CN1150824A - 来自米曲霉的areA基因和其中areA基因已被修饰的真菌 - Google Patents
来自米曲霉的areA基因和其中areA基因已被修饰的真菌 Download PDFInfo
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- CN1150824A CN1150824A CN95193631.XA CN95193631A CN1150824A CN 1150824 A CN1150824 A CN 1150824A CN 95193631 A CN95193631 A CN 95193631A CN 1150824 A CN1150824 A CN 1150824A
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Abstract
本发明涉及不产生蛋白酶的真菌。本发明的真菌可作为宿主用于生产可被通常产生的蛋白酶降解的蛋白质，因而本发明还包括利用本发明的真菌以高产率生产所研究的蛋白质的方法。本发明也包括生产这样的真菌的方法以及用于这些方法的DNA构建体。
Description
发明领域
本发明涉及不产生蛋白酶的真菌。本发明的真菌可作为宿主用于生产可被通常产生的蛋白酶降解的蛋白质，因而本发明还包括利用本发明的真菌以高产率生产所研究的蛋白质的方法。本发明也包括产生这些真菌的方法以及用于这些方法的DNA构建体。发明背景
真菌特别是丝状真菌被广泛应用于商业领域，因为他们具有分泌极大量蛋白质的能力。
在曲霉属的丝状真菌种类中，在生产内源蛋白质的商业用途方面已有很长的历史，近来也用于生产异源蛋白质。
用于生产蛋白质的大多数微生物的一个缺点在于内在生成蛋白酶，它会使所研究的蛋白质产物因蛋白水解而发生降解。
人们已经设想出避免这个缺点的多种方法。在另外一些解决方法中，有人提议可删除或破坏多种蛋白酶的编码基因。遗憾的是，真菌可产生大量的蛋白酶，这使该方法多少有些不够实用。
因而我们非常需要一种不产生或只产生极少量蛋白酶的丝状真菌菌株。
多年来人们知道在无冠构巢曲霉中介导氮代谢物阻抑的调控基因areA可影响胞外蛋白酶的产生[Arst & Cove，molec，gen.Genet.126，(1973)111-141]。
人们对来自无冠构巢曲霉的areA基因进行了克隆[Caddick等人，EMBO Journa(5，(1986)1087-1090]，并对其进行了多种修饰以评估在由该基因编码的激活蛋白中的不同区域的功能[Stankovitch等人，Mol.Microbiol.7，(1993)81-87]。另外，编码烟曲霉中相应功能的基因最近显然已进行了克隆[Heasel等人，第二届欧洲真菌遗传学会议，1994年4月28至5月1日，摘要本，E11]。
从文献中也得知了基因型arg B areA1的无冠构巢曲霉菌株作为生产t-PA的宿主的专一用途[Upshall等人，Biotechnology 5，(1987)1301-1304]。在这个例子中，只有argB基因型通过其精氨酸原养型被用作选择标记物，而areA基因型不过是一个并发物。
本发明的目的在于缓解对无蛋白酶丝状真菌的需求。发明概述
本发明涉及一种真菌，其中由重组DNA技术产生的areA基因已进行了修饰，使得其不能以提供功能性AreA激活物的方式进行表达。
本发明还涉及生产这些真菌的方法，是通过areA基因的缺失而获得的。
其可以通过一种方法而获得，该方法包括
i)对来自所研究真菌的areA基因进行克隆，
ii)生产含有areA基因的DNA构建体，其中基因内在部分已被取代、删除，或已插入额外的DNA，
iii)用该构建体来转化所述真菌，和
iv)选择出为areA-的转化体。
由上述对areA基因克隆而得到的信息也可与人们熟悉的反义技术结合用于构建可合成互补于由areA基因转录的mRNA的RNA分子的表达质粒，并以此来转化所研究的真菌。
本发明还涉及拟用于上述方法的DNA构建体。
本发明亦涉及生产所需蛋白质或基因产物(特别是分泌性蛋白质)的方法，即：用DNA构建体(至少含有所研究的蛋白质或基因产物的DNA编码序列)修饰并选择性转化真菌宿主，在适宜的条件下在适宜的生长培养基中进行培养，回收所需要的基因产物并提纯。
当实施本发明时，人们惊奇地发现，本发明的真菌产生了这样的分泌性蛋白质，而且产率大大提高。
还令人惊讶的是，能够提供米曲霉areA-菌株良好生长的唯一氮源为谷氨酰胺。
最后，本发明涉及通过上述方法生产的蛋白质产物。附图简述
以下部分将参照实施例及附图进一步详述本发明，其中
图1显示了HowB101构建的步骤，
图2显示了pSK5及pSK9构建的步骤，
图3a及3b显示了pToC266构建的步骤，
图4显示了pMT1606构建的步骤，
图5显示了pToC56构建的步骤。定义
在本说明书中用到了以下定义。
areAΔ意指一种菌株，其中缺失了areA基因。
areA-意指一种菌株，其不产生功能性AreA激活物。术语“丧失功能”也经常用于这方面。
“反义技术”描述了如美国专利5,190,931中所公开的几种方法。
发明详述
如所述，本发明的第一方面涉及真菌，其中通过重组DNA技术得到的areA基因已被修饰，使得其不能以提供功能性AreA激活物的方式进行表达。
这个目的尤其可通过areA基因的缺失或破坏而达到。
areA基因的克隆见实施例中所述。
来自其它真菌的AreA同源物也可以进行克隆，方法既可以是与已知基因之一的交叉杂交，也可以是areA突变体的互补作用；例如，本申请中所述的无冠构巢曲霉areA-18或米曲霉areA缺失的菌株。
删除或破坏基因的方法具体描述于WO90/00192(Genencor)。
取代基因中DNA的方法也是人们普遍知道的，其可以通过取代基因的一个或多个连续部分而实现，但其也可以通过位点针对性诱变产生编码无功能的AreA激活变异体的DNA序列而获得。
达到这种目的的另一种方法是使用反义技术。
反义技术及其如何应用详见上述美国专利5,190,931(纽约大学)。
获得所述失活作用的另一个方法是在areA基因内部插入额外的DNA，从而引起机能障碍性激活蛋白的表达。
与该方法相关，由克隆提供的信息也能用于建造可以整合入areA基因中的DNA构建体，甚至可用另一个基因(如pvrG基因)来取代它。
避免areA激活物出现的另一个方法是干扰用于调控areA基因本身表达的表达信号的调控作用。
依据本发明，真菌优选属于选自下列的属：曲霉属、木霉属、腐质霉属、假丝酵母属、Acremonium、镰孢属及青霉属。
在这些属中，选自下列的菌种是优选的：米曲霉、黑曲霉、泡盛曲霉、海枣曲霉、日本曲霉、臭曲霉、无冠构巢曲霉、T.reesei、T.harzianum、H.insulens、柔毛腐质霉、禾木科镰孢、腐皮镰孢、产黄青霉等等。
如所述，本发明还包括生产本发明第一方面的真菌的方法，且其中所述失活作用已通过areA基因的缺失而获得，该方法包括
i)来自所研究真菌的areA基因同源物的克隆化，
ii)生产含有areA基因的DNA构建体，其中内在部分已被取代、删除，或者已插入额外的DNA，
iii)用该构建体来转化上述真菌，和
iv)选择出为areA-的转化体。
还包括产生该真菌的方法，其中失活作用已通过反义技术获得。这种方法包括
i)一种表达质粒的构建，该质粒可引起互补于转录自areA基因的MRA的RNA分子的合成，
ii)用所述表达质粒及适宜的标记物来转化宿主真菌，可在独立的质粒上进行，或在相同的质粒上进行，
iii)利用上述标记物来选择转化体，和
iv)筛选出在AreA产物的合成中表现出还原作用的菌株转化体，例如可通过对各种氮源上生长的分析而获得。
本发明的另一个方面包括用于上述方法的DNA构建体。
就前者的方法而言，所述DNA构建体可含有areA基因，其中内在部分已被取代、删除，或已插入额外的DNA。
DNA构建体还可含有编码所研究的蛋白质产物的DNA序列，如后面所述及。
就后者的反义方法而言，DNA构建体可含有与功能性启动子相连的areA基因的反向DNA序列，使得mRNA至少与来自areA基因的mRNA部分互补。
本发明的另一方面涉及生产所需要的基因产物(优选为分泌性基因产物)的方法，即依据本发明的真菌在适宜的条件下在适宜的培养基中进行培养，回收所需的基因产物并提纯。
在由异源基因所表达的基因产物方面，编码所需基因产物的DNA序列可以是用于产生所述真菌的DNA构建体的一部分。
不过，一般而言，本发明中真菌的独立转化作用的发生是为了使真菌能够生产所需要的产物。
转化真菌的方法是本领域人士所熟悉的，例如可参见EP0184A2(Gist-Brocades N.V.)及欧洲专利申请87103806(NovoNordisk A/S)。
对于固有产物来说，当然这并不是必需的，但是为了增加产量，提供拟掺入宿主的编码所研究蛋白质的基因可能是有好处的。
所需基因产物通常为肽或蛋白质，优选为酶。
