgaaagaaaaagctgctggtaaaccgaaagaa420aaagctgctggtaaaccgaaagaaaaagctgctggtaaaccgaaagaaaaagctgctggt480aaataatgactcgag495<210>18<223>taf12-k-(pkek-ggaak)10metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilelys65707580prolysglulysglyglyalaalalysprolysglulysglyglyala859095alalysprolysglulysglyglyalaalalysprolysglulysgly100105110glyalaalalysprolysglulysglyglyalaalalysprolysglu115120125lysglyglyalaalalysprolysglulysglyglyalaalalyspro130135140lysglulysglyglyalaalalysprolysglulysglyglyalaala145150155160lysprolysglulysglyglyalaalalys165170<210>19<223>taf12-k-(pkek-ggaak)10catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240aaaccgaaagaaaaaggtggtgctgctaaaccgaaagaaaaaggtggtgctgctaaaccg300aaagaaaaaggtggtgctgctaaaccgaaagaaaaaggtggtgctgctaaaccgaaagaa360aaaggtggtgctgctaaaccgaaagaaaaaggtggtgctgctaaaccgaaagaaaaaggt420ggtgctgctaaaccgaaagaaaaaggtggtgctgctaaaccgaaagaaaaaggtggtgct480gctaaaccgaaagaaaaaggtggtgctgctaaataatgactcgag525<210>20<223>taf12-k-(pkek-wtk)10metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilelys65707580prolysglulystrpthrlysprolysglulystrpthrlysprolys859095glulystrpthrlysprolysglulystrpthrlysprolysglulys100105110trpthrlysprolysglulystrpthrlysprolysglulystrpthr115120125lysprolysglulystrpthrlysprolysglulystrpthrlyspro130135140lysglulystrpthrlys145150<210>21<223>taf12-k-(pkek-wtk)10catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240aaaccgaaagaaaaatggaccaaaccgaaagaaaaatggaccaaaccgaaagaaaaatgg300accaaaccgaaagaaaaatggaccaaaccgaaagaaaaatggaccaaaccgaaagaaaaa360tggaccaaaccgaaagaaaaatggaccaaaccgaaagaaaaatggaccaaaccgaaagaa420aaatggaccaaaccgaaagaaaaatggaccaaataatgactcgag465<210>22<223>taf12-k-(pkek-ppk)10metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilelys65707580prolysglulysproprolysprolysglulysproprolysprolys859095glulysproprolysprolysglulysproprolysprolysglulys100105110proprolysprolysglulysproprolysprolysglulyspropro115120125lysprolysglulysproprolysprolysglulysproprolyspro130135140lysglulysproprolys145150<210>23<223>taf12-k-(pkek-ppk)10catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240aaaccgaaagaaaaaccgccgaaaccgaaagaaaaaccgccgaaaccgaaagaaaaaccg300ccgaaaccgaaagaaaaaccgccgaaaccgaaagaaaaaccgccgaaaccgaaagaaaaa360ccgccgaaaccgaaagaaaaaccgccgaaaccgaaagaaaaaccgccgaaaccgaaagaa420aaaccgccgaaaccgaaagaaaaaccgccgaaataatgactcgag465<210>24<223>pagp分子伴侣metasnalaaspglutrpmetthrthrphearggluasnilealagln151015thrtrpglnglnprogluhistyraspleutyrileproalailethr202530trphisalaargphealatyrasplysglulysthraspargtyrasn354045gluargprotrpglyglyglypheglyleuserargtrpaspglulys505560glyasntrphisglyleutyralametalaphelysaspsertrpasn65707580lystrpgluproilealaglytyrglytrpgluserthrtrpargpro859095leualaaspgluasnphehisleuglyleuglyphethralaglyval100105110thralaargaspasntrpasntyrileproleuprovalleuleupro115120125leualaservalglytyrglyprovalthrpheglnmetthrtyrile130135140proglythrtyrasnasnglyasnvaltyrphealatrpmetargphe145150155160glnphe<210>25<223>taf12-pagp-k-(pkek-aak)10metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilemet65707580asnalaaspglutrpmetthrthrphearggluasnilealaglnthr859095trpglnglnprogluhistyraspleutyrileproalailethrtrp100105110hisalaargphealatyrasplysglulysthraspargtyrasnglu115120125argprotrpglyglyglypheglyleuserargtrpaspglulysgly130135140asntrphisglyleutyralametalaphelysaspsertrpasnlys145150155160trpgluproilealaglytyrglytrpgluserthrtrpargproleu165170175alaaspgluasnphehisleuglyleuglyphethralaglyvalthr180185190alaargaspasntrpasntyrileproleuprovalleuleuproleu195200205alaservalglytyrglyprovalthrpheglnmetthrtyrilepro210215220glythrtyrasnasnglyasnvaltyrphealatrpmetargphegln225230235240phelysprolysglulysalaalalysprolysglulysalaalalys245