CN1626237A - 一个蛋白质家族的抗菌用途 - Google Patents
一个蛋白质家族的抗菌用途 Download PDFInfo
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- CN1626237A CN1626237A CNA2003101173546A CN200310117354A CN1626237A CN 1626237 A CN1626237 A CN 1626237A CN A2003101173546 A CNA2003101173546 A CN A2003101173546A CN 200310117354 A CN200310117354 A CN 200310117354A CN 1626237 A CN1626237 A CN 1626237A
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Abstract
本发明公开了一个蛋白质家族的抗菌用途。本发明利用抑制性差减杂交的方法，获得了一个新的天然Glyrichin蛋白质家族，该家族的蛋白质具有抗菌的活性，可用于制备细菌性感染疾病的治疗药物，或用于筛选促进或拮抗该家族蛋白质的抗体、多肽或其它配体，或用于化妆品添加剂，食品防腐剂，畜禽饲料添加剂，种子包衣以及提高转基因植物或动物抗病虫害的能力。
Description
技术领域
本发明涉及一个蛋白质家族的用途，具体地说涉及一个蛋白质家族的抗菌用途。
背景技术
20多年来，医药界没有发现任何新的抗菌素家族，但是抗药性现象的发展却极为迅速，人们甚至可以用肉眼观察到细菌的基因突变。更为可怕的是，对付金葡萄球菌的障碍刚刚攻克，美国医生就发现了对可有效抑制细菌的最新抗菌素-万古霉素具有抗性的菌株。致病菌的抗药性问题已经日益严重地威胁着人们的健康。寻找全新类型的抗生素是解决抗药性问题的一条有效途径。抗菌肽作为生物天然免疫的活性分子，被各种生物用来抵御来自外界病菌的侵染，在生物界中广泛存在。人为分离出来的天然抗菌肽表现出抗菌活性高，抗菌谱广，种类多，可供选择的范围广，靶菌株不易产生抗性突变等特点，因而在医药工业、食品工业和农业上被认为有着广阔的应用前景。抗菌肽是指由基因编码，在核糖体上合成的相对分子质量通常在10kDa以下，具有抗菌活性的多肽类物质，也叫多肽抗生素。大部分抗菌肽具有热稳定性，在100℃下加热10～15min仍能保持其活性。抗菌肽对较大的离子强度和较高或较低的pH值均具有较强的抗性。多数抗菌肽的等电点大于7，表现出较强的阳离子特征。同时，部分抗菌肽尚具备抵抗胰蛋白酶或胃蛋白酶水解的能力。此外，研究发现不同家族的抗菌肽之间在序列上极少存在同源性，而同一家族的不同成员之间序列上却存在高度的保守性，这意味着其功能也非常保守。抗菌肽除了具有抗细菌或真菌的作用，有些还具有抗原虫、病毒或癌细胞的功能。抗菌肽在生物界中广泛存在。迄今为止，人们已经从细菌、真菌，到两栖类、昆虫、高等植物、哺乳动物、直至人类体内发现了多达700种以上的多肽抗生素。目前，已有多种抗菌肽正在进行临床前的可行性研究；此外，抗菌肽转基因动物、转基因植物，抗菌肽在食品防腐，鲜花保鲜，化妆品，种子包衣和动物饲料添加剂等方面的应用研究也正在进行之中。
鉴于抗菌肽在医药工业、食品工业和农业上有着广阔的应用前景，通过各种途径从生物界中寻找新型的天然抗菌肽就成为目前世界范围内热点之一。
本发明的发明人通过试验首次发现了一种蛋白质，将其命名为Glyrichin蛋白，然后利用生物信息学技术，通过同源性分析，发现了一个包含多个蛋白质在内的蛋白质家族，Glyrichin蛋白家族。目前，这些蛋白质的功能还不清楚，也没有这些蛋白质的抗菌特性的报道。
发明内容
本发明公开了一个天然蛋白质家族的用途。这个蛋白质家族中的蛋白质包括来自酵母菌、线虫、果蝇、按蚊、斑马鱼、小鼠到人等物种的高度同源体蛋白质，具有如表1中序列1～14所示的氨基酸序列。
本发明所述的蛋白质家族的用途包括以下方面：
(1)直接做为人或畜细菌性感染疾病的治疗药物；
(2)用于筛选促进或拮抗Glyrichin蛋白功能的抗体、多肽或其它配体；
(3)用于化妆品添加剂；
(4)用于食品防腐剂；
(5)用于畜禽或水产饲料添加剂；
(6)用于种子包衣；
(7)用于转基因植物或动物以提高其抗病虫害的能力。
(8)用于鲜花保鲜。
本发明所述的蛋白质家族包括下列蛋白质：