在酶中，其优选的是选自蛋白酶，如胰蛋白酶和凝乳酶、脂酶、几丁质酶、纤维素酶、木聚糖酶、漆酶、果胶酶等等。
另一类所需的基因产物一般为具有治疗活性的肽或蛋白质。
在具有治疗活性的肽或蛋白质中，蛋白质优选地选自胰岛素、生长激素、胰高血糖素、生长激素抑制素、干扰素、PDGF、VII因子、VIII因子、尿激酶、t-PA、CSF、乳铁蛋白、TPO等等。
以下给出的实施例将对本发明做进一步的详尽阐述。不过，这些实施例不应以任何方式限制如所附权利要求书中所限定的本发明的范围。实施例材料及方法菌株
米曲霉，IFO4177：可从日本大阪发酵研究所；日本大阪17-25Juso Hammachi Z-chome Yodogawa-Ku得到。
ToC913：该菌株的构建如实施例所述。基因：
areA：该基因编码能控制氮代谢的调控蛋白。
pyrG：该基因编码乳清酸核苷-S-磷酸脱羧酶，这是一种尿苷合成中所涉及的酶。
bar：该基因最初分离自吸水链霉菌，并为phosphinothricin乙酰转移酶编码。该酶可修饰phosphinothricin(＝glufosinate)，从而使这种对细菌、真菌及植物有毒性的化合物失活。质粒
pUC118：Viera and Mesing J.Meth.Enzymol.1987 153 3-11
pSO2：该质粒的构建如实施例所述。
pJers4：pUC118中pSO2的2.0kb亚克隆。pJers4含有功能性米曲霉pyrG基因
pSO5：该来自pSO2的质粒的构建如实施例所述。
pToC56：该质粒的构建如欧洲专利申请87103806所述。
pToC266：该质粒的构建如实施例所述。
pMT1606：该来自pBP1T(B.Straubinger等人，Fungal GeneticsNewsletter 39(1992)：82-83)及p775(欧洲专利申请87103806)的质粒的构建如实施例所述。
p777：该质粒的构建如欧洲专利申请87103806所述。
pHW470：该质粒的构建如实施例所述。实施例1米曲霉areAΔ菌株的构建
areAΔ菌株是通过下述步骤构建的。对米曲霉pyrG基因进行克隆，分离出米曲霉pyrG突变株。对来自米曲霉的areA基因进行克隆。用携带着pyrG基因(插在areA基因的DNA片段上游与下游之间)来转化pyrG基因突变体。在质粒上并没有areA的编码区域。根据转化体在没有尿苷和存在氯酸盐的条件下生长的能力将它们选择出来。这种双重选择既可以选择功能性pyrG基因，也可以选择areA负型。最后利用Southern分析来筛选用这种选择程序所得到的菌株以鉴定其中染色体areA基因被pyrG基因所取代的菌株。米曲霉pyrG基因的克隆化
通过与黑曲霉pyrG基因的交叉杂交对米曲霉pyrG基因进行克隆(W.van Hartingsvelolt等人，Mol.Gen.Genet 206：71-75(1987))。利用来自黑曲霉pyrG基因的1kb DNA片段在低严格性条件下探查部分SauIIIA消化的米曲霉IFO4177 DNA的λ文库。将来自阳性克隆的3.8kb HindIII片段亚克隆到pUC118载体中。所生成的质粒pSO2通过黑曲霉pyrG突变体的互补作用而表现出含有pyrG基因。米曲霉pyrG负型菌株的构建
pyrG缺失质粒pSO5在每一末端含有约1kb的pyrG侧翼序列，该质粒是从质粒pSO2构建而来的。用该构建体转化米曲霉IFO4177，并通过对5-氟-乳清酸的抗性(一种具有pyrG突变体特性的表型)而选择出转化体。通过Southern分析表明了转化体HowB101在pyrG基因座具有预期的缺失。做为pyrG突变体，HowB101需要有尿苷才能生长。通过在无尿苷条件下生长能力的选择，用wtpyrG基因可转化HowB101。
HowB101构建中所涉及的步骤如图1所述。areA基因的克隆化
通过与无冠构巢曲霉areA基因的交叉杂交克隆了米曲霉areA基因[B.Kudla等人，EMBOJ.9：1355-1364(1990)]。通过用SauIIIA部分消化染色体DNA并将得到的DNA片段克隆到载体λGEM-II(得自Promega)中，制备出米曲霉IFO4177的基因组文库。在40％的甲酰胺中，于37℃进行该文库与无冠构巢曲霉areA基因的交叉杂交。从这些片段中分离出杂交性λ克隆，再亚克隆到载体pBluescript SK+(得自Stratagene)中，得到如图2所述的质粒pSK5及pSK9。经克隆的基因能补足无冠构巢曲霉areA突变体，这说明其的确是米曲霉areA同源物。对5643bp的克隆进行序列测定，对米曲霉及无冠构巢曲霉areA基因序列的比较表明：它们具有较高的同源性。米曲霉areA基因的序列如SEQ ID NO.1所示。areA缺失质粒的构建
为了从米曲霉染色体中删除掉areA基因，构建了质粒pToC266。pToC266含有源于areA基因(分离自pSK5)上游的-2.1kb的DNA片段以及源于areA基因(分离自pSK9)下游的-1.4kb的DNA片段。这两个片段在基因组中相隔约3.2kb。编码区域则位于该基因的这个部分。将来自pJers4的米曲霉pyrG基因插入areA上游及下游DNA片段之间。pToC266的构建如图3a及3b所示。pToC266具有一独特位点，在用做转化作用之前，可用该限制性内切酶的切割使其线性化。米曲霉areAΔ菌株的选择
用线性化pToC266来转化米曲霉HowB101。在基本平板[CoveBiochem.biophg.Acta(1966)113 51-56]上选择出转化体，该平板含有5％的氯酸钠、0.5mM的硫酸铵和1％的葡萄糖。对转化体进行双重选择，即从通过可在不加尿苷的条件下生长而得出pyrG基因以及氯酸盐抗性两方面来选择。氯酸盐抗性是无冠构巢曲霉areA突变体表型之一[H.N.Arst及D.J.Cove，MGG 126：111-141(1973)]。在相同类型的平板上对生长性差的转化体重复分离两次。在不同的氯源上对三种独立的转化体ToC913、ToC919及ToC920进行生长测试。它们在谷氨酰胺上生长良好，但在其它测试的氮源(包括氨)上生长较差。Southern分析表明，这三个菌株都缺失了areA结构基因，后者已被pyrG基因所取代了。
areAΔ菌株也可以通过在含有谷氨酰胺作为氮源的基本平板上对线性化pToC266转化体的选择而得到。在一个这样的实验中，25个转化体中有一个是areAΔ菌株。实施例2pMT1606的构建
构建-含有来自吸水链霉菌的bar基因[C.J.Thompson等人，EMBOJ.6：2519-2523(1987)]的质粒，bar基因被插入在米曲霉TAKA-淀粉酶启动子之后，其后是含有转录终止子的片段和来自黑曲霉gla基因的聚腺苷酸化信号。
质粒pMT1606可用于选择米曲霉的glufosinate抗性突变体。通过从质粒pBP1T[B.Straubinger等人，Fungel Genetics Newslet-tel39：82-83(1993)]中分离出bar基因并将其克隆到真菌表达质粒p775(见欧洲专利申请87103806)中而构建出pMT1606。图4示pMT1606的构建。实施例3ToC913(米曲霉IFO4177areAΔ)中的凝乳酶的生产。
通过pMT1606的共转化作用用质粒pToC56转化米曲霉areAΔ菌株ToC913(图5)，质粒pToC56是一种用于哺乳动物酶凝乳酶的真菌表达质粒。质粒pToC56的构建如欧洲专利申请87103806所述。
通过其产生凝乳酶的能力，依据在基本培养基(含有10mM的氨及1mg/ml的glufosinate)上的生长而选择转化体，并依据pToC56的存在而进行筛选。使三个转化体在摇瓶中的基本培养基(含有麦芽糖糊精及谷氨酰胺)上于30℃生长4天。使在IFO4177(如欧洲专利87103806所述而得到)中的两个pToC56转化体以及未转化的IFO4177和ToC913与ToC913转化体共同生长。
每天抽取发酵液样品并用于SDS-Page及Western印迹法。将印迹膜与凝乳酶特异性兔抗体并随后与偶联在过氧化酶上的山羊兔抗体一起温育。膜的染色表明，在发酵作用发生的第一天和第二天，来自IFO4177转化体的上清液含有少量的凝乳酶或其降解产物，而在其后的发酵中并不含这些物质。
ToC913转化体含有至少10倍以上的整体凝乳酶。上清液中的凝乳酶量在头2-3天内是增加的，其后则维持不变。
将IFO4177、ToC913、ToC913中pToC56的转化体以及IFO4177中的转化体发酵的第3天和第4天的上清液用于等电子聚焦凝胶，进行电泳操作。PH梯度为3.5-9.5。电泳之后，用PH为7.0的缓冲液(含2mMZnz+)洗涤凝胶，再用含0.5％酪蛋白的琼脂涂盖。在45℃下对凝胶进行温育，直至可见到蛋白酶活性为止。
在IFO4177的样品中，可以看到三条具有蛋白酶活性的带，一个具碱性pI，两个具酸性pI。
