CN110484551B - 金属硫蛋白表达载体及其应用 - Google Patents
金属硫蛋白表达载体及其应用 Download PDFInfo
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- CN110484551B CN110484551B CN201910699097.2A CN201910699097A CN110484551B CN 110484551 B CN110484551 B CN 110484551B CN 201910699097 A CN201910699097 A CN 201910699097A CN 110484551 B CN110484551 B CN 110484551B
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- metallothionein
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Abstract
本发明公开一种金属硫蛋白表达载体及其应用,所述金属硫蛋白表达载体包括由金属硫蛋白的编码序列作为目标蛋白***类伴侣样蛋白的下游所形成的融合蛋白表达载体,其中,所述类伴侣样蛋白为序列如SEQ ID NO:1~3任一所示的链球菌蛋白G的B结构域PGB。本发明构建的金属硫蛋白表达载体,能够促进金属硫蛋白的表达并提高表达量,且经由该融合蛋白表达载体所表达的PGB‑MT融合蛋白在His‑Tag‑镍柱分离纯化后,能够被Fax酶切水解,并进一步通过常规方法纯化MT,为MT蛋白的科学研究和工业化生产提供了新技术。
Description
技术领域
本发明涉及基因工程和蛋白质表达技术领域,特别涉及重组人碱性成纤维细胞生长因子融合蛋白构建技术领域,具体涉及一种金属硫蛋白表达载体及其应用。
背景技术
金属硫蛋白(Metallothionein,MT)具有多种强大的生理功能,给其在治疗学上的应用提供了坚实基础。同时,MT也是进化上非常古老和保守的小分子蛋白性生物保护剂,从微生物到灵长类,都有自己的MT,这是因为重金属铅汞镉、自由基对所有动物、微生物的组织结构和机能都有伤害作用。其中,MT在人体医学中的应用体现在以下方面:
1、有害重金属解毒作用:皮肤是人体最大的器官,外界灰尘和皮肤污染物中(如含铅化妆品)的有害重金属较多,常常来自矿山粉尘、污染的公路或土地扬起的灰尘、含铅燃油的排烟、污染汽油车的尾气、污染的水源等等。人类皮肤具有微量吸收和累积能力,通过接触而被吸收,MT具有结合有害重金属,阻止有害重金属进入细胞影响细胞代谢机能,被MT结合包裹后的有害重金属不显示毒性。
2、MT的抗氧化作用:皮肤是人体的暴露器官,各种辐射(如太阳光的紫外等)首先作用于皮肤,导致皮肤和皮下的水分子发生电离效应,产生自由基;自由基是一种强氧化物,能使细胞膜通透性增加、细胞的酶和蛋白质氧化失活、DNA突变等,导致细胞代谢障碍、细胞衰老加速、细胞提早老化凋亡。同时使得皮肤的胶原蛋白(胶原纤维)、弹性蛋白(弹性纤维)氧化(即老化)而失去弹性,使得皮肤松弛,失去弹性,产生大量皱纹。因为MT主要集中在皮肤中,所以在护肤、延缓衰老、缓解皮肤过敏的抗衰美容日化产品中有重要用途。除此之外,MT抗氧化保护细胞的作用还可用在:①抗衰老、防癌:通过刺激机体淋巴细胞释放淋巴因子,激活免疫细胞活性,达到或恢复正常免疫监视功能,从而起到防癌作用,例如美国人将兔肝MT做成膳食补充剂。②癌症放化疗的辅助治疗手段:放射线和某些化疗药都能加速自由基的产生,MT可以中和自由基(提供电子,而本身不抢夺电子),从而减轻放化疗不良反应。③防止动脉粥样硬化:MT通过减少脂质过氧化物,阻碍氧化脂质对血管内皮细胞的损伤。④减轻和预防吸烟对人体的危害:吸烟会产生大量自由基,MT能中和自由基。⑤阻止或减少皮肤脂褐素和腊样质的产生:脂褐素和腊样质可致皮肤色斑和粗糙,自由基对这两类粘稠状物质形成起着至关重要的作用,在自由基与过氧化酶作用下,脂质形成脂质过氧化物,后者与某些蛋白质和磷脂聚合交联而形成脂褐素和腊样质。
3、参与微量元素补充、储存、运输和代谢,如锌、铜等,1分子MT与5~7个锌离子结合并被吸收,可预防机体由缺锌导致的对机体的不良影响。
4、辅助治疗糖尿病、冠心病、消化道溃疡、风湿及类风湿关节炎,防治老年性痴呆、皮肤过敏症等疾病。
其次,MT也可以应用于动物饲料中,例如饲料中添加清洁MT,可以帮助饲养低有害重金属含量的家畜、家禽,同时还可以提高养殖动物的免疫力,减少疫病发生。因此MT对肉食食品或禽蛋供应市场,也存在巨大的需求潜力。
此外,MT在植物抗逆性中也有应用,例如,植物金属硫蛋白在植物体内转运重金属并解除其毒性,转运必需微量金属作为金属供体,调节微量必需金属的体内稳态或金属库,还参与植物的多种胁迫抗性的形成和活性氧清除,对调节植物胞内氧化还原状态起到信号的作用;植物金属硫蛋白还受植物激素的调控,在植物的形态构建中发挥重要作用。因此植物金属硫蛋白是植物抗逆过程中的重要信号,具有重要的生理功能。所以,有望开发出植物生物刺激素或页面肥,用于植物抗病害能力提升,提高植物产量。
MT又称巯基金属结合蛋白,是一类非酶蛋白质,除含有镉(Cd)、锌(Zn)的MT外,在自然界也存在含有铜(Cu)、汞(Hz)、金(Au)、铋(Bi)等元素的MT。MT的蛋白质分子内不含有芳香族氨基酸和组氨酸。让MT中50%的金属离子发生解离的pH值分别为:Zn-MT:3.5~4.5;Cd-MT:2.5~3.5;Cu-MT<1.0。因MT缺乏芳香族氨基酸,故在280nm处无吸收峰,而具有与金属相关的吸收峰,去金属的MT在190nm处有一个吸收峰。无论是自然产生还是诱导产生的MT,其氨基酸组成基本相同,主要不同在所含金属及其含量。目前MT产品主要从兔肝、马肾、猪肝和微生物(如粗脉孢菌)等中提取。
关于MT的功能和开发利用,自1978年到1999年先后在瑞士、日本、美国共举行了四次国际专题研讨会,第五次国际专题研讨会于2005年10月在北京召开。1987年我国将MT列入[863]计划,“八五”、“九五”重大攻关项目,1994年列入国家级“火炬计划”;2003年MT项目被国家科技部列为“国家科技成果重点推广计划项目,1995年***将MT列入向世界各国推荐的21世纪生物技术产品。由此可见国内外对MT的研究、开发、推广、应用都给予了高度重视。
MT的发现和研究超过半个世纪,适于上世纪60年代。尽管MT的需求量很大,尤其人类MT,有可能发展到人类同源蛋白制药。然而,目前的工业化生产技术仍然没有得到广泛突破。MT的动物源性提取,依旧占据原料来源主要市场。然而,动物保护和动物疫病的传播(如疯牛病、动物病毒病等),使得各国都很谨慎使用和控制国外进口。在非控制饲养的方便来源的动物肝肾中提取MT,不仅产量低,而存在有害重金属携带可能。
关于MT的生物发酵生产方面,国内外曾报道用枯草杆菌、链球菌、酵母菌等菌种采取高重金属胁迫适应筛选菌株方法生产,上世纪也曾报道基因工程重组载体导入宿主大肠杆菌、酵母菌诱导发酵生产,但效果不尽人意,主要是MT产量过低或重金属污染而未能投入工业化发酵提取。近年来,有国内专利报道了小泛素修饰蛋白(SUMO)融合蛋白载体大肠杆菌表达MT1,但实际应用时产量仍然不高;国内盘古基因生物工程有专利报道采用人类游离脂肪酸结合蛋白(hFABP)融合蛋白载体大肠杆菌发酵生产MT1~4,在小试过程中提高了MT产量,但仍然存在放大生产后分离工艺引起的效率和产量问题。这是由于hMT(人金属硫蛋白)分子内有20个半胱氨酸(Cystine),占据近1/3的hMT氨基酸构成,却没有分子内二硫键,酸变性后,自复性能力很强,而过多的半胱氨酸游离[-SH]容易还原聚集,造成分子悬浮不稳定,蛋白丢失,只有解决了这个问题,提高产量的效率才能体现出来。
发明内容
本发明的主要目的是提出一种金属硫蛋白表达载体及其应用,旨在提高金属硫蛋白的可溶性表达。
为实现上述目的,本发明提出一种金属硫蛋白表达载体,包括由金属硫蛋白的编码序列作为目标蛋白***类伴侣样蛋白的下游所形成的融合蛋白表达载体,其中,所述类伴侣样蛋白为序列如SEQ ID NO:1~3任一所示的链球菌蛋白G的B结构域PGB。
可选地,所述金属硫蛋白为金属硫蛋白-1、金属硫蛋白-2、金属硫蛋白-3或金属硫蛋白-4。
可选地,所述金属硫蛋白-1包括金属硫蛋白-1A、金属硫蛋白-1B、金属硫蛋白-1E、金属硫蛋白-1F、金属硫蛋白-1G、金属硫蛋白-1H、金属硫蛋白-1M、金属硫蛋白-1R和金属硫蛋白-1X中的任意一种;和/或,
由所述金属硫蛋白-1的编码序列所编码的金属硫蛋白-1的氨基酸序列与天然金属硫蛋白的氨基酸序列的同源性不低于80%。
可选地,所述金属硫蛋白-1为人金属硫蛋白-1F,所述人金属硫蛋白-1F的氨基酸编码序列如SEQ ID NO:4所示;或者,
所述金属硫蛋白-2为人金属硫蛋白-2,所述人金属硫蛋白-2的氨基酸编码序列如SEQ ID NO:5所示;或者,
所述金属硫蛋白-3为人金属硫蛋白-3,所述人金属硫蛋白-3的氨基酸编码序列如SEQ ID NO:6所示;或者,
所述金属硫蛋白-4为人金属硫蛋白-4,所述人金属硫蛋白-4的氨基酸编码序列如SEQ ID NO:7所示。
可选地,所述类伴侣样蛋白为序列如SEQ ID NO:3所示的链球菌细胞壁的蛋白G的B3结构域PGB3。
可选地,所述PGB的编码序列下游包含一段柔性接头序列,所述柔性接头序列下游包含一段分离纯化标签序列,所述分离纯化标签序列与所述目标蛋白的编码序列之间包含一段肽链切割识别序列;和/或,
所述PGB编码序列的N端添加有如SEQ ID NO:8所示的起始适配序列,所编码的PGB的氨基酸序列与天然PGB氨基酸序列的同源性不低于80%。
可选地,所述柔性接头序列包括6~30个氨基酸;
所述分离纯化标签序列包含2~11个组氨酸;
所述肽链切割识别序列包括凝血因子Xa、凝血酶、肠激酶、TEV酶和羟胺中任意一种的切割识别序列。
可选地,所述柔性接头序列包括22个氨基酸,序列如SEQ ID NO:10所示;
所述分离纯化标签序列标签包含9个内置组氨酸,序列如SEQ ID NO:11所示;
所述肽链切割识别序列为凝血因子Xa的切割识别序列,序列如SEQ ID NO:12所示。
可选地,所述金属硫蛋白表达载体经过密码子与整体序列优化,且优化后的DNA编码序列如SEQ ID NO:17~10中的任一所示。
本发明还提出一种一种包含如上所述的金属硫蛋白表达载体的金属硫蛋白重组表达载体或表达工程菌株。
本发明提供的技术方案中,通过将金属硫蛋白的编码序列***链球菌蛋白G的B结构域编码序列下游而构建获得PGB-MT融合蛋白表达载体,能够促进金属硫蛋白的表达并提高表达量,且经由该融合蛋白表达载体所表达的PGB-MT融合蛋白在His-Tag-镍柱分离纯化后,能够被Fax酶切水解,并进一步通过常规方法纯化MT,为MT蛋白的科学研究和工业化生产提供了新技术。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅为本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为本发明提供的金属硫蛋白表达载体的表达框示意图;
图2为本发明提供的融合蛋白表达载体的第一实施例中PGB3-hMT1F融合蛋白表达框示意图;
图3为实施例1中制得的pET28-PGB3-hMT1F表达载体物理图;
图4为实施例1中制得的pET28-hMT1F对照测试载体物理图;
图5为本发明提供的融合蛋白表达载体的第二实施例中PGB3-hMT2融合蛋白表达框示意图;
图6为实施例4中制得的pET28-PGB3-hMT2表达载体物理图;
图7为实施例4中制得的pET28-hMT2对照测试载体物理图;
图8为本发明提供的融合蛋白表达载体的第三实施例中PGB3-hMT3融合蛋白表达框示意图;
图9为实施例7中制得的pET28-PGB3-hMT3表达载体物理图;
图10为实施例7中制得的pET28-hMT3对照测试载体物理图;
图11为本发明提供的融合蛋白表达载体的第四实施例中PGB3-hMT4融合蛋白表达框示意图;
图12为实施例10中制得的pET28-PGB3-hMT4表达载体物理图;
图13为实施例10中制得的pET28-hMT4对照测试载体物理图。
本发明目的的实现、功能特点及优点将结合实施例,参照附图做进一步说明。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
近年来,有国内专利报道了小泛素修饰蛋白(SUMO)融合蛋白载体大肠杆菌表达MT1,但实际应用时产量仍然不高;国内盘古基因生物工程有专利报道采用人类游离脂肪酸结合蛋白(hFABP)融合蛋白载体大肠杆菌发酵生产MT1~4,在小试过程中提高了MT产量,但仍然存在放大生产后分离工艺引起的效率和产量问题。
本发明提出一种MT表达载体,以链球菌蛋白G的B结构域和金属硫蛋白融合形成融合蛋白表达载体,以提高MT的可溶性表达及产量。在本发明提供的MT表达载体的一实施例中,所述MT表达载体包括由MT的编码序列作为目标蛋白***类伴侣样蛋白的下游所形成的融合蛋白表达载体,其中,所述类伴侣样蛋白为序列如SEQ ID NO:1~3任一所示的链球菌蛋白G的B结构域PGB,具体地,SEQ ID NO:1所示为链球菌G的B1结构域PGB1,SEQ ID NO:2所示为链球菌G的B2结构域PGB2,SEQ ID NO:3所示为链球菌G的B3结构域PGB3。
链球菌属蛋白G(Streptococcal Protein G,SPG或PG)的B1~B3结构域是一种小分子蛋白,PGB1具有促进小分子蛋白表达和折叠的作用,有助于制备小分子肽的核磁共振分析样品,并且允许蛋白PGB1共存在的条件下,直接对目标蛋白进行NMR关联能谱分析(NMR1H-l5N correlation spectroscopy);并且,经过进一步研究发现,PGB2和PGB3自身也有良好的自折叠效应,并且PGB1、PGB2、PGB3都能诱导分子大于自身的某些蛋白提高可溶性表达量或蛋白表达总量。
因此,本发明提供的技术方案中,通过将MT的编码序列***PGB编码序列下游而构建获得PGB-MT融合蛋白表达载体,能够促进MT的表达并提高表达量,且经由该融合蛋白表达载体所表达的PGB-MT融合蛋白在His-Tag-镍柱分离纯化后,能够被Fax酶切水解,并进一步通过常规方法纯化MT,为MT蛋白的科学研究和工业化生产提供了新技术。
金属硫蛋白存在于各种动物、植物或微生物中,在本发明的技术方案中,所述MT优选为动物MT,例如可以是人类MT或者来自其他动物的MT,更优选为人类MT。进一步地,所述MT可以为MT-1、MT-2、MT-3或MT-4,上述四种类型的MT均可以作为本发明中所述的MT,将其编码序列***到PGB的编码序列中而构建获得PGB-MT融合蛋白,实现促进MT表达斌提高表达量的目的。
MT1、MT2、MT3和MT4均为MT的亚群之一,其中MT1又由于自身分子变异,不仅有MT1,还存在MT1亚类,目前已经被发现并公开的MT1亚类有以下九种:金属硫蛋白-1A(MT1A)、金属硫蛋白-1B(MT1B)、金属硫蛋白-1E(MT1E)、金属硫蛋白-1F(MT1F)、金属硫蛋白-1G(MT1G)、金属硫蛋白-1H(MT1H)、金属硫蛋白-1M(MT1M)、金属硫蛋白-1R(MT1R)和金属硫蛋白-1X(MT1X),在本实施例中,所述MT1优选为上述九种MT1亚类中的任意一种。进一步地,由所述MT1的编码序列所编码的MT1的氨基酸序列与天然金属硫蛋白的氨基酸序列的同源性不低于80%。
作为一种较佳的实施方式,所述MT-1为hMT1(人类MT1),主要由肝脏合成,分布在血液和全身器官中,尤其分布在体内重金属超标的肾脏中,具有去重金属、抗氧化的作用,更优选为所述MT1为hMT1F,在本发明的第一实施例中,所述hMT1F的氨基酸编码序列如SEQID NO:4所示,所编码的hMT1F的氨基酸序列与天然hMT1F的氨基酸序列的同源性不低于80%。Z在本发明的其他实施例中,当所述MT为MT1A、MT1B、MT1E、MT1G、MT1H、MT1M、MT1R或MT1X时,其序列对应如SEQ ID NO:38~45所示。
或者,所述MT-2为hMT2(人类MT2),对于hMT2而言,至今只有一种是被发现并公开的,所以hMT2又可记为hMT2A,主要由肝脏合成,分布在血液和全身器官中,尤其分布在体内重金属超标的肾脏中,具有去重金属、抗氧化的作用,在本发明的第二实施例中,所述hMT2的氨基酸编码序列如SEQ ID NO:5所示,所编码的hMT2的氨基酸序列与天然hMT2的氨基酸序列的同源性不低于80%。
又或者,所述MT-3为hMT3(人类MT3),主要分布在人类神经***,尤其是脑组织中,生物医学研究发现,hMT3可能是神经元的保护剂和生理调节剂,能抑制老年性痴呆小鼠模型的β淀粉样蛋白沉积和神经原纤维缠结(NFT)相关病理变化,可应用于健脑保健或制药以防治老年痴呆或儿童益智,在本发明的第三实施例中,所述hMT3的氨基酸编码序列如SEQID NO:6所示,所编码的hMT3的氨基酸序列与天然hMT3的氨基酸序列的同源性不低于80%。
还或者,所述MT-4为hMT4(人类MT4),主要分布在人类皮肤组织中,可应用与护肤类产品,在本发明的第四实施例中,所述hMT4的氨基酸编码序列如SEQ ID NO:7所示,所编码的hMT4的氨基酸序列与天然hMT4的氨基酸序列的同源性不低于80%。
在上述四个实施例中,所述类伴侣样蛋白均可选用上述PGB1、PGB2和PGB3中的任意一种,为便于描述,以下均以所述类伴侣样蛋白选用序列如SEQ ID NO:3所示的链球菌细胞壁的蛋白G的B3结构域PGB3为例对上述四个实施例进行说明,所构建的为PGB3-hMT表达载体。其中,第一实施例所构建的为PGB3-hMT1F表达载体,第二实施例所构建的为PGB3-hMT2表达载体,第三实施例所构建的为PGB3-hMT3表达载体,第四实施例所构建的为PGB3-hMT4表达载体。
进一步地,所述PGB3的编码序列下游包含一段柔性接头序列,所述柔性接头序列包括6~30个氨基酸,在本实施例中优选为所述柔性接头序列包括22个氨基酸,序列如SEQID NO:10所示;所述柔性接头序列下游包含一段分离纯化标签序列,所述分离纯化标签序列包含2~11个组氨酸,所述组氨酸可以是内置或外置组氨酸,也可以是其他类型的分离纯化标签序列,例如6x-His-Tag、T7-Tag、Strept-Tag、CBD等,在本实施例中优选为所述分离纯化标签序列标签为9个内置组氨酸(9xHis-Tag)组成,序列如SEQ ID NO:11所示;所述分离纯化标签序列与所述目标蛋白的编码序列之间包含一段肽链切割识别序列,所述肽链切割识别序列可以是凝血因子Xa、凝血酶、肠激酶、TEV酶和羟胺中任意一种的切割识别序列,在本实施例中优选为所述肽链切割识别序列为凝血因子Xa的切割识别序列,序列如SEQ IDNO:12所示。
更进一步地,所述PGB编码序列的N端添加有如SEQ ID NO:8所示的起始适配序列,所编码的PGB的氨基酸序列与天然PGB氨基酸序列的同源性不低于80%。
基于上述实施方案,本发明第一实施例中所构建的PGB3-hMT1F表达载体的氨基酸序列如SEQ ID NO:13所示;本发明第二实施例中所构建的PGB3-hMT2表达载体的氨基酸序列如SEQ ID NO:14所示;本发明第三实施例中所构建的PGB3-hMT3表达载体的氨基酸序列如SEQ ID NO:15所示;本发明第四实施例中所构建的PGB3-hMT4表达载体的氨基酸序列如SEQ ID NO:16所示。
更优选地,上述四个实施例中构建的表达载体还经过密码子与整体序列优化,其中,第一实施例中PGB3-hMT1F表达载体优化后的DNA编码序列如SEQ ID NO:17所示;第二实施例中PGB3-hMT2表达载体优化后的DNA编码序列如SEQ ID NO:18所示;第三实施例中PGB3-hMT3表达载体优化后的DNA编码序列如SEQ ID NO:19所示;第四实施例中PGB3-hMT4表达载体优化后的DNA编码序列如SEQ ID NO:20所示。当然,优化后的DNA编码序列不仅限于此,在本发明的其他实施例中,也可以通过其他的常规密码子优化方式进行序列优化,以翻译后的蛋白质氨基酸序列同源性不低于80%为限。本发明提供的金属硫蛋白表达载体的表达框示意图如图1所示,第一至第四实施例中的表达载体经优化后的融合蛋白的阅读框架示意图分别如图2、图5、图8和图11所示,在图中,RBS表示核糖体结合区,PGB3表示链球菌蛋白G的B3结构域,FL表示柔性接头区,PRS表示肽链切割识别区,9xHis-Tag表示9个组氨酸组成的内置标签区,hMT1F表示优化后的hMT1F编码区,hMT2表示优化后的hMT2编码区,hMT33表示优化后的hMT3编码区,hMT4表示优化后的hMT4编码区。本发明构建的PGB3-hMT融合蛋白表达载体能够促进hMT表达并提高表达量,且所表达的PGB3-hMT融合蛋白在His-Tag-镍柱分离纯化后,能够被FXa酶切水解,并进一步通过常规方法纯化hMT;此外,所表达的PGB3-hMT融合蛋白还可以用兔IgG或兔IgG的Fc段固化磁珠分离纯化,或兔IgG/Fc段配体-固相载体亲和色谱柱分离纯化。
本发明还提出一种包含如上所述的MT表达载体(即PGB-MT表达载体)的MT重组表达载体,所述重组表达载体可选用例如pET28等表达质粒作为亲本表达载体,将PGB-MT融合蛋白的编码基因***到pET28表达质粒的NcoI和XhoI酶切位点之间,从而构建成pET28-PGB-MT重组表达载体。此处所提供的pET28-PGB-MT重组表达载体可应用于表达PGB-MT融合蛋白。
本发明还进一步提出一种包含如上所述的MT表达载体(即PGB-MT表达载体)的MT表达工程菌株,所述表达工程菌株可由PGB-MT表达载体先构建成重组表达载体,例如上述提供的pET28-PGB-MT重组表达载体,然后将所述重组表达载体转化表达菌株,进而获得MT表达工程菌株,其具体制备方法可参照现有技术进行(或者参照本文下述具体实施例),在此不做作赘述。
以下结合具体实施例和附图对本发明的技术方案作进一步详细说明,应当理解,以下实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1 hMT1F表达载体的构建
以hMT1F为例,构建PGB3-hMT1F融合蛋白表达载体,步骤如下:
步骤一、PGB3-hMT1F融合蛋白序列拼接、密码子与整体序列优化和全序列人工合成:
(1)PGB3-hMT1F融合蛋白氨基酸序列拼接:将[MAS]适配序列(SEQ ID NO:8)添加到PGB3(SEQ ID NO:3)的N-端(SEQ ID NO:9),在PGB3的C-端依次加上:柔性接头(FL)氨基酸序列(SEQ ID NO:10)、9xHis组氨酸标签(9xHis-Tag)序列(SEQ ID NO:11)、FXa PRS序列(SEQ ID NO:12)、hMT1F氨基酸序列(SEQ ID NO:4),形成完整的PGB3-hMT1F融合蛋白氨基酸序列,如SEQ ID NO:13所示。
(2)PGB3-hMT1F融合蛋白密码子整体优化:将上一步的PGB3-hMT1F融合蛋白氨基酸序列输入NCBI在线软件DNAWorks,逆翻译成密码子优化了的DNA编码序列(ORF),如SEQID NO:17所示;同时设计人工基因合成引物。在第一条引物的5’-端添加[-CC-]形成DNA限制性内切酶NcoI酶切位点[5’-CCATGG-3’],并添加4个保护碱基形成[5’-TTTTCCATGG-3’]序列;在最后一个引物的5’端,依次添加4个保护碱基[5’-TTTT-3’]、XhoI酶切位点[5’-CTCGAG-3’]、终止密码TAA的反向互补序列[5’-TTA-3’],形成[5’-TTTTCTCGAGTTA-3’]序列,置于最后一个引物的5’端(引物序列如SEQ ID NO:21~32所示)。
(3)全序列人工基因合成:
①将引物以去离子水配成5μM浓度,各取1μL加入200μL PCR管,用TakaraPyrobest PCR Kit,做成50μL反应体积。
②引物重叠延伸多聚酶链式反应(Overlap PCR)条件:预变性95℃/3min;变性94℃/20sec,复性56℃/20sec,延伸72℃/30sec;16个循环,补全延伸72℃/1min,降至4℃取出。
③全长扩增多聚酶链式反应(Full-length PCR):从全序列人工基因合成第②步PCR产物中取1μL作模板,置入一个新的PCR管,取上游引物4μL(5μM浓度),下游最末端引物4μL(5μM浓度)依次加入PCR管内,引物如SEQ ID NO:21,32所示。以Takara Pyrobest PCRKit,做成50μL反应体积。PCR条件:预变性95℃/3min;变性94℃/30sec,复性60℃/30sec,延伸72℃/1min;25个循环,补全延伸72℃/3min,降至4℃待取出。
④电泳鉴定合成的DNA片段,硅胶柱纯化PCR片段待酶切使用。
步骤二、PGB3-hMT1F融合蛋白载体重组:
(1)将步骤一合成得到的已纯化的PGB3-hMT1F全长PCR产物,经限制性内切酶NcoI和XhoI双酶切消化、琼脂糖凝胶纯化,获得两端分别是NcoI和XhoI的粘性接头的***片段。