250255prolysglulysalaalalysprolysglulysalaalalysprolys260265270glulysalaalalysprolysglulysalaalalysprolysglulys275280285alaalalysprolysglulysalaalalysprolysglulysalaala290295300lysprolysglulysalaalalys305310<210>26<223>taf12-pagp-k-(pkek-aak)10catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240atgaacgctgacgaatggatgaccaccttccgtgaaaacatcgctcagacctggcagcag300ccggaacactacgacctgtacatcccggctatcacctggcacgctcgtttcgcttacgac360aaagaaaaaaccgaccgttacaacgaacgtccgtggggtggtggtttcggtctgtctcgt420tgggacgaaaaaggtaactggcacggtctgtacgctatggctttcaaagactcttggaac480aaatgggaaccgatcgctggttacggttgggaatctacctggcgtccgctggctgacgaa540aacttccacctgggtctgggtttcaccgctggtgttaccgctcgtgacaactggaactac600atcccgctgccggttctgctgccgctggcttctgttggttacggtccggttaccttccag660atgacctacatcccgggtacctacaacaacggtaacgtttacttcgcttggatgcgtttc720cagttcaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaa780aaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaa840gaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccg900aaagaaaaagctgctaaaccgaaagaaaaagctgctaaataatgactcgag951<210>27<223>taf12-pagp-k-(pkek-aak)20metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilemet65707580asnalaaspglutrpmetthrthrphearggluasnilealaglnthr859095trpglnglnprogluhistyraspleutyrileproalailethrtrp100105110hisalaargphealatyrasplysglulysthraspargtyrasnglu115120125argprotrpglyglyglypheglyleuserargtrpaspglulysgly130135140asntrphisglyleutyralametalaphelysaspsertrpasnlys145150155160trpgluproilealaglytyrglytrpgluserthrtrpargproleu165170175alaaspgluasnphehisleuglyleuglyphethralaglyvalthr180185190alaargaspasntrpasntyrileproleuprovalleuleuproleu195200205alaservalglytyrglyprovalthrpheglnmetthrtyrilepro210215220glythrtyrasnasnglyasnvaltyrphealatrpmetargphegln225230235240phelysprolysglulysalaalalysprolysglulysalaalalys245250255prolysglulysalaalalysprolysglulysalaalalysprolys260265270glulysalaalalysprolysglulysalaalalysprolysglulys275280285alaalalysprolysglulysalaalalysprolysglulysalaala290295300lysprolysglulysalaalalysprolysglulysalaalalyspro305310315320lysglulysalaalalysprolysglulysalaalalysprolysglu325330335lysalaalalysprolysglulysalaalalysprolysglulysala340345350alalysprolysglulysalaalalysprolysglulysalaalalys355360365prolysglulysalaalalysprolysglulysalaalalys370375380<210>28<223>taf12-pagp-k-(pkek-aak)20catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240atgaacgctgacgaatggatgaccaccttccgtgaaaacatcgctcagacctggcagcag300ccggaacactacgacctgtacatcccggctatcacctggcacgctcgtttcgcttacgac360aaagaaaaaaccgaccgttacaacgaacgtccgtggggtggtggtttcggtctgtctcgt420tgggacgaaaaaggtaactggcacggtctgtacgctatggctttcaaagactcttggaac480aaatgggaaccgatcgctggttacggttgggaatctacctggcgtccgctggctgacgaa540aacttccacctgggtctgggtttcaccgctggtgttaccgctcgtgacaactggaactac600atcccgctgccggttctgctgccgctggcttctgttggttacggtccggttaccttccag660atgacctacatcccgggtacctacaacaacggtaacgtttacttcgcttggatgcgtttc720cagttcaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaa780aaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaa840gaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccg900aaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaa960ccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgct1020aaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagct1080gctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaa1140gctgctaaataatgactcgag1161<210>29<223>ksi分子伴侣methisthrprogluhisilethralavalvalglnargphevalala151015alaleuasnalaglyaspleuaspglyilevalalaleuphealaasp202530aspalathrvalgluaspprovalglysergluproargserglythr354045alaalailearggluphetyralaasnserleulysleuproleuala505560valgluleuthrglngluvalargalavalalaasnglualaalaphe65707580alaphethrvalserpheglutyrglnglyarglysthrvalvalala859095proileasphispheargpheasnglyalaglylysvalvalserile100105110argalaleupheglyglulysasnilehisalacys115120<210>30<223>taf12-ksi-k-(pkek-aak)10metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilemet65707580histhrprogluhisilethralavalvalglnargphevalalaala859095leuasnalaglyaspleuaspglyilevalalaleuphealaaspasp100105110alathrvalgluaspprovalglysergluproargserglythrala115120125alailearggluphetyralaasnserleulysleuproleualaval130135140gluleuthrglngluvalargalavalalaasnglualaalapheala145150155160phethrvalserpheglutyrglnglyarglysthrvalvalalapro165170175ileasphispheargpheasnglyalaglylysvalvalserilearg180185190alaleupheglyglulysasnilehisalacyslysprolysglulys195200205alaalalysprolysglulysalaalalysprolysglulysalaala210215220lysprolysglulysalaalalysprolysglulysalaalalyspro225230235240lysglulysalaalalysprolysglulysalaalalysprolysglu245250255lysalaalalysprolysglulysalaalalysprolysglulysala260265270alalys<210>31<223>taf12-ksi-k-(pkek-aak)10catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240atgcacaccccggaacacatcaccgctgttgttcagcgtttcgttgctgctctgaacgct300ggtgacctggacggtatcgttgctctgttcgctgacgacgctaccgttgaagacccggtt360ggttctgaaccgcgttctggtaccgctgctatccgtgaattctacgctaactctctgaaa420ctgccgctggctgttgaactgacccaggaagttcgtgctgttgctaacgaagctgctttc480gctttcaccgtttctttcgaataccagggtcgtaaaaccgttgttgctccgatcgaccac540ttccgtttcaacggtgctggtaaagttgtttctatccgtgctctgttcggtgaaaaaaac600atccacgcttgcaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccg660aaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaa720ccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgct780aaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaataatgactcgag837<210>32<223>taf12-ksi-k-(pkek-aak)20metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilemet65707580histhrprogluhisilethralavalvalglnargphevalalaala859095leuasnalaglyaspleuaspglyilevalalaleuphealaaspasp100105110alathrvalgluaspprovalglysergluproargserglythrala115120125alailearggluphetyralaasnserleulysleuproleualaval130135140gluleuthrglngluvalargalavalalaasnglualaalapheala145150155160phethrvalserpheglutyrglnglyarglysthrvalvalalapro165170175ileasphispheargpheasnglyalaglylysvalvalserilearg180185190alaleupheglyglulysasnilehisalacyslysprolysglulys195200205alaalalysprolysglulysalaalalysprolysglulysalaala210215220lysprolysglulysalaalalysprolysglulysalaalalyspro225230235240lysglulysalaalalysprolysglulysalaalalysprolysglu245250255lysalaalalysprolysglulysalaalalysprolysglulysala260265270alalysprolysglulysalaalalysprolysglulysalaalalys275280285prolysglulysalaalalysprolysglulysalaalalysprolys290295300glulysalaalalysprolysglulysalaalalysprolysglulys305310315320alaalalysprolysglulysalaalalysprolysglulysalaala325330335lysprolysglulysalaalalys340<210>33<223>taf12-ksi-k-(pkek-aak)20catatgtctccggaaaacaaccaggttctgaccaaaaaaaaactgcaggacctggttcgt60gaagttgacccgaacgaacagctggacgaagacgttgaagaaatgctgctgcagatcgct120gacgacttcatcgaatctgttgttaccgctgcttgccagctggctcgtcaccgtaaatct180tctaccctggaagttaaagacgttcagctgcacctggaacgtcagtggaacatgtggatc240atgcacaccccggaacacatcaccgctgttgttcagcgtttcgttgctgctctgaacgct300ggtgacctggacggtatcgttgctctgttcgctgacgacgctaccgttgaagacccggtt360ggttctgaaccgcgttctggtaccgctgctatccgtgaattctacgctaactctctgaaa420ctgccgctggctgttgaactgacccaggaagttcgtgctgttgctaacgaagctgctttc480gctttcaccgtttctttcgaataccagggtcgtaaaaccgttgttgctccgatcgaccac540ttccgtttcaacggtgctggtaaagttgtttctatccgtgctctgttcggtgaaaaaaac600atccacgcttgcaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccg660aaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaa720ccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgct780aaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagct840gctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaa900gctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaa960aaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaa1020gaaaaagctgctaaataatgactcgag1047<210>34<223>gygbmetglulysvalproglyglumetgluilegluargarggluargser151015glugluleuserglualagluarglysalavalglnalamettrpala202530argleutyralaasncysgluaspvalglyvalalaileleuvalarg354045phephevalasnpheproseralalysglntyrpheserglnphelys505560hismetgluaspproleuglumetgluargserproglnleuarglys65707580hisalacysargvalmetglyalaleuasnthrvalvalgluasnleu859095hisaspproasp100<210>35<211>390<223>taf12-gygb-k-(pkek-aak)30metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilemet65707580glulysvalproglyglumetgluilegluargarggluargserglu859095gluleuserglualagluarglysalavalglnalamettrpalaarg100105110leutyralaasncysgluaspvalglyvalalaileleuvalargphe115120125phevalasnpheproseralalysglntyrpheserglnphelyshis130135140metgluaspproleuglumetgluargserproglnleuarglyshis145150155160alacysargvalmetglyalaleuasnthrvalvalgluasnleuhis165170175aspproasplysprolysglulysalaalalysprolysglulysala180185190alalysprolysglulysalaalalysprolysglulysalaalalys195200205prolysglulysalaalalysprolysglulysalaalalysprolys210215220glulysalaalalysprolysglulysalaalalysprolysglulys225230235240alaalalysprolysglulysalaalalysprolysglulysalaala245250255lysprolysglulysalaalalysprolysglulysalaalalyspro260265270lysglulysalaalalysprolysglulysalaalalysprolysglu275280285lysalaalalysprolysglulysalaalalysprolysglulysala290295300alalysprolysglulysalaalalysprolysglulysalaalalys305310315320prolysglulysalaalalysprolysglulysalaalalysprolys325330335glulysalaalalysprolysglulysalaalalysprolysglulys340345350alaalalysprolysglulysalaalalysprolysglulysalaala355360365lysprolysglulysalaalalysprolysglulysalaalalyspro370375380lysglulysalaalalys385390<210>36<223>taf12-gygb-k-(pkek-aak)30catatgtccccggaaaacaaccaggtgctgaccaaaaaaaaactgcaggacctggtgcgc60gaagtggacccgaacgaacagctggacgaagacgtggaagaaatgctgctgcagatcgcc120gacgacttcatcgaatccgtggtgaccgccgcctgccagctggcccgccaccgcaaatcc180tccaccctggaagtgaaagacgtgcagctgcacctggaacgccagtggaacatgtggatc240atggaaaaagtgccgggcgaaatggaaatcgaacgccgcgaacgctccgaagaactgtcc300gaagccgaacgcaaagccgtgcaggccatgtgggcccgcctgtacgccaactgcgaagac360gtgggcgtggccatcctggtgcgcttcttcgtgaacttcccgtccgccaaacagtacttc420tcccagttcaaacacatggaagacccgctggaaatggaacgctccccgcagctgcgcaaa480cacgcctgccgcgtgatgggcgccctgaacaccgtggtggaaaacctgcacgacccggac540aaaccgaaagaaaaagccgccaaaccgaaagaaaaagccgccaaaccgaaagaaaaagcc600gccaaaccgaaagaaaaagccgccaaaccgaaagaaaaagccgccaaaccgaaagaaaaa660gccgccaaaccgaaagaaaaagccgccaaaccgaaagaaaaagccgccaaaccgaaagaa720aaagccgccaaaccgaaagaaaaagccgccaaaccgaaagaaaaagccgccaaaccgaaa780gaaaaagccgccaaaccgaaagaaaaagccgccaaaccgaaagaaaaagccgccaaaccg840aaagaaaaagccgccaaaccgaaagaaaaagccgccaaaccgaaagaaaaagccgccaaa900ccgaaagaaaaagccgccaaaccgaaagaaaaagccgccaaaccgaaagaaaaagccgcc960aaaccgaaagaaaaagccgccaaaccgaaagaaaaagccgccaaaccgaaagaaaaagcc1020gccaaaccgaaagaaaaagccgccaaaccgaaagaaaaagccgccaaaccgaaagaaaaa1080gccgccaaaccgaaagaaaaagccgccaaaccgaaagaaaaagccgccaaaccgaaagaa1140aaagccgccaaaccgaaagaaaaagccgccaaataatgactcgag1185<210>37<211>215<223>ksi-k-(pkek-ggaak)10methisthrprogluhisilethralavalvalglnargphevalala151015alaleuasnalaglyaspleuaspglyilevalalaleuphealaasp202530aspalathrvalgluaspprovalglysergluproargserglythr354045alaalailearggluphetyralaasnserleulysleuproleuala505560valgluleuthrglngluvalargalavalalaasnglualaalaphe65707580alaphethrvalserpheglutyrglnglyarglysthrvalvalala859095proileasphispheargpheasnglyalaglylysvalvalserile100105110argalaleupheglyglulysasnilehisalacyslysprolysglu115120125lysglyglyalaalalysprolysglulysglyglyalaalalyspro130135140lysglulysglyglyalaalalysprolysglulysglyglyalaala145150155160lysprolysglulysglyglyalaalalysprolysglulysglygly165170175alaalalysprolysglulysglyglyalaalalysprolysglulys180185190glyglyalaalalysprolysglulysglyglyalaalalysprolys195200205glulysglyglyalaalalys210215<210>38<223>ksi-k-(pkek-ggaak)10catatgcacaccccggaacacatcaccgctgttgttcagcgtttcgttgctgctctgaac60gctggtgacctggacggtatcgttgctctgttcgctgacgacgctaccgttgaagacccg120gttggttctgaaccgcgttctggtaccgctgctatccgtgaattctacgctaactctctg180aaactgccgctggctgttgaactgacccaggaagttcgtgctgttgctaacgaagctgct240ttcgctttcaccgtttctttcgaataccagggtcgtaaaaccgttgttgctccgatcgac300cacttccgtttcaacggtgctggtaaagttgtttctatccgtgctctgttcggtgaaaaa360aacatccacgcttgcaaaccgaaagaaaaaggtggtgctgctaaaccgaaagaaaaaggt420ggtgctgctaaaccgaaagaaaaaggtggtgctgctaaaccgaaagaaaaaggtggtgct480gctaaaccgaaagaaaaaggtggtgctgctaaaccgaaagaaaaaggtggtgctgctaaa540ccgaaagaaaaaggtggtgctgctaaaccgaaagaaaaaggtggtgctgctaaaccgaaa600gaaaaaggtggtgctgctaaaccgaaagaaaaaggtggtgctgctaaataatgactcgag660<210>39<223>ksi-k-(pkek-ggsask)20methisthrprogluhisilethralavalvalglnargphevalala151015alaleuasnalaglyaspleuaspglyilevalalaleuphealaasp202530aspalathrvalgluaspprovalglysergluproargserglythr354045alaalailearggluphetyralaasnserleulysleuproleuala505560valgluleuthrglngluvalargalavalalaasnglualaalaphe65707580alaphethrvalserpheglutyrglnglyarglysthrvalvalala859095proileasphispheargpheasnglyalaglylysvalvalserile100105110argalaleupheglyglulysasnilehisalacyslysprolysglu115120125lysglyglyseralaserlysprolysglulysglyglyseralaser130135140lysprolysglulysglyglyseralaserlysprolysglulysgly145150155160glyseralaserlysprolysglulysglyglyseralaserlyspro165170175lysglulysglyglyseralaserlysprolysglulysglyglyser180185190alaserlysprolysglulysglyglyseralaserlysprolysglu195200205lysglyglyseralaserlysprolysglulysglyglyseralaser210215220lysprolysglulysglyglyseralaserlysprolysglulysgly225230235240glyseralaserlysprolysglulysglyglyseralaserlyspro245250255lysglulysglyglyseralaserlysprolysglulysglyglyser260265270alaserlysprolysglulysglyglyseralaserlysprolysglu275280285lysglyglyseralaserlysprolysglulysglyglyseralaser290295300lysprolysglulysglyglyseralaserlysprolysglulysgly305310315320glyseralaserlys325<210>40<223>ksi-k-(pkek-ggsask)20catatgcacaccccggaacacatcaccgctgttgttcagcgtttcgttgctgctctgaac60gctggtgacctggacggtatcgttgctctgttcgctgacgacgctaccgttgaagacccg120gttggttctgaaccgcgttctggtaccgctgctatccgtgaattctacgctaactctctg180aaactgccgctggctgttgaactgacccaggaagttcgtgctgttgctaacgaagctgct240ttcgctttcaccgtttctttcgaataccagggtcgtaaaaccgttgttgctccgatcgac300cacttccgtttcaacggtgctggtaaagttgtttctatccgtgctctgttcggtgaaaaa360aacatccacgcttgcaaaccgaaagaaaaaggtggttctgcttctaaaccgaaagaaaaa420ggtggttctgcttctaaaccgaaagaaaaaggtggttctgcttctaaaccgaaagaaaaa480ggtggttctgcttctaaaccgaaagaaaaaggtggttctgcttctaaaccgaaagaaaaa540ggtggttctgcttctaaaccgaaagaaaaaggtggttctgcttctaaaccgaaagaaaaa600ggtggttctgcttctaaaccgaaagaaaaaggtggttctgcttctaaaccgaaagaaaaa660ggtggttctgcttctaaaccgaaagaaaaaggtggttctgcttctaaaccgaaagaaaaa720ggtggttctgcttctaaaccgaaagaaaaaggtggttctgcttctaaaccgaaagaaaaa780ggtggttctgcttctaaaccgaaagaaaaaggtggttctgcttctaaaccgaaagaaaaa840ggtggttctgcttctaaaccgaaagaaaaaggtggttctgcttctaaaccgaaagaaaaa900ggtggttctgcttctaaaccgaaagaaaaaggtggttctgcttctaaaccgaaagaaaaa960ggtggttctgcttctaaataatgactcgag990<210>41<223>pagp-k-(pkek-aak)10metasnalaaspglutrpmetthrthrphearggluasnilealagln151015thrtrpglnglnprogluhistyraspleutyrileproalailethr202530trphisalaargphealatyrasplysglulysthraspargtyrasn354045gluargprotrpglyglyglypheglyleuserargtrpaspglulys505560glyasntrphisglyleutyralametalaphelysaspsertrpasn65707580lystrpgluproilealaglytyrglytrpgluserthrtrpargpro859095leualaaspgluasnphehisleuglyleuglyphethralaglyval100105110thralaargaspasntrpasntyrileproleuprovalleuleupro115120125leualaservalglytyrglyprovalthrpheglnmetthrtyrile130135140proglythrtyrasnasnglyasnvaltyrphealatrpmetargphe145150155160glnphelysprolysglulysalaalalysprolysglulysalaala165170175lysprolysglulysalaalalysprolysglulysalaalalyspro180185190lysglulysalaalalysprolysglulysalaalalysprolysglu195200205lysalaalalysprolysglulysalaalalysprolysglulysala210215220alalysprolysglulysalaalalys225230<210>42<223>pagp-k-(pkek-aak)10catatgaacgctgacgaatggatgaccaccttccgtgaaaacatcgctcagacctggcag60cagccggaacactacgacctgtacatcccggctatcacctggcacgctcgtttcgcttac120gacaaagaaaaaaccgaccgttacaacgaacgtccgtggggtggtggtttcggtctgtct180cgttgggacgaaaaaggtaactggcacggtctgtacgctatggctttcaaagactcttgg240aacaaatgggaaccgatcgctggttacggttgggaatctacctggcgtccgctggctgac300gaaaacttccacctgggtctgggtttcaccgctggtgttaccgctcgtgacaactggaac360tacatcccgctgccggttctgctgccgctggcttctgttggttacggtccggttaccttc420cagatgacctacatcccgggtacctacaacaacggtaacgtttacttcgcttggatgcgt480ttccagttcaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaa540gaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccg600aaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaa660ccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaataatgactcgag714<210>43<223>ksi-pagp-k-(pkek-aak)10methisthrprogluhisilethralavalvalglnargphevalala151015alaleuasnalaglyaspleuaspglyilevalalaleuphealaasp202530aspalathrvalgluaspprovalglysergluproargserglythr354045alaalailearggluphetyralaasnserleulysleuproleuala505560valgluleuthrglngluvalargalavalalaasnglualaalaphe65707580alaphethrvalserpheglutyrglnglyarglysthrvalvalala859095proileasphispheargpheasnglyalaglylysvalvalserile100105110argalaleupheglyglulysasnilehisalacysmetasnalaasp115120125glutrpmetthrthrphearggluasnilealaglnthrtrpglngln130135140progluhistyraspleutyrileproalailethrtrphisalaarg145150155160phealatyrasplysglulysthraspargtyrasngluargprotrp165170175glyglyglypheglyleuserargtrpaspglulysglyasntrphis180185190glyleutyralametalaphelysaspsertrpasnlystrpglupro195200205ilealaglytyrglytrpgluserthrtrpargproleualaaspglu210215220asnphehisleuglyleuglyphethralaglyvalthralaargasp225230235240asntrpasntyrileproleuprovalleuleuproleualaserval245250255glytyrglyprovalthrpheglnmetthrtyrileproglythrtyr260265270asnasnglyasnvaltyrphealatrpmetargpheglnphelyspro275280285lysglulysalaalalysprolysglulysalaalalysprolysglu290295300lysalaalalysprolysglulysalaalalysprolysglulysala305310315320alalysprolysglulysalaalalysprolysglulysalaalalys325330335prolysglulysalaalalysprolysglulysalaalalysprolys340345350glulysalaalalys355<210>44<223>ksi-pagp-k-(pkek-aak)10catatgcacaccccggaacacatcaccgctgttgttcagcgtttcgttgctgctctgaac60gctggtgacctggacggtatcgttgctctgttcgctgacgacgctaccgttgaagacccg120gttggttctgaaccgcgttctggtaccgctgctatccgtgaattctacgctaactctctg180aaactgccgctggctgttgaactgacccaggaagttcgtgctgttgctaacgaagctgct240ttcgctttcaccgtttctttcgaataccagggtcgtaaaaccgttgttgctccgatcgac300cacttccgtttcaacggtgctggtaaagttgtttctatccgtgctctgttcggtgaaaaa360aacatccacgcttgcatgaacgctgacgaatggatgaccaccttccgtgaaaacatcgct420cagacctggcagcagccggaacactacgacctgtacatcccggctatcacctggcacgct480cgtttcgcttacgacaaagaaaaaaccgaccgttacaacgaacgtccgtggggtggtggt540ttcggtctgtctcgttgggacgaaaaaggtaactggcacggtctgtacgctatggctttc600aaagactcttggaacaaatgggaaccgatcgctggttacggttgggaatctacctggcgt660ccgctggctgacgaaaacttccacctgggtctgggtttcaccgctggtgttaccgctcgt720gacaactggaactacatcccgctgccggttctgctgccgctggcttctgttggttacggt780ccggttaccttccagatgacctacatcccgggtacctacaacaacggtaacgtttacttc840gcttggatgcgtttccagttcaaaccgaaagaaaaagctgctaaaccgaaagaaaaagct900gctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaa960gctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaa1020aaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaataatga1080ctcgag1086<210>45<223>pap3.30分子伴侣metleuserlysserleugluasphisglualaglyvalcysproleu151015glycysprotyrcysleualaasptyrgluargiletrpglyvalarg202530valvalserservalalaalaserasnasplysvalgluvalasppro354045asnglyilelysalaglyaspproglyalalysleuasplysglylys505560valaspvalglyileilepheglualapheproargalaleutyrala65707580valalaglnvalalaasnpheglyalaserlystyrserargglygly859095trpargphevalgluasnglyileglnargtyraspalaalaphegly100105110arghisleuleugluarghislysglygluvalleuaspproglnser115120125serleuprohisargtyrhisgluvaltrpasnalaleualaalaleu130135140gluleuvalileglnglnglugluglyserasnglythrserthrgly145150155160serglugly<210>46<223>pap3.30-k-(pkek-aak)10metleuserlysserleugluasphisglualaglyvalcysproleu151015glycysprotyrcysleualaasptyrgluargiletrpglyvalarg202530valvalserservalalaalaserasnasplysvalgluvalasppro354045asnglyilelysalaglyaspproglyalalysleuasplysglylys505560valaspvalglyileilepheglualapheproargalaleutyrala65707580valalaglnvalalaasnpheglyalaserlystyrserargglygly859095trpargphevalgluasnglyileglnargtyraspalaalaphegly100105110arghisleuleugluarghislysglygluvalleuaspproglnser115120125serleuprohisargtyrhisgluvaltrpasnalaleualaalaleu130135140gluleuvalileglnglnglugluglyserasnglythrserthrgly145150155160sergluglylysprolysglulysalaalalysprolysglulysala165170175alalysprolysglulysalaalalysprolysglulysalaalalys180185190prolysglulysalaalalysprolysglulysalaalalysprolys195200205glulysalaalalysprolysglulysalaalalysprolysglulys210215220alaalalysprolysglulysalaalalys225230<210>47<223>pap3.30-k-(pkek-aak)10catatgctgtctaaatctctggaagaccacgaagctggtgtttgcccgctgggttgcccg60tactgcctggctgactacgaacgtatctggggtgttcgtgttgtttcttctgttgctgct120tctaacgacaaagttgaagttgacccgaacggtatcaaagctggtgacccgggtgctaaa180ctggacaaaggtaaagttgacgttggtatcatcttcgaagctttcccgcgtgctctgtac240gctgttgctcaggttgctaacttcggtgcttctaaatactctcgtggtggttggcgtttc300gttgaaaacggtatccagcgttacgacgctgctttcggtcgtcacctgctggaacgtcac360aaaggtgaagttctggacccgcagtcttctctgccgcaccgttaccacgaagtttggaac420gctctggctgctctggaactggttatccagcaggaagaaggttctaacggtacctctacc480ggttctgaaggtaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccg540aaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaa600ccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgct660aaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaataatgactcgag717<210>48<223>gdf15metalaargasnglyasphiscysproleuglyproglyargcyscys151015argleuhisthrvalargalaserleugluaspleuglytrpalaasp202530trpvalleuserproarggluvalglnvalthrmetcysileglyala354045cysproserglnpheargalaalaasnmethisalaglnilelysthr505560serleuhisargleulysproaspthrvalproalaprocyscysval65707580proalasertyrasnprometvalleuileglnlysthraspthrgly859095valserleuglnthrtyraspaspleuleualalysaspcyshiscys100105110ile<210>49<223>gdf15-k-(pkek-aak)10metalaargasnglyasphiscysproleuglyproglyargcyscys151015argleuhisthrvalargalaserleugluaspleuglytrpalaasp202530trpvalleuserproarggluvalglnvalthrmetcysileglyala354045cysproserglnpheargalaalaasnmethisalaglnilelysthr505560serleuhisargleulysproaspthrvalproalaprocyscysval65707580proalasertyrasnprometvalleuileglnlysthraspthrgly859095valserleuglnthrtyraspaspleuleualalysaspcyshiscys100105110ilelysprolysglulysalaalalysprolysglulysalaalalys115120125prolysglulysalaalalysprolysglulysalaalalysprolys130135140glulysalaalalysprolysglulysalaalalysprolysglulys145150155160alaalalysprolysglulysalaalalysprolysglulysalaala165170175lysprolysglulysalaalalys180<210>50<223>gdf15-k-(pkek-aak)10catatggctcgtaacggtgaccactgcccgctgggtccgggtcgttgctgccgtctgcac60accgttcgtgcttctctggaagacctgggttgggctgactgggttctgtctccgcgtgaa120gttcaggttaccatgtgcatcggtgcttgcccgtctcagttccgtgctgctaacatgcac180gctcagatcaaaacctctctgcaccgtctgaaaccggacaccgttccggctccgtgctgc240gttccggcttcttacaacccgatggttctgatccagaaaaccgacaccggtgtttctctg300cagacctacgacgacctgctggctaaagactgccactgcatcaaaccgaaagaaaaagct360gctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaa420gctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaa480aaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaa540gaaaaagctgctaaataatgactcgag567<210>51<223>gygb-k-(pkek-aak)10metglulysvalproglyglumetgluilegluargarggluargser151015glugluleuserglualagluarglysalavalglnalamettrpala202530argleutyralaasncysgluaspvalglyvalalaileleuvalarg354045phephevalasnpheproseralalysglntyrpheserglnphelys505560hismetgluaspproleuglumetgluargserproglnleuarglys65707580hisalacysargvalmetglyalaleuasnthrvalvalgluasnleu859095hisaspproasplysprolysglulysalaalalysprolysglulys100105110alaalalysprolysglulysalaalalysprolysglulysalaala115120125lysprolysglulysalaalalysprolysglulysalaalalyspro130135140lysglulysalaalalysprolysglulysalaalalysprolysglu145150155160lysalaalalysprolysglulysalaalalys165170<210>52<223>gygb-k-(pkek-aak)10catatggaaaaagttccgggtgaaatggaaatcgaacgtcgtgaacgttctgaagaactg60tctgaagctgaacgtaaagctgttcaggctatgtgggctcgtctgtacgctaactgcgaa120gacgttggtgttgctatcctggttcgtttcttcgttaacttcccgtctgctaaacagtac180ttctctcagttcaaacacatggaagacccgctggaaatggaacgttctccgcagctgcgt240aaacacgcttgccgtgttatgggtgctctgaacaccgttgttgaaaacctgcacgacccg300gacaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaa360gctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaa420aaagctgctaaaccgaaagaaaaagctgctaaaccgaaagaaaaagctgctaaaccgaaa480gaaaaagctgctaaaccgaaagaaaaagctgctaaataatgactcgag528<210>53<223>taf12-k-(pkek)10metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilelys65707580prolysglulysprolysglulysprolysglulysprolysglulys859095prolysglulysprolysglulysprolysglulysprolysglulys100105110prolysglulysprolysglulys115120<210>54<223>taf12-k-(pkek)10catatgtccccggaaaacaaccaggtcctgaccaagaagaagctgcaggacctggtccgc60gaagtcgacccgaacgaacagctggacgaagacgtcgaagaaatgctgctgcagatcgcc120gacgacttcatcgaatccgtcgtcaccgccgcctgccagctggcccgccaccgcaagtcc180tccaccctggaagtcaaggacgtccagctgcacctggaacgccagtggaacatgtggatc240aagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaa300aagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaa360aagtaatgactcgag375<210>55<223>taf12-k-(pkek)20metserprogluasnasnglnvalleuthrlyslyslysleuglnasp151015leuvalarggluvalaspproasngluglnleuaspgluaspvalglu202530glumetleuleuglnilealaaspasppheilegluservalvalthr354045alaalacysglnleualaarghisarglysserserthrleugluval505560lysaspvalglnleuhisleugluargglntrpasnmettrpilelys65707580prolysglulysprolysglulysprolysglulysprolysglulys859095prolysglulysprolysglulysprolysglulysprolysglulys100105110prolysglulysprolysglulysprolysglulysprolysglulys115120125prolysglulysprolysglulysprolysglulysprolysglulys130135140prolysglulysprolysglulysprolysglulysprolysglulys145150155160<210>56<223>taf12-k-(pkek)20catatgtccccggaaaacaaccaggtcctgaccaagaagaagctgcaggacctggtccgc60gaagtcgacccgaacgaacagctggacgaagacgtcgaagaaatgctgctgcagatcgcc120gacgacttcatcgaatccgtcgtcaccgccgcctgccagctggcccgccaccgcaagtcc180tccaccctggaagtcaaggacgtccagctgcacctggaacgccagtggaacatgtggatc240aagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaa300aagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaa360aagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaa420aagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaaaagccgaaggaa480aagtaatgactcgag495当前第1页12
当前第1页1 2 
网友询问留言 已有0条留言
  • 还没有人留言评论。精彩留言会获得点赞!
1