表1 Glyrichin蛋白家族包含的蛋白质
| 序号 | 物种 | 来源 | GenBankTM注册号 |
| 1 | 人 | Homo sapiens | CAC11117 |
| 2 | 小鼠 | Mus musculus | AAK18748 |
| 3 | 斑马鱼 | Danio rerio | AAH59661 |
| 4 | 按蚊 | Anopheles gambiae | XP_320383 |
| 5 | 果蝇 | Drosophila melanogas | NP_648757 |
| 6 | 线虫 | Caenorhabditis elegans | NP_496804 |
| 7 | 线虫 | Caenorhabditis elegans | B88349 |
| 8 | 芽殖酵母 | Schizosaccharomycespombe | CAB89006 |
| 9 | 酿酒酵母 | Saccharomycescerevisiae | AAB68201 |
| 10 | 拟南芥 | Arabidopsis thaliana | NP_566324 |
| 11 | 疟原虫 | Plasmodium falciparum3D7 | NP_705421 |
| 12 | 疟原虫 | Plasmodium yoeliiyoelii | EAA17587 |
| 13 | 稻瘟病菌 | Magnaporthe grisea | EAA47634 |
| 14 | 脉孢菌 | Neurospora crassa | EAA34843 |
本发明所述的蛋白质家族还包括具有在上述蛋白质的氨基酸序列基础上发生氨基酸残基的插入、取代、缺失、添加等所产生的，具有与原序列相同功能的序列的蛋白质。
发明人利用抑制性差减杂交的方法，对LTC(long term culture)培养前后的小鼠骨髓基质细胞差异表达基因进行了大量筛选，获得了131个差异表达的EST克隆。生物信息学分析证实，这些克隆代表了26种已知或部分已知功能的基因和7条全新的基因，其中5条具有完整的开放阅读框架，3条已在GenBank注册，鼠源的mGlyrichin就是其中之一，其基因全长cDNA序列521bp，其GenBankTM注册号为AY028425，参见《实验血液学杂志)》2002第10期第177-182页。
在得到鼠源mGlyrichin后，发明人根据鼠源Glyrichin基因设计一对简并引物，通过PCR扩增人胎肝cDNA文库，获得人源Glyrichin完整的ORF序列共240bp，以及根据ORF序列推测的氨基酸序列。发明人利用生物信息学手段对序列进行了详细分析，进行了Glyrichin家族成员间的同源性比较-多序列对齐。Glyrichin家族各成员的序列均来自GenBank，它们的注册号分别为：人Homo sapiens_CAC11117、小鼠Mus muscul us_AAK18748、斑马鱼Danio rerio_AAH59661、按蚊Anopheles gambiae_XP_320383、果蝇Drosophila melanogas_NP_648757、线虫Caenorhabditisel egans_B88349和NP_496804、芽殖酵母Schizosaccharomyces pombe_CAB89006、酿酒酵母Saccharomyces cerevisiae_AAB68201、拟南芥Arabidopsisthaliana_NP_566324和疟原虫Plasmodium falciparum 3D7_NP_705421和Plasmodium yoelii yoelii_EAA17587等。用DNASTAR Inc.公司的分析软件MegAlign作图，多序列比对图如附图1所示，反映出Glyrichin家族各成员的共同特点是存在一个高度保守的、大约60aa长度的核心结构域，在该结构域的上游或下游的序列则同源性较低。到目前为止，该家族所有成员功能均未知。