在IFO4177的pToC56转化体的样品中，可见到来自凝乳酶的微弱反应，它被见于未转化的IFO4177中的酸性带之一部分地覆盖，具强酸性pI的蛋白酶几乎未见到，而具碱性pI的蛋白酶却伴随着具几乎中性的pI的一个或多个带而清晰可见。
在ToC913样品中，未检测到任何蛋白酶活性，而ToC913的pToC56转化体的样品却表现出强烈的凝乳酶信号。在该转化体样品中未检测到其它蛋白酶。实施例4ToC913(米曲霉IFO4177areAΔ)中人胰蛋白酶I的生产
使用标准程序及由M.Emi等人发表的序列[Gene(1986)41：305-310，参见丹麦专利申请no.693/95]来分离出编码人胰蛋白酶原I(TRYI)的cDNA。将BamHI位点(GGATCC)直接引入其间具有短序列ACC的起始密码子[ATG(Met)]的上游。
在如欧洲专利申请no.87103806所述的真菌表达质粒p777中用该BamHI位点使cDNA与Taka-淀粉酶启动子中的BamHI接头融合。cDNA的3′末端在终止密码子下游的41bp与p777中的Nru1位点融合。这样将TRYICDNA插在米曲霉Taka-淀粉酶启动子与黑曲霉葡糖淀粉酶转录终止子之间。所生成的质粒称为pHW470(参见丹麦专利申请no.693/95)。
利用质粒pMT1606共转从作用将pHW470转化到ToC913中。通过分生孢子对BASTA抗性转化体进行两次重复分离。在30℃条件下使8个转化体生长4天，培养基为YPM[YPD(Sherman，F，等人(1981)，见“酵母遗传学方法”，冷泉港实验室，纽约冷泉港)，其中用2％的麦芽糖代替葡萄糖]。利用SDS-PAGE，随后利用Western印迹法和与抗猪胰蛋白酶的兔抗体一起温育来分析上清液中人胰蛋白酶的含量。然后将印迹膜与偶联在过氧化酶上的山羊抗兔抗体一起温育并与3′-氨基-9-乙基咔唑进行反应。三个转化体的上清液含有预期大小的染色带。三种阳性上清液中的胰蛋白浓度为2-5mg/l。
通过将上清液样品与L-苯甲酰-精氨酰-对硝基苯胺(L-BAPNA)一起温育进一步证实了胰蛋白酶的存在。三种免疫阳性菌株样品裂解了底物，引起了黄色的显色。来自ToC913及IFO4177的样品并未显示对该底物的丝毫活性。我们对该检测中人胰蛋白酶的特异活性尚不得而知，因而不可能从这些数据中计算出上清液中胰蛋白酶的浓度。
也在野生型菌株IFO4177中制备出pHW470转化体。我们看到了20多个L-BAPAN阳性转化体，但尚不可能从这些转化体的上清液中检测到任何免疫活性带。在该检测中检测限度约为0.5mg/l。
序列表(1)一般信息(i)申请人
(A)名称：Novo Nordisk A/S
(B)街道：Novo Alle
(C)城市：Bagsvaerd
(E)国家：丹麦
(F)邮编：Dk-2880
(G)电话：+45 44422668
(H)传真：+45 44493256(ii)发明名称：新型微生物(iii)序列数：2(iV)计算机可读形式：
(A)媒介类型：软盘
(B)计算机：IBM PC兼容
(C)操作系统：PC-DOS/MS-DOS
(D)软件：Patent In Release #1.0，Version #1.25(EPO)(2)SEQ ID NO：1的信息：(i)序列特性
(A)长度5643个碱基对
(B)类型：核酸
(C)链型：单
(D)拓扑学：线性(ii)分子类型：DNA(基因组)(iii)假拟：无(iii)反义：无(vi)来源：
(A)生物体：米曲霉
(B)菌株：IFO4177(ix)特征： (A)名称/关键词：内含子(B)位置：2701..2769(ix)特征：(A)名称/关键词：CDS(B)位置：连接处(2282..2700，2770..4949)(xi)序列描述：SEQ ID NO：1：AAGCTTCGTC CTCGCATCTC GGCCGGGTGA GTAAGGTATG GTATTATTCA TGAAGGGATC 60TCGTTGGTTA CCGTTGTCTA TCCCTAAACA AAGGATTCAA GAGAACAACT CGGAATGCTC 120CCTCCGCTTA AACCCCTTGA CTCACTGATG GTGTATGTAC TATGGGTACG ACGTTCGGGA 180TGTGGACTAC CAACCAGAGA GTGATTAGAG AGTCCGGGTT CTCAGTCCAT GATTTTTGCA 240TCTTTGAAAC AGACGATGCG GAGCGGTCAT TGGCGGAGTT TACTCCCAAA TACGGCCGAA 300CGGGGTACTT TAAGTGGAAT CTCCGATTTT GGATCTAAGC TCATGAAGGA AAAGTACTAC 360TAATGCGTAC CTGTGCCTAA TGTTAGTGCT AGTTCGTCTG TTGCATTTTA CCCGTCGGTT 420AAGACGAATG GATCCGTTCA GGTTTTAAAA TAACTATCTA TGAAATATTT TAGATTTCCC 480GACATAGTGG TTGGGATGTC TCGATTAACA CTAGGTACAT CAGGCTCAAT TGATTTTGGT 540TTTAACGAAA CATGATATAG GTCAGGGTCG TGGACCACCC TCCGCCAGGG ATCAGGGGAC 600GGTTACATGC GAAGGATTCT GATTATATTC ATGATTATGT CAAGCCTTTT CTCTCGTGTG 660AAGAGGAGCA GAGAATCCGT ACGGGTTTAA TTTAATTTAG CGCCCTGCAG CTTCGAGAAC 720ATCCCCAGCA ACGTTAAAAA CCACGAGCTA AAATGGGTCG CCACCGGAAG CACTCGAGTC 780GAGAGATCGG TCGGCTCAGT ATTCGTAATA CCTGCGTTCC AGACGGTTTT GGTCGTTGGT 840TTCACTCAGG GAACTTAATT CCAGCGGGAC CCAATATAAT TTGAATGATT CATGATACAT 900CCATTCGTTT GAACCGATCC TGCAAGAGTT CTGTCTGATT TGGTCAACAT AGTTTTCCTC 960TGGGGGAGAC TGGGGAAGAG TCAACACAAT GGTCAGGGAG AGAAGAATGA AAGCTCTCGC 1020AAGTGGATGA TCATGCTACG TACTGTAGGA ATAAAATTAA TTAATGCGAG GCTGCAAGTA 1080TCCCTGCGCC GATTTTCTCT TCTTACGGCG GGAACCAAAA AATGTGACGC TGTGATTTTC 1140TGGAAAAGGT AAGGATGTTT AGTTTCCCAG GATTATTACT GGTTCCGTAT GTGTATGTGT 1200ATGGATATCA TTCCGTATGG ATACGCCCGT TTCCTCCGCC CAGAACCAGT CCGTCATCCA 1260TCCTCCACTC TTTCTTCTCT TAGAGCCTTT CCACCTCTCT TCACTTTCTT TTTCTTTCCC 1320CCCTCCCTCT TTGCTTTCCC TCTCCCAGTA TTATTCTTAT ATTATCGGTT TGACCGTCGC 1380CTCAGTATCG GCCCCCCGTG AATCACTTTT CGTTTCTCTT GTATTTTACT TTCCTATCTG 1440GGATTGCTCC TCGATTAGCA GCTCTACTTC ATTCGGCCAT GTGCGTCTAG AGGGTCTAGC 1500CCCTCTCTCT CTTTGCACTG ACTGTCAGCC ATACCATAGT ATCATCCCGG AATTAAGAAA 1560AAAAAAGAAA TTATTCTACC TCCGATCTGG ACAAATTATA ACCAGGAGAA AATCAAGCGA 1620AAGAGGGGCA AAGGAGGAGA CACCATTAAA ACTGGGTCTG GTTTGATTCA TGACATACAT 1680TCGTCGTCTT GAATTTCAAT AGGTACGGAC TGATGCATTC CACTCGAGCC TTTTTAGCTG 1740CGTGTCCGTC TCCAATCGCA CTTCTTTTCT TATTTCCTTG TGGGATAAAT TGATTATTTA 1800CCGTTTCGTT TTCTCTATAT TGCGGTGGTG GTGCGACCCA TCCAACTATT ATTATTATAA 1860TTGGAATTTG ATTTGGATTT