CN1355850A - 来源于马尼拉水蛭的透明质酸酶,分离、纯化和重组生产方法 - Google Patents
来源于马尼拉水蛭的透明质酸酶,分离、纯化和重组生产方法 Download PDFInfo
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Abstract
本发明涉及一种来源于热带水蛭马尼拉水蛭(Hirudinariamanillensis)的新型透明质酸酶的分离、纯化和表征。因此,根据本发明,这种新酶被称为“马尼拉水蛭酶(manillase)”。本发明还涉及马尼拉水蛭酶的重组生产方法,包括DNA和氨基酸序列以及表达载体和宿主系统的公开。最后,本发明涉及马尼拉水蛭酶用于治疗目的的用途,例如,用于治疗心肌疾病、血栓事件和肿瘤。
Description
本发明涉及一种来源于热带水蛭马尼拉水蛭(Hirudinariamanillensis)的新型透明质酸酶的分离、纯化和表征。因此,根据本发明,这种新酶被称为“马尼拉水蛭酶(manillase)”。本发明还涉及马尼拉水蛭酶的重组生产方法,包括DNA和氨基酸序列以及表达载体和宿主系统的公开。最后,本发明涉及马尼拉水蛭酶用于治疗目的的用途,例如,用于治疗心肌疾病、血栓事件和肿瘤。
透明质酸或hyaluronan(HA)是线性不分支的高分子量(2-6×106)葡糖胺聚糖,由重复二糖结构GlcNAc(β1-4)GlcUA组成。其羧基在胞外液体的优势pH下完全离子化,无论是正常的或病理的。HA与硫酸软骨素、硫酸角质素和肝素均属于葡糖胺聚糖类(Jeanloz R.W.,风湿性关节炎(Arthr Rheum.)1960,3,233-237)。与其他未修饰的葡糖胺聚糖(GAG)不同,它没有硫酸取代或共价连接的肽,其链长度和分子量通常极高。HA普遍分布于结缔组织中,在引入改进的固定方法(Hellstrom S.等人,1990,组织化学杂志(Histochem.J.)22,677-682)和利用hyaluronan结合肽(HABP)的特异组织化学方法后,实际上在身体的所有部分中均发现。它在发育和成熟过程中也存在于神经外胚层来源的组织中。
术语透明质酸酶根据本发明通常是指一种酶,其作用于透明质酸,而无论其对其他底物的活性如何。
透明质酸酶首先从微生物中而后从哺乳动物睾丸中分离,后者现在是其主要来源(Meyer,K.《酶》,1971,307)。
根据反应机制,透明质酸酶被分为主要三类。
第一类微生物酶包括通过β-消除作用于其底物,产生A-4,5-不饱和二糖。因此该酶必须被命名为透明质酸裂合酶,EC 4.2.99.1。
第二类,透明质酸氨基葡糖苷酶或睾丸型透明质酸酶(EC 3.2.1.35)作为一种内切N-乙酰-β-D-己糖胺酶,将HA降解为较小的片段,首先是在游离还原末端含有己糖胺部分的四糖。与睾丸透明质酸酶有类似性质的酶已经从蝌蚪、蛇毒、蜂毒、大量动物组织、人血清和其他来源中获得。众所周知,来自睾丸的透明质酸酶也具有转糖基酶活性(WeissmanB.等人,生物化学杂志(J.Biol.Chem.)1954,208,417-429)。属于这类透明质酸酶的酶显示不仅对于透明质酸而且对于软骨素-4-硫酸、软骨素-6-硫酸、软骨素和硫酸皮肤素的酶活性。
第三类由透明质酸葡糖苷酸酶(EC 3.2.1.36)组成,其作为一种内切-β-葡糖苷酸酶。该酶从欧洲医蛭(Hirudo medicinalis)中分离(Yuki H.和Fishman W.H.,生物化学杂志1963,238,1877-79),对HA绝对特异。硫酸软骨素、皮肤素和肝素不是该透明质酸酶的底物。它只将透明质酸降解为在游离还原末端含有葡糖醛酸的四糖(Linker A.等人,生物化学杂志1960,235,924-27)。与哺乳动物内切-β-葡糖胺酶相反,肝素对该水蛭透明质酸酶的活性没有影响。因此,它能与肝素一起对患者施用,其衍生物被广泛用作抗凝剂。EP 0 193 330中公开了来源于水牛水蛭的蛭科亚科(包括Hirudinaria、Illebdella、Poecilodbella、Sanguisoga属)种(Poecilodbella granulosa)的透明质酸特异的内切-β-葡糖苷酸酶(称为“orgelase”),其分子量约为28.5。
透明质酸酶有许多体内和体外用途。静脉内施用透明质酸酶已被提议用于心肌梗塞的治疗(Kloner R.A.等人,循环(Circulation)1978,58,220-226;Wolf R.A.等人,美国心脏病杂志(Am.J.Cardial.)1984,53,941-944;Taira A.等人,血管学(Angiology)1990,41,1029-1036)。心肌梗塞代表非机械损伤的常见形式;即严重的细胞损伤和死亡,在此情况下是由突然细胞缺氧引起的。在大鼠中诱导的实验性心肌梗塞中(Waldenstrom A.等人,1991,临床研究杂志(J.Clin.Invest.)88,1622-1628),损伤的(梗塞区)心肌的HA含量3天后在24小时内几乎增加到正常的3倍,并伴随有间质性水肿。梗塞区的相对含水量也进行性增高,到第3天达到最大值,并且与HA积累强烈相关。升高的HA含量与水肿的相同关联已经在实验心脏和肾移植排斥中(Hallgren R.等人,临床研究杂志(J.Clin.Invest.)1990,85,668-673;Hallgren R.等人,实验医学杂志(J.Exp.Med.)1990,171,2063-2076),人肾移植排斥(Well A.等人,移植(Transplantation),1990,50,240-243)、肺病(Bjermer A.等人,英国医学杂志(Brit.Med.J.)1987,295,801-806)和特发性间质纤维化(Bjermer A.等人,胸(Thorax),1989,44,126-131)中发现。所有这些研究不仅提供了急性炎症中HA升高的证据,而且证明其在液体局部滞留中的部分主要引起组织肿胀,并影响心脏的机械和电生理功能。
这些结果能解释临床实验中使用的透明质酸酶的作用机理。据报道,透明质酸酶处理限制大鼠、狗和人中心肌缺血期间的细胞损伤(Maclean D.等人,科学,1976,194,199)。HA的降解之后可以是组织水积累的减少,组织压的降低和最终较好的灌注。
已经显示,来自水蛭的透明质酸酶以及含透明质酸酶提取物能用于其他治疗目的。因此,透明质酸酶治疗单独或与环孢霉素结合,导致移植物存活期延长(Johnson C.等人,国际移植(Transplant Inter.),待发表)。透明质酸酶(“扩散因子”)最广义上讲可用于提高组织通透性,以增强其他药理制剂的扩散(例如在癌症治疗中与细胞抑制剂结合)。此外,也能证明,透明质酸酶可用于肿瘤治疗,用作血管生成抑制剂,并作为肿瘤治疗中局部药物施用的助剂,用于青光眼和其他眼病的治疗,并作为其他治疗剂如局部麻醉剂和抗生素的助剂。Farr等人(1997,Wiener Medizinische Wochenschrift,15,347页)的综述文章及此处引用的文献中给出了治疗及有关用途的综述。因此,需要一种活性化合物如透明质酸酶。然而,已知的和可获得的透明质酸酶或者不稳定(来自欧洲医蛭的透明质酸酶,Linker等人,1960,生物化学杂志235,924页;Yuki和Fishman,1963,生物化学杂志238,1877页)或者显示相当低的比活(EP 0 193 330,Budds等人,1987,生物化学与生理学(Comp.Biochem.Physiol.)87B,3,497页)。而且,已知的透明质酸酶均未能以重组形式获得,而这是广泛商业应用的必要条件。
本发明现在第一次公开了从马尼拉水蛭中分离、纯化的一种新型透明质酸酶,以及通过生物工程技术获得的该酶的重组形式。
因此,本发明的一个目的在于提供从水蛭种马尼拉水蛭中分离的纯化蛋白质,它具有透明质酸酶的生物活性,其活性不受肝素影响,特征在于根据糖基化具有53-60kD的分子量。这种新的蛋白质,被称为“马尼拉水蛭酶”,以其天然形式糖基化,具有约58kD(±2kD)的分子量,有4种糖基形式。然而,非糖基化的蛋白质也是本发明的目的,它们可按照标准技术通过酶促或化学切割糖残基而获得。经SDS-PAGE测定,本发明的非糖基化酶具有约54(±2)的分子量。
直接比较显示,EP 0 193 330中公开的透明质酸酶(“orgelase”)在相同条件下具有约28的分子量,并且含有许多杂质如血红蛋白。
根据本发明的天然马尼拉水蛭酶的最适pH为6.0-7.0,等电点为7.2-8.0,具有图7所示的氨基酸序列。
令人吃惊的是,通过制备性纯化方法获得的马尼拉水蛭酶(见下文)具有100-150、优选地110-140(WHO)kU/mg蛋白质的极高的比活,而orgelase的比活只有约1.2kU/mg。而且,orgelase与马尼拉水蛭酶相比有更低的最适pH(5.2-6.0)。马尼拉水蛭酶象orgelase一样,不受肝素影响。
此外,本发明的一个目的在于提供一种分离并纯化马尼拉水蛭酶的方法,其包括下列步骤:
(i)用酸性缓冲液匀浆马尼拉水蛭种的水蛭的头和离心,
(ii)硫酸铵沉淀步骤(i)的上清液,
(iii)阳离子交换层析,
(iv)伴刀豆球蛋白A亲和层析,
(v)疏水作用层析,
(vi)在用透明质酸片段包被的基质上亲和层析,
(vii)凝胶渗透层析,和任选地,
(viii)纯化蛋白质的酶促或化学去糖基化。
以上公开的方法步骤保证了能获得具有这种高生物酶活性的根据本发明的蛋白质。因此,本发明的另一目的在于提供一种具有透明质酸酶生物活性的蛋白质,其活性不受肝素影响,其分子量根据糖基化为53-60,可通过上述和权利要求书中所述的方法步骤获得,优选地具有>100kU/mg蛋白质的酶比活。术语“单位”在上下文中是指“国际单位”(IU)。
本发明公开了一种制备重组马尼拉水蛭酶的方法,包括各自的DNA分子、载体和转化的宿主细胞。因此,本发明的一个目的在于提供一种编码具有天然马尼拉水蛭酶性质的蛋白质的DNA序列。
也能显示,能选择至少另外三种具有略微不同DNA序列的克隆,它们编码具有马尼拉水蛭酶(透明质酸酶)性质、具有略微不同氨基酸序列的蛋白质。
所述克隆具有图8、9、10所示的DNA序列(上面的序列),这也是本发明的一个目的,以及含有所述序列的表达载体,和用所述载体转化的宿主细胞。
另外,本发明的目的在于提供一种重组蛋白,其具有透明质酸酶生物活性,分子量根据糖基化为55-59kD,具有图8、9、10所示的任一氨基酸序列(下面的序列),或与所述序列至少80%同源的序列。术语“马尼拉水蛭酶”包括具有上述性质的所有这些蛋白质。
天然的以及重组的蛋白质可以用作能直接对患者施用或在药用组合物中使用的药物。因此,本发明的另一个目的在于提供一种如上下文中所述的、可用作药物的重组或天然蛋白质,和含有该蛋白质和药学可接受的稀释剂、载体或赋形剂的各自的药用组合物。
本发明的药用组合物还可含有极其多样性的活性药用化合物。优选的药剂是不抑制或影响根据本发明的蛋白质的生物和药学活性的抗凝剂。这些抗凝剂可以是,例如,肝素、蛭素或双香豆素,优选地是肝素。因此,本发明的一个目的在于提供一种另外含有药学活性化合物、优选地肝素的药用组合物。
对于在人类或兽医治疗中的应用,根据本发明的蛋白质优选地作为分散剂(“扩散”因子)或支持通过组织和皮肤的渗透。因此,马尼拉水蛭酶能在例如肿瘤化学治疗领域中用作其他物质(如局部麻醉剂)的一种助剂,用于与急性心肌缺血或梗塞有关的疾病的治疗,用于青光眼及其他眼病的治疗,例如提高眼中生理液体的循环,用于皮肤和组织移植物的处理,用以去除淤血并改善循环,作为通过皮肤、膜、其他组织的药物输送系统,作为去除环绕某些致病微生物或某些肿瘤和癌组织的透明质酸囊的药剂,作为血管生成抑制剂,能用作抗血栓和抗肿瘤剂。
因此,如上下文所述的马尼拉水蛭酶在生产特别用于治疗心肌、心血管和血栓疾病和肿瘤的药物中的应用是本发明的一个目的。
在此使用时,术语“药学可接受的载体”是指惰性的、无毒的固体或液体填料、稀释剂或包裹材料,它们不与活性化合物或患者不利地反应。合适的优选的液体载体在本领域周知,如无菌水、盐水、葡萄糖水、糖溶液、乙醇、二醇类和油,包括石油、动物、植物或合成来源的油,例如花生油、豆油和矿物油。
根据本发明的制剂可作为单位剂量施用,其含有一般用于肠胃外施用的常规无毒药学可接受的载体、稀释剂、佐剂和载体。
术语“肠胃外”在此包括皮下、静脉内、关节内和气管内注射和输注技术。其他施用如口服和局部施用也是合适的。肠胃外组合物和组合最优选地根据已知方法以大丸剂(bolus)形式或作为固定滴注静脉内施用。用于口服的片剂和胶囊含有常规赋形剂,如结合剂、填料、稀释剂、成片剂、滑润剂、分解剂和湿润剂。片剂可按照本领域周知的方法包被。