(2)制备载体pET28a:
①取pET28空载体2μg,用限制性内切酶NcoI和XhoI双酶切。
②琼脂糖凝胶电泳分离、胶回收纯化获得线性化的pET28载体。
(3)连接、转化与重组质粒序列鉴定:
①用T4 DNA连接酶将步骤二(1)的PGB3-hMT1F***片段与步骤二(2)制备好的pET28载体连接,22℃,30min。
②转化大肠杆菌感受态菌株DH5α,在含卡那霉素(Kan)的琼脂培养皿上培养过夜,然后挑取单菌落,PCR法鉴定阳性克隆,常规制备DNA质粒。
③将阳性克隆质粒送交DNA序列分析,选取和保留正确序列的重组克隆供表达测试。
④所获得的重组表达载体命名为pET28-PGB3-hMT1F(其物理图如图3所示,序列如SEQ ID NO:33所示)。
步骤三、对照测试载体pET28-hMT1F的构建
(1)hMT1F***子的PCR扩增
扩增的hMT1F编码序列为密码子优化序列,与pET28-PGB3-hMT1F载体中的hMT1F编码序列相同(序列如SEQ ID NO:34所示)。为便于***pET28载体,在hMT1F ORF的5’-端和3’-端分别添加NcoI和XhoI酶切位点。
①hMT1F PCR引物设计:
上游引物:[5’-TTTTCCATGGACCCGAATTGTTCTTGTGC-3’](SEQ ID NO:35),
下游引物:[5’-TTTTCTCGAGTTAGTCACAACAGCTGCAT-3’](SEQ ID NO:36)。
②hMT1F***子的PCR及其参数:
以pET-PGB3-hMT1F DNA质粒10ng为PCR模板,按Takara Pyrobest PCR试剂盒使用说明制备PCR反应体系50μL,PCR参数为:预变性95℃/3min,变性94℃/30sec,退火56℃/30sec,延伸72℃/30sec,30个循环。
(2)hMT1F PCR产物的限制性内切酶酶切
①PCR产物经琼脂糖凝胶电泳鉴定条带、切胶、胶回收试剂盒纯化。
②回收的PCR产物用NcoI和XhoI双酶切过夜,电泳、切胶纯化。
(3)pET28载体制备:用NcoI和XhoI双酶切处理过夜,电泳分离,切胶纯化。
(4)重组连接:T4 DNA连接酶连接线性化pET28载体与***片段hMT1F。
(5)将连接产物转化大肠杆菌感受态细胞DH5α,挑取单菌落扩增鉴定重组子,测序分析获得正确的克隆。
(6)正确克隆命名为pET28-hMT1F(物理图如图4所示,序列如SEQ ID NO:37所示)。
实施例2测试表达工程菌株BL21(DE3)/pET28-PGB3-hMT1F的建立
(1)将实施例1获得的表达载体pET28-PGB3-hMT1F和pET28-hMT1F的DNA质粒转化大肠杆菌BL21(DE3)感受态细胞,获得卡那霉素(Kan)抗性的菌落。
(2)分别挑取单菌落数个,LB培养基5mL,37℃/200RPM,过夜培养,Kan筛选,硅胶柱质粒小量制备,送DNA测序进一步验证表达菌株BL21(DE3)/pET28-PGB3-hMT1F和BL21(DE3)/pET28-hMT1F的克隆正确性。
(3)将正确克隆再行稀释,划Kan琼脂平板,挑单克隆LB培养基(含Kan)增菌。经稀释后再次划Kan琼脂平板,挑单菌落增菌,如此重复2~3次,最大限度的剔除跟随阳性菌落生长的不含目标质粒的BL21(DE3)细胞,以保证表达菌株的表达效率,减少营养竞争。
(4)将最后获得的菌液,做成含15%甘油菌液,分装成100μL/支,冻存于-80℃冰箱,供表达测试或小试发酵使用。
实施例3 PGB3-hMT1F表达载体的小试表达测试
(1)将实施例2获得的表达菌株BL21(DE3)/pET28-PGB3-hMT1F和BL21(DE3)/pET28-hMT1F解冻,划含Kan的LB琼脂平皿,37℃倒置过夜培养,获得Kan抗性菌落。
(2)分别挑取单菌落数个,LB培养基40mL,37℃/220RPM,Kan提供筛选压,增菌至OD(600)=0.8,加IPTG至0.5mM诱导12h。分别收集菌液1mL,离心收集菌体,1×PBS 1mL洗涤一次,再悬浮于0.5mL 1×PBS,超声破碎菌体,离心收取上清,保留沉淀,在沉淀物中加入0.25mL 8M尿素溶液,震荡悬浮沉淀物,然后再加入1×PBS 0.25mL,混匀。
(3)各取20μL上清液和沉淀物悬浮液与20μL 2x蛋白电泳上样缓冲液混合,95℃加热变性10min,Eppendorf离心机12000RPM/5min,然后置于冰上等待上样。
(4)各取10μL上样,15%SDS-PAGE电泳,考马斯亮蓝染色、脱色,观察细菌总蛋白质条带、可溶性PGB3-hMT1F融合蛋白条带(上清液)和包涵体PGB3-hMT1F融合蛋白条带(沉淀物尿素溶解)、对照载体的hMT1F蛋白可溶性和包涵体表达条带的表达情况,以及它们之间的比例。表达数据采集采用Tanon 1600R凝胶成像分析***进行条带密度扫描分析。
测试分析结果为:重组PGB3-hMT1F融合蛋白,154aa(SEQ ID NO:13),理论分子量16.39kDa。根据SDS-PAGE电泳条带密度测算hMT1F在不同表达载体中的表达状况,结果如下表1所示:PGB3-hMT1F融合蛋白表达量占菌体总蛋白的18%左右,可溶性蛋白约占88%,说明PGB3与hMT1F融合,可促使hMT1F表达,提高表达量,可能还参与了阻碍hMT1F分子聚集丢失。
表1 hMT1F在两种不同表达载体中的表达状况
实施例4 hMT2表达载体的构建
步骤一、PGB3-hMT2融合蛋白序列拼接、密码子与整体序列优化和全序列人工合成:
(1)PGB3-hMT2融合蛋白氨基酸序列拼接:将[MAS]适配序列(SEQ ID NO:8)添加到PGB3(SEQ ID NO:3)的N-端(SEQ ID NO:9),在PGB3的C-端依次加上:柔性接头(FL)氨基酸序列(SEQ ID NO:10)、9xHis组氨酸标签(9xHis-Tag)序列(SEQ ID NO:11)、FXa PRS序列(SEQ ID NO:12)、hMT2氨基酸序列(SEQ ID NO:5),形成完整的PGB3-hMT2融合蛋白氨基酸序列,如SEQ ID NO:14所示。
(2)PGB3-hMT2融合蛋白密码子整体优化:将上一步的PGB3-hMT2融合蛋白氨基酸序列输入NCBI在线软件DNAWorks,逆翻译成密码子优化了的DNA编码序列(ORF),如SEQ IDNO:18所示;同时设计人工基因合成引物。在第一条引物的5’-端添加[-CC-]形成DNA限制性内切酶NcoI酶切位点[5’-CCATGG-3’],并添加4个保护碱基形成[5’-TTTTCCATGG-3’]序列;在最后一个引物的5’端,依次添加4个保护碱基[5’-TTTT-3’]、XhoI酶切位点[5’-CTCGAG-3’]、终止密码TAA的反向互补序列[5’-TTA-3’],形成[5’-TTTTCTCGAGTTA-3’]序列,置于最后一个引物的5’端(引物序列如SEQ ID NO:46~57所示)。
(3)全序列人工基因合成:
①将引物以去离子水配成5μM浓度,各取1μL加入200μL PCR管,用TakaraPyrobest PCR Kit,做成50μL反应体积。
②引物重叠延伸多聚酶链式反应(Overlap PCR)条件:预变性95℃/3min;变性94℃/20sec,复性56℃/20sec,延伸72℃/30sec;16个循环,补全延伸72℃/1min,降至4℃取出。
③全长扩增多聚酶链式反应(Full-length PCR):从全序列人工基因合成第②步PCR产物中取1μL作模板,置入一个新的PCR管,取上游引物4μL(5μM浓度),下游最末端引物4μL(5μM浓度)依次加入PCR管内,引物如SEQ ID NO:46,57所示。以Takara Pyrobest PCRKit,做成50μL反应体积。PCR条件:预变性95℃/3min;变性94℃/30sec,复性60℃/30sec,延伸72℃/1min;25个循环,补全延伸72℃/3min,降至4℃待取出。
④电泳鉴定合成的DNA片段,硅胶柱纯化PCR片段待酶切使用。
步骤二、PGB3-hMT2融合蛋白载体重组:
(1)将步骤一合成得到的已纯化的PGB3-hMT2全长PCR产物,经限制性内切酶NcoI和XhoI双酶切消化、琼脂糖凝胶纯化,获得两端分别是NcoI和XhoI的粘性接头的***片段。
(2)制备载体pET28a:
①取pET28空载体2μg,用限制性内切酶NcoI和XhoI双酶切。
②琼脂糖凝胶电泳分离、胶回收纯化获得线性化的pET28载体。
(3)连接、转化与重组质粒序列鉴定:
①用T4 DNA连接酶将步骤二(1)的PGB3-hMT2***片段与步骤二(2)制备好的pET28载体连接,22℃,30min。
②转化大肠杆菌感受态菌株DH5α,在含卡那霉素(Kan)的琼脂培养皿上培养过夜,然后挑取单菌落,PCR法鉴定阳性克隆,常规制备DNA质粒。
③将阳性克隆质粒送交DNA序列分析,选取和保留正确序列的重组克隆供表达测试。
④所获得的重组表达载体命名为pET28-PGB3-hMT2(其物理图如图6所示,序列如SEQ ID NO:58所示)。
步骤三、对照测试载体pET28-hMT2的构建
(1)hMT2***子的PCR扩增
扩增的hMT2编码序列为密码子优化序列,与pET28-PGB3-hMT2载体中的hMT2编码序列相同(SEQ ID NO:59)。为便于***pET28载体,在hMT2 ORF的5’-端和3’-端分别添加NcoI和XhoI酶切位点。
①hMT2 PCR引物设计:
上游引物:[5’-TTTTCCATGGACCCGAACTGCAGCTG-3’](SEQ ID NO:60),
下游引物:[5’-TTTTCTCGAGTTACGCACAGCAGGA-3’](SEQ ID NO:61)。
②hMT2***子的PCR及其参数:
以pET-PGB3-hMT2 DNA质粒10ng为PCR模板,按Takara Pyrobest PCR试剂盒使用说明制备PCR反应体系50μL,PCR参数为:预变性95℃/3min,变性94℃/30sec,退火56℃/30sec,延伸72℃/30sec,30个循环。
(2)hMT2 PCR产物的限制性内切酶酶切
①PCR产物经琼脂糖凝胶电泳鉴定条带、切胶、胶回收试剂盒纯化。
②回收的PCR产物用NcoI和XhoI双酶切过夜,电泳、切胶纯化。
(3)pET28载体制备:用NcoI和XhoI双酶切处理过夜,电泳分离,切胶纯化。
(4)重组连接:T4 DNA连接酶连接线性化pET28载体与***片段hMT2。
(5)将连接产物转化大肠杆菌感受态细胞DH5α,挑取单菌落扩增鉴定重组子,测序分析获得正确的克隆。
(6)正确克隆命名为pET28-hMT2(物理图如图7所示,序列如SEQ ID NO:62所示)。
实施例5测试表达工程菌株BL21(DE3)/pET28-PGB3-hMT2的建立
将实施例4获得的表达载体pET28-PGB3-hMT2和pET28-hMT2构建成表达菌株BL21(DE3)/pET28-PGB3-hMT2和BL21(DE3)/pET28-hMT2,方法与实施例2相同。
实施例6 PGB3-hMT2表达载体的小试表达测试
对实施例5获得的表达菌株BL21(DE3)/pET28-PGB3-hMT2和BL21(DE3)/pET28-hMT2进行表达测试,方法与实施例3相同。
测试分析结果为:重组PGB3-hMT2融合蛋白,154aa(SEQ ID NO:14),理论分子量16.7kDa。根据SDS-PAGE电泳条带密度测算hMT2在不同表达载体中的表达状况,结果如下表2所示:PGB3-hMT2融合蛋白表达量占菌体总蛋白的20%左右,可溶性蛋白约占75%,说明PGB3与hMT2融合,可促使hMT2表达,提高表达量,可能还参与了阻碍hMT2分子聚集丢失。
表2 hMT2在两种不同表达载体中的表达状况
实施例7 hMT3表达载体的构建
步骤一、PGB3-hMT3融合蛋白序列拼接、密码子与整体序列优化和全序列人工合成:
(1)PGB3-hMT3融合蛋白氨基酸序列拼接:将[MAS]适配序列(SEQ ID NO:8)添加到PGB3(SEQ ID NO:3)的N-端(SEQ ID NO:9),在PGB3的C-端依次加上:柔性接头(FL)氨基酸序列(SEQ ID NO:10)、9xHis组氨酸标签(9xHis-Tag)序列(SEQ ID NO:11)、FXa PRS序列(SEQ ID NO:12)、hMT3氨基酸序列(SEQ ID NO:6),形成完整的PGB3-hMT3融合蛋白氨基酸序列,如SEQ ID NO:15所示。
(2)PGB3-hMT3融合蛋白密码子整体优化:将上一步的PGB3-hMT3融合蛋白氨基酸序列输入NCBI在线软件DNAWorks,逆翻译成密码子优化了的DNA编码序列(ORF),如SEQ IDNO:19所示;同时设计人工基因合成引物。在第一条引物的5’-端添加[-CC-]形成DNA限制性内切酶NcoI酶切位点[5’-CCATGG-3’],并添加4个保护碱基形成[5’-TTTTCCATGG-3’]序列;在最后一个引物的5’端,依次添加4个保护碱基[5’-TTTT-3’]、XhoI酶切位点[5’-CTCGAG-3’]、终止密码TAA的反向互补序列[5’-TTA-3’],形成[5’-TTTTCTCGAGTTA-3’]序列,置于最后一个引物的5’端(引物序列如SEQ ID NO:63~74所示)。
(3)全序列人工基因合成:
①将引物以去离子水配成5μM浓度,各取1μL加入200μL PCR管,用TakaraPyrobest PCR Kit,做成50μL反应体积。
②引物重叠延伸多聚酶链式反应(Overlap PCR)条件:预变性95℃/3min;变性94℃/20sec,复性56℃/20sec,延伸72℃/30sec;16个循环,补全延伸72℃/1min,降至4℃取出。
③全长扩增多聚酶链式反应(Full-length PCR):从全序列人工基因合成第②步PCR产物中取1μL作模板,置入一个新的PCR管,取上游引物4μL(5μM浓度),下游最末端引物4μL(5μM浓度)依次加入PCR管内,引物如SEQ ID NO:63,74所示。以Takara Pyrobest PCRKit,做成50μL反应体积。PCR条件:预变性95℃/3min;变性94℃/30sec,复性60℃/30sec,延伸72℃/1.5min;30个循环,补全延伸72℃/5min,降至4℃待取出。
④电泳鉴定合成的DNA片段,硅胶柱纯化PCR片段待酶切使用。
步骤二、PGB3-hMT3融合蛋白载体重组:
(1)将步骤一合成得到的已纯化的PGB3-hMT3全长PCR产物,经限制性内切酶NcoI和XhoI双酶切消化、琼脂糖凝胶纯化,获得两端分别是NcoI和XhoI的粘性接头的***片段。
(2)制备载体pET28a:
①取pET28空载体2μg,用限制性内切酶NcoI和XhoI双酶切。
②琼脂糖凝胶电泳分离、胶回收纯化获得线性化的pET28载体。
(3)连接、转化与重组质粒序列鉴定:
①用T4 DNA连接酶将步骤二(1)的PGB3-hMT3***片段与步骤二(2)制备好的pET28载体连接,22℃,30min。
②转化大肠杆菌感受态菌株DH5α,在含卡那霉素(Kan)的琼脂培养皿上培养过夜,然后挑取单菌落,PCR法鉴定阳性克隆,常规制备DNA质粒。
③将阳性克隆质粒送交DNA序列分析,选取和保留正确序列的重组克隆供表达测试。
④所获得的重组表达载体命名为pET28-PGB3-hMT3(其物理图如图9所示,序列如SEQ ID NO:75所示)。
步骤三、对照测试载体pET28-hMT3的构建
(1)hMT3***子的PCR扩增
扩增的hMT3编码序列为密码子优化序列,与pET28-PGB3-hMT3载体中的hMT3编码序列相同(SEQ ID NO:76)。为便于***pET28载体,在hMT3 ORF的5’-端和3’-端分别添加NcoI和XhoI酶切位点。
①hMT3 PCR引物设计:
上游引物:[5’-TTTTCCATGGACCCGGAAACCTGCC-3’](SEQ ID NO:77),
下游引物:[5’-TTTTCTCGAGTTACTGACAGCAGGAACATTTC-3’](SEQ ID NO:78)。
②hMT3***子的PCR及其参数:
以pET-PGB3-hMT3 DNA质粒10ng为PCR模板,按Takara Pyrobest PCR试剂盒使用说明制备PCR反应体系50μL,PCR参数为:预变性95℃/3min,变性94℃/30sec,退火56℃/30sec,延伸72℃/30sec,30个循环。
(2)hMT3 PCR产物的限制性内切酶酶切
①PCR产物经琼脂糖凝胶电泳鉴定条带、切胶、胶回收试剂盒纯化。
②回收的PCR产物用NcoI和XhoI双酶切过夜,电泳、切胶纯化。
(3)pET28载体制备:用NcoI和XhoI双酶切处理过夜,电泳分离,切胶纯化。
(4)重组连接:T4 DNA连接酶连接线性化pET28载体与***片段hMT3。
(5)将连接产物转化大肠杆菌感受态细胞DH5α,挑取单菌落扩增鉴定重组子,测序分析获得正确的克隆。
(6)正确克隆命名为pET28-hMT3(物理图如图10所示,序列如SEQ ID NO:79所示)。
实施例8测试表达工程菌株BL21(DE3)/pET28-PGB3-hMT3的建立
将实施例7获得的表达载体pET28-PGB3-hMT3和pET28-hMT3构建成表达菌株BL21(DE3)/pET28-PGB3-hMT3和BL21(DE3)/pET28-hMT3,方法与实施例2相同。
实施例9 PGB3-hMT3表达载体的小试表达测试
对实施例8获得的表达菌株BL21(DE3)/pET28-PGB3-hMT3和BL21(DE3)/pET28-hMT3进行表达测试,方法与实施例3相同。
测试分析结果为:重组PGB3-hMT3融合蛋白,162aa(SEQ ID NO:15),理论分子量17.56kDa。根据SDS-PAGE电泳条带密度测算hMT3在不同表达载体中的表达状况,结果如下表3所示:PGB3-hMT3融合蛋白表达量占菌体总蛋白的21%左右,可溶性蛋白约占80%,说明PGB3与hMT3融合,可促使hMT3表达,提高表达量,可能还参与了阻碍hMT3分子聚集丢失。
表3 hMT3在两种不同表达载体中的表达状况
实施例10 hMT4表达载体的构建
步骤一、PGB3-hMT4融合蛋白序列拼接、密码子与整体序列优化和全序列人工合成:
(1)PGB3-hMT4融合蛋白氨基酸序列拼接:将[MAS]适配序列(SEQ ID NO:8)添加到PGB3(SEQ ID NO:3)的N-端(SEQ ID NO:9),在PGB3的C-端依次加上:柔性接头(FL)氨基酸序列(SEQ ID NO:10)、9xHis组氨酸标签(9xHis-Tag)序列(SEQ ID NO:11)、FXa PRS序列(SEQ ID NO:12)、hMT4氨基酸序列(SEQ ID NO:7),形成完整的PGB3-hMT4融合蛋白氨基酸序列,如SEQ ID NO:16所示。
(2)PGB3-hMT4融合蛋白密码子整体优化:将上一步的PGB3-hMT4融合蛋白氨基酸序列输入NCBI在线软件DNAWorks,逆翻译成密码子优化了的DNA编码序列(ORF),如SEQ IDNO:20所示;同时设计人工基因合成引物。在第一条引物的5’-端添加[-CC-]形成DNA限制性内切酶NcoI酶切位点[5’-CCATGG-3’],并添加4个保护碱基形成[5’-TTTTCCATGG-3’]序列;在最后一个引物的5’端,依次添加4个保护碱基[5’-TTTT-3’]、XhoI酶切位点[5’-CTCGAG-3’]、终止密码TAA的反向互补序列[5’-TTA-3’],形成[5’-TTTTCTCGAGTTA-3’]序列,置于最后一个引物的5’端(引物序列如SEQ ID NO:80~91所示)。
(3)全序列人工基因合成:
①将引物以去离子水配成5μM浓度,各取1μL加入200μL PCR管,用TakaraPyrobest PCR Kit,做成50μL反应体积。
②引物重叠延伸多聚酶链式反应(Overlap PCR)条件:预变性95℃/3min;变性94℃/20sec,复性56℃/20sec,延伸72℃/30sec;16个循环,补全延伸72℃/1min,降至4℃取出。
③全长扩增多聚酶链式反应(Full-length PCR):从全序列人工基因合成第②步PCR产物中取1μL作模板,置入一个新的PCR管,取上游引物4μL(5μM浓度),下游最末端引物4μL(5μM浓度)依次加入PCR管内,引物如SEQ ID NO:80,91所示。以Takara Pyrobest PCRKit,做成50μL反应体积。PCR条件:预变性95℃/3min;变性94℃/30sec,复性60℃/30sec,延伸72℃/1min;25个循环,补全延伸72℃/3min,降至4℃待取出。
④电泳鉴定合成的DNA片段,硅胶柱纯化PCR片段待酶切使用。
步骤二、PGB3-hMT4融合蛋白载体重组:
(1)将步骤一合成得到的已纯化的PGB3-hMT4全长PCR产物,经限制性内切酶NcoI和XhoI双酶切消化、琼脂糖凝胶纯化,获得两端分别是NcoI和XhoI的粘性接头的***片段。
(2)制备载体pET28a:
①取pET28空载体2μg,用限制性内切酶NcoI和XhoI双酶切。
②琼脂糖凝胶电泳分离、胶回收纯化获得线性化的pET28载体。
(3)连接、转化与重组质粒序列鉴定:
①用T4 DNA连接酶将步骤二(1)的PGB3-hMT4***片段与步骤二(2)制备好的pET28载体连接,22℃,30min。
②转化大肠杆菌感受态菌株DH5α,在含卡那霉素(Kan)的琼脂培养皿上培养过夜,然后挑取单菌落,PCR法鉴定阳性克隆,常规制备DNA质粒。
③将阳性克隆质粒送交DNA序列分析,选取和保留正确序列的重组克隆供表达测试。
④所获得的重组表达载体命名为pET28-PGB3-hMT4(其物理图如图12所示,序列如SEQ ID NO:92所示)。
步骤三、对照测试载体pET28-hMT4的构建
(1)hMT4***子的PCR扩增
扩增的hMT4编码序列为密码子优化序列,与pET28-PGB3-hMT4载体中的hMT4编码序列相同(SEQ ID NO:93)。为便于***pET28载体,在hMT4 ORF的5’-端和3’-端分别添加NcoI和XhoI酶切位点。
①hMT4 PCR引物设计:
上游引物:[5’-TTTTCCATGGACCCGCGTGAATGC-3’](SEQ ID NO:94),
下游引物:[5’-TTTTCTCGAGTTACGGGCAACAGGA-3’](SEQ ID NO:95)。
②hMT4***子的PCR及其参数:
以pET-PGB3-hMT4 DNA质粒10ng为PCR模板,按Takara Pyrobest PCR试剂盒使用说明制备PCR反应体系50μL,PCR参数为:预变性95℃/3min,变性94℃/30sec,退火56℃/30sec,延伸72℃/30sec,30个循环。
(2)hMT4 PCR产物的限制性内切酶酶切
①PCR产物经琼脂糖凝胶电泳鉴定条带、切胶、胶回收试剂盒纯化。
②回收的PCR产物用NcoI和XhoI双酶切过夜,电泳、切胶纯化。
(3)pET28载体制备:用NcoI和XhoI双酶切处理过夜,电泳分离,切胶纯化。
(4)重组连接:T4 DNA连接酶连接线性化pET28载体与***片段hMT4。
(5)将连接产物转化大肠杆菌感受态细胞DH5α,挑取单菌落扩增鉴定重组子,测序分析获得正确的克隆。
(6)正确克隆命名为pET28-hMT4(物理图如图13所示,序列如SEQ ID NO:96所示)。
实施例11测试表达工程菌株BL21(DE3)/pET28-PGB3-hMT4的建立
将实施例10获得的表达载体pET28-PGB3-hMT4和pET28-hMT4构建成表达菌株BL21(DE3)/pET28-PGB3-hMT4和BL21(DE3)/pET28-hMT4,方法与实施例2相同。
实施例12 PGB3-hMT4表达载体的小试表达测试
对实施例11获得的表达菌株BL21(DE3)/pET28-PGB3-hMT4和BL21(DE3)/pET28-hMT4进行表达测试,方法与实施例3相同。
测试分析结果为:重组PGB3-hMT4融合蛋白,155aa(SEQ ID NO:16),理论分子量16.7kDa。根据SDS-PAGE电泳条带密度测算hMT4在不同表达载体中的表达状况,结果如下表4所示:PGB3-hMT4融合蛋白表达量占菌体总蛋白的17%左右,可溶性蛋白约占88%,说明PGB3与hMT4融合,可促使hMT4表达,提高表达量,可能还参与了阻碍hMT4分子聚集丢失。
表4 hMT4在两种不同表达载体中的表达状况
以上仅为本发明的优选实施例,并非因此限制本发明的专利范围,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包括在本发明的专利保护范围内。
SEQUENCE LISTING
<110> 因之彩生物科技(武汉)有限公司
<120> 一种人金属硫蛋白表达载体及其应用
<130> 20190709