TGATTCCTGT GACGGATCTC AGACCAAGTG CCTAAACTAT 1920AACTGACTTG GACCCCCTTC AGATCCTAGC TTCCCGATTC TTTTCCACCA CTGCTGCATC 1980CTCTTCCTGC ACGCAGCGTT CGTTTAGGGC GGGTAGACTG GAATTTATTC CTTGCGCCAC 2040GGACCAATCG CTCCCTCGAC GCTCTCATTC CTGCGTCGAG CTCTTTTTCC CTCGACTCTC 2100ATTGCTTGCT GGGCTGGTTC TTGAACCTCT TCAATCGTCC TTATCTCTTT CCCCCCATCC 2160GGCCTGTGAT TCCTATCTTT CCTTTTTTTC TTCCCTTTCT TGTTTGATCC CCCCTCCTCC 2220CCGTCTTATC GCCTACTATC GTGATCCCCG CCCTTCCCAA TAAAGAGTAG GGCGTGTGAA 2280C ATG TCC GGG TTA ACC CTC GGG CGA GGC CCT GGG GGC GTG CGA CCG 2326Met Ser Gly Leu Thr Leu Gly Arg Gly Pro Gly Gly Val Arg Pro
1 5 10 15ACT CAA ACC GCA ACT TTT ACC ACC CAC CAC CCG TCC GCC GAT GCT GAC 2374Thr Gln Thr Ala Thr Phe Thr Thr His His Pro Ser Ala Asp Ala Asp
20 25 30CGC TCC TCC AAC AAC CTC CCC CCT ACC TCC TCG CAG CTG TCC GAT GAC 2422Arg Ser Ser Asn Asn Leu Pro Pro Thr Ser Ser Gln Leu Ser Asp Asp
35 40 45TTT TCT TTC GGT TCC CCT CTG AGC CCC GCC GAC TCA CAG GCC CAT GAC 2470Phe Ser Phe Gly Ser Pro Leu Ser Pro Ala Asp Ser Gln Ala His Asp
50 55 60GGC CTA CTT CAG GAC TCC CTC TTC CCT GAA TGG GGG TCT GGT GCG CCT 2518Gly Leu Leu Gln Asp Ser Leu Phe Pro Glu Trp Gly Ser Gly Ala Pro
65 70 75CGA CCC GGC ATT GAC AGT CCG GAT GAG ATG CAG AGG CAA GAT CCG CTA 2566Arg Pro Gly Ile Asp Ser Pro Asp Glu Met Gln Arg Gln Asp Pro Leu80 85 90 95GCG ACT CAA ATA TGG AAG CTC TAT TCT AGG ACC AAG GCC CAG TTG CCC 2614Ala Thr Gln Ile Trp Lys Leu Tyr Ser Arg Thr Lys Ala Gln Leu Pro
100 105 110AAC CAG GAG CGT ATG GAA AAC CTG ACC TGG CGG ATG ATG GCG ATG AGT 2662Asn Gln Glu Arg Met Glu Asn Leu Thr Trp Arg Met Met Ala Met Ser
115 120 125TTG AAA CGT AAG GAG CGG GAA CGT GCT CAA CAG TCC AT GTAGGTGTTC 2710Leu Lys Arg Lys Glu Arg Glu Arg Ala Gln Gln Ser Met
130 135 140TCCCTCTGTA GAGGAACGGC TGGACCCGCT CATCATTAAT TTTTTTTTTG TCTGTGAAG G 2770TTT CCT GCG AGA CGC GGT AGC GCT GGC CCC AGT GGT ATC GCT CAA CTG 2818Phe Pro Ala Arg Arg Gly Ser Ala Gly Pro Ser Gly Ile Ala Gln Leu
145 150 155CGC ATT TCC GAC CCG CCC GTT GCC ACC GGT AAC CCT CAG TCA ACC GAC 2866Arg Ile Ser Asp Pro Pro Val Ala Thr Gly Asn Pro Gln Ser Thr Asp
160 165 170CTG ACC GCC GAC CCT ATG AAC CTC GAC GAT TTC ATC GTG CCC TTC GAA 2914Leu Thr Ala Asp Pro Met Asn Leu Asp Asp Phe Ile Val Pro Phe Glu
175 180 185TCT CCT TCG GAC CAC CCC TCG CCC AGT GCC GTC AAG ATT TCC GAC TCC 2962Ser Pro Ser Asp His Pro Ser Pro Ser Ala Val Lys Ile Ser Asp Ser
190 195 200ACG GCG TCC GCG GCC ATT CCC ATC AAG TCC CGG AAA GAC CAG CTG AGA 3010Thr Ala Ser Ala Ala Ile Pro Ile Lys Ser Arg Lys Asp Gln Leu Arg205 210 215 220GAT TCT ACC CCG GTG CCG GCC TCG TTC CAC CAT CCG GCT CAG GAT CAA 3058Asp Ser Thr Pro Val Pro Ala Ser Phe His His Pro Ala Gln Asp Gln
225 230 235CGG AAG AAC AGT GAA TTT GGC TAC GTC CCC CGT CGC GTG CGC AAG ACG 3106Arg Lys Asn Ser Glu Phe Gly Tyr Val Pro Arg Arg Val Arg Lys Thr
240 245 250AGT ATC GAC GAG CGT CAA TTT TTC TCA CTG CAG GTG CCG ACC CGA AAG 3154Ser Ile Asp Glu Arg Gln Phe Phe Ser Leu Gln Val Pro Thr Arg Lys
255 260 265CGA CCG GCC GAA TCC TCG CCC CAG GTA CCC CCC GTT TCC AAC TCG ATG 3202Arg Pro Ala Glu Ser Ser Pro Gln Val Pro Pro Val Ser Asn Ser Met
270 275 280TTG GCC CAC GAT CCG GAC CTC GCT TCC GGC GTG CCC GAT TAT GCC TTG 3250Leu Ala His Asp Pro Asp Leu Ala Ser Gly Val Pro Asp Tyr Ala Leu285 290 295 300GAC GCC CCG TCC TCG GCC TTT GGC TTC CAT CAG GGT AAC CAC CAT CCG 3298Asp Ala Pro Ser Ser Ala Phe Gly Phe His Gln Gly Asn His His Pro
305 310 315GTC AAT CAT CAC AAC CAC ACC TCC CCC GGG GCA CCG TTT GGC TTG GAT 3346Val Asn His His Asn His Thr Ser Pro Gly Ala Pro Phe Gly Leu Asp
320 325 330ACG TTC GGC CTG GGA GAT GAT CCA ATC TTG CCC TCC GCG GGC CCC TAC 3394Thr Phe Gly Leu Gly Asp Asp Pro Ile Leu Pro Ser Ala Gly Pro Tyr
335 340 345CAG TCG CAA TTC ACC TTC TCA CCC AGC GAG TCT CCG ATG GCC TCC GGT 3442Gln Ser Gln Phe Thr Phe Ser Pro Ser Glu Ser Pro Met Ala Ser Gly