口服液体制剂可以是水或油状悬液、溶液、乳剂、糖浆或酏剂的形式,或者可以是干燥产品,在使用前可用水或另一种合适的载体重建。这些液体制剂可含有常规添加剂,如悬浮剂、乳化剂、非水载体和防腐剂。
局部施用可以是水或油状悬液、溶液、乳剂、软膏或优选地乳膏的形式。
根据本发明的单位剂量可含有每日需要量的根据本发明的蛋白质,或其亚多剂量,补足希望的剂量。对于指定患者(哺乳动物,包括人类),治疗可接受的剂量和剂量率取决于多种因素,如使用的特定活性材料的活性,年龄、体重、全身健康、性别、饮食、施用时间和途径、清除率、的性质。
因此,在所治疗的患者中(体内)含抗凝剂(如肝素)的组合物和组合中,本发明蛋白质(马尼拉水蛭酶)的药学有效的每日剂量约为0.01-100mg/kg体重(根据100kU/mg的比活),优选地为0.1-10mg/kg体重。根据施用形式,单次剂量可含有0.5-10mg马尼拉水蛭酶。
当与马尼拉水蛭酶一起施用时,例如肝素的浓度一般为一天500-4000U(IU),但是,必要时可增加或减少。
本发明的马尼拉水蛭酶的纯化如实施例所详述实现。表1显示马尼拉水蛭酶的制备性纯化方案。表2显示根据本发明的蛋白质的富集过程,表3显示马尼拉水蛭酶与已知的水蛭透明质酸酶的比较。
一种切割透明质酸的酶,称为马尼拉水蛭酶,已经从马尼拉水蛭水蛭的头中分离,并纯化为同质。该透明质酸酶如下纯化:用酸抽提,硫酸铵沉淀,随后在阳离子交换剂、伴刀豆球蛋白A Sepharose、丙基-Fractogel、hyaluronan片段-Sepharose和二醇-LiChrospher柱上连续层析。hyaluronan片段利用牛睾丸透明质酸酶切割天然hyaluronan制备。纯化并表征该片段后,如下所述制备亲和基质。这些亲和基质第一次用于透明质酸酶的纯化。这种高效层析是一种快速、有效地纯化hyaluronan结合蛋白的技术。每一纯化步骤后酶活性的恢复相当高。三次独立的制备性纯化的结果相当。它们产生高活性样品,根据纯化程度为20-160kU/mg。在比较实验中,如现有技术所述分离已知的透明质酸酶,并将其性质与根据本发明的蛋白质相比较(表3)。
根据表1的方案纯化的透明质酸酶不同于其他作者描述的其他水蛭透明质酸酶。在非解离条件下(任何β-巯基乙醇)获得类似的分子量,表明马尼拉水蛭酶与哺乳动物来源的许多透明质酸酶制剂一样,是一种单亚基酶。经MALDI测定,这种终制剂是表观分子量为58±2的单亚基酶(图1),等电点为7.2-8.0。表1:马尼拉水蛭酶的制备纯化
原材料的制备来自孟加拉的水蛭~15kg
↓
分离活动物
冷冻这些动物
↓
头的制备
~1kg水蛭头
↓
匀浆与抽提*
酸沉淀
离心**
阶段I-样品36%硫酸铵沉淀上清液
离心、透析**
阶段II-样品阳离子交换EMD(SO3 -)*
层析透析***
↓
Con A-亲和层析
透析***
↓丙基-Fractogel层析*
透析***
↓透明质酸片段(HA)-亲和层析
透析***
↓
二醇-LiChrospher层析****→140 000 WHO单位
↓反相层析分析****
表2:马尼拉水蛭酶从1kg水蛭头中的纯化(富集)
纯化步骤 | 总蛋白质Mg | 总活性kU | %回收 | 比活U/mg | 纯化(倍数) |
阶段I抽提和酸沉淀之后的上清液 | 31700 | 633.3 | 100 | 20 | 1 |
阶段II36%硫酸铵沉淀后的上清液 | 9530 | 443.3 | 70 | 45 | 2.25 |
阳离子交换层析 | 426.7 | 332.5 | 52.5 | 770 | 38.5 |
Con A-亲和层析 | 41.0 | 166.2 | 26.2 | 4.000 | 200 |
丙基-Fractogel层析 | 11.9 | 133.0 | 21.0 | 11000 | 550 |
透明质酸片段-Sepharose亲和层析 | 1.9 | 66.4 | 10.5 | 35000 | 1750 |
二醇-LiChrospher | 0.307 | 33.2 | 5.2 | 108000 | 5400 |
表3:马尼拉水蛭酶与已知水蛭透明质酸酶的比较
“马尼拉水蛭酶”Hirudinariamanillens本发明 | 透明质酸酶欧洲医蛭比较实验 | 透明质酸酶欧洲医蛭Linker等人;(生物化学杂志,1960) | “Orgelase”P.granulosaEP0 193 330Budds等人 | |
比活WHO(IU)单位/mg | 140000 | ~20000半纯化的 | ≤100 | ≤100 |
同质性SDS-PAGEMALDI | 1种蛋白质匀质4种糖基形式 | 蛋白质的混合物 | 未获得结果 | 许多蛋白质的混合物,主要杂质:血红蛋白 |
分子量 | 58.3kD±2kD | n.d. | 未报道 | 28.5kD±3kD |
氨基酸序列 | 测定 | n.d. | 未报道 | 未测定 |
最适pH | 6.0-7.0 | 6.0-7.0 | 未报道 | 5.2-6.0 |
pI | 7.5-8.0 | n.d. | n.d. | n.d. |
疏水性 | 在2M硫酸铵时与丙基HIC结合 | 在2M硫酸铵时不与丙基HIC结合 | ||
肝素引起的活性降低 | 无影响 | 未测定 | 无影响 | 无影响 |
稳定性 | ||||
+4℃时 | 7天后稳定保留~75%活性 | 不稳定温育7天后活性丢失100% | ||
+37℃时 | 7天后稳定,保留~60%活性 | 不稳定温育7天后活性丢失100% | 相对稳定 | |
在狗血清存在下在+37℃时的稳定性 | 7天后稳定,保留~100%活性 | 不稳定温育1天后活性丢失100% | 未报道 | 未检测 |
表中的星号是指关于活性测定和生物化学表征的信息(*_*****)。
使用的活性测定和生物化学表征方法取决于分析样品中马尼拉水蛭酶的浓度。因此,它们在连续纯化步骤中通过适当技术连续扩展。
* -活性测定-浊度降低检测
** -活性测定-浊度降低检测
-蛋白质含量测定(E280,Pierce BCA方法)
-SDS-PAGE(SDS-聚丙烯酰胺凝胶电泳)
-血红蛋白测定
***-活性测定-浊度降低检测
-蛋白质含量测定(E280,Pierce BCA方法)
-SDS-PAGE-Western印迹(抗人血红蛋白抗体)
****-活性测定-浊度降低检测
-蛋白质含量测定(E280,Pierce BCA方法)
-SDS-PAGE-Western印迹,抗人血红蛋白抗体
-SDS-PAGE-Western印迹,抗Con A抗体
-SDS-PAGE-Western印迹,抗肽抗体*****-MALDI
-蛋白质含量测定(Pierce BCA方法)
-SDS-PAGE-Western印迹-抗肽抗体
马尼拉水蛭酶与伴刀豆球蛋白A的结合显示,透明质酸酶是一种糖蛋白,其糖成分终止于α-D-吡喃甘露糖基或α-D-吡喃葡糖基和空间相关的残基。马尼拉水蛭酶活性样品在SDS-PAGE中显示RF值几乎相同的两条带。较长的SDS-PAGE和不同的电泳条件可用于更好地分离这些条带。在这些实验中,能检测到另外两条较弱的带(图2)。它们全部的N端部分(30个氨基酸)分别测序,再次显示N端无差异。用内切-F-糖苷酶(PNGase)去糖基化后,观察到所有4条带产生一条带,MW降低约3。
因此,观察到的电泳迁移率的差异极有可能是由于马尼拉水蛭酶分子糖基化模式的差异。神经氨酸酶、O-内切-糖苷酶和神经氨酸酶加O-糖苷酶处理对纯化的酶的分子量无影响(图3)。这些结果表明,马尼拉水蛭酶含有至少一种N-连接的寡糖链。O-连接的糖链不能用所用的方法检测到。
作为结束的纯化步骤,进行RP-层析。尽管还不能检测到酶活性,但能除去盐和肽蛋白酶抑制剂(图4)。利用MALDI进一步表征含有蛋白质的级分。利用MALDI测定的马尼拉水蛭酶分子量为58.3。
肝素对该透明质酸酶的活性没有影响(图5)。马尼拉水蛭酶比欧洲医蛭透明质酸酶稳定许多倍(图6)。而且,部分纯化的马尼拉水蛭酶的样品在狗和大鼠血浆中显示极高的稳定性,范围为-20到+37。
HA-亲和基质的制备已经在文献中描述(Tengblad A.,生物化学与生物物理学学报(Biochim.Biophys.Acta),1979,578,281-289)。该HA-基质用于从相同来源中纯化软骨透明质酸盐结合蛋白或蛋白聚糖蛋白质-硫酸角质素核(Christner J.E.,分析生物化学(Anal.Biochem.),1978,90,22-32)。利用亲和基质纯化的HA-结合蛋白(HABP)进一步用于关于透明质酸盐受体(Green S.J.等人,细胞科学杂志(J.Cell Science),1988,89,145-156;Chan F.L.等人,细胞生物学杂志(J.Cell.Biol.),1997,107,289-301)或hyaluronan(Waldenstrom A.等人,1991,临床研究杂志,88,1622-1628;Waldenstrom A.等人,欧洲临床研究杂志(Eur.J.Clin.Invest.),1993,23,277-282)在组织中分布的组织化学研究。
然而,我们实验室发展的这种凝胶的制备方法使人们能生产可准确确定HA片段浓度(1-15mg/ml)的凝胶。这又使得人们能使用这些凝胶,利用它们对hyaluronan的不同亲和力,不仅用于hyaluronan结合蛋白的纯化,而且用于其分离。这种选择性制备能利用不同长度的HA片段来控制。这种分离可使人们能更好地表征生物学相关的多种受体(例如在肿瘤学中)。
按照所述方法制备的HA-基质能用于:
1)已知HA-结合蛋白的纯化
2)未知HA-结合蛋白的纯化
3)新HA-结合蛋白的鉴定
4)透明质酸酶的纯化
用本发明所述方法获得的HA-片段能利用现代分析方法(NMR,MALDI-MS)表征,并应用于对蛋白质-蛋白质相互作用的研究。此外,这些片段也能应用于关于血管生成和新血管生成过程的研究。
附图简述:
图1:蛋白质标准、马尼拉水蛭酶样品的SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE-CBB染色)(在二醇-LiChrospher层析后)。
1-宽范围蛋白质标准
2-马尼拉水蛭酶,4μg
3-Orgelase,6μg
4-血红蛋白,40μg
图2:a)SDS-PAGE(CBB染色)和
b)HA-亲和层析后对4种马尼拉水蛭酶活性样品的SDS-PAGE-Western印迹(3-6道)。在该实验中使用兔P3-2A多克隆抗肽抗体。
图3:下列样品的SDS-PAGE(CBB):
1-LW-MM-低分子量标记(BioRad)
2-马尼拉水蛭酶
3-N-糖苷酶F(PNGase F)
4-用PNGase F处理后的马尼拉水蛭酶
5-用O-糖苷酶处理后的马尼拉水蛭酶
6-用O-糖苷酶和神经氨酸酶处理后的马尼拉水蛭酶
7-O-糖苷酶和神经氨酸酶
8-分子量标记(MWM-预染色的,BioRad)
图4:下列样品的反相层析:
a)核糖核酸酶标准
b)马尼拉水蛭酶样品(比活140kU/mg)
图5:肝素对马尼拉水蛭酶(-O-)和牛睾丸透明质酸酶(-·-)的透明质酸酶活性的影响
X轴:IU肝素;Y轴:保留的%活性
图6:透明质酸酶在缓冲液和血浆中的稳定性测定:
(a)马尼拉水蛭酶(4℃),
(b)马尼拉水蛭酶(-20℃)
(c)马尼拉水蛭酶(37℃)
(d)牛睾丸透明质酸酶(Y)和欧洲医蛭透明质酸酶(A)
X轴:温育天数;Y轴:WHO(IU)单位
图7:通过测定从马尼拉水蛭中分离并纯化的根据本发明的蛋白质的序列而获得的天然马尼拉水蛭酶的氨基酸序列(对应于SEQ IDNo.1)。
图8:重组马尼拉水蛭酶克隆(克隆21)的核苷酸(上面的行)和氨基酸序列;(对应于SEQ ID No.2,3)。
图9:重组马尼拉水蛭酶克隆(克隆31)的核苷酸(上面的行)和氨基酸序列;(对应于SEQ IDNo.4,5)。
图10:重组马尼拉水蛭酶克隆(克隆31)的核苷酸(上面的行)和氨基酸序列;(对应于SEQ ID No.6,7)。
图11:用于马尼拉水蛭酶的大肠杆菌表达载体
图12:用于马尼拉水蛭酶的Baculo供体质粒
图13:用于马尼拉水蛭酶的酵母表达载体
本发明通过下列实施例详细描述。然而,这些实施例不将本发明限制于实验中使用和在实施例中所述的普通材料、方法、物理参数、化合物、生物材料、表达载体和宿主等。如果没有另外提出,使用现有技术中周知的标准技术和通常可获得的材料。
实施例1(一般说明)
使用马尼拉水蛭的粗提物进行多次预实验,以建立纯化方法。
选择并验证下列方法:硫酸铵沉淀法,阳离子和阴离子交换层析,利用肝素-Fractogel、Con A-Sepharose的亲和层析,在辛基-Sepharose、丙基-、苯基-、丁基-Fractogel上的疏水作用层析(HIC),制备等电聚焦和制备电泳。
结果显示,酸和铵沉淀、阳离子交换、Con A-Sepharose、丙基-Fractogel HIC和二醇-LiChrospher和透明质酸片段-Sepharose(HA-Sepharose)层析适用于马尼拉水蛭酶的纯化。我们实验室制备的HA-Sepharose基质成功地用于该糖苷酶的纯化。
除非另外指出,所有制备都在低温下进行。
纯化按照以上所示的方案进行(表1)。
实施例2:用于纯化的原材料的制备;水蛭头的制备。
在孟加拉国收集的马尼拉水蛭水蛭立即骤冻,然后贮存于-40℃至-80℃中。它们在冷冻状态下切下头部,头的重量约占体重的5%。
实施例3:马尼拉水蛭酶从水蛭头的提取方法
在典型纯化中,1kg冷冻水蛭头在Waring搅拌器中用2500ml含0.025%硫柳汞和17mg/ml海藻糖的冷0.1M乙酸缓冲液pH4.0(MerckKGaA,Art.No.1.08216)匀浆。轻轻搅拌匀浆,并立即加入下列蛋白酶抑制剂:
1.PMSF 1.7mg/ml 10.0mM
2.亮抑蛋白酶肽 10.0μg/ml 20.0μM
3.胃酶抑制剂A 0.7μg/ml 1μM
4.EGTA 380.35μg/ml 1.0mM
5.p-APMSF 40.0μg/ml 20.0μM
在低温下连续搅拌4小时,并以4900转/分离心20分钟。收集上清液溶液(上清液I),并与随后通过提取组织沉淀获得的上清液II合并。
*样品的活性测定和生物化学表征利用活性测定-浊度降低检测和蛋白质含量测定(E280,Pierce BCA方法,SDS-PAGE)进行。
测定水蛭匀浆的酶活性是不可能的,因为血红蛋白(用血红蛋白测定试剂盒测定,Merck KGaA,13851)和其他蛋白质含量极高。而且,透明质酸酶活性在酸沉淀之前的阶段不能测定。这些提取物的最终比活(每mg蛋白质的活性)约为10-30 WHO单位。根据SDS-PAGE,粗提物含有大量不同的蛋白质,主要的分子量为~120、55~60、45、31、28、22、15和14-10。
实施例4:阶段I物质的硫酸铵沉淀步骤
然后,选择硫酸铵沉淀步骤作为马尼拉水蛭酶纯化的第一步,这导致该酶富集~5倍。
通过在+4℃下缓慢加入固体硫酸铵(Merck KGaA)至36%w/v,从阶段I的粗提物中沉淀酶学惰性的物质。搅拌该混合物1小时并离心。弃去沉淀物。上清液对流动的去离子水透析过夜,并对20mM磷酸缓冲液pH6.0透析24小时。这些提取物的终比活约为40-150 WHO单位。根据SDS-PAGE,阶段II提取物含有大量不同的蛋白质。
实施例5:阳离子交换层析
以分批吸附方式使用阳离子交换剂。富含酶的透析样品(阶段II)与1升Fractogel EMD SO3 -650(S)阳离子交换剂,Merck KGaA,Art.No.16882温育过夜。在通过离心结束温育后,用缓冲液洗涤阳离子交换剂,再次离心,并使HPLC-Superformace柱充满凝胶。用20mM磷酸缓冲液pH4.9洗涤该柱后,用含有0-1M线性NaCl梯度的相同磷酸钠缓冲液pH6.0从该柱上洗脱下结合的蛋白质。每3分钟收集级分(9ml),监测280nm的吸光度。马尼拉水蛭酶在0.15-0.18M NaCl浓度时被洗脱。测定所有级分的活性和蛋白质含量,合并级分,并对含有叠氮钠和17mg/ml海藻糖的20mM磷酸缓冲液pH6.0透析过夜。
进行蛋白质浓度、“合并液”的比活的测定和SDS-PAGE分析。尽管有极高的产量(活性)和高比活(每mg蛋白质的WHO活性单位,相当于IU),样品的SDS-PAGE分析结果仍显示许多蛋白质的混合物。利用Fractogel EMD SO3 -650(S)_(Merck KGaA,德国)的阳离子交换层析导致约10-50的极高的纯化倍数。该步骤在减少血红蛋白杂质上极其有效。而且,我们发现,分批方法是处理大体积阶段II上清液(5-16升)的极有用的初始步骤。
实施例6:伴刀豆球蛋白A-Sepharose亲和层析
利用Con A凝集素亲和层析在阳离子交换后进行对富含酶的合并液的进一步纯化。可从Pharmacia Biotech,Art.17-0440-01购得的Con A-Sepharose_用乙酸缓冲液0.1M+0.5M NaCl pH8.0;0.1M硼酸+0.1%Triton×100 pH6.0,最后用0.1M乙酸缓冲液+0.5M NaCl pH6.0洗涤。样品对20mM乙酸缓冲液+0.5M NaCl+1mM CaCl2+1mM MgCl2pH6.0+1mM MnCl2透析过夜,在室温下加于1000ml ConA柱上,并用510ml 100mM乙酸缓冲液+0.5M NaCl+1mM CaCl2+1mM MgCl2pH6.0+1mM MnCl2洗脱2小时。
随后利用含有0.5M甲基-α-D-吡喃甘露糖苷的相同缓冲液解吸。在280nm下连续监测洗脱。测定已收集的3ml级分的透明质酸酶活性。合并活性级分,对含有叠氮钠和17mg/ml海藻糖的20mM磷酸缓冲液pH6.0透析过夜。
进行蛋白质浓度、“合并液”的比活的测定和SDS-PAGE分析。该步骤在除去剩余血红蛋白上极有效。ConA层析导致4-10的纯化倍数。该倍数因原材料的质量而不同。
实施例7:丙基Fractogel疏水作用层析
向透明质酸酶活性Con A-合并液中加入硫酸铵至2M的终浓度。然后将样品与150ml丙基-Fractogel EMD丙基650(S)_,Merck KGaA,德国,Art.No.1.10085在室温下温育1小时,用含有2M硫酸铵的0.1M磷酸缓冲液pH7.0平衡。待温育结束后,用相同缓冲液洗涤凝胶两次,制备HPLC-Superformance(2.6cm×60cm)柱。用0.1M磷酸缓冲液pH7.0洗脱结合的蛋白质。每3分钟收集6ml级分,直接对去离子水透析(2-3小时),然后对20mM磷酸缓冲液pH6.0透析。测定级分的透明质酸酶活性。合并活性级分,并对含有叠氮钠和17mg/ml海藻糖的20mM磷酸缓冲液pH6.0透析过夜。进行合并液的蛋白质和活性测定。
该层析步骤的纯化倍数约为3-5。前一步骤中从载体凝胶中释放的少量Con A以及其他蛋白质杂质被去除。
实施例8:透明质酸寡糖亲和柱的制备
(a)用牛睾丸透明质酸酶对hyaluronan(HA)的水解
通过在甲苯存在下在4℃下混合过夜,将透明质酸7g溶解于1.25升含0.15 NaCl和0.5mM EDTA的0.1M乙酸钠缓冲液pH5.2中。之后将含HA溶液的pH调节为5.2,并在升温到37℃后加入牛睾丸透明质酸酶(Merck KGaA;700 WHO单位/mg)。对于7g HA,使用在用前立即溶解于50ml上述缓冲液的210mg酶。使水解在恒定搅拌下在37℃下进行30分钟,通过在沸水浴中100℃加热5分钟终止。通过以10000g离心30分钟澄清反应混合液,弃去含有变性蛋白质的沉淀,上清液通过上面放置有一个玻璃纤维预过滤器的0.2μm滤膜。利用通过如下具有不同分子截断值的树Diaflo超滤膜(Amicon)过滤,分级分离含有HA寡糖(HAOS)的澄清溶液。
(b)通过超滤对HAOS的分级分离
由上一步获得的含HAOS溶液通过30YM Diaflo超滤膜过滤。保存存留液用于其他研究,而滤过液通过10YM Diaflo超滤膜进行第二次超滤。再保存存留液用于其他研究,而通过10YM的溶液通过3YM Diaflo膜进行最后一次超滤。之后,含HA-OS的存留液,约10ml溶液,用于进一步的纯化。该级分:HAOS 3-10如下纯化并进一步用于与Sepharose偶联。
(c)HAOS 3-10的纯化
HA-OS 3-10用Biogel P2_柱纯化(脱盐)。该柱(4cm×100cm)用Biogel2基质_装填,200-400目(BioRad),用5倍柱体积的水洗涤(Milli Q,Millipore)。由前一步获得的HAOS 3-10级分(15ml;1.5g寡糖)加于该柱上。该柱用水洗脱,收集15ml级分,分析HA寡糖的存在。合并在盐(用AgNO3检测)之前洗脱的含寡糖级分,并再次用3 YM Diaflo膜浓缩。
(d)HAOS 3-10的分析
为了确定Sepharose的偶联效率,洗涤凝胶(同一批)并悬浮于水中以制备50%浆液。从用作对照的Sepharose HAOS 3-10结合物和Sepharose悬液中一式三份取出100μl等份,加入在盖有特氟隆螺旋帽的管中的2.5ml 2.2N三氟乙酸(TFA,Merck KgaA)中。为了水解,用氩冲洗该混合液,并在100℃下温育16小时。在水解结束时,样品在氮气下干燥,重悬浮于水中,用于测定葡糖胺和糖醛酸。为了确定每次水解糖醛酸和葡糖胺分解的程度,包括含有已知量UA或GIcNAc的对照样品,并在相同条件下温育。
在上述条件下,在两次独立实验中每1ml排出的Sepharose凝胶偶联5、8、9、11和15mg HAOS 3-10。该结果基于UA和葡糖胺测定。
(e)使用的测定
分析样品中糖醛酸的含量按照Bitter T.和Muir H.M.,分析生物化学,1962,4,330-334测定。
己糖胺含量用Rondle C.J.M.和Morgan W.T.J.,生物化学杂志,1995,61,586-593的方法分析。
实施例9:透明质酸片段Sepharose层析(HA-Sepharose层析)
含有8-10mg/ml的层析基质如下所述制备。含酶样品对20mM乙酸缓冲液+0.15M NaCl pH4.0透析,并加于25ml HA-Sepharose柱上。在用相同缓冲液洗涤后,用含0.15-1M NaCl梯度的20mM乙酸缓冲液进行洗脱。
1ml级分在透明质酸酶活性测定试验中检测,合并,对含有叠氮钠和17mg/ml海藻糖的20mM磷酸缓冲液pH6.0透析过夜。进行合并液的蛋白质和活性测定。该层析步骤的纯化因数约为3。
实施例10:二醇-LiChrospher层析
对Milli-Q-H2O透析的20ml活性样品加于二醇-LiChrospher柱上。然后用15ml Milli-Q-H2O平衡该柱,用2ml水洗涤5分钟。用20mM乙酸缓冲液pH5.9(梯度,0-5mM NaCl)进行活性样品洗脱15分钟,用含5mM NaCl的梯度20mM-100mM乙酸缓冲液pH5.5进行35分钟。测定级分的透明质酸酶活性。合并活性级分并对含有叠氮钠和17mg/ml海藻糖的20mM磷酸缓冲液pH6.0透析过夜。进行合并液的蛋白质和活性测定。纯化因数:3。
实施例11:RP 18e层析
该纯化步骤只能用作最后一步,其目的在于获得不含盐和其他蛋白质杂质(例如肽蛋白酶抑制剂)的样品。透明质酸酶活性完全丧失,因为马尼拉水蛭酶对该步骤中使用的有机溶剂没有抗性。马尼拉水蛭酶样品加于RP 18e柱上。收集0.25mg/min级分。在0.1%TFA存在下进行洗脱,使用水到99%乙腈的梯度。RP纯化的样品能直接用于氨基酸测序、MALDI测定、糖结构分析,和作为标准用于其他批次马尼拉水蛭酶的纯化。
实施例12:活性测定——浊度降低检测
用浊度降低测定进行透明质酸酶活性测定。可购得的hyaluronan(分离自不同的动物组织和液体,如人脐带、公鸡冠)和透明质酸酶(来源于牛睾丸、猪睾丸、蜂毒的内切-β-氨基葡糖苷酶;来源于Streptomyceshyalurolyticus的裂合酶)制剂用于建立合适的活性测定条件。我们实验室中部分纯化了来源于欧洲医蛭的内切-β-葡糖醛酸糖苷酶。
通过将HA溶解于0.3M磷酸缓冲液pH5.3中制备hyaluronan贮存液(浓度为2mg/ml)。该溶液在测试前直接用相同缓冲液稀释至0.2mg/ml的浓度。含酶样品用含0.01%牛白蛋白和77mM NaCl的20mM磷酸缓冲液(酶稀释缓冲液)稀释为适量的酶(0.5-5 WHO单位)。向0.1ml这些样品中加入0.1ml hyaluronan(0.2mg/ml)溶液,混合并在37℃下温育45分钟。测试一式两份进行。通过用1.0ml白蛋白试剂(溶于80mM乙酸/40mM乙酸钠缓冲液pH3.75中的0.1%白蛋白)稀释终止该反应。在室温或37℃温育10分钟后,读取600nm的光密度,活性通过与标准比较(SLT-程序)表示为WHO(IU)单位。牛睾丸透明质酸酶的WHO制剂(HumphreyJ.H.,世界卫生组织公告(Bull.World Health Org.)1957,16,291-294)用作标准。
实施例13:蛋白质估测
通过测量溶液在280nm的紫外吸光度确定柱洗脱液的蛋白质含量。合并级分的蛋白质浓度利用Pierce微量测定法测定。BSA溶液用作参照蛋白质。
实施例14:SDS-PAGE电泳
按照Laemmli方法进行电泳(自然,1970,227,680-685)。使用下列凝胶:4-20%梯度或12.5%分离胶及4%积层胶。样品在十二烷基硫酸钠和β-巯基乙醇存在下进行电泳。在用考马斯亮蓝染色和/或银染(按照Pharmacia说明)后显示蛋白质。