<160> 96
<170> PatentIn version 3.5
<210> 1
<211> 55
<212> PRT
<213> 人工合成
<400> 1
Thr Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr
1 5 10 15
Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr
20 25 30
Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr
35 40 45
Lys Thr Phe Thr Val Thr Glu
50 55
<210> 2
<211> 55
<212> PRT
<213> 人工合成
<400> 2
Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr
1 5 10 15
Thr Lys Ala Val Asp Ala Glu Thr Ala Glu Lys Ala Phe Lys Gln Tyr
20 25 30
Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr Asp Asp Ala Thr
35 40 45
Lys Thr Phe Thr Val Thr Glu
50 55
<210> 3
<211> 55
<212> PRT
<213> 人工合成
<400> 3
Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr
1 5 10 15
Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr
20 25 30
Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr
35 40 45
Lys Thr Phe Thr Val Thr Glu
50 55
<210> 4
<211> 61
<212> PRT
<213> 人工合成
<400> 4
Met Asp Pro Asn Cys Ser Cys Ala Ala Gly Val Ser Cys Thr Cys Ala
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ser Lys Cys Ala Gln Gly Cys
35 40 45
Val Cys Lys Gly Ala Ser Glu Lys Cys Ser Cys Cys Asp
50 55 60
<210> 5
<211> 61
<212> PRT
<213> 人工合成
<400> 5
Met Asp Pro Asn Cys Ser Cys Ala Ala Gly Asp Ser Cys Thr Cys Ala
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Ile Cys Lys Gly Ala Ser Asp Lys Cys Ser Cys Cys Ala
50 55 60
<210> 6
<211> 68
<212> PRT
<213> 人工合成
<400> 6
Met Asp Pro Glu Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys
1 5 10 15
Ala Asp Ser Cys Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys Lys Lys
20 25 30
Ser Cys Cys Ser Cys Cys Pro Ala Glu Cys Glu Lys Cys Ala Lys Asp
35 40 45
Cys Val Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu Ala Glu Lys Cys
50 55 60
<210> 7
<211> 62
<212> PRT
<213> 人工合成
<400> 7
Met Asp Pro Arg Glu Cys Val Cys Met Ser Gly Gly Ile Cys Met Cys
1 5 10 15
Gly Asp Asn Cys Lys Cys Thr Thr Cys Asn Cys Lys Thr Cys Arg Lys
20 25 30
Ser Cys Cys Pro Cys Cys Pro Pro Gly Cys Ala Lys Cys Ala Arg Gly
35 40 45
Cys Ile Cys Lys Gly Gly Ser Asp Lys Cys Ser Cys Cys Pro
50 55 60
<210> 8
<211> 3
<212> PRT
<213> 人工合成
<400> 8
Met Ala Ser
1
<210> 9
<211> 58
<212> PRT
<213> 人工合成
<400> 9
Met Ala Ser Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly
1 5 10 15
Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe
20 25 30
Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp
35 40 45
Asp Ala Thr Lys Thr Phe Thr Val Thr Glu
50 55
<210> 10
<211> 22
<212> PRT
<213> 人工合成
<400> 10
Lys Pro Glu Val Ile Asp Ala Ser Glu Leu Thr Pro Ala Val Thr Asp
1 5 10 15
Ala Pro Thr Glu Pro Glu
20
<210> 11
<211> 9
<212> PRT
<213> 人工合成
<400> 11
His His His His His His His His His
1 5
<210> 12
<211> 4
<212> PRT
<213> 人工合成
<400> 12
Ile Glu Gly Arg
1
<210> 13
<211> 154
<212> PRT
<213> 人工合成
<400> 13
Met Ala Ser Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly
1 5 10 15
Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe
20 25 30
Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp
35 40 45
Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp
50 55 60
Ala Ser Glu Leu Thr Pro Ala Val Thr Asp Ala Pro Thr Glu Pro Glu
65 70 75 80
His His His His His His His His His Ile Glu Gly Arg Met Asp Pro
85 90 95
Asn Cys Ser Cys Ala Ala Gly Val Ser Cys Thr Cys Ala Gly Ser Cys
100 105 110
Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser Cys Cys Ser
115 120 125
Cys Cys Pro Val Gly Cys Ser Lys Cys Ala Gln Gly Cys Val Cys Lys
130 135 140
Gly Ala Ser Glu Lys Cys Ser Cys Cys Asp
145 150
<210> 14
<211> 154
<212> PRT
<213> 人工合成
<400> 14
Met Ala Ser Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly
1 5 10 15
Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe
20 25 30
Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp
35 40 45
Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp
50 55 60
Ala Ser Glu Leu Thr Pro Ala Val Thr Asp Ala Pro Thr Glu Pro Glu
65 70 75 80
His His His His His His His His His Ile Glu Gly Arg Met Asp Pro
85 90 95
Asn Cys Ser Cys Ala Ala Gly Asp Ser Cys Thr Cys Ala Gly Ser Cys
100 105 110
Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser Cys Cys Ser
115 120 125
Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys Ile Cys Lys
130 135 140
Gly Ala Ser Asp Lys Cys Ser Cys Cys Ala
145 150
<210> 15
<211> 162
<212> PRT
<213> 人工合成
<400> 15
Met Ala Ser Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly
1 5 10 15
Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe
20 25 30
Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp
35 40 45
Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp
50 55 60
Ala Ser Glu Leu Thr Pro Ala Arg His Lys Glu Lys Thr Lys Glu Lys
65 70 75 80
Thr His His His His His His His His His Ile Glu Gly Arg Met Asp
85 90 95
Pro Glu Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys Ala Asp
100 105 110
Ser Cys Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys Lys Lys Ser Cys
115 120 125
Cys Ser Cys Cys Pro Ala Glu Cys Glu Lys Cys Ala Lys Asp Cys Val
130 135 140
Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu Ala Glu Lys Cys Ser Cys
145 150 155 160
Cys Gln
<210> 16
<211> 155
<212> PRT
<213> 人工合成
<400> 16
Met Ala Ser Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly
1 5 10 15
Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe
20 25 30
Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp
35 40 45
Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Pro Glu Val Ile Asp
50 55 60
Ala Ser Glu Leu Thr Pro Ala Val Thr Asp Ala Pro Thr Glu Pro Glu
65 70 75 80
His His His His His His His His His Ile Glu Gly Arg Met Asp Pro
85 90 95
Arg Glu Cys Val Cys Met Ser Gly Gly Ile Cys Met Cys Gly Asp Asn
100 105 110
Cys Lys Cys Thr Thr Cys Asn Cys Lys Thr Cys Arg Lys Ser Cys Cys
115 120 125
Pro Cys Cys Pro Pro Gly Cys Ala Lys Cys Ala Arg Gly Cys Ile Cys
130 135 140
Lys Gly Gly Ser Asp Lys Cys Ser Cys Cys Pro
145 150 155
<210> 17
<211> 465
<212> DNA
<213> 人工合成
<400> 17
atggcgtcta cctacaaact ggttatcaac ggtaaaaccc tgaaaggtga aactaccacc 60
gaagcggttg acgcggcgac cgcggaaaaa gttttcaaac agtacgcgaa cgacaacggt 120
gttgacggcg aatggaccta cgacgacgcg accaaaacct tcaccgttac cgagaaaccg 180
gaagttatcg atgcgtctga actgaccccg gcagtgaccg acgcgccgac cgagccggaa 240
catcatcacc accaccacca tcatcatatc gaaggtcgta tggacccgaa ttgttcttgt 300
gctgcgggtg tttcttgtac ctgcgcgggt tcttgcaaat gcaaagaatg taaatgcacc 360
tcttgtaaaa agtcttgctg ctcttgttgc ccggttggtt gctctaaatg cgcacaaggt 420
tgcgtttgta aaggtgcgag cgaaaaatgc agctgttgtg actaa 465
<210> 18
<211> 465
<212> DNA
<213> 人工合成
<400> 18
atggcgtcta cctacaaact ggttatcaac ggtaaaaccc tgaaaggtga aactaccacc 60
gaagcggttg acgcggcgac cgcggaaaaa gttttcaaac agtacgcgaa cgacaacggt 120
gttgacggtg aatggaccta cgacgacgcg accaaaacct tcactgttac cgaaaagccg 180
gaagttatcg atgcgtctga actgacccca gcggtaaccg acgcgccgac cgaaccggaa 240
caccatcatc atcatcatca ccaccacatc gaaggtcgta tggacccgaa ctgcagctgt 300
gcagcgggtg actcttgtac ctgcgcgggt agctgcaaat gcaaagaatg caagtgcacc 360
tcttgcaaaa aatcttgctg ctcttgttgc ccggttggtt gcgcgaaatg tgcgcaaggc 420
tgcatctgca aaggtgcgtc cgacaaatgc tcctgctgtg cgtaa 465
<210> 19
<211> 489
<212> DNA
<213> 人工合成
<400> 19
atggcgtcta cctacaaact ggttatcaac ggtaaaaccc tgaaaggtga aaccactacc 60
gaagcggttg acgcggcgac cgcggaaaaa gttttcaaac agtacgcgaa cgacaacggt 120
gttgacggcg aatggaccta cgacgacgcg accaaaacct tcaccgttac cgaaaaaccg 180
gaagttatcg atgcgtctga actgacccca gcgcgtcata aggaaaaaac caaagaaaag 240
actcatcacc accatcacca ccatcatcac atcgaaggtc gtatggaccc ggaaacctgc 300
ccgtgcccgt ctggtggttc ttgcacctgc gcggactcct gcaaatgcga aggttgtaaa 360
tgtacgtctt gcaaaaaatc ttgttgctct tgctgcccag ccgaatgtga aaaatgcgcg 420
aaagactgcg tttgtaaagg tggcgaggca gcggaagcag aagctgagaa atgttcctgc 480
tgtcagtaa 489
<210> 20
<211> 468
<212> DNA
<213> 人工合成
<400> 20
atggctagca cctacaaact ggttatcaac ggtaaaaccc tgaaaggtga aactaccacc 60
gaagcggttg acgccgcgac cgcggaaaaa gttttcaaac agtacgcgaa cgacaacggt 120
gttgacggcg aatggaccta cgacgatgcg accaaaacct tcaccgtaac cgaaaaaccg 180
gaggttatcg acgcgagcga actgacccca gccgttaccg acgcgccgac cgaaccggaa 240
caccaccatc atcaccatca tcaccacatc gaaggtcgta tggacccgcg tgaatgcgtt 300
tgcatgtctg gtggtatctg catgtgcggc gacaattgca aatgcaccac ctgcaactgc 360
aagacctgcc gtaaatcttg ctgtccatgc tgccctccgg gttgcgcgaa atgcgcgcgt 420
ggttgcatct gcaaaggtgg ttctgacaaa tgctcctgtt gcccgtaa 468
<210> 21
<211> 38
<212> DNA
<213> 人工合成
<400> 21
ttttccatgg cgtctaccta caaactggtt atcaacgg 38
<210> 22
<211> 60
<212> DNA
<213> 人工合成
<400> 22
caaccgcttc ggtggtagtt tcacctttca gggttttacc gttgataacc agtttgtagg 60
<210> 23
<211> 60
<212> DNA
<213> 人工合成
<400> 23
ctaccaccga agcggttgac gcggcgaccg cggaaaaagt tttcaaacag tacgcgaacg 60
<210> 24
<211> 60
<212> DNA
<213> 人工合成
<400> 24
tcgcgtcgtc gtaggtccat tcgccgtcaa caccgttgtc gttcgcgtac tgtttgaaaa 60
<210> 25
<211> 60
<212> DNA
<213> 人工合成
<400> 25
gacctacgac gacgcgacca aaaccttcac cgttaccgag aaaccggaag ttatcgatgc 60
<210> 26
<211> 60
<212> DNA
<213> 人工合成
<400> 26
tcggtcggcg cgtcggtcac tgccggggtc agttcagacg catcgataac ttccggtttc 60
<210> 27
<211> 60
<212> DNA
<213> 人工合成
<400> 27
gacgcgccga ccgagccgga acatcatcac caccaccacc atcatcatat cgaaggtcgt 60
<210> 28
<211> 60
<212> DNA
<213> 人工合成
<400> 28
caagaaacac ccgcagcaca agaacaattc gggtccatac gaccttcgat atgatgatgg 60
<210> 29
<211> 60
<212> DNA
<213> 人工合成
<400> 29
tgctgcgggt gtttcttgta cctgcgcggg ttcttgcaaa tgcaaagaat gtaaatgcac 60
<210> 30
<211> 60
<212> DNA
<213> 人工合成
<400> 30
accgggcaac aagagcagca agacttttta caagaggtgc atttacattc tttgcatttg 60
<210> 31
<211> 60
<212> DNA
<213> 人工合成
<400> 31
tgctcttgtt gcccggttgg ttgctctaaa tgcgcacaag gttgcgtttg taaaggtgcg 60
<210> 32
<211> 56
<212> DNA
<213> 人工合成
<400> 32
ttttctcgag ttagtcacaa cagctgcatt tttcgctcgc acctttacaa acgcaa 56
<210> 33
<211> 5698
<212> DNA
<213> 人工合成
<400> 33
cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 60
tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 120
gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 180
cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 240
taattttgtt taactttaag aaggagatat accatggcgt ctacctacaa actggttatc 300
aacggtaaaa ccctgaaagg tgaaactacc accgaagcgg ttgacgcggc gaccgcggaa 360
aaagttttca aacagtacgc gaacgacaac ggtgttgacg gcgaatggac ctacgacgac 420
gcgaccaaaa ccttcaccgt taccgagaaa ccggaagtta tcgatgcgtc tgaactgacc 480
ccggcagtga ccgacgcgcc gaccgagccg gaacatcatc accaccacca ccatcatcat 540
atcgaaggtc gtatggaccc gaattgttct tgtgctgcgg gtgtttcttg tacctgcgcg 600
ggttcttgca aatgcaaaga atgtaaatgc acctcttgta aaaagtcttg ctgctcttgt 660
tgcccggttg gttgctctaa atgcgcacaa ggttgcgttt gtaaaggtgc gagcgaaaaa 720
tgcagctgtt gtgactaact cgagcaccac caccaccacc actgagatcc ggctgctaac 780
aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc 840
cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga 900
ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 960
gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 1020
cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 1080
ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 1140
cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 1200
tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 1260
cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 1320
aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca ggtggcactt 1380
ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 1440
atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta 1500
ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa 1560
aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact 1620
cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag 1680
aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc 1740
cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa 1800
ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga 1860
caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata 1920
ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca 1980
gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc 2040
ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta 2100
cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt 2160
gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc 2220
atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca 2280
ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta 2340
acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 2400
agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 2460
ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 2520
cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 2580
gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 2640
cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 2700
gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 2760
caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 2820
aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 2880
tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 2940
gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 3000
ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 3060
atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 3120
cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg 3180
gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt 3240
acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 3300
gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 3360
tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 3420
ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt 3480
cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca 3540
gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt 3600
tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga 3660
aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac 3720
gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg 3780
gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc 3840
ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt 3900
acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc 3960
agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac 4020
cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg 4080
ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga 4140
aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg 4200
cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta 4260
cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc 4320
accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct 4380
aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 4440
acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 4500
ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc 4560
accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga 4620
aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg 4680
tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt 4740
gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc 4800
agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct 4860
atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc 4920
gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga 4980
tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc 5040
tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg 5100
gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga 5160
ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg 5220
ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc 5280
agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt 5340
gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt 5400
ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg 5460
gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc 5520
tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg 5580
atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag 5640
gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagt 5698
<210> 34
<211> 186
<212> DNA
<213> 人工合成
<400> 34
atggacccga attgttcttg tgctgcgggt gtttcttgta cctgcgcggg ttcttgcaaa 60
tgcaaagaat gtaaatgcac ctcttgtaaa aagtcttgct gctcttgttg cccggttggt 120
tgctctaaat gcgcacaagg ttgcgtttgt aaaggtgcga gcgaaaaatg cagctgttgt 180
gactaa 186
<210> 35
<211> 29
<212> DNA
<213> 人工合成
<400> 35
ttttccatgg acccgaattg ttcttgtgc 29
<210> 36
<211> 29
<212> DNA
<213> 人工合成
<400> 36
ttttctcgag ttagtcacaa cagctgcat 29
<210> 37
<211> 5419
<212> DNA
<213> 人工合成
<400> 37
cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 60
tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 120
gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 180
cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 240
taattttgtt taactttaag aaggagatat accatggacc cgaattgttc ttgtgctgcg 300
ggtgtttctt gtacctgcgc gggttcttgc aaatgcaaag aatgtaaatg cacctcttgt 360
aaaaagtctt gctgctcttg ttgcccggtt ggttgctcta aatgcgcaca aggttgcgtt 420
tgtaaaggtg cgagcgaaaa atgcagctgt tgtgactaac tcgagcacca ccaccaccac 480
cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 540
gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 600
aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg 660
cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 720
ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 780
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 840
aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 900
ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 960
tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 1020
ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 1080
ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 1140
tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc 1200
atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc 1260
cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg 1320
tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca 1380
aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc 1440
aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca 1500
aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat 1560
acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac 1620
actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat 1680
gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa 1740
tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct 1800
gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc 1860
ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta 1920
tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc 1980
cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt 2040
gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 2100
aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 2160
aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 2220
tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 2280
gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 2340
cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 2400
acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 2460
cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 2520
cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 2580
aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 2640
gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 2700
atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 2760
tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 2820
gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 2880
agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 2940
catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca 3000
ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg 3060
acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 3120
ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc 3180
ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt 3240
ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt 3300
taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca 3360
tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg 3420
aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg 3480
accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc 3540
cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg 3600
acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc 3660
aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat 3720
tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga 3780
tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt 3840
tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa 3900
gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga 3960
gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga 4020
cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca 4080
tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac 4140
tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4200
cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg 4260
ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg 4320
ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat 4380
gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg 4440
gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca 4500
gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc 4560
cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag 4620
ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc 4680
tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa 4740
ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg 4800
caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca 4860
ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt 4920
tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg 4980
acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac 5040
tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc 5100
gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa 5160
acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca 5220
ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg 5280
cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa 5340
gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa 5400
ggagatggcg cccaacagt 5419
<210> 38
<211> 61
<212> PRT
<213> 人工合成
<400> 38