为研究Glyrichin家族各成员的亲缘关系，对这12条序列进行了进化树分析，如附图2所示，显示出Glyrichin基因从低等到高等的进化历程及各成员间亲缘关系的远近。Glyrichin家族成员间保守的甘氨酸富含区(Glyrich domain)如附图3所示。Glyrich结构域在Glyrichin家族不同成员间的序列一致性如附图4所示，可以看出每两个成员间的亲缘关系的远近。核酸序列比较结果显示hGlyrichin基因定位于人染色体20q11.21区域，由两个外显子组成，编码79个氨基酸的小分子蛋白。氨基酸水平BlastP同源性比较显示该基因在进化上十分保守，存在于从真菌(如脉孢菌、酵母菌等)、植物(如拟南芥等)、疟原虫、线虫、果蝇、按蚊、斑马鱼、小鼠到人等物种中，组成Glyrichin同源性蛋白家族；在不同种属间显示出很高的同源性，已知人源Glyrichin与鼠源相比100％同源，与斑马鱼Glyrinchin相比具有90％同源性，与果蝇的同源性有62％，与酵母的同源性也有46％(图1)；家族内不同成员间的亲缘关系以进化树表示(图2)。hGlyrichin的氨基酸序列特征见表2，为Protean分析结果。
表2 hGlyrichin的序列特征
| 项目 | 内容 |
| 分子量 | 8181.9 |
| 多肽长度(aa) | 79 |
| 等电点 | 9.36 |
| 电荷数(pH7.0) | 4.79 |
| Gly含量(％) | 21.52 |
Glyrichin家族成员的序列特征见表3。发明人进行了hGlyrichin二级结构预测，hGlyrichin的结构特点见附图5，为SignalP分析结果。
表3 Glyrichin家族成员的序列特征
| 物种属名 | 多肽长度(aa) | 等电点 | 电荷数(pH7.0) | 甘氨基酸含量(％) |
| Homo sapiens | 79 | 9.36 | 4.79 | 21.52 |
| Mus musculus | 79 | 9.36 | 4.79 | 21.52 |
| Danio rerio | 80 | 9.81 | 4.82 | 21.2 |
| Anopheles gambiae | 128 | 9.73 | 10.08 | 14.1 |
| Drosophila melanogaster | 79 | 9.66 | 4.82 | 20.3 |
| Caenorhabditis elegans | 145 | 10.79 | 12.01 | 14.48 |
| Schizosaccharomyces pombe | 107 | 10.04 | 4.05 | 13.08 |
| Saccharomyces cerevisiae | 112 | 9.74 | 5.04 | 13.27 |
| Plasmodium falciparum 3D7 | 168 | 9.66 | 5.15 | 15.94 |
| Plasmodium Yoelii Yoelii | 167 | 9.16 | 4.15 | 15.28 |
| Magnapor the grisea | 107 | 11.75 | 8.24 | 13.08 |
| Arabidopsis thaliana | 74 | 9.84 | 6.18 | 14.86 |
鉴于Glyrichin家族各成员进化上高度保守的特点以及其功能未知的事实，发明人对该基因家族的功能开展研究，拟证实Glyrichin家族成员的功能及其用途。
目前已知Glyrichin家族共包括14个成员。这14个成员分布在从真菌到人的所有基因组已完成测序的物种中。本领域的普通技术人员可以轻易地据此推断出它也存在于其他基因组尚未完成测序的物种中。Glyrichin家族各个成员结构上共同的特点是存在一个甘氨酸(Glycine)富含区，长度在59-68个氨基酸之间。该区域基本涵盖了人源和鼠源Glyrichin全部的序列。存在于其他物种中的Glyrichin除了共同的甘氨酸富含区外，还在此保守区的氨基端或羧基端存在其他序列(如信号肽等)，但不具有保守性(见图3、图4)。这说明甘氨酸保守区决定了Glyrichin家族成员最基本的生物活性；也从另一方面说明生物在进化过程中逐渐淘汰了冗余序列，使分子变得更短小、结构更紧凑。
在获得人源Glyrichin多核苷酸序列后，发明人以人源和鼠源Glyrichin为例，对该基因家族的功能开展研究，结果证明人源和鼠源Glyrichin是一个在人和小鼠多种组织中广泛表达的具抗菌活性的天然免疫分子，是一种天然存在的抗菌肽，有望发展成为在医药工业、食品工业、化妆品行业、农业、畜牧业和渔业等领域具有广泛用途的新型多肽抗生素。这也是Glyrichin家族各个成员的基本功能和活性。
表2的统计数据表明人/鼠源Glyrichin多肽的分子量小于10kDa，等电点为9.36，在pH值为7的条件下带4.79个正电荷。表3的SignalP分析表明h/mGlyrichin分子中存在一个强疏水区。Glyrichin家族其他成员的等电点均大于7，在pH＝7时都带正电荷，且都存在强的疏水区域(图5)。上述结果表明Glyrichin家族具有已知抗菌肽典型的结构特征。对人源Glyrichin进行Northern blot分析和体外转录与翻译分析，以[α-32P]dATP标记的hGlyrichin为探针检测其转录本大小和表达谱，结果见图6A。所用的4种肿瘤细胞株分别为HepG2、HeLa、HEK293、Jurket，显示人Glyrichin基因在所测试的4种不同组织来源的肿瘤细胞株中都有表达，提示它可能是一种广泛表达的天然抗菌肽。同时揭示只存在一个转录本，且大小约为600bp。将hGlyrichin基因重组到含有T7启动子的pT7载体中，进行人源Glyrichin体外转录和翻译，hGlyrichin蛋白体外翻译后的大小约为8.8kDa，同理论推断的结果一致，表明该基因可以在体外正常地转录和翻译，见图6B。
接下来发明人使用两种方法测试抗菌活性：将hGlyrichin基因导入细菌中然后诱导表达观察抑菌效应和分离纯化Glyrichin蛋白求最小抑菌浓度。首先，将hGlyrichin不带任何标签(即不同任何蛋白融合)重组到原核表达载体pET-22b(+)中，将pET-22b(+)-Glyrichin转化到大肠杆菌BL21中，比较IPTG存在和不存在两种情况下细菌生长情况。Glyrichin在不加IPTG诱导剂时不表达，而只在培养液中加入IPTG诱导后才大量表达；进一步地，在诱导条件下，如果Glyrichin不具有抗菌活性，则同样不会影响细菌生长；但是如果Glyrichin是抗菌肽，则会明显影响细菌生长；在后一种情况下，随着培养时间延长，IPTG不断消耗，Glyrichin的表达逐渐停止，理论上此时细菌的生长应逐渐恢复。每隔45分钟测量OD600一次，共测量20次。以时间为横轴，OD600对数值为纵轴做生长曲线。转化了pET-22b(+)空载体的BL21在0.5mM IPTG诱导和不诱导条件下绘制半对数生长曲线见图7A，转化了不具抗菌活性的无关基因pET-22b(+)-UBF和pET-22b(+)-PTP的BL21在0.5mM IPTG诱导和不诱导条件下的半对数生长曲线见图7B，转化了待测的pET-22b(+)-hGlyrichin阳性克隆1和8的BL21在0.5mM IPTG诱导和不诱导条件下的半对数生长曲线见图7C，待测的pET-22b(+)-hGlyrichin阳性克隆1的两次重复试验的曲线图见图7D。
单转空载体pET-22b的菌株在加入和不加入IPTG两种情况下细菌的生长曲线基本吻合(图7A)；在单转两个已知不具有抗菌活性基因UBF和PTP的情况下，加入和不加入IPTG两种情况下细菌的生长同样不受影响(图7B)；但是，在转入Glyrichin后，加入和不加入IPTG两种情况下细菌的生长曲线明显不同，在前一种情况下细菌的生长明显受到抑制(图7C，D)；从图中还可以看出，随着培养时间延长IPTG消耗殆尽，细菌的生长又得以恢复，而其他测试基因都没有此现象。上述结果充分说明这种抑菌效应是Glyrichin蛋白本身的作用。在接下来的另一组实验中，hGlyrichin基因被重组到pGEX-4T2原核表达载体中，然后转化到BL21中诱导表达GST-hGlyrichin融合蛋白(图8)；首先根据小鼠Glyrichin的cDNA序列设计引物，以人胎肝mRNA进行RT-PCR扩增，纯化后PCR产物经测序和限制性内切酶消化后在T4连接酶的作用下，插入到用同样内切酶消化后的pGEX4-4T2原核表达载体中，经转化JM109大肠杆菌后，以IPTG进行体外诱导表达5小时，用全菌体蛋白进行SDS-PAGE电泳。然后，纯化游离hGlyrichin表达产物并检测其杀菌活性，将上所述诱导表达得到GST-Glyrichin融合蛋白，经GST纯化柱纯化后用Enterokinase切割，得到游离的hGlyrichin蛋白，利用96孔板法检测多肽的抗菌活性，测定最小抑菌浓度(MIC)，结果表明GST-Glyrichin融合蛋白可以在大肠杆菌菌株JM109中大量表达，不能抑制细菌生长，不具有抗菌活性，游离的Glyrichin蛋白具有在较低浓度下抑制革兰氏阳性菌和革兰氏阴性菌的生长，具有抗菌活性，见表4。