实施例15:等电聚焦
为了对马尼拉水蛭酶制剂进行等电聚焦研究,采用供应商(Pharmacia)提供的方法。聚焦后,固定凝胶并进行银染(按照Pharmacia方案)。
实施例16:由兔免疫血清制备免疫球蛋白(抗-ConA、抗血红蛋白和抗-肽兔抗体)
利用下列免疫原产生兔血清:伴刀豆球蛋白A凝集素、血红蛋白与肽-KLH偶联物的混合物。肽序列与马尼拉水蛭酶的14氨基酸N端部分相同(KEIAVTIDDKNVIA)。
按照标准Pharmacia说明用蛋白A Sepharose(Pharmacia,17-0780-01)柱纯化血清。利用SDS-PAGE和ELISA试验核实IgG样品的纯度。
实施例17:Western-免疫印迹测定
合适的样品等份和预染色的已知分子量的蛋白质标记如上所述进行SDS-PAGE。使用预染色的BioRad分子量标记。在转移缓冲液存在下将蛋白质从聚丙烯酰胺凝胶向固定的聚二氟乙烯(PVDF)膜电泳转移(0.8mA/cm2)100分钟。PVDF膜与封闭液(PBS,pH7.5+2%脱脂奶)在室温下温育1小时。然后,该膜与用封闭液适当稀释的抗体在室温下温育2小时。该膜用TBS+0.05%吐温20,pH7.5洗涤,并与BioRad的山羊抗兔碱性磷酸偶联物(第二抗体)在室温下温育2小时。该膜用TBS+0.05%吐温20洗涤两次,并与BCIP碱性磷酸酶底物液温育10分钟。加入终止缓冲液终止反应。
实施例18:氨基酸测序
马尼拉水蛭酶的N端33个氨基酸残基的序列通过Edman降解获得。在马尼拉水蛭酶活性样品进行SDS-PAGE后,将条带转移到PDVF膜上,用考马斯蓝染色,切下并测序。对于利用RP-柱层析在最后一个纯化步骤后获得的样品,发现有相同的氨基序列。
实施例19:酶活性的pH依赖性
(对于从马尼拉水蛭和欧洲医蛭头中分离的透明质酸酶)
在本实验中使用的透明质酸酶样品从马尼拉水蛭或欧洲医蛭头中提取,并利用硫酸铵沉淀和阳离子交换层析部分纯化。含有500 WHO单位/ml的每一样品在-20℃、+4℃和37℃和pH 2.6-9.0下温育(对于pH2.6-5为20mM乙酸;对于pH5-9为20mM磷酸缓冲液)。酶活性在1、2和7天温育期后测定。在酸性和碱性极端pH时,对于两种透明质酸酶观察到相同程度的活性抑制。然而,在较长的温育期间,马尼拉水蛭酶比欧洲医蛭透明质酸酶更稳定:例如在pH7.0和4℃和37℃下温育7天后,马尼拉水蛭酶分别保留初始活性的75%和60%。在相同条件下温育的欧洲医蛭透明质酸酶在1天后已经失活。
实施例20:在狗血清存在下透明质酸酶的稳定性测定(对于从马尼拉水蛭和欧洲医蛭头中分离的透明质酸酶)
5kU/ml马尼拉水蛭酶样品、欧洲医蛭和牛睾丸透明质酸酶用狗或大鼠柠檬酸化血浆稀释至250U/ml的终浓度。然后,这三种溶液在-20℃、+4℃和37℃下温育0-7天。在该实验中包括用缓冲液稀释的含相同透明质酸酶的对照。最后,测定透明质酸酶活性。
实施例21:污染酶活性
在水蛭透明质酸酶纯化过程的每一阶段,都按照Twining S.S.(分析生物化学,1984,143,30-34)利用通用蛋白酶底物(BoehringerMannheim,目录号1080 733)检测制剂中能降解蛋白质的其他酶。
实施例22:肝素对透明质酸酶活性的影响
透明质酸酶切割hyaluronan导致还原糖的释放。释放的糖的量通过改进的Park法(Park J.和Johnson M.;生物化学杂志1949,181,149)比色测定。为了确定肝素对马尼拉水蛭酶和牛睾丸透明质酸酶活性的影响,进行两种活性测定:一种在肝素存在下,第二种不含肝素。透明质酸酶样品25μl(3.2 WHO单位)与含有0-24单位肝素的25μl肝素(Liquemin,Fa.Hoffmann LaRoche)溶液在37℃下温育30分钟。然后,加入50μl hyaluronan(2.5mg/ml),在37℃下继续温育30分钟。通过在100℃下加热2分钟终止反应。然后,向灭活的消化液中加入100μl碳酸盐-氰化物和100μl铁氰酸钾溶液。样品在沸水浴中加热15分钟,然后在冰浴中冷却。之后,向反应混合液中加入0.75μl硫酸铁铵溶液。在室温下温育15分钟后,用Shimadzu分光光度计测量690nm的显色。在每次试验中包括合适的空白和无酶对照。对于所分析的样品类型,用希望的还原糖(葡萄糖醛酸或N-乙酰-葡糖胺)作为标准。
实施例23:马尼拉水蛭酶的去糖基化
利用PNGase F酶(BioLabs Art.No.701L)按照使用说明书使马尼拉水蛭酶样品去糖基化。去糖基化在变性和自然条件下进行。O-聚糖酶、神经氨酸酶和神经氨酸酶+O-聚糖酶处理根据Boehringer Mannheim标准要求进行。所有样品都用SDS-PAGE和活性测定试验表征。
实施例24:大肠杆菌表达载体的构建(图11)
为了在大肠杆菌中表达,我们使用一种修饰形式的质粒pASK75,其携带tet启动区。{Skerra,基因151,(1994),131-135页}。通过在XbaI与HindIII位点之间克隆一个新接头进行修饰。该新接头含有ompA前导序列、另一个多克隆位点和6×His尾代替strep-尾。
在pAsK75中克隆的接头序列。Xbal119 CTAGATAACG AGGGCAAAAA ATGAAAAAGA CAGCTATCGC GATTGCAGTG GCACTGGCTG
TATTGC TCCCGTTTTT TACTTTTTCT GTCGATAGCG CTAACGTCAC CGTGACCGAC 179 GTTTCGCTAC CGTAGCGCAG GC AT CGA TGA ATT CGA GCT CGG TAC CCG GGG
CAAAGCGATG GCATCGCGTC CG TA GCT ACT TAA GCT CGA GCC ATG GGC CCC 230 ATC CCT CGA GGT CGA CCT GCA GGC AGC GCTATGAGAGGATCGCATCACCATCACCA
为了构建马尼拉水蛭酶的表达载体,利用PCR方法导入5’ClaI和3’Ec047III限制位点是必要的。因此使用两条引物:
5’ATC GAT AAA GAG ATT GCC GTG AC和
5’GTT GTT TCC GAT GCT AAA GCG CTPCR产物首先克隆到PCRII载体系统(Invitrogen)中并测序。
第二步,利用限制位点5’ClaI和3’Eco47III将马尼拉水蛭酶基因克隆到修饰的pASK75载体中。
在第二次PCR反应中表达并证明该重组马尼拉水蛭酶的活性后,除去His尾,将马尼拉水蛭酶基因的起始密码子与ompA前导序列直接融合。该PCR反应的引物是:
5’ACC GTA GCG CAG GCC AAA GAG ATT GCC GTG和
3’CAC GGC AAT CTC TTT GGC CTG CGC TAC GGT。
实施例25:Baculo供体质粒的构建(图12)
为了在Baculo病毒表达系统中表达马尼拉水蛭酶,使用来自GibcoLife Technologies的Bac-To-BacTM杆状病毒表达系统。为了得到分泌系统,将蜜蜂蜂毒肽前导序列与马尼拉水蛭酶基因融合,并引入限制位点5’BamHI和3’KpnI。进行一次PCR反应。
5’引物:
CGG ATC CAT GAA ATT CTT AGT CAA CGT TGC CCT TGTTTT TAT GGT CGT ATA CAT TTC TTA CAT CTA TGC GAA AGAGAT TGC CGT GAC
3’引物:
AAT GTT GAA GCA TAA GGT ACC
将PCR产物克隆到PCRII载体(Invitrogen)中并测序。然后利用限制位点5’BamHI和3’KpnI将蜂毒肽-马尼拉水蛭酶融合体克隆到pFastBac载体中(图12)。
实施例26:酵母表达载体的构建(图13)
为了在酵母中表达,我们使用毕赤酵母(Pichia)多拷贝表达载体(Invitrogen)。为了构建用于马尼拉水蛭酶的表达系统,我们使用对马尼拉水蛭酶基因的PCR扩增法,这样产生相容的限制末端(5’EcoRI,3’NotI),用于向合适的载体(pPIC9K)中连接。因此使用下列引物:
5’GTA GAA TTC AAA GAG ATT GCC GTG ACA
3’GAT GCT AAT GTT GAA GCA TAA TGA GCG GCC GC
在转化毕赤酵母原生质球之前,表达载体必须用SalI线性化。
实施例26:在大肠杆菌中的表达
用含有与ompA前导序列融合的sarastatin结构基因的表达载体pRG72转化W3110感受态细胞。细胞生长至对数中期,然后加入200μg aTC/l诱导启动子。1小时后能清楚地检测到重组马尼拉水蛭酶。
实施例27:重组杆状病毒和马尼拉水蛭酶的产生
利用Bac-To-Bac表达系统的表达
用供体质粒pTD13转化DH10Bac感受态细胞,该细胞含有具有一个mini-attTn7靶位点的杆粒和辅助质粒。供体质粒上的mini-Tn7元件在辅助质粒提供的转座蛋白存在下能转座到杆粒上的mini-attTn7位点上。根据lacZ基因的破坏鉴定含有重组杆粒的集落。由选择的含有重组杆粒的大肠杆菌克隆制备高分子量mini-prep DNA,然后用该DNA转染昆虫细胞。
在表达试剂盒说明手册中能见到详细叙述。
实施例28:在酵母中的表达
为了确定已经整合马尼拉水蛭酶基因,必须对His+Mut+-突变体筛查菌落。
用单菌落接种1升摇瓶中的100ml培养基。生长条件是:28-30℃,250转/分,可达OD 2-6。为了诱导表达,首先离心培养物,倾弃上清液,将细胞沉淀重悬浮于1/5原始培养体积的新培养基中。每24小时加入100%甲醇至0.5%的终浓度以保持诱导。最多6天后,通过SDS-PAGE和活性测定分析上清液。
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20 25 30aag ggt ctt tgg agc ttt gtt gat att acc tct cca aaa ttg ttc aaa 144Lys Gly Leu Trp Ser Phe Val Asp Ile Thr Ser Pro Lys Leu Phe Lys
35 40 45ttg ctg gaa gga ctt tct cct gga tac ttc agg gtt ggc gga acg ttt 192Leu Leu Glu Gly Leu Ser Pro Gly Tyr Phe Arg Val Gly Gly Thr Phe
50 55 60gcc aat tgg ctg ttt ttt gac ttg gac gaa aat aat aag tgg aag gat 240Ala Asn Trp Leu Phe Phe Asp Leu Asp Glu Asn Asn Lys Trp Lys Asp65 70 75 80tat tgg gct ttt aaa gac aaa acc ccc gaa act gcg aca ata aca agg 288Tyr Trp Ala Phe Lys Asp Lys Thr Pro Glu Thr Ala Thr Ile Thr Arg
85 90 95aga tgg ctg ttc aga aaa caa aat aat ctg aaa aag gag act ttt gac 336Arg Trp Leu Phe Arg Lys Gln Asn Asn Leu Lys Lys Glu Thr Phe Asp
100 105 110aat tta gtg aaa cta aca aag gga agc aag atg aga ttg tta ttc gat 384Asn Leu Val Lys Leu Thr Lys Gly Ser Lys Met Arg Leu Leu Phe Asp
115 120 125ttg aat gcc gaa gtg agg act ggt tat gaa att gga aag aag atg aca 432Leu Asn Ala Glu Val Arg Thr Gly Tyr Glu Ile Gly Lys Lys Met Thr
130 135 140tcc act tgg gat tca tcg gag gct gaa aag tta ttt aaa tat tgt gtg 480Ser Thr Trp Asp Ser Ser Glu Ala Glu Lys Leu Phe Lys Tyr Cys Val145 150 155 160tca aaa ggt tac gga gac aat atc gat tgg gaa ctt gga aat gaa ccg 528Ser Lys Gly Tyr Gly Asp Asn Ile Asp Trp Glu Leu Gly Asn Glu Pro