Met Asp Pro Asn Cys Ser Cys Ala Thr Gly Gly Ser Cys Thr Cys Thr
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Met Ser Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Ile Cys Lys Gly Ala Ser Glu Lys Cys Ser Cys Cys Ala
50 55 60
<210> 39
<211> 61
<212> PRT
<213> 人工合成
<400> 39
Met Asp Pro Asn Cys Ser Cys Thr Thr Gly Gly Ser Cys Ala Cys Ala
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Cys
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Val Cys Lys Gly Ser Ser Glu Lys Cys Arg Cys Cys Ala
50 55 60
<210> 40
<211> 61
<212> PRT
<213> 人工合成
<400> 40
Met Asp Pro Asn Cys Ser Cys Ala Thr Gly Gly Ser Cys Thr Cys Ala
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Val Cys Lys Gly Ala Ser Glu Lys Cys Ser Cys Cys Ala
50 55 60
<210> 41
<211> 61
<212> PRT
<213> 人工合成
<400> 41
Met Asp Pro Asn Cys Ser Cys Ala Ala Gly Val Ser Cys Thr Cys Ala
1 5 10 15
Ser Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Ile Cys Lys Gly Ala Ser Glu Lys Cys Ser Cys Cys Ala
50 55 60
<210> 42
<211> 61
<212> PRT
<213> 人工合成
<400> 42
Met Asp Pro Asn Cys Ser Cys Glu Ala Gly Gly Ser Cys Ala Cys Ala
1 5 10 15
Gly Ser Cys Lys Cys Lys Lys Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Leu Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Ile Cys Lys Gly Ala Ser Glu Lys Cys Ser Cys Cys Ala
50 55 60
<210> 43
<211> 61
<212> PRT
<213> 人工合成
<400> 43
Met Asp Pro Asn Cys Ser Cys Thr Thr Gly Val Ser Cys Ala Cys Thr
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala His Gly Cys
35 40 45
Val Cys Lys Gly Thr Leu Glu Asn Cys Ser Cys Cys Ala
50 55 60
<210> 44
<211> 61
<212> PRT
<213> 人工合成
<400> 44
Met Asp Pro Asn Cys Ser Cys Ala Thr Gly Gly Ser Cys Ser Cys Ala
1 5 10 15
Ser Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Met Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Val Cys Lys Gly Ala Ser Glu Lys Cys Ser Cys Cys Ala
50 55 60
<210> 45
<211> 61
<212> PRT
<213> 人工合成
<400> 45
Met Asp Pro Asn Cys Ser Cys Ser Pro Val Gly Ser Cys Ala Cys Ala
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Ile Cys Lys Gly Thr Ser Asp Lys Cys Ser Cys Cys Ala
50 55 60
<210> 46
<211> 52
<212> DNA
<213> 人工合成
<400> 46
ttttccatgg cgtctaccta caaactggtt atcaacggta aaaccctgaa ag 52
<210> 47
<211> 60
<212> DNA
<213> 人工合成
<400> 47
cggtcgccgc gtcaaccgct tcggtggtag tttcaccttt cagggtttta ccgttgataa 60
<210> 48
<211> 60
<212> DNA
<213> 人工合成
<400> 48
tgacgcggcg accgcggaaa aagttttcaa acagtacgcg aacgacaacg gtgttgacgg 60
<210> 49
<211> 60
<212> DNA
<213> 人工合成
<400> 49
gtaacagtga aggttttggt cgcgtcgtcg taggtccatt caccgtcaac accgttgtcg 60
<210> 50
<211> 60
<212> DNA
<213> 人工合成
<400> 50
cgaccaaaac cttcactgtt accgaaaagc cggaagttat cgatgcgtct gaactgaccc 60
<210> 51
<211> 60
<212> DNA
<213> 人工合成
<400> 51
atgatggtgt tccggttcgg tcggcgcgtc ggttaccgct ggggtcagtt cagacgcatc 60
<210> 52
<211> 60
<212> DNA
<213> 人工合成
<400> 52
cgaaccggaa caccatcatc atcatcatca ccaccacatc gaaggtcgta tggacccgaa 60
<210> 53
<211> 60
<212> DNA
<213> 人工合成
<400> 53
tacccgcgca ggtacaagag tcacccgctg cacagctgca gttcgggtcc atacgacctt 60
<210> 54
<211> 60
<212> DNA
<213> 人工合成
<400> 54
ttgtacctgc gcgggtagct gcaaatgcaa agaatgcaag tgcacctctt gcaaaaaatc 60
<210> 55
<211> 60
<212> DNA
<213> 人工合成
<400> 55
gcacatttcg cgcaaccaac cgggcaacaa gagcagcaag attttttgca agaggtgcac 60
<210> 56
<211> 60
<212> DNA
<213> 人工合成
<400> 56
ggttgcgcga aatgtgcgca aggctgcatc tgcaaaggtg cgtccgacaa atgctcctgc 60
<210> 57
<211> 37
<212> DNA
<213> 人工合成
<400> 57
ttttctcgag ttacgcacag caggagcatt tgtcgga 37
<210> 58
<211> 5698
<212> DNA
<213> 人工合成
<400> 58
cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 60
tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 120
gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 180
cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 240
taattttgtt taactttaag aaggagatat accatggcgt ctacctacaa actggttatc 300
aacggtaaaa ccctgaaagg tgaaactacc accgaagcgg ttgacgcggc gaccgcggaa 360
aaagttttca aacagtacgc gaacgacaac ggtgttgacg gtgaatggac ctacgacgac 420
gcgaccaaaa ccttcactgt taccgaaaag ccggaagtta tcgatgcgtc tgaactgacc 480
ccagcggtaa ccgacgcgcc gaccgaaccg gaacaccatc atcatcatca tcaccaccac 540
atcgaaggtc gtatggaccc gaactgcagc tgtgcagcgg gtgactcttg tacctgcgcg 600
ggtagctgca aatgcaaaga atgcaagtgc acctcttgca aaaaatcttg ctgctcttgt 660
tgcccggttg gttgcgcgaa atgtgcgcaa ggctgcatct gcaaaggtgc gtccgacaaa 720
tgctcctgct gtgcgtaact cgagcaccac caccaccacc actgagatcc ggctgctaac 780
aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc 840
cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga 900
ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 960
gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 1020
cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 1080
ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 1140
cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 1200
tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 1260
cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 1320
aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca ggtggcactt 1380
ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 1440
atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta 1500
ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa 1560
aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact 1620
cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag 1680
aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc 1740
cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa 1800
ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga 1860
caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata 1920
ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca 1980
gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc 2040
ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta 2100
cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt 2160
gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc 2220
atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca 2280
ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta 2340
acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 2400
agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 2460
ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 2520
cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 2580
gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 2640
cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 2700
gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 2760
caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 2820
aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 2880
tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 2940
gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 3000
ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 3060
atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 3120
cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg 3180
gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt 3240
acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 3300
gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 3360
tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 3420
ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt 3480
cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca 3540
gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt 3600
tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga 3660
aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac 3720
gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg 3780
gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc 3840
ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt 3900
acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc 3960
agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac 4020
cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg 4080
ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga 4140
aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg 4200
cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta 4260
cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc 4320
accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct 4380
aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 4440
acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 4500
ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc 4560
accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga 4620
aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg 4680
tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt 4740
gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc 4800
agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct 4860
atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc 4920
gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga 4980
tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc 5040
tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg 5100
gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga 5160
ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg 5220
ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc 5280
agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt 5340
gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt 5400
ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg 5460
gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc 5520
tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg 5580
atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag 5640
gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagt 5698
<210> 59
<211> 186
<212> DNA
<213> 人工合成
<400> 59
atggacccga actgcagctg tgcagcgggt gactcttgta cctgcgcggg tagctgcaaa 60
tgcaaagaat gcaagtgcac ctcttgcaaa aaatcttgct gctcttgttg cccggttggt 120
tgcgcgaaat gtgcgcaagg ctgcatctgc aaaggtgcgt ccgacaaatg ctcctgctgt 180
gcgtaa 186
<210> 60
<211> 26
<212> DNA
<213> 人工合成
<400> 60
ttttccatgg acccgaactg cagctg 26
<210> 61
<211> 25
<212> DNA
<213> 人工合成
<400> 61
ttttctcgag ttacgcacag cagga 25
<210> 62
<211> 5419
<212> DNA
<213> 人工合成
<400> 62
cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 60
tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 120
gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 180
cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 240
taattttgtt taactttaag aaggagatat accatggacc cgaactgcag ctgtgcagcg 300
ggtgactctt gtacctgcgc gggtagctgc aaatgcaaag aatgcaagtg cacctcttgc 360
aaaaaatctt gctgctcttg ttgcccggtt ggttgcgcga aatgtgcgca aggctgcatc 420
tgcaaaggtg cgtccgacaa atgctcctgc tgtgcgtaac tcgagcacca ccaccaccac 480
cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 540
gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 600
aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg 660
cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 720
ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 780
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 840
aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 900
ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 960
tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 1020
ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 1080
ttacaatttc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 1140
tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc 1200
atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc 1260
cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg 1320
tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca 1380
aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc 1440
aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca 1500
aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat 1560
acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac 1620
actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat 1680
gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa 1740
tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct 1800
gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc 1860
ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta 1920
tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc 1980
cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt 2040
gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 2100
aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 2160
aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 2220
tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 2280
gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 2340
cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 2400
acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 2460
cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 2520
cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 2580
aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 2640
gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 2700
atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 2760
tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 2820
gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 2880
agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 2940
catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca 3000
ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg 3060
acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 3120
ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc 3180
ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt 3240
ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt 3300
taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca 3360
tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg 3420
aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg 3480
accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc 3540
cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg 3600
acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc 3660
aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat 3720
tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga 3780
tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt 3840
tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa 3900
gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga 3960
gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga 4020
cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca 4080
tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac 4140
tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4200
cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg 4260
ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg 4320
ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat 4380
gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg 4440
gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca 4500
gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc 4560
cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag 4620
ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc 4680
tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa 4740
ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg 4800
caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca 4860
ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt 4920
tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg 4980
acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac 5040
tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc 5100
gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa 5160
acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca 5220
ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg 5280
cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa 5340
gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa 5400
ggagatggcg cccaacagt 5419
<210> 63
<211> 54
<212> DNA
<213> 人工合成
<400> 63
ttttccatgg cgtctaccta caaactggtt atcaacggta aaaccctgaa aggt 54
<210> 64
<211> 60
<212> DNA
<213> 人工合成
<400> 64
ttccgcggtc gccgcgtcaa ccgcttcggt agtggtttca cctttcaggg ttttaccgtt 60
<210> 65
<211> 60
<212> DNA
<213> 人工合成
<400> 65
cggcgaccgc ggaaaaagtt ttcaaacagt acgcgaacga caacggtgtt gacggcgaat 60
<210> 66
<211> 60
<212> DNA
<213> 人工合成
<400> 66
ttttcggtaa cggtgaaggt tttggtcgcg tcgtcgtagg tccattcgcc gtcaacaccg 60
<210> 67
<211> 60
<212> DNA
<213> 人工合成
<400> 67
aaccttcacc gttaccgaaa aaccggaagt tatcgatgcg tctgaactga ccccagcgcg 60
<210> 68
<211> 60
<212> DNA
<213> 人工合成
<400> 68
tggtgatggt ggtgatgagt cttttctttg gttttttcct tatgacgcgc tggggtcagt 60
<210> 69
<211> 60
<212> DNA
<213> 人工合成
<400> 69
actcatcacc accatcacca ccatcatcac atcgaaggtc gtatggaccc ggaaacctgc 60
<210> 70
<211> 60
<212> DNA
<213> 人工合成
<400> 70
tgcaggagtc cgcgcaggtg caagaaccac cagacgggca cgggcaggtt tccgggtcca 60
<210> 71
<211> 60
<212> DNA
<213> 人工合成
<400> 71
gcgcggactc ctgcaaatgc gaaggttgta aatgtacgtc ttgcaaaaaa tcttgttgct 60
<210> 72
<211> 60
<212> DNA
<213> 人工合成
<400> 72
tctttcgcgc atttttcaca ttcggctggg cagcaagagc aacaagattt tttgcaagac 60
<210> 73
<211> 60
<212> DNA
<213> 人工合成
<400> 73
atgtgaaaaa tgcgcgaaag actgcgtttg taaaggtggc gaggcagcgg aagcagaagc 60
<210> 74
<211> 52
<212> DNA
<213> 人工合成
<400> 74
ttttctcgag ttactgacag caggaacatt tctcagcttc tgcttccgct gc 52
<210> 75
<211> 5722
<212> DNA
<213> 人工合成
<400> 75
cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 60
tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 120
gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 180
cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 240
taattttgtt taactttaag aaggagatat accatggcgt ctacctacaa actggttatc 300
aacggtaaaa ccctgaaagg tgaaaccact accgaagcgg ttgacgcggc gaccgcggaa 360
aaagttttca aacagtacgc gaacgacaac ggtgttgacg gcgaatggac ctacgacgac 420
gcgaccaaaa ccttcaccgt taccgaaaaa ccggaagtta tcgatgcgtc tgaactgacc 480
ccagcgcgtc ataaggaaaa aaccaaagaa aagactcatc accaccatca ccaccatcat 540
cacatcgaag gtcgtatgga cccggaaacc tgcccgtgcc cgtctggtgg ttcttgcacc 600
tgcgcggact cctgcaaatg cgaaggttgt aaatgtacgt cttgcaaaaa atcttgttgc 660
tcttgctgcc cagccgaatg tgaaaaatgc gcgaaagact gcgtttgtaa aggtggcgag 720
gcagcggaag cagaagctga gaaatgttcc tgctgtcagt aactcgagca ccaccaccac 780
caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc 840
gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg 900
ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa 960
gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 1020
ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 1080
ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 1140
aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 1200
gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 1260
cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 1320
attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 1380
cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 1440
ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg 1500
agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 1560
agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 1620
tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 1680
tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 1740
ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 1800
tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 1860
aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg 1920
aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 1980
aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 2040
aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 2100
tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 2160
ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 2220
ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt 2280
tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt 2340
attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 2400
agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 2460
aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 2520
ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 2580
gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 2640
aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 2700
aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 2760
gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 2820
aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 2880
aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 2940
cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 3000
cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 3060
tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 3120
tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 3180
ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 3240
ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat 3300
acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg 3360
ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 3420
tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc 3480
tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg 3540
cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca 3600
tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt 3660
tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg 3720
atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc 3780
gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg 3840
ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg 3900
ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg 3960
ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt 4020
cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca 4080
cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac 4140
gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg 4200
caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc 4260
agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg 4320
cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg 4380
gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct 4440
cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 4500
gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag 4560
acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc 4620
acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa 4680
catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc 4740
ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc 4800
gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca 4860
ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc 4920
cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt 4980
tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag 5040
aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta 5100
gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc 5160
ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt 5220
cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc 5280
gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac 5340
gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc 5400
gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg 5460
gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc 5520
acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt 5580
ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag 5640
gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg 5700
caaggagatg gcgcccaaca gt 5722
<210> 76
<211> 207
<212> DNA
<213> 人工合成
<400> 76
atggacccgg aaacctgccc gtgcccgtct ggtggttctt gcacctgcgc ggactcctgc 60
aaatgcgaag gttgtaaatg tacgtcttgc aaaaaatctt gttgctcttg ctgcccagcc 120
gaatgtgaaa aatgcgcgaa agactgcgtt tgtaaaggtg gcgaggcagc ggaagcagaa 180
gctgagaaat gttcctgctg tcagtaa 207
<210> 77
<211> 25
<212> DNA
<213> 人工合成
<400> 77
ttttccatgg acccggaaac ctgcc 25
<210> 78
<211> 32
<212> DNA
<213> 人工合成
<400> 78
ttttctcgag ttactgacag caggaacatt tc 32
<210> 79
<211> 5440
<212> DNA
<213> 人工合成
<400> 79
cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 60
tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 120
gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 180
cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 240
taattttgtt taactttaag aaggagatat accatggacc cggaaacctg cccgtgcccg 300
tctggtggtt cttgcacctg cgcggactcc tgcaaatgcg aaggttgtaa atgtacgtct 360
tgcaaaaaat cttgttgctc ttgctgccca gccgaatgtg aaaaatgcgc gaaagactgc 420
gtttgtaaag gtggcgaggc agcggaagca gaagctgaga aatgttcctg ctgtcagtaa 480
ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct 540
gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 600
gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc 660
cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 720
ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 780
ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 840
tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 900
cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 960
tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 1020
ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 1080
attttaacaa aatattaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg 1140
gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat 1200
tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca 1260
ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc 1320
cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa 1380
cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg 1440
actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc 1500
cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat 1560
tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc 1620
gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga 1680
tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca 1740
tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag 1800
tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga 1860
aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg 1920
acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc 1980
ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt 2040
atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca 2100
ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 2160
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 2220
tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 2280
tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 2340
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 2400
tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 2460
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 2520
acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 2580
ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 2640
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 2700
ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 2760
ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 2820
taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 2880
cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2940
tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 3000
gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 3060
gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 3120
acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 3180
cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 3240
tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 3300
ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 3360
tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 3420
cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 3480
tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 3540
ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 3600
acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 3660
agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 3720
gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 3780
tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg 3840
cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt 3900
gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt 3960
cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga 4020
agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg 4080
ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta 4140
cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 4200
attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt 4260
tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag 4320
agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg 4380
gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata 4440
tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga 4500
tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt 4560
tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg 4620
cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg 4680
cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc 4740
gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga 4800
aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc 4860
ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta 4920
caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg 4980
gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg 5040
gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg 5100
taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg 5160
gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg 5220
tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat 5280
gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt 5340
atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc 5400
cgcaaggaat ggtgcatgca aggagatggc gcccaacagt 5440
<210> 80
<211> 38
<212> DNA
<213> 人工合成
<400> 80
ttttccatgg ctagcaccta caaactggtt atcaacgg 38
<210> 81
<211> 60
<212> DNA
<213> 人工合成
<400> 81
caaccgcttc ggtggtagtt tcacctttca gggttttacc gttgataacc agtttgtagg 60
<210> 82
<211> 60
<212> DNA
<213> 人工合成
<400> 82
ctaccaccga agcggttgac gccgcgaccg cggaaaaagt tttcaaacag tacgcgaacg 60
<210> 83
<211> 60
<212> DNA
<213> 人工合成
<400> 83
tcgcatcgtc gtaggtccat tcgccgtcaa caccgttgtc gttcgcgtac tgtttgaaaa 60
<210> 84
<211> 60
<212> DNA
<213> 人工合成
<400> 84
ggacctacga cgatgcgacc aaaaccttca ccgtaaccga aaaaccggag gttatcgacg 60
<210> 85
<211> 60
<212> DNA
<213> 人工合成
<400> 85
tcggtcggcg cgtcggtaac ggctggggtc agttcgctcg cgtcgataac ctccggtttt 60
<210> 86
<211> 60
<212> DNA
<213> 人工合成
<400> 86
gacgcgccga ccgaaccgga acaccaccat catcaccatc atcaccacat cgaaggtcgt 60
<210> 87
<211> 60
<212> DNA
<213> 人工合成
<400> 87
cagataccac cagacatgca aacgcattca cgcgggtcca tacgaccttc gatgtggtga 60
<210> 88
<211> 60
<212> DNA
<213> 人工合成
<400> 88
ttgcatgtct ggtggtatct gcatgtgcgg cgacaattgc aaatgcacca cctgcaactg 60
<210> 89
<211> 60
<212> DNA
<213> 人工合成
<400> 89
cccggagggc agcatggaca gcaagattta cggcaggtct tgcagttgca ggtggtgcat 60
<210> 90
<211> 60
<212> DNA
<213> 人工合成
<400> 90
catgctgccc tccgggttgc gcgaaatgcg cgcgtggttg catctgcaaa ggtggttctg 60
<210> 91
<211> 53
<212> DNA
<213> 人工合成
<400> 91
ttttctcgag ttacgggcaa caggagcatt tgtcagaacc acctttgcag atg 53
<210> 92
<211> 5701
<212> DNA
<213> 人工合成
<400> 92
cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 60
tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 120
gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 180
cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 240
taattttgtt taactttaag aaggagatat accatggcgt ctacctacaa actggttatc 300
aacggtaaaa ccctgaaagg tgaaactacc accgaagcgg ttgacgccgc gaccgcggaa 360
aaagttttca aacagtacgc gaacgacaac ggtgttgacg gcgaatggac ctacgacgat 420
gcgaccaaaa ccttcaccgt aaccgaaaaa ccggaggtta tcgacgcgag cgaactgacc 480
ccagccgtta ccgacgcgcc gaccgaaccg gaacaccacc atcatcacca tcatcaccac 540
atcgaaggtc gtatggaccc gcgtgaatgc gtttgcatgt ctggtggtat ctgcatgtgc 600
ggcgacaatt gcaaatgcac cacctgcaac tgcaagacct gccgtaaatc ttgctgtcca 660
tgctgccctc cgggttgcgc gaaatgcgcg cgtggttgca tctgcaaagg tggttctgac 720
aaatgctcct gttgcccgta actcgagcac caccaccacc accactgaga tccggctgct 780
aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa 840
ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc 900
ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 960
cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 1020
cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 1080
tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 1140
gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 1200
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 1260
attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 1320
tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca 1380
cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 1440
tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 1500
ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 1560
gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 1620
actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 1680
gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 1740
ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 1800
aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 1860
ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 1920
atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 1980
gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 2040
ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 2100
ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 2160
attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 2220
tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 2280
acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 2340
ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 2400
ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 2460
agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 2520
cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 2580
caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 2640
tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 2700
ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 2760
ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 2820
gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 2880
gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 2940
tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 3000
cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 3060
gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 3120
ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat 3180
gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc 3240
agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg 3300
actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 3360
gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 3420
agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt 3480
ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct 3540
ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct 3600
gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga 3660
tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg 3720
aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc 3780
agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc 3840
atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac 3900
tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc 3960
agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc 4020
aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg 4080
ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga 4140
cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg 4200
tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc 4260
ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg 4320
cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg 4380
cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg 4440
gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 4500
gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc 4560
ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg 4620
cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg 4680
tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc 4740
attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca 4800
ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc 4860
gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc 4920
gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc 4980
agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt 5040
gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca 5100
atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga 5160
agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc 5220
acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg 5280
tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt 5340
tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc 5400
gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca 5460
ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga 5520
ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc 5580
gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt 5640
gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag 5700
t 5701
<210> 93
<211> 189
<212> DNA
<213> 人工合成
<400> 93
atggacccgc gtgaatgcgt ttgcatgtct ggtggtatct gcatgtgcgg cgacaattgc 60
aaatgcacca cctgcaactg caagacctgc cgtaaatctt gctgtccatg ctgccctccg 120
ggttgcgcga aatgcgcgcg tggttgcatc tgcaaaggtg gttctgacaa atgctcctgt 180
tgcccgtaa 189
<210> 94
<211> 24
<212> DNA
<213> 人工合成
<400> 94
ttttccatgg acccgcgtga atgc 24
<210> 95
<211> 25
<212> DNA
<213> 人工合成
<400> 95
ttttctcgag ttacgggcaa cagga 25
<210> 96
<211> 5422
<212> DNA
<213> 人工合成
<400> 96
cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 60
tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 120
gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 180
cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 240
taattttgtt taactttaag aaggagatat accatggacc cgcgtgaatg cgtttgcatg 300
tctggtggta tctgcatgtg cggcgacaat tgcaaatgca ccacctgcaa ctgcaagacc 360
tgccgtaaat cttgctgtcc atgctgccct ccgggttgcg cgaaatgcgc gcgtggttgc 420
atctgcaaag gtggttctga caaatgctcc tgttgcccgt aactcgagca ccaccaccac 480
caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc 540
gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg 600
ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa 660
gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 720
ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 780
ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 840
aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 900
gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 960
cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 1020
attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 1080
cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 1140
ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg 1200
agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 1260
agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 1320
tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 1380
tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 1440
ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 1500
tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 1560
aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg 1620
aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 1680
aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 1740
aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 1800
tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 1860
ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 1920
ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt 1980
tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt 2040
attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 2100
agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 2160
aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 2220
ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 2280
gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 2340
aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 2400
aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 2460
gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 2520
aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 2580
aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 2640
cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 2700
cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 2760
tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 2820
tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 2880
ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 2940
ccgcatatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat 3000
acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg 3060
ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 3120
tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc 3180
tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg 3240
cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca 3300
tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt 3360
tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg 3420
atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc 3480
gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg 3540
ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg 3600
ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg 3660
ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt 3720
cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca 3780
cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac 3840
gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg 3900
caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc 3960
agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg 4020
cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg 4080
gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct 4140
cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 4200
gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag 4260
acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc 4320
acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa 4380
catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc 4440
ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc 4500
gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca 4560
ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc 4620
cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt 4680
tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag 4740
aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta 4800
gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc 4860
ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt 4920
cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc 4980
gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac 5040
gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc 5100
gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg 5160
gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc 5220
acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt 5280
ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag 5340
gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg 5400
caaggagatg gcgcccaaca gt 5422
Claims (9)
1.一种金属硫蛋白表达载体,其特征在于,包括由金属硫蛋白的编码序列作为目标蛋白***类伴侣样蛋白的下游所形成的融合蛋白表达载体,其中,所述类伴侣样蛋白为序列如SEQ ID NO:3所示的链球菌蛋白G的B3结构域PGB3。
2.如权利要求1所述的金属硫蛋白表达载体,其特征在于,所述金属硫蛋白为金属硫蛋白-1、金属硫蛋白-2、金属硫蛋白-3或金属硫蛋白-4。
3.如权利要求2所述的金属硫蛋白表达载体,其特征在于,所述金属硫蛋白-1包括金属硫蛋白-1A、金属硫蛋白-1B、金属硫蛋白-1E、金属硫蛋白-1F、金属硫蛋白-1G、金属硫蛋白-1H、金属硫蛋白-1M、金属硫蛋白-1R和金属硫蛋白-1X中的任意一种。
4.如权利要求3所述的金属硫蛋白表达载体,其特征在于,所述金属硫蛋白-1为人金属硫蛋白-1F,所述人金属硫蛋白-1F的氨基酸编码序列如SEQ ID NO:4所示;或者,
所述金属硫蛋白-2为人金属硫蛋白-2,所述人金属硫蛋白-2的氨基酸编码序列如SEQID NO:5所示;或者,
所述金属硫蛋白-3为人金属硫蛋白-3,所述人金属硫蛋白-3的氨基酸编码序列如SEQID NO:6所示;或者,
所述金属硫蛋白-4为人金属硫蛋白-4,所述人金属硫蛋白-4的氨基酸编码序列如SEQID NO:7所示。
5.如权利要求1所述的金属硫蛋白表达载体,其特征在于,所述PGB3的编码序列下游包含一段柔性接头序列,所述柔性接头序列下游包含一段分离纯化标签序列,所述分离纯化标签序列与所述目标蛋白的编码序列之间包含一段肽链切割识别序列;和/或,
所述PGB3编码序列的N端添加有如SEQ ID NO:8所示的起始适配序列。
6.如权利要求5所述的金属硫蛋白表达载体,其特征在于,
所述柔性接头序列包括6~30个氨基酸;
所述分离纯化标签序列包含2~11个组氨酸;
所述肽链切割识别序列包括凝血因子Xa、凝血酶、肠激酶、TEV酶和羟胺中任意一种的切割识别序列。
7.如权利要求6所述的金属硫蛋白表达载体,其特征在于,所述柔性接头序列包括22个氨基酸,序列如SEQ ID NO:10所示;
所述分离纯化标签序列标签包含9个内置组氨酸,序列如SEQ ID NO:11所示;
所述肽链切割识别序列为凝血因子Xa的切割识别序列,序列如SEQ ID NO:12所示。
8.如权利要求1所述的金属硫蛋白表达载体,其特征在于,所述金属硫蛋白表达载体经过密码子与整体序列优化,且优化后的DNA编码序列如SEQ ID NO:17~20中的任一所示。
9.一种包含如权利要求1至8任意一项所述的金属硫蛋白表达载体的金属硫蛋白表达工程菌株。
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