350 355 360CAT CCG TTT GCG AAC CTC TAT TCG CAT ACC CCG GTG GCT TCG TCC CTC 3490His Pro Phe Ala Asn Leu Tyr Ser His Thr Pro Val Ala Ser Ser Leu365 370 375 380AAC TCG ACG GAT TTC TTC TCT CCA CCG CCA TCA GGC TAC CAG TCC ACG 3538Asn Ser Thr Asp Phe Phe Ser Pro Pro Pro Ser Gly Tyr Gln Ser Thr
385 390 395GCA TCC ACG CCG CAG CCC ACC TAC GAC GGG GAC CAT TCC GTT TAT TTC 3586Ala Ser Thr Pro Gln Pro Thr Tyr Asp Gly Asp His Ser Val Tyr Phe
400 405 410GAT ATG CCG TCG GGC GAC GCG CGC ACC CAG CGC CGC ATT CCG AAC TAT 3634Asp Met Pro Ser Gly Asp Ala Arg Thr Gln Arg Arg Ile Pro Asn Tyr
415 420 425ATT TCG CAT CGG TCC AAC TTG TCT GCT TCG CTG CAG CCT CGG TAT ATG 3682Ile Ser His Arg Ser Asn Leu Ser Ala Ser Leu Gln Pro Arg Tyr Met
430 435 440TTC AAC CAG AAC AAC CAT GAA CAG GCC AGT TCG TCG ACG GTG CAT TCG 3730Phe Asn Gln Asn Asn His Glu Gln Ala Ser Ser Ser Thr Val His Ser445 450 455 460CCG AGC TAC CCC ATT CCC CAG CCG CAA CAT GTG GAC CCC ACT CAG GTG 3778Pro Ser Tyr Pro Ile Pro Gln Pro Gln His Val Asp Pro Thr Gln Val
465 470 475TTG AAC GCC ACC AAT TAC TCG ACC GGC AAC TCC CAC CAT ACC GGC GCC 3826Leu Asn Ala Thr Asn Tyr Ser Thr Gly Asn Ser His His Thr Gly Ala
480 485 490ATG TTT TCA TTT GGA GCC GAT TCA GAT AAC GAG GAT GAC GAT GGT CAT 3874Met Phe Ser Phe Gly Ala Asp Ser Asp Asn Glu Asp Asp Asp Gly His
495 500 505CAG CTG TCC GAG CGG GCT GGT CTG GCG ATG CCG ACT GAA TAT GGG GAC 3922Gln Leu Ser Glu Arg Ala Gly Leu Ala Met Pro Thr Glu Tyr Gly Asp
510 515 520GAG GAC GGG TTC TCG TCG GGC ATG CAG TGG GAT GGG CAG TTC CCG GGC 3970Glu Asp Gly Phe Ser Ser Gly Met Gln Trp Asp Gly Gln Phe Pro Gly525 530 535 540TCC TTC CAT TCG CTG CCG GGC TTT GGC CCT CAA CAT CGC AAG CAT GTT 4018Ser Phe His Ser Leu Pro Gly Phe Gly Pro Gln His Arg Lys His Val
545 550 555ACC ATC GGG TCC ACG GAC ATG ATG GAC ACC CCC GAG GAG TGG AAT CAC 4066Thr Ile Gly Ser Thr Asp Met Met Asp Thr Pro Glu Glu Trp Asn His
560 565 570GGT GGC AGT TTG GGT CGG ACT CAT GGG TCG GTG GCT TCG GTC AGT GAG 4114Gly Gly Ser Leu Gly Arg Thr His Gly Ser Val Ala Ser Val Ser Glu
575 580 585GTG CGC AAC CGA GAG CAG GAC CCT CGC CGG CAG AAG ATT GCC CGC ACC 4162Val Arg Asn Arg Glu Gln Asp Pro Arg Arg Gln Lys Ile Ala Arg Thr
590 595 600ACG TCC ACC CCC AAT ACG GCC CAG CTG TTG CGC CAA AGC ATG CAC TCT 4210Thr Ser Thr Pro Asn Thr Ala Gln Leu Leu Arg Gln Ser Met His Ser605 610 615 620AAT AAC AAT ACG TCT CAT ACC TCC CCT AAT ACG CCG CCC GAG TCC GCC 4258Asn Asn Asn Thr Ser His Thr Ser Pro Asn Thr Pro Pro Glu Ser Ala
625 630 635CTG AGC AGC GCA GTT CCG TCC CGC CCG GCC AGT CCC GGG GGC AGC AAG 4306Leu Ser Ser Ala Val Pro Ser Arg Pro Ala Ser Pro Gly Gly Ser Lys
640 645 650AAC GGC GAC CAA GGC AGC AAC GGA CCG ACC ACC TGC ACG AAC TGC TTC 4354Asn Gly Asp Gln Gly Ser Asn Gly Pro Thr Thr Cys Thr Asn Cys Phe
655 660 665ACT CAA ACC ACT CCG CTG TGG CGT CGG AAC CCA GAG GGC CAG CCA CTG 4402Thr Gln Thr Thr Pro Leu Trp Arg Arg Asn Pro Glu Gly Gln Pro Leu
670 675 680TGC AAT GCC TGC GGG TTG TTT TTG AAA TTG CAC GGT GTC GTG CGC CCT 4450Cys Asn Ala Cys Gly Leu Phe Leu Lys Leu His Gly Val Val Arg Pro685 690 695 700CTG TCC CTG AAA ACG GAC GTT ATC AAA AAG CGC AAC CGT AGC AGT GCC 4498Leu Ser Leu Lys Thr Asp Val Ile Lys Lys Arg Asn Arg Ser Ser Ala
705 710 715AAC AGC TTG GCG GTT GGG ACC TCC CGT GCG TCG AAG AAG ACA GCC CGC 4546Asn Ser Leu Ala Val Gly Thr Ser Arg Ala Ser Lys Lys Thr Ala Arg
720 725 730AAG AAC TCG GTG CAG CAA GCA TCC GTC ACG ACT CCG ACA TCA AGC CGC 4594Lys Asn Ser Val Gln Gln Ala Ser Val Thr Thr Pro Thr Ser Ser Arg
735 740 745GCT CAG AAT GGG ACT TCC TTC GAA TCC CCG CCC GCC GGC TTT AGT GCT 4642Ala Gln Asn Gly Thr Ser Phe Glu Ser Pro Pro Ala Gly Phe Ser Ala
750 755 760GCC GCG GGA CGG TCG AAT GGG GTG GTA CCC ATT GCC GCC GCT CCT CCG 4690Ala Ala Gly Arg Ser Asn Gly Val Val Pro Ile Ala Ala Ala Pro Pro765 770 775 780AAG GCA GCT CCC TCC GCA GCC GCC TCC CCT AGC ACG GGC CAG ACC CGC 4738Lys Ala Ala Pro Ser Ala Ala Ala Ser Pro Ser Thr Gly Gln Thr Arg