165 170 175gac cac acc tca gct cac aat tta act gaa aag cag gtt gga gaa gat 576Asp His Thr Ser Ala His Asn Leu Thr Glu Lys Gln Val Gly Glu Asp
180 185 190ttt aaa gca ctg cat aaa gtt cta gag aaa tat cca act ctt aac aag 624Phe Lys Ala Leu His Lys Val Leu Glu Lys Tyr Pro Thr Leu Asn Lys
195 200 205gga tcg ctc gtt ggt cca gat gta ggg tgg atg ggc gtc agt wac gtc 672Gly Ser Leu Val Gly Pro Asp Val Gly Trp Met Gly Val Ser Xaa Val
210 215 220aag gga ttg gca gac gag gcr ggt gac cat gta ack gct ttt aca ctc 720Lys Gly Leu Ala Asp Glu Xaa Gly Asp His Val Xaa Ala Phe Thr Leu225 230 235 240cac caa tat tat ttc gat gga aac acy tct gat gta tca ata tat ctt 768His Gln Tyr Tyr Phe Asp Gly Asn Xaa Ser Asp Val Ser Ile Tyr Leu
245 250 255gat gcc aca tac ttt aag aag ctg caa caa cta ttt gat aaa gtg aaa 816Asp Ala Thr Tyr Phe Lys Lys Leu Gln Gln Leu Phe Asp Lys Val Lys
260 265 270gat gtt ttg aaa gat tct cca cat aaa gac gaa cca tta tgg ctt gga 864Asp Val Leu Lys Asp Ser Pro His Lys Asp Glu Pro Leu Trp Leu Gly
275 280 285gaa aca agt tct gga tac aac agc ggc aca gaa gat gta tcc gat cga 912Glu Thr Ser Ser Gly Tyr Asn Ser Gly Thr Glu Asp Val Ser Asp Arg
290 295 300tat gtt tca gga ttt cta aca tta gac aag ttg ggt ctc agt gca gcc 960Tyr Val Ser Gly Phe Leu Thr Leu Asp Lys Leu Gly Leu Ser Ala Ala305 310 315 320aac aat gta aag gtt gtt ata aga cag aca ata tac aat gga tat tat 1008Asn Asn Val Lys Val Val Ile Arg Gln Thr Ile Tyr Asn Gly Tyr Tyr
325 330 335ggt ctc ctt gac aaa aac act tta gag ccg aat ccg gat tac tgg tta 1056Gly Leu Leu Asp Lys Asn Thr Leu Glu Pro Asn Pro Asp Tyr Trp Leu
340 345 350atg cat gtt cat aat tct ttg gtc gga aat aca gtt ttt aaa gtt gac 1104Met His Val His Asn Ser Leu Val Gly Ash Thr Val Phe Lys Val Asp
355 360 365gtt agt gat cca act aat aaa gca aga gtt tac gcg caa tgt acc aaa 1152Val Ser Asp Pro Thr Asn Lys Ala Arg Val Tyr Ala Gln Cys Thr Lys
370 375 380aca aat agc aaa cat act caa agc aga tat tac aag ggc tct ttg aca 1200Thr Asn Ser Lys His Thr Gln Ser Arg Tyr Tyr Lys Gly Ser Leu Thr385 390 395 400atc ttt gca ctt aat gtt gga gat gga gat gta acg tta aag atc ggt 1248Ile Phe Ala Leu Asn Val Gly Asp Gly Asp Val Thr Leu Lys Ile Gly
405 410 415caa tac agc ggt aaa aaa att tat tca tac att ctg aca cct gaa gga 1296Gln Tyr Ser Gly Lys Lys Ile Tyr Ser Tyr Ile Leu Thr Pro Glu Gly
420 425 430gga caa ctt aca tca cag aaa gtt ctc ttg aat gga aag gaa ttg aac 1344Gly Gln Leu Thr Ser Gln Lys Val Leu Leu Asn Gly Lys Glu Leu Asn
435 440 445tta gtg tct gat cag tta cca gaa cta aat gca gat gaa tcc aaa aca 1392Leu Val Ser Asp Gln Leu Pro Glu Leu Asn Ala Asp Glu Ser Lys Thr
450 455 460tct ttc acc tta tcc cca aag aca ttt ggt ttt ttt gtt gtt tcc gat 1440Ser Phe Thr Leu Ser Pro Lys Thr Phe Gly Phe Phe Val Val Ser Asp465 470 475 480gct aat gtt gaa gca tgy aar aar 1464Ala Asn Val Glu Ala Cys Lys Lys
485<210>3<211>488<212>PRT<213>水蛭<400>3Lys Glu Ile Ala Val Thr Ile Asp Asp Lys Asn Val Ile Ala Ser Ala1 5 10 15Ser Gly Ser Phe Leu Gly Val Ala Phe Asp Ala Ser Leu Phe Ser Pro
20 25 30Lys Gly Leu Trp Ser Phe Val Asp Ile Thr Ser Pro Lys Leu Phe Lys
35 40 45Leu Leu Glu Gly Leu Ser Pro Gly Tyr Phe Arg Val Gly Gly Thr Phe
50 55 60Ala Asn Trp Leu Phe Phe Asp Leu Asp Glu Asn Asn Lys Trp Lys Asp65 70 75 80Tyr Trp Ala Phe Lys Asp Lys Thr Pro Glu Thr Ala Thr Ile Thr Arg
85 90 95Arg Trp Leu Phe Arg Lys Gln Asn Asn Leu Lys Lys Glu Thr Phe Asp
100 105 110Asn Leu Val Lys Leu Thr Lys Gly Ser Lys Met Arg Leu Leu Phe Asp
115 120 125Leu Asn Ala Glu Val Arg Thr Gly Tyr Glu Ile Gly Lys Lys Met Thr
130 135 140Ser Thr Trp Asp Ser Ser Glu Ala Glu Lys Leu Phe Lys Tyr Cys Val145 150 155 160Ser Lys Gly Tyr Gly Asp Asn Ile Asp Trp Glu Leu Gly Asn Glu Pro
165 170 175Asp His Thr Ser Ala His Asn Leu Thr Glu Lys Gln Val Gly Glu Asp
180 185 190Phe Lys Ala Leu His Lys Val Leu Glu Lys Tyr Pro Thr Leu Asn Lys
195 200 205Gly Ser Leu Val Gly Pro Asp Val Gly Trp Met Gly Val Ser Xaa Val
210 215 220Lys Gly Leu Ala Asp Glu Xaa Gly Asp His Val Xaa Ala Phe Thr Leu225 230 235 240His Gln Tyr Tyr Phe Asp Gly Asn Xaa Ser Asp Val Ser Ile Tyr Leu
245 250 255Asp Ala Thr Tyr Phe Lys Lys Leu Gln Gln Leu Phe Asp Lys Val Lys
260 265 270Asp Val Leu Lys Asp Ser Pro His Lys Asp Glu Pro Leu Trp Leu Gly
275 280 285Glu Thr Ser Ser Gly Tyr Asn Ser Gly Thr Glu Asp Val Ser Asp Arg
290 295 300Tyr Val Ser Gly Phe Leu Thr Leu Asp Lys Leu Gly Leu Ser Ala Ala305 310 315 320Asn Asn Val Lys Val Val Ile Arg Gln Thr Ile Tyr Asn Gly Tyr Tyr
325 330 335Gly Leu Leu Asp Lys Asn Thr Leu Glu Pro Asn Pro Asp Tyr Trp Leu
340 345 350Met His Val His Asn Ser Leu Val Gly Asn Thr Val Phe Lys Val Asp
355 360 365Val Ser Asp Pro Thr Asn Lys Ala Arg Val Tyr Ala Gln Cys Thr Lys
370 375 380Thr Asn Ser Lys His Thr Gln Ser Arg Tyr Tyr Lys Gly Ser Leu Thr385 390 395 400Ile Phe Ala Leu Asn Val Gly Asp Gly Asp Val Thr Leu Lys Ile Gly
405 410 415Gln Tyr Ser Gly Lys Lys Ile Tyr Ser Tyr Ile Leu Thr Pro Glu Gly
420 425 430Gly Gln Leu Thr Ser Gln Lys Val Leu Leu Asn Gly Lys Glu Leu Asn
435 440 445Leu Val Ser Asp Gln Leu Pro Glu Leu Asn Ala Asp Glu Ser Lys Thr
450 455 460Ser Phe Thr Leu Ser Pro Lys Thr Phe Gly Phe Phe Val Val Ser Asp465 470 475 480Ala Asn Val Glu Ala Cys Lys Lys