表4 游离hGlyrichin蛋白的最小抑菌浓度(MIC)
| 菌株名称 | 最小抑菌浓度(ug/ml) | |
| 革兰氏阴性菌 | 大肠杆菌DH5α | 0.2 |
| 革兰氏阳性菌 | 枯草杆菌DB430 | 3 |
在本发明中，术语“Glyrichin家族”是指具有Glyrich结构域且在该结构域区段氨基酸同源性相互间在30％以上的多肽，包括本发明中已经提到的14个多肽和其他尚未报道的多肽。以进化生物学的观点，它们来自于同一个祖先。术语“Glyrich结构域”特指表1中序列1～14中最为保守的一段多肽，其氨基酸长度在59-68之间，等电点大于7，pH7.0条件下带正电荷，Glycine含量在20种氨基酸中含量为最高，并且含有至少一个疏水区的一段多肽。它们的序列及结构特点如图4和图5，表2和表3所界定。
术语“Glyrichin多肽”指具有Glyrichin蛋白抗菌活性的表1中序列1～14的多肽。该术语还包括具有与Glyrichin蛋白相同功能的表1中序列1～14的变异形式。这些变异形式包括(但并不限于)：若干个(通常为1-20个，较佳地为1-10个，更佳地1-5个，最佳地1-3个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括Glyrichin蛋白的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱变突变体、在高或底的严紧度条件下能与Glyrichin多肽的抗血清获得的多肽和蛋白。本发明还提供了包含Glyrichin多肽或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了Glyrichin多肽的可溶性片段。通常，该片段具有Glyrichin多肽序列的至少约25个连续氨基酸，通常至少约35个连续氨基酸，更佳地至少约45个连续氨基酸，最佳地至少约60个连续氨基酸。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸丝氨酸、磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在本发明中，“Glyrichin蛋白保守性变异多肽”指与表1中序列1～14的氨基酸序列相比，有至多10个，较多地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表5进行氨基酸替换而产生。
表5 Glyrichin蛋白可取代氨基酸列表
| 最初的残基 | 代表性的取代 | 优选的取代 |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala：Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
本文所用“简并的变异体”在本发明中是指编码具有表1中序列1-14的蛋白质，但与表1中序列1-14中相应编码区序列有差别的核酸序列。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
在本发明中，术语“Glyrichin家族成员”、“Glyrichin蛋白”或“Glyrichin多肽”可相互换使用，都指具有天然抗菌肽Glyrichin家族各成员氨基酸序列(表1中序列1-14)的蛋白或多肽。它们包括含有或不含起始甲硫氨酸的天然抗菌肽Glyrichin家族成员，以及含有或不含有信号肽的Glyrichin成员蛋白。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或Glyrichin蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列表达或生产重组的Glyrichin多肽。一般来说有以下步骤：
(1)用本发明的编码Glyrichin多肽的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养的宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