785 790 795AAC CCG ATC CAG GCT GCC CCG AAA CGT CAA CGA CGG CTG GAA AAG GCC 4786Asn Pro Ile Gln Ala Ala Pro Lys Arg Gln Arg Arg Leu Glu Lys Ala
800 805 810ACG GAG ATG GAA ACG GAC GAG GCT AAC AAG TCC GCG GGA GGC CGA TCC 4834Thr Glu Met Glu Thr Asp Glu Ala Asn Lys Ser Ala Gly Gly Arg Ser
815 820 825AAG GTG GTG CCT CTG GCA CCC GCC ATG CCA CCG GCA GCA GCC AAT CCG 4882Lys Val Val Pro Leu Ala Pro Ala Met Pro Pro Ala Ala Ala Asn Pro
830 835 840GCG AAC CAT AGT ATT GCC GGA GGC CAA GGG GCT AGT CAG GAA TGG GAG 4930Ala Asn His Ser Ile Ala Gly Gly Gln Gly Ala Ser Gln Glu Trp Glu845 850 855 860TGG TTG ACG ATG AGT CTGTAATGGC CGCGCTTACC TCTCTACTTC TCTACACTCG 4985Trp Leu Thr Met Ser Leu
865TTTCTTAATA TCTTTCTTGA ACCCCCCCTT ATATTTTCCC ACCGTTGATG CTACGCCATG 5045ACCGATAGAG ATGATGAATA CTGCAACCAA TGGAATCTCG CTAGACGAGA GGTGTTAGAT 5105GACGTGGCCC GCGATGCACT TAATGAGATA CGAGGAGGTG CAATGCGTTG GTTACGCTAG 5165TTTAATGGTA ACATGACGAG GGATATTCGC TCTGTTATTT CGGGCTTTGA TCTGTTTCAG 5225TCTGCGATTT AACAGCGACT GATCCTCTGC TGTGACAATA CACAGCTTGT CTTGTGGTTC 5285TGTTGTGGCT TTCTGTTTGT TTGGCTGATT TGATTTATGC TTGATACAAT CGCGTCTGTC 5345CGGACCCCGG CCTTTGTTTT GTTTTCAGTT CTGATTCTTC ACTGTTTCTG ATTCTCTTGT 5405TCATGTTTTT GATTTGTTCA AGGCTTGGGG CCGGGCAGAA GTGCGCATCT CTGCTTTGTG 5465TTTTCCGTCA CCGTGCATAG ACGCTGTATG TATATGCTAC AGCAAGATTC TACTTATCCA 5525GTCTGAGCCT GTATTCATTG AAGTGTAGCC AGCTGTCGAA TGAGCTTTTT AACGATATTG 5585TTTTGTTGAG TAGTCAACAA GTAGTATCTG TATATTCCGG AGTCTAAGTA AGACACTT 5643(2)SEQ ID NO：2的信息(i)序列特性：
(A)长度：866个氨基酸
(B)类型：氨基酸
(D)拓扑学：线性(ii)分子类型：蛋白质(xi)序列描述：SEQ ID NO：2Met Ser Gly Leu Thr Leu Gly Arg Gly Pro Gly Gly Val Arg Pro Thr1 5 10 15Gln Thr Ala Thr Phe Thr Thr His His Pro Ser Ala Asp Ala Asp Arg
20 25 30Ser Ser Asn Asn Leu Pro Pro Thr Ser Ser Gln Leu Ser Asp Asp Phe
35 40 45Ser Phe Gly Ser Pro Leu Ser Pro Ala Asp Ser Gln Ala His Asp Gly
50 55 60Leu Leu Gln Asp Ser Leu Phe Pro Glu Trp Gly Ser Gly Ala Pro Arg65 70 75 80Pro Gly Ile Asp Ser Pro Asp Glu Met Gln Arg Gln Asp Pro Leu Ala
85 90 95Thr Gln Ile Trp Lys Leu Tyr Ser Arg Thr Lys Ala Gln Leu Pro Asn
100 105 110Gln Glu Arg Met Glu Asn Leu Thr Trp Arg Met Met Ala Met Ser Leu
115 120 125Lys Arg Lys Glu Arg Glu Arg Ala Gln Gln Ser Met Phe Pro Ala Arg
130 135 140Arg Gly Ser Ala Gly Pro Ser Gly Ile Ala Gln Leu Arg Ile Ser Asp145 150 155 160Pro Pro Val Ala Thr Gly Asn Pro Gln Ser Thr Asp Leu Thr Ala Asp
165 170 175Pro Met Asn Leu Asp Asp Phe Ile Val Pro Phe Glu Ser Pro Ser Asp
180 185 190His Pro Ser Pro Ser Ala Val Lys Ile Ser Asp Ser Thr Ala Ser Ala
195 200 205Ala Ile Pro Ile Lys Ser Arg Lys Asp Gln Leu Arg Asp Ser Thr Pro
210 215 220Val Pro Ala Ser Phe His His Pro Ala Gln Asp Gln Arg Lys Asn Ser225 230 235 240Glu Phe Gly Tyr Val Pro Arg Arg Val Arg Lys Thr Ser Ile Asp Glu
245 250 255Arg Gln Phe Phe Ser Leu Gln Val Pro Thr Arg Lys Arg Pro Ala Glu
260 265 270Ser Ser Pro Gln Val Pro Pro Val Ser Asn Ser Met Leu Ala His Asp
275 280 285Pro Asp Leu Ala Ser Gly Val Pro Asp Tyr Ala Leu Asp Ala Pro Ser
290 295 300Ser Ala Phe Gly Phe His Gln Gly Asn His His Pro Val Asn His His305 310 315 320Asn His Thr Ser Pro Gly Ala Pro Phe Gly Leu Asp Thr Phe Gly Leu
325 330 335Gly Asp Asp Pro Ile Leu Pro Ser Ala Gly Pro Tyr Gln Ser Gln Phe
340 345 350Thr Phe Ser Pro Ser Glu Ser Pro Met Ala Ser Gly His Pro Phe Ala
355 360 365Asn Leu Tyr Ser His Thr Pro Val Ala Ser Ser Leu Asn Ser Thr Asp
370 375 380Phe Phe Ser Pro Pro Pro Ser Gly Tyr Gln Ser Thr Ala Ser Thr Pro385 390 395 400Gln Pro Thr Tyr Asp Gly Asp His Ser Val Tyr Phe Asp Met Pro Ser
405 410 415Gly Asp Ala Arg Thr Gln Arg Arg Ile Pro Asn Tyr Ile Ser His Arg
420 425 430Ser Asn Leu Ser Ala Ser Leu Gln Pro Arg Tyr Met Phe Asn Gln Asn
435 440 445Asn His Glu Gln Ala Ser Sar Ser Thr Val His Ser Pro Ser Tyr Pro