485<210>4<211>1464<212>DNA<213>水蛭<220><221>CDS<222>(1)..(1464)<220><221>变化<222>(1348)..(1350)<223>Xaa=Val或Met<400>4aaa gag att gcc gtg aca att gac gat aag aat gtg att gca tct gcc 48Lys Glu Ile Ala Val Thr Ile Asp Asp Lys Asn Val Ile Ala Ser Ala1 5 10 15agt gag tct ttc cat gga gtt gcc ttt gat gcg tct cta ttt tcg ccc 96Ser Glu Ser Phe His Gly Val Ala Phe Asp Ala Ser Leu Phe Ser Pro
20 25 30aag ggt ctt tgg agc ttt gtt gat att acc tct cca aaa ttg ttc aaa 144Lys Gly Leu Trp Ser Phe Val Asp Ile Thr Ser Pro Lys Leu Phe Lys
35 40 45ttg ctg gaa gga ctt tct cct gga tac ttc agg gtt ggc gga acg ttt 192Leu Leu Glu Gly Leu Ser Pro Gly Tyr Phe Arg Val Gly Gly Thr Phe
50 55 60gcc aat cgg ctg ttt ttt gac ttg gac gaa aat aat aag tgg aar gat 240Ala Asn Arg Leu Phe Phe Asp Leu Asp Glu Asn Asn Lys Trp Lys Asp65 70 75 80tat tgg gct ttt aaa gac aaa acc ccc gaa act gcg aca ata aca agg 288Tyr Trp Ala Phe Lys Asp Lys Thr Pro Glu Thr Ala Thr Ile Thr Arg
85 90 95aga tgg ctg ttc aga aaa caa aat aat ctg aaa aag gag act ttt gac 336Arg Trp Leu Phe Arg Lys Gln Asn Asn Leu Lys Lys Glu Thr Phe Asp
100 105 110aat tta gtg aaa cta aca aag gga agc aag atg aga ttg tta ttc gat 384Asn Leu Val Lys Leu Thr Lys Gly Ser Lys Met Arg Leu Leu Phe Asp
115 120 125ttg aat gcc gaa gtg agg act ggt tat gaa att gga aag aag atg aca 432Leu Asn Ala Glu Val Arg Thr Gly Tyr Glu Ile Gly Lys Lys Met Thr
130 135 140tcc act tgg gat tca tcg gag gct gaa aag tta ttt aaa tat tgt gtg 480Ser Thr Trp Asp Ser Ser Glu Ala Glu Lys Leu Phe Lys Tyr Cys Val145 150 155 160tca aaa ggt tac gga gac aat atc gat tgg gaa ctt ggg aat gga ccg 528Ser Lys Gly Tyr Gly Asp Asn Ile Asp Trp Glu Leu Gly Asn Gly Pro
165 170 175gac cac acc tca gct cac aat tta act gaa aag cag gtt gga gaa gat 576Asp His Thr Ser Ala His Asn Leu Thr Glu Lys Gln Val Gly Glu Asp
180 185 190ttt aaa gca ctg cat aaa gtt cta gag aaa tat cca act ctt aac aag 624Phe Lys Ala Leu His Lys Val Leu Glu Lys Tyr Pro Thr Leu Asn Lys
195 200 205gga tcg ctc gtt ggt cca gat gta ggg tgg atg ggc gtc agt tac gtc 672Gly Ser Leu Val Gly Pro Asp Val Gly Trp Met Gly Val Ser Tyr Val
210 215 220aag gga ttg gca gac gag gca ggt gac cat gta act gct ttt aca ctc 720Lys Gly Leu Ala Asp Glu Ala Gly Asp His Val Thr Ala Phe Thr Leu225 230 235 240cac caa tat tat ttc gat gga aac acc tct gat gta tca ata tat ctt 768His Gln Tyr Tyr Phe Asp Gly Asn Thr Ser Asp Val Ser Ile Tyr Leu
245 250 255gat gcc aca tac ttt aag aag ctg caa caa cta ttt gat aaa gtg aaa 816Asp Ala Thr Tyr Phe Lys Lys Leu Gln Gln Leu Phe Asp Lys Val Lys
260 265 270gat gtt ttg aaa gat tct cca cat aaa gac aaa cca tta tgg ctt gga 864Asp Val Leu Lys Asp Ser Pro His Lys Asp Lys Pro Leu Trp Leu Gly
275 280 285gaa aca agt tct gga tac aac agc ggc aca gaa gat gta tcc gat cga 912Glu Thr Ser Ser Gly Tyr Asn Ser Gly Thr Glu Asp Val Ser Asp Arg
290 295 300tat gtt tca gga ttt cta aca tta gac aag ttg ggt ctc agt gca gcc 960Tyr Val Ser Gly Phe Leu Thr Leu Asp Lys Leu Gly Leu Ser Ala Ala305 310 315 320aac aat gta aag gtt gtt ata aga cag aca ata tac agt gga tat tat 1008Asn Asn Val Lys Val Val Ile Arg Gln Thr Ile Tyr Ser Gly Tyr Tyr
325 330 335ggt ccc ctt gac aaa aac act tta gag cca aat ccg gat tac tgg tta 1056Gly Pro Leu Asp Lys Asn Thr Leu Glu Pro Asn Pro Asp Tyr Trp Leu
340 345 350atg cat gtt cat aat tct ttg gtc gga aat aca gtt ttt aaa gtt gac 1104Met His Val His Asn Ser Leu Val Gly Asn Thr Val Phe Lys Val Asp
355 360 365gtt agt gat cca act aat aaa gca aga gtt tac gcg caa tgt acc aaa 1152Val Ser Asp Pro Thr Asn Lys Ala Arg Val Tyr Ala Gln Cys Thr Lys
370 375 380aca aat agc aaa cat act caa agc aga tat tac aag ggc tct ttg aca 1200Thr Asn Ser Lys His Thr Gln Ser Arg Tyr Tyr Lys Gly Ser Leu Thr385 390 395 400atc ttt gca ctt aat gtt gga gat gaa gat gta acg tta aag atc ggt 1248Ile Phe Ala Leu Asn Val Gly Asp Glu Asp Val Thr Leu Lys Ile Gly
405 410 415caa tac agc ggt aaa aaa att tat tca tac att ctg aca cct gaa gga 1296Gln Tyr Ser Gly Lys Lys Ile Tyr Ser Tyr Ile Leu Thr Pro Glu Gly
420 425 430gga caa ctt aca tca cag aaa gtt ctc ttg aat gga aag gaa ttg aac 1344Gly Gln Leu Thr Ser Gln Lys Val Leu Leu Asn Gly Lys Glu Leu Asn
435 440 445tta rtg tct gat cag tta cca caa cta aat gca gat gaa tcc aaa aca 1392Leu Xaa Ser Asp Gln Leu Pro Gln Leu Asn Ala Asp Glu Ser Lys Thr
450 455 460tct ttc acc tta tcc cca aag aca ttt ggt ttt ttt gtt gtt tcc gat 1440Ser Phe Thr Leu Ser Pro Lys Thr Phe Gly Phe Phe Val Val Ser Asp465 470 475 480gct aat gtt gaa gca tgy aar aar 1464Ala Asn Val Glu Ala Cys Lys Lys
485<2l0>5<211>488<212>PRT<213>水蛭<400>5Lys Glu Ile Ala Val Thr Ile Asp Asp Lys Asn Val Ile Ala Ser Ala1 5 10 15Ser Glu Ser Phe His Gly Val Ala Phe Asp Ala Ser Leu Phe Ser Pro
20 25 30Lys Gly Leu Trp Ser Phe Val Asp Ile Thr Ser Pro Lys Leu Phe Lys
35 40 45Leu Leu Glu Gly Leu Ser Pro Gly Tyr Phe Arg Val Gly Gly Thr Phe
50 55 60Ala Asn Arg Leu Phe Phe Asp Leu Asp Glu Asn Asn Lys Trp Lys Asp65 70 75 80Tyr Trp Ala Phe Lys Asp Lys Thr Pro Glu Thr Ala Thr Ile Thr Arg
85 90 95Arg Trp Leu Phe Arg Lys Gln Asn Asn Leu Lys Lys Glu Thr Phe Asp
100 105 110Asn Leu Val Lys Leu Thr Lys Gly Ser Lys Met Arg Leu Leu Phe Asp
115 120 125Leu Asn Ala Glu Val Arg Thr Gly Tyr Glu Ile Gly Lys Lys Met Thr
130 135 140Ser Thr Trp Asp Ser Ser Glu Ala Glu Lys Leu Phe Lys Tyr Cys Val145 150 155 160Ser Lys Gly Tyr Gly Asp Asn Ile Asp Trp Glu Leu Gly Asn Gly Pro
165 170 175Asp His Thr Ser Ala His Asn Leu Thr Glu Lys Gln Val Gly Glu Asp
180 185 190Phe Lys Ala Leu His Lys Val Leu Glu Lys Tyr Pro Thr Leu Asn Lys
195 200 205Gly Ser Leu Val Gly Pro Asp Val Gly Trp Met Gly Val Ser Tyr Val
210 215 220Lys Gly Leu Ala Asp Glu Ala Gly Asp His Val Thr Ala Phe Thr Leu225 230 235 240His Gln Tyr Tyr Phe Asp Gly Asn Thr Ser Asp Val Ser Ile Tyr Leu
