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CN100432107C - Protein for promoting erythrocyte growth factor activity and its use - Google Patents

Protein for promoting erythrocyte growth factor activity and its use Download PDF

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CN100432107C
CN100432107C CNB2005100867084A CN200510086708A CN100432107C CN 100432107 C CN100432107 C CN 100432107C CN B2005100867084 A CNB2005100867084 A CN B2005100867084A CN 200510086708 A CN200510086708 A CN 200510086708A CN 100432107 C CN100432107 C CN 100432107C
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epo
pleckstrin homology
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CN1955192A (en
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来鲁华
曹傲能
刘森
刘士勇
常智杰
王银银
程龙
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Peking University
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Abstract

本发明涉及一种具有促红细胞生长因子活性的蛋白质及其制备方法。该蛋白质是在保持pleckstrin同源性结构域的三级结构不变的情况下,将其氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加衍生而来,具有促红细胞生长因子的活性。本发明的技术方案是利用蛋白质功能嫁接方法将红细胞生成素EPO的功能嫁接到pleckstrin同源性结构域骨架上,进行突变体设计、表达及纯化,并通过实验测定突变体与EPO受体的结合能力和EPO活性。本发明的蛋白质可作为EPO替代品,用于治疗贫血,慢性肾功能衰竭(CRF),HIV感染/ZDU治疗病人,类风湿性关节炎,癌性贫血以及其它任何采用EPO治疗的症状。The invention relates to a protein with erythrocyte growth-stimulating factor activity and a preparation method thereof. The protein is derived from its amino acid sequence by substituting, deleting or adding one or several amino acid residues while keeping the tertiary structure of the pleckstrin homology domain unchanged, and has the activity of erythrocyte growth-stimulating factor . The technical scheme of the present invention is to use the protein function grafting method to graft the function of erythropoietin EPO onto the skeleton of the pleckstrin homology domain, carry out mutant design, expression and purification, and measure the binding of the mutant to the EPO receptor through experiments capacity and EPO activity. The protein of the present invention can be used as an EPO substitute for the treatment of anemia, chronic renal failure (CRF), HIV infection/ZDU treatment patients, rheumatoid arthritis, cancerous anemia and any other symptoms treated with EPO.

Description

具有促红细胞生长因子活性的蛋白质及其用途 Proteins with erythrocyte growth-stimulating factor activity and uses thereof

技术领域 technical field

本发明属于生物技术领域,涉及具有促红细胞生长因子活性的蛋白质,具体涉及广泛存在于生物体内的pleckstrin同源性结构域及其具有促红细胞生长因子活性的突变体。The invention belongs to the field of biotechnology and relates to a protein with erythrocyte growth-stimulating factor activity, in particular to a pleckstrin homology domain widely present in organisms and a mutant with erythrocyte growth-stimulating factor activity.

背景技术 Background technique

蛋白质是生物体中完成各种生命功能的主要物质。蛋白质结构和功能规律的研究是生命科学的中心课题之一,它不仅包括对天然蛋白质结构和功能规律分析研究,还包括利用现有知识进行蛋白质设计。蛋白质设计的最终目标是从头设计具有任意指定结构和功能的蛋白质。无论是使已有的蛋白质具有新的功能还是在全新设计的蛋白质中引入功能,对于研究蛋白质结构与功能的关系都具有十分重要的理论意义和潜在的应用价值。蛋白质功能设计包括蛋白质功能的改造和新功能的开发。我们发展了适用于非同源非连续位点的蛋白质功能嫁接方法,并应用于促红细胞生成素(EPO)功能的嫁接。Protein is the main substance that completes various life functions in organisms. The study of protein structure and function rules is one of the central topics of life sciences, which includes not only the analysis of natural protein structure and function rules, but also the use of existing knowledge for