450 455 460Ile Pro Gln Pro Gln His Val Asp Pro Thr Gln Val Leu Asn Ala Thr465 470 475 480Asn Tyr Ser Thr Gly Asn Ser His His Thr Gly Ala Met Phe Ser Phe
485 490 495Gly Ala Asp Ser Asp Asn Glu Asp Asp Asp Gly His Gln Leu Ser Glu
500 505 510Arg Ala Gly Leu Ala Met Pro Thr Glu Tyr Gly Asp Glu Asp Gly Phe
515 520 525Ser Ser Gly Met Gln Trp Asp Gly Gln Phe Pro Gly Ser Phe His Ser
530 535 540Leu Pro Gly Phe Gly Pro Gln His Arg Lys His Val Thr Ile Gly Ser545 550 555 560Thr Asp Met Met Asp Thr Pro Glu Glu Trp Asn His Gly Gly Ser Leu
565 570 575Gly Arg Thr His Gly Ser Val Ala Ser Val Ser Glu Val Arg Asn Arg
580 585 590Glu Gln Asp Pro Arg Arg Gln Lys Ile Ala Arg Thr Thr Ser Thr Pro
595 600 605Asn Thr Ala Gln Leu Leu Arg Gln Ser Met His Ser Asn Asn Asn Thr
610 615 620Ser His Thr Ser Pro Asn Thr Pro Pro Glu Ser Ala Leu Ser Ser Ala625 630 635 640Val Pro Ser Arg Pro Ala Ser Pro Gly Gly Ser Lys Asn Gly Asp Gln
645 650 655Gly Ser Asn Gly Pro Thr Thr Cys Thr Asn Cys Phe Thr Gln Thr Thr
660 665 670Pro Leu Trp Arg Arg Asn Pro Glu Gly Gln Pro Leu Cys Asn Ala Cys
675 680 685Gly Leu Phe Leu Lys Leu His Gly Val Val Arg Pro Leu Ser Leu Lys
690 695 700Thr Asp Val Ile Lys Lys Arg Asn Arg Ser Ser Ala Asn Ser Leu Ala705 710 715 720Val Gly Thr Ser Arg Ala Ser Lys Lys Thr Ala Arg Lys Asn Ser Val
725 730 735Gln Gln Ala Ser Val Thr Thr Pro Thr Ser Ser Arg Ala Gln Asn Gly
740 745 750Thr Ser Phe Glu Ser Pro Pro Ala Gly Phe Ser Ala Ala Ala Gly Arg
755 760 765Ser Asn Gly Val Val Pro Ile Ala Ala Ala Pro Pro Lys Ala Ala Pro
770 775 780Ser Ala Ala Ala Ser Pro Ser Thr Gly Gln Thr Arg Asn Pro Ile Gln785 790 795 800Ala Ala Pro Lys Arg Gln Arg Arg Leu Glu Lys Ala Thr Glu Met Glu
805 810 815Thr Asp Glu Ala Asn Lys Ser Ala Gly Gly Arg Ser Lys Val Val Pro
820 825 830Leu Ala Pro Ala Met Pro Pro Ala Ala Ala Asn Pro Ala Asn His Ser
835 840 845Ile Ala Gly Gly Gln Gly Ala Ser Gln Glu Trp Glu Trp Leu Thr Met
850 855 860Ser Leu865
Claims (23)
1.一种真菌，其中由重组DNA技术得到的areA基因已得到修饰，方法为使其不能以提供功能性AreA激活物的方式加以表达。
2.权利要求1的真菌，其中所述失活作用已通过全部或部分areA基因的缺失而得到。
3.权利要求1的真菌，其中所述失活作用已通过干扰调控areA基因本身表达的表达信号的调控作用而得到。
4.权利要求1的真菌，其中所述失活作用已通过使用反义技术而得到。
5.权利要求1的真菌，其中所述失活作用已通过在areA基因中插入额外的DNA而得到。
6.权利要求1-5任一项的真菌，为一种丝状真菌，优选的是属于选自曲霉属、木霉属、腐质霉属、假丝酵母属、Acremonium、镰孢属和青霉属的某一个属。
7.权利要求6的真菌，其属于选自米曲霉、黑曲霉、泡盛曲霉、海枣曲霉、日本曲霉、臭曲霉、无冠构巢曲霉、T.reesei、T.harzianum、H.insulens、柔毛腐质霉、禾木科镰孢、腐皮镰孢、产黄青霉等等的菌种。
8.生产依据权利要求1之真菌的方法，其中所述失活作用已通过AreA基因的缺失而获得，该方法包括：
(i)对来自所研究真菌的areA基因进行克隆，
(ii)生产含有areA基因的DNA构建体，其中基因内在部分已被取代、删除、或者已插入额外的DNA，
(iii)用该构建体转化所述真菌，和
(iv)选择出为areA-的转化体。
9.生产依据权利要求1之真菌的方法，其中所述失活作用已利用反义技术而获得，该方法包括
(i)一种表达质粒的构建，该质粒可引起互补于转录自areA基因的mRNA的RNA分子的合成，
(ii)用所述表达质粒及适宜的标记物来转化宿主真菌，即可以是在独立的质粒上，也可以在同样的质粒上进行，
(iii)利用所述标记物选择转化体，
(iv)从所选择的转化体筛选出在AreA产物合成中表现出还原作用的菌株。
10.一种生产所需基因产物的方法，该方法包括在适宜的生长培养基中，在适宜的条件下培养依据权利要求1-7任一项的真菌，对所需的基因产物进行回收并提纯。
11.一种生产所需基因产物的方法，该方法包括使依据权利要求1-7任一项的真菌转化以功能性方式将编码所需基因产物的DNA序列整合到真菌的基因组中，在适宜的生长培养基中和适宜的条件下培养真菌，回收所需的基因产物并提纯。
12.一种生产所需多肽的方法，包括在适宜的生长培养基中培养真菌，并从所述培养物中回收所述多肽，所述真菌携带着能够引起在所述真菌中表达所述多肽或其前体的重组DNA构建体，与野生型的所述真菌相比，该真菌还具有可产生较低量功能性AreA的特征。
13.依据权利要求12的方法，其中，与野生型相比，所述真菌已得到修饰以产生较少量的AreA，其方法包括：利用当整合入所述真菌基因组时能减少功能性AreA产生的DNA构建体来转化所述真菌的亲代。
14.依据权利要求12的方法，其中所述多肽被所述真菌分泌至胞外介质中。
15.依据权利要求12的方法，其中与类似真菌相比，所述真菌可产生较高量的所述多肽，而所述类似真菌产生的AreA量与野生型的所述真菌产生的量近似，在其它所有方面该类似真菌都与所述真菌相同。
16.权利要求10或11-15的方法，其中所述基因产物是分泌性蛋白质。
17.权利要求10-16任一项的方法，其中所述需要的基因产物是一种工业用途的肽或蛋白质，优选为酶。
18.权利要求17的方法，其中所述酶选自蛋白酶、脂酶、几丁质酶、纤维素酶、凝乳酶。
19.权利要求10-16任一项的方法，其中所述需要的基因产物是治疗上有效的肽或蛋白质。
20.权利要求19的方法，其中所述治疗上有效的肽或蛋白质选自胰岛素、生长激素、胰高血糖素、生长激素抑制素、干扰素、PDGF、VII因子、VIII因子、尿激酶、tPA、EPO或TPO。
21.按照方法10-20任意一种所生产的基因产物。
22.编码来自米曲霉的areA基因的DNA序列(SEQ ID NO.1)或其功能性等位基因。
23.来自米曲霉的AreA激活物(SEQ ID NO.2)。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK71794 | 1994-06-17 | ||
| DK0717/94 | 1994-06-17 |
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| Publication Number | Publication Date |
|---|---|