245 250 255Asp Ala Thr Tyr Phe Lys Lys Leu Gln Gln Leu Phe Asp Lys Val Lys
260 265 270Asp Val Leu Lys Asp Ser Pro His Lys Asp Lys Pro Leu Trp Leu Gly
275 280 285Glu Thr Ser Ser Gly Tyr Asn Ser Gly Thr Glu Asp Val Ser Asp Arg
290 295 300Tyr Val Ser Gly Phe Leu Thr Leu Asp Lys Leu Gly Leu Ser Ala Ala305 310 315 320Asn Asn Val Lys Val Val Ile Arg Gln Thr Ile Tyr Ser Gly Tyr Tyr
325 330 335Gly Pro Leu Asp Lys Asn Thr Leu Glu Pro Asn Pro Asp Tyr Trp Leu
340 345 350Met His Val His Asn Ser Leu Val Gly Asn Thr Val Phe Lys Val Asp
355 360 365Val Ser Asp Pro Thr Asn Lys Ala Arg Val Tyr Ala Gln Cys Thr Lys
370 375 380Thr Asn Ser Lys His Thr Gln Ser Arg Tyr Tyr Lys Gly Ser Leu Thr385 390 395 400Ile Phe Ala Leu Asn Val Gly Asp Glu Asp Val Thr Leu Lys Ile Gly
405 410 415Gln Tyr Ser Gly Lys Lys Ile Tyr Ser Tyr Ile Leu Thr Pro Glu Gly
420 425 430Gly Gln Leu Thr Ser Gln Lys Val Leu Leu Asn Gly Lys Glu Leu Asn
435 440 445Leu Xaa Ser Asp Gln Leu Pro Gln Leu Asn Ala Asp Glu Ser Lys Thr
450 455 460Ser Phe Thr Leu Ser Pro Lys Thr Phe Gly Phe Phe Val Val Ser Asp465 470 475 480Ala Asn Val Glu Ala Cys Lys Lys
485<210>6<211>1464<212>DNA<213>水蛭<220><221>CDS<222>(1)..(1464)<400>6aaa gag att gcc gtg aca att gac gat aag aat gtg att gca tct gtc 48Lys Glu Ile Ala Val Thr Ile Asp Asp Lys Asn Val Ile Ala Ser Val1 5 10 15agt gag tct ttc cat gga gtt gcc ttt gat gcg tct cta ttc tcg ccc 96Ser Glu Ser Phe His Gly Val Ala Phe Asp Ala Ser Leu Phe Ser Pro
20 25 30aag ggt cct tgg agc ttt gtt aat att acc tct cca aaa ttg ttc aaa 144Lys Gly Pro Trp Ser Phe Val Asn Ile Thr Ser Pro Lys Leu Phe Lys
35 40 45ttg ctg gaa gga ctt tct cct gga tac ttc agg gtt ggc gga acg ttt 192Leu Leu Glu Gly Leu Ser Pro Gly Tyr Phe Arg Val Gly Gly Thr Phe
50 55 60gcc aat tgg ctg ttt ttt gac ttg gac gaa aat aat aag tgg aag gat 240Ala Asn Trp Leu Phe Phe Asp Leu Asp Glu Asn Asn Lys Trp Lys Asp65 70 75 80tat tgg gct ttt aaa gac aaa acc ccc gaa act gcg aca ata aca agg 288Tyr Trp Ala Phe Lys Asp Lys Thr Pro Glu Thr Ala Thr Ile Thr Arg
85 90 95aga tgg ctg ttc aga aaa caa aat aat ctg aaa aag gag act ttt gac 336Arg Trp Leu Phe Arg Lys Gln Asn Asn Leu Lys Lys Glu Thr Phe Asp
100 105 110gat tta gtg aaa cta aca aag gga agc aag atg aga ttg tta ttc gat 384Asp Leu Val Lys Leu Thr Lys Gly Ser Lys Met Arg Leu Leu Phe Asp
115 120 125ttg aat gcc gaa gtg agg act ggt tat gaa att gga aag aag acg aca 432Leu Asn Ala Glu Val Arg Thr Gly Tyr Glu Ile Gly Lys Lys Thr Thr
130 135 140tcc act tgg gat tca tcg gag gct gaa aag tta ttt aaa tat tgt gtg 480Ser Thr Trp Asp Ser Ser Glu Ala Glu Lys Leu Phe Lys Tyr Cys Val145 150 155 160tca aaa ggt tac gga gac aat atc gat tgg gaa ctt gga aat gaa ccg 528Ser Lys Gly Tyr Gly Asp Asn Ile Asp Trp Glu Leu Gly Asn Glu Pro
165 170 175gac cac acc tca gct cac aat tta act gaa aag cag gtt gga gaa gat 576Asp His Thr Ser Ala His Asn Leu Thr Glu Lys Gln Val Gly Glu Asp
180 185 190ttc aaa gca ctg cat aaa gtt tta gag aaa tat cca act ctt aac aag 624Phe Lys Ala Leu His Lys Val Leu Glu Lys Tyr Pro Thr Leu Asn Lys
195 200 205gga tcg ccc gtt ggt cca gat gta ggg tgg atg ggc gtc agc tac gtc 672Gly Ser Pro Val Gly Pro Asp Val Gly Trp Met Gly Val Ser Tyr Val
210 215 220aag gga ttg gca gac ggg gca ggt gac ctt gta act gct ttt aca cta 720Lys Gly Leu Ala Asp Gly Ala Gly Asp Leu Val Thr Ala Phe Thr Leu225 230 235 240cac caa tat tat ttc gat gga aac acc tct gat gta tca aca tat ctt 768His Gln Tyr Tyr Phe Asp Gly Asn Thr Ser Asp Val Ser Thr Tyr Leu
245 250 255gat gcc tca tac ttt aaa aag ctg caa cag ctg ttt gat aaa gtg aaa 816Asp Ala Ser Tyr Phe Lys Lys Leu Gln Gln Leu Phe Asp Lys Val Lys
260 265 270gat gtt ttg aaa aat tct cca cat aaa gac aaa cca tta tgg ctt gga 864Asp Val Leu Lys Asn Ser Pro His Lys Asp Lys Pro Leu Trp Leu Gly
275 280 285gag aca agt tct gga tgc aac agc ggc aca aaa gat gta tcc gat cga 912Glu Thr Ser Ser Gly Cys Asn Ser Gly Thr Lys Asp Val Ser Asp Arg
290 295 300tat gtt tca gga ttt cta aca tta gac aag ttg ggt ctc agt gca gcc 960Tyr Val Ser Gly Phe Leu Thr Leu Asp Lys Leu Gly Leu Ser Ala Ala305 310 315 320aac aat gta aag gtt gtt ata aga cag aca ata tac aat gga tat tat 1008Asn Asn Val Lys Val Val Ile Arg Gln Thr Ile Tyr Asn Gly Tyr Tyr
325 330 335ggt ctc ctt gat aaa aac act tta gag cca aat cct gat tac tgg tta 1056Gly Leu Leu Asp Lys Asn Thr Leu Glu Pro Asn Pro Asp Tyr Trp Leu
340 345 350atg cat gtt cac aat tct ttg gtc gga aat aca gtt ttt aaa gtt gac 1104Met His Val His Asn Ser Leu Val Gly Asn Thr Val Phe Lys Val Asp
355 360 365gtt ggt gat cca act aat aaa acg aga gtc tat gca caa tgt acc aag 1152Val Gly Asp Pro Thr Asn Lys Thr Arg Val Tyr Ala Gln Cys Thr Lys
370 375 380aca aat agc aaa cac act caa ggc aag tat tac aag ggc tct ttg aca 1200Thr Asn Ser Lys His Thr Gln Gly Lys Tyr Tyr Lys Gly Ser Leu Thr385 390 395 400atc ttt gca ctt aat gtt gga gat gaa gaa gta acg tta aag atc gat 1248Ile Phe Ala Leu Asn Val Gly Asp Glu Glu Val Thr Leu Lys Ile Asp
405 410 415caa tac ggc ggt aaa aaa att tat tca tac att ctg aca cct gaa gga 1296Gln Tyr Gly Gly Lys Lys Ile Tyr Ser Tyr Ile Leu Thr Pro Glu Gly
420 425 430gga caa ctt aca tca cag aaa gtt ctc ttg aat gga aag gaa ttg aac 1344Gly Gln Leu Thr Ser Gln Lys Val Leu Leu Asn Gly Lys Glu Leu Asn
435 440 445tta gtg tct gat cag tta cca gaa cta aat gca gat gaa tcc aaa aca 1392Leu Val Ser Asp Gln Leu Pro Glu Leu Asn Ala Asp Glu Ser Lys Thr