本发明中，Glyrichin多核苷酸序列可插入到重组表达载体中，术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病逆转录病毒或其它载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含Glyrichin编码DNA序列和合适的转录/翻译控制信号的表达载体。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
本领域普通技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。获得的转化子可用常规方法培养，表达本发明的基因编码的多肽。在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。在使用本发明Glyrichin蛋白时，还可同时使用其它药剂，如青霉素等抗菌素。
发明人通过实验，首次发现了Glyrichin蛋白家族，得到了其保守序列，并证明了Glyrichin蛋白具有能抑制细菌生长的作用。Glyrichin蛋白可以应用于医药领域，以及食品防腐，鲜花保鲜，化妆品，种子包衣和动物饲料添加剂等方面，使用本发明的Glyrichin蛋白家族进行抗菌，具有抗菌活性高，抗菌谱广，靶菌株不易产生抗性突变等特点，有广阔的应用前景。
附图说明
图1为Glyrichin家族成员同源性比较图，左边为基因来源及注册号，右边为相应序列。
图2为Glyrichin家族成员同源性比较中的12条序列进化树分析图。
图3为保守的Glyrich结构域，保守的氨基酸用反显字表示。
图4为Glyrich结构域在不同成员间的序列一致性。
图5为Glyrichin家族的结构特点图。
图6为人源Glyrichin Northern blot分析和体外转录与翻译分析，其中
图6A为以hGlyrichin为探针检测其转录本大小和表达谱，
图6B为人源Glyrichin体外转录和翻译试验的结果。
图7为内源性诱导表达的h/mGlyrichin抑制了大肠杆菌BL21的生长图。其中
图7A为pET-22b(+)空载体，
图7B为pET-22b(+)-UBF和pET-22b(+)-PTP，
图7C为pET-22b-hGlyrichin阳性克隆1和8；
图7D为pET-22b-hGlyrichin阳性克隆1的两次重复实验结果。
图8为hGlyrichin原核表达产物纯化的PAGE电泳结果。其中泳道1为蛋白质分子量标准，泳道2为空载体诱导表达，泳道3为空载体未诱导表达，泳道4为hGlyrichin克隆基因诱导表达，泳道5为hGlyrichin克隆基因未诱导表达。
具体实施方式
下面以人源Glyrichin基因的克隆、表达、纯化和抗菌活性测试实施例为例，进一步阐述如何利用Glyrichin家族各成员本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人的分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1人源Glyrichin基因的制备及检测
1、人源Glyrichin基因完整ORF序列的获得
根据鼠源Glyrichin基因设计如下一对简并引物扩增人胎肝cDNA文库，获得人源Glyrichin完整的ORF序列共240bp，以及根据ORF序列推测的氨基酸序列：
5’引物Pa：5-CGATGCCGGTGGCCGTGGGTCCCT-3
3’引物Pb：5-TTAGCATCGTATGCCCATTCCA-3
PCR扩增条件为：94℃、4分钟、1循环；94℃、40秒，60℃、50秒，72℃、1分钟，30循环；72℃、7分钟、1循环。PCR产物经过Winzard PCRpreps purification kit纯化，用T4连接酶重组到pGEM-T载体中，然后转化大肠杆菌JM109，测序鉴定。
2、Northern blot分析
培养人4种肿瘤细胞株HepG2、HeLa、Jurket和HEK293，然后用Winzardplus RNA purification kit提取总RNA。各取20微克总RNA，在1.2％甲醛变性琼脂糖凝胶上分离，并转到Hybond-N+尼龙膜。HGlyrichin探针用Promega公司的Prime-a-gene试剂盒标记。杂交结果表明Glyrichin基因在所测试的4种不同组织来源的肿瘤细胞株中都有表达；同时揭示只存在一个转录本，且大小约为600bp。
3、体外转录和翻译试验