| CN1150824A true CN1150824A (zh) | 1997-05-28 |
| CN1253574C CN1253574C (zh) | 2006-04-26 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN95193631.XA Expired - Lifetime CN1253574C (zh) | 1994-06-17 | 1995-06-19 | 来自米曲霉的areA基因和其中areA基因已被修饰的真菌 |
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| Country | Link |
|---|---|
| US (1) | US6025185A (zh) |
| EP (1) | EP0770139B1 (zh) |
| JP (1) | JPH10501414A (zh) |
| CN (1) | CN1253574C (zh) |
| AT (1) | ATE256191T1 (zh) |
| AU (1) | AU2733895A (zh) |
| DE (1) | DE69532283T2 (zh) |
| DK (1) | DK0770139T3 (zh) |
| FI (1) | FI965031A (zh) |
| WO (1) | WO1995035385A1 (zh) |
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| CN105492604A (zh) * | 2013-08-23 | 2016-04-13 | 诺维信公司 | 受调节的pepc表达 |
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| US6806062B1 (en) | 1998-10-05 | 2004-10-19 | Novozymes A/S | Fungal transcriptional activator useful in methods for producing polypeptides |
| CA2345356C (en) * | 1998-10-06 | 2012-10-02 | Mark Aaron Emalfarb | Transformation system in the field of filamentous fungal hosts |
| AU6188599A (en) | 1998-10-26 | 2000-05-15 | Novozymes A/S | Constructing and screening a dna library of interest in filamentous fungal cells |
| WO2001068864A1 (en) * | 2000-03-14 | 2001-09-20 | Novozymes A/S | Fungal transcriptional activator useful in methods for producing polypeptides |
| EP2505651A3 (en) | 2006-12-10 | 2013-01-09 | Dyadic International, Inc. | Isolated fungus with reduced protease activity |
| US9862956B2 (en) | 2006-12-10 | 2018-01-09 | Danisco Us Inc. | Expression and high-throughput screening of complex expressed DNA libraries in filamentous fungi |
| US8551751B2 (en) * | 2007-09-07 | 2013-10-08 | Dyadic International, Inc. | BX11 enzymes having xylosidase activity |
| US8278507B2 (en) | 2009-06-02 | 2012-10-02 | Novozymes, Inc. | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
| US8143021B2 (en) | 2009-07-07 | 2012-03-27 | Novozymes, Inc. | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
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| WO2012149344A1 (en) | 2011-04-29 | 2012-11-01 | Novozymes, Inc. | Methods for enhancing the degradation or conversion of cellulosic material |
| EP2773656B1 (en) | 2011-10-31 | 2019-06-19 | Novozymes, Inc. | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
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1995
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- 1995-06-19 DK DK95922443T patent/DK0770139T3/da active
- 1995-06-19 JP JP8501508A patent/JPH10501414A/ja active Pending
- 1995-06-19 AU AU27338/95A patent/AU2733895A/en not_active Abandoned
- 1995-06-19 CN CN95193631.XA patent/CN1253574C/zh not_active Expired - Lifetime
- 1995-06-19 EP EP95922443A patent/EP0770139B1/en not_active Expired - Lifetime
- 1995-06-19 US US08/750,458 patent/US6025185A/en not_active Expired - Lifetime
- 1995-06-19 DE DE69532283T patent/DE69532283T2/de not_active Expired - Lifetime
- 1995-06-19 AT AT95922443T patent/ATE256191T1/de not_active IP Right Cessation
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105492604A (zh) * | 2013-08-23 | 2016-04-13 | 诺维信公司 | 受调节的pepc表达 |
| CN105492604B (zh) * | 2013-08-23 | 2021-01-05 | 诺维信公司 | 受调节的pepc表达 |
Also Published As
| Publication number | Publication date |
|---|---|
| DK0770139T3 (da) | 2004-04-13 |
| JPH10501414A (ja) | 1998-02-10 |
| AU2733895A (en) | 1996-01-15 |
| DE69532283T2 (de) | 2004-09-23 |
| CN1253574C (zh) | 2006-04-26 |
| ATE256191T1 (de) | 2003-12-15 |
| FI965031A0 (fi) | 1996-12-16 |
| WO1995035385A1 (en) | 1995-12-28 |
| EP0770139A1 (en) | 1997-05-02 |
| EP0770139B1 (en) | 2003-12-10 |
| DE69532283D1 (de) | 2004-01-22 |
| FI965031A (fi) | 1996-12-16 |
| US6025185A (en) | 2000-02-15 |
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