450 455 460tct ttc acc tta tcc cca aag aca ttt ggt ttt ttt gtt gtt tcc gat 1440Ser Phe Thr Leu Ser Pro Lys Thr Phe Gly Phe Phe Val Val Ser Asp465 470 475 480gct aat gtt gaa gca tgy aar aar 1464Ala Asn Val Glu Ala Cys Lys Lys
485<210>7<211>488<212>PRT<213>水蛭<400>7Lys Glu Ile Ala Val Thr Ile Asp Asp Lys Asn Val Ile Ala Ser Val1 5 10 15Ser Glu Ser Phe His Gly Val Ala Phe Asp Ala Ser Leu Phe Ser Pro
20 25 30Lys Gly Pro Trp Ser Phe Val Asn Ile Thr Ser Pro Lys Leu Phe Lys
35 40 45Leu Leu Glu Gly Leu Ser Pro Gly Tyr Phe Arg Val Gly Gly Thr Phe
50 55 60Ala Asn Trp Leu Phe Phe Asp Leu Asp Glu Asn Asn Lys Trp Lys Asp65 70 75 80Tyr Trp Ala Phe Lys Asp Lys Thr Pro Glu Thr Ala Thr Ile Thr Arg
85 90 95Arg Trp Leu Phe Arg Lys Gln Asn Asn Leu Lys Lys Glu Thr Phe Asp
100 105 110Asp Leu Val Lys Leu Thr Lys Gly Ser Lys Met Arg Leu Leu Phe Asp
115 120 125Leu Asn Ala Glu Val Arg Thr Gly Tyr Glu Ile Gly Lys Lys Thr Thr
130 135 140Ser Thr Trp Asp Ser Ser Glu Ala Glu Lys Leu Phe Lys Tyr Cys Val145 150 155 160Ser Lys Gly Tyr Gly Asp Asn Ile Asp Trp Glu Leu Gly Asn Glu Pro
165 170 175Asp His Thr Ser Ala His Asn Leu Thr Glu Lys Gln Val Gly Glu Asp
180 185 190Phe Lys Ala Leu His Lys Val Leu Glu Lys Tyr Pro Thr Leu Asn Lys
195 200 205Gly Ser Pro Val Gly Pro Asp Val Gly Trp Met Gly Val Ser Tyr Val
210 215 220Lys Gly Leu Ala Asp Gly Ala Gly Asp Leu Val Thr Ala Phe Thr Leu225 230 235 240His Gln Tyr Tyr Phe Asp Gly Asn Thr Ser Asp Val Ser Thr Tyr Leu
245 250 255Asp Ala Ser Tyr Phe Lys Lys Leu Gln Gln Leu Phe Asp Lys Val Lys
260 265 270Asp Val Leu Lys Asn Ser Pro His Lys Asp Lys Pro Leu Trp Leu Gly
275 280 285Glu Thr Ser Ser Gly Cys Asn Ser Gly Thr Lys Asp Val Ser Asp Arg
290 295 300Tyr Val Ser Gly Phe Leu Thr Leu Asp Lys Leu Gly Leu Ser Ala Ala305 310 315 320Asn Asn Val Lys Val Val Ile Arg Gln Thr Ile Tyr Asn Gly Tyr Tyr
325 330 335Gly Leu Leu Asp Lys Asn Thr Leu Glu Pro Asn Pro Asp Tyr Trp Leu
340 345 350Met His Val His Asn Ser Leu Val Gly Asn Thr Val Phe Lys Val Asp
355 360 365Val Gly Asp Pro Thr Asn Lys Thr Arg Val Tyr Ala Gln Cys Thr Lys
370 375 380Thr Asn Ser Lys His Thr Gln Gly Lys Tyr Tyr Lys Gly Ser Leu Thr385 390 395 400Ile Phe Ala Leu Asn Val Gly Asp Glu Glu Val Thr Leu Lys Ile Asp
405 410 415Gln Tyr Gly Gly Lys Lys Ile Tyr Ser Tyr Ile Leu Thr Pro Glu Gly
420 425 430Gly Gln Leu Thr Ser Gln Lys Val Leu Leu Asn Gly Lys Glu Leu Asn
435 440 445Leu Val Ser Asp Gln Leu Pro Glu Leu Asn Ala Asp Glu Ser Lys Thr
450 455 460Ser Phe Thr Leu Ser Pro Lys Thr Phe Gly Phe Phe Val Val Ser Asp465 470 475 480Ala Asn Val Glu Ala Cys Lys Lys
485<210>8<211>23<212>DNA<213>人工序列<220><223>人工序列描述:5′-引物<400>8atcgataaag agattgccgt gac 23<210>9<211>23<212>DNA<213>人工序列<220><223>人工序列描述:3′-引物<400>9gttgtttccg atgctaaagc gct 23<210>10<211>30<212>DNA<213>人工序列<220><223>人工序列描述:5′-引物<400>10accgtagcgc aggccaaaga gattgccgtg 30<210>11<211>30<212>DNA<213>人工序列<220><223>人工序列描述:3′-引物<400>11cacggcaatc tctttggcct gcgctacggt 30<210>12<211>87<212>DNA<213>人工序列<220><223>人工序列描述:5′-引物<400>12cggatccatg aaattcttag tcaacgttgc ccttgttttt atggtcgtat acatttctta 60catctatgcg aaagagattg ccgtgac 87<210>13<211>21<212>DNA<213>人工序列<220><223>人工序列描述:3′-引物<400>13aatgttgaag cataaggtac c 21<210>14<211>27<212>DNA<213>人工序列<220><223>人工序列描述:5′-引物<400>14gtagaattca aagagattgc cgtgaca 27<210>15<211>32<212>DNA<213>人工序列<220><223>人工序列描述:3′-引物<400>15gatgctaatg ttgaagcata atgagcggcc gc 32
Claims (20)
1.一种从水蛭种马尼拉水蛭中分离的纯化的蛋白质,其具有透明质酸酶的生物活性,其活性不受肝素影响,其特征在于根据糖基化其分子量为53-60。
2.根据权利要求1的糖基化蛋白质,其分子量为58(±2)。
3.根据权利要求1的非糖基化蛋白质,其分子量为54(±2)。
4.根据权利要求1-3任一项的蛋白质,其等电点为7.2-8.0。
5.根据权利要求1-4任一项的蛋白质,其具有图7和SEQ ID No.1所示的氨基酸序列。
6.根据权利要求1-5的蛋白质,其酶比活>100kU/mg蛋白质。
7.一种分离并纯化如权利要求1-6所述蛋白质的方法,其包括下列步骤:
(i)用酸性缓冲液匀浆马尼拉水蛭种的水蛭的头和离心,
(ii)硫酸铵沉淀步骤(i)的上清液,
(iii)阳离子交换层析,
(iv)伴刀豆球蛋白A亲和层析,
(v)疏水作用层析,
(vi)在用透明质酸片段包被的基质上亲和层析,
(vii)凝胶渗透层析,和任选地,
(viii)纯化蛋白质的酶促或化学去糖基化。
8.一种具有透明质酸酶生物活性的蛋白质,其活性不受肝素影响,根据糖基化其分子量为53-60,可通过权利要求7的步骤获得。
9.根据权利要求8的蛋白质,其酶比活>100kU/mg蛋白质。
10.一种编码权利要求1和9的蛋白质的DNA序列。
11.一种编码权利要求8的蛋白质的DNA序列,其含有图8(SEQ IDNo.2)、图9(SEQ ID No.4)和图10(SEQ ID No.6)所示的任一核苷酸序列。
12.一种重组蛋白,其具有透明质酸酶生物活性,由权利要求11的任一DNA序列编码。
13.一种重组蛋白,其具有透明质酸酶生物活性,根据糖基化其分子量为55-59,含有图8、9和10(SEQ ID No.3、5、7)所示的任一氨基酸序列或与该序列至少80%同源的序列。
14.含有权利要求10或11的DNA序列的一种表达载体。
15.用权利要求14的载体转化的一种适于表达权利要求12或13的蛋白质的宿主细胞。
16.根据权利要求1-6、8、9、12和13任一项的一种蛋白质,作为一种药物。
17.一种药用组合物,其含有权利要求16的蛋白质和药学可接受的稀释剂、载体或赋形剂。
18.一种药用组合物,其还含有一种药理活性化合物。
19.根据权利要求18的一种药用组合物,其中药理活性化合物是肝素。
20.根据权利要求1-6、8、9、12和13任一项的蛋白质在生产可治疗心肌、心血管和血栓疾病和肿瘤的药物中的用途。
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GB8504025D0 (en) * | 1985-02-16 | 1985-03-20 | Biopharm Ltd | Hyaluronidase |
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US5747027A (en) * | 1995-04-07 | 1998-05-05 | The Regents Of The University Of California | BH55 hyaluronidase |
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JP2003502045A (ja) | 2003-01-21 |
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AU6149300A (en) | 2001-01-02 |
SK17612001A3 (sk) | 2002-04-04 |
US7049124B1 (en) | 2006-05-23 |
EP1185666A1 (en) | 2002-03-13 |
AR024335A1 (es) | 2002-10-02 |
WO2000077221A1 (en) | 2000-12-21 |
KR20020011138A (ko) | 2002-02-07 |
PT1185666E (pt) | 2006-09-29 |
DE60027562D1 (de) | 2006-06-01 |
ATE324452T1 (de) | 2006-05-15 |
DE60027562T2 (de) | 2006-11-02 |
BR0011500A (pt) | 2002-03-12 |
DK1185666T3 (da) | 2006-07-03 |
HUP0201452A2 (en) | 2002-08-28 |
MXPA01012806A (es) | 2002-09-02 |
PL352060A1 (en) | 2003-07-28 |
CA2376467A1 (en) | 2000-12-21 |
HK1046297A1 (zh) | 2003-01-03 |
ZA200200233B (en) | 2003-06-25 |
CZ20014383A3 (cs) | 2002-03-13 |
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