通过体外转录和翻译试验，证实了hGlyrichin基因编码的蛋白体外翻译后的大小约为8.8kDa，同理论推断的结果一致。将hGlyrichin全长的ORF序列通过下列引物，PCR扩增获得。
5’引物：5’-CGGGATCCCGATGCCGGTGGCCGTGGGTCCCT-3’
3’引物：5′-gctcgagttagcatcggatgcccatcc-3′
然后用BamHI和SacI酶切；将pT7载体用同样的酶切割，然后用T4 DNA连接酶连接，构建成功pT7-hGlyrichin表达载体。环状的载体在如下体系中进行转录和翻译反应：
25μl体系：
兔网织红细胞裂解液：12.5μl
反应缓冲液： 1.0μl
氨基酸混合物： 0.5μl
S35-Mat： 1.0μl(50μli)
DN 2.0μl(0.5μg/μl)
RNase inhibiton： 0.5μl
T7 polyase： 0.5μl
去离子水： 7.0μl
总体积： 25.0μl
30℃90分钟反应。反应结束后取5μl样品，加入10μl Loading Buffer，进行SDS-PAGE电泳，固定液中固定30分钟，干燥剂中浸泡5分钟，用干燥架固定凝胶，干燥凝胶过夜。放射自显影，-20℃压片24小时，洗片，观察结果如图6B。
实施例2 内源性诱导表达的h/mGlyrichin
对大肠杆菌BL21的生长抑制试验
将hGlyrichin不带任何标签(即不同任何蛋白融合)重组到原核表达载体pET-22b(+)的EcoRI和XhoI位点之间，转化大肠杆菌BL21，通过酶切鉴定出阳性克隆。挑取阳性克隆1和8，接种到AMP抗性的液体LB培养基中，于37℃摇床过夜；次日以1∶100接种，继续摇菌至OD值为0.03时，向每只试管中加入IPTG至终浓度为0.5mM，阴性对照组则加入相应体积的PBS；于30℃、250rpm摇菌，每隔45分钟取出1毫升菌液测OD600值，连续测20次。以时间为横轴，OD600对数值为纵轴做生长曲线。图7A为转化了pET-22b(+)空载体的BL21在0.5mM IPTG诱导和不诱导条件下的半对数生长曲线；图7B为转化了不具抗菌活性的无关基因pET-22b(+)-UBF和pET-22b(+)-PTP的BL21在0.5mM IPTG诱导和不诱导条件下的半对数生长曲线；图7C为转化了待测的pET-22b(+)-hGlyrichin阳性克隆1和8的BL21在0.5mM IPTG诱导和不诱导条件下的半对数生长曲线；图7D为待测的pET-22b(+)-hGlyrichin阳性克隆1的两次重复。实验结果表明在转入Glyrichin后，在IPTG诱导表达Glyrichin后，细菌的生长明显受到抑制；随着培养时间延长IPTG消耗殆尽，细菌的生长又得以恢复，而其他测试基因都没有此现象，充分说明Glyrichin蛋白本身具有抗菌活性。
实施例3游离hGlyrichin表达产物的纯化及杀菌活性检测
根据小鼠Glyrichin的cDNA序列设计引物，在引物中引入Enterokinase的酶切位点，以人胎肝mRNA进行RT-PCR扩增，纯化后PCR产物经测序和限制性内切酶消化后在T4连接酶的作用下，插入到用同样内切酶消化后的pGEX4-4T2原核表达载体中，经转化JM109大肠杆菌后，以IPTG进行体外诱导表达5小时，收集菌体；用PBS重悬，反复冻融裂解；于4℃、12000rpm离心20分钟，取上清进行SDS-PAGE电泳。确定GST-Glyrichin融合蛋白在上清中表达后，用GST亲和柱纯化，然后用中酶切下来的游离hGlyrichin蛋白具有杀菌活性。如上所述诱导表达得到GST-Glyrichin融合蛋白，经GST纯化柱纯化后用Enterokinase切割，得到游离的hGlyrichin蛋白，用于最小抑菌浓度(MIC)测定。在抗菌活性测定中，稀释菌液的浓度为104-105CFU/ml，按每孔80微升菌液接种于96孔板中，将多肽以一定比例稀释，每孔加入5微升；将96孔板之于37℃培养过夜，紫外分析仪检测OD600值，结果如图8所示，游离的Glyrichin具有在较低浓度下抑制细菌生长的活性，而GST-Glyrichin融合蛋白没有抑制细菌生长的活性。
Claims (9)
1、一个蛋白质家族在医药领域、化妆品、食品、畜禽水产饲料、种子包衣以及提高转基因动植物抗病虫害的能力中的应用，所说的蛋白质家族包括具有下列GenBankTM注册号的蛋白质：CAC11117，AAK18748，AAH59661，XP_320383，NP_648757，NP_496804，B88349，CAB89006，AAB68201，NP_566324，NP_705421，EAA17587，EAA47634，EAA34843。
2、根据权利要求1所述的用途，其特征在于用于制备细菌性感染疾病的治疗药物。
3、根据权利要求1所述的用途，其特征在于用于筛选权利要求1所述蛋白质家族中蛋白质的配体。
4、根据权利要求1所述的用途，其特征在于用于化妆品添加剂。
5、根据权利要求1所述的用途，其特征在于用于食品防腐剂。
6、根据权利要求1所述的用途，其特征在于用于畜禽或水产饲料添加剂。
7、根据权利要求1所述的用途，其特征在于用于种子包衣。
8、根据权利要求1所述的用途，其特征在于用于提高转基因植物或动物抗病虫害的能力。
9、根据权利要求1所述的用途，其特征在于用于鲜花保鲜。
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| US5221732A (en) * | 1988-12-06 | 1993-06-22 | The United States Of America As Represented By The Department Of Health And Human Services | Antimicrobial magainin modified peptides |
| WO1999043802A2 (en) * | 1998-02-27 | 1999-09-02 | Sagami Chemical Research Center | HUMAN PROTEINS HAVING TRANSMEMBRANE DOMAINS AND DNAs ENCODING THESE PROTEINS |
| US7060479B2 (en) * | 1999-12-08 | 2006-06-13 | Serono Genetics Institute, S.A. | Full-length human cDNAs encoding potentially secreted proteins |
| WO2001060860A2 (en) * | 2000-02-17 | 2001-08-23 | Millennium Predictive Medicine, Inc. | Genes differentially expressed in human prostate cancer and their use |
| AU2002220079A1 (en) * | 2000-11-15 | 2002-05-27 | Emory University | Acylated antimicrobial peptides |
-
2003
- 2003-12-11 CN CNB2003101173546A patent/CN100484568C/zh not_active Expired - Fee Related
-
2004
- 2004-12-10 JP JP2006543349A patent/JP2008504003A/ja active Pending
- 2004-12-10 US US10/582,420 patent/US20070054356A1/en not_active Abandoned
- 2004-12-10 WO PCT/CN2004/001435 patent/WO2005056591A1/zh active Application Filing
- 2004-12-10 EP EP04802449A patent/EP1693381A4/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| WO2005056591A1 (fr) | 2005-06-23 |
| EP1693381A1 (en) | 2006-08-23 |
| US20070054356A1 (en) | 2007-03-08 |
| CN100484568C (zh) | 2009-05-06 |
| JP2008504003A (ja) | 2008-02-14 |
| EP1693381A4 (en) | 2007-10-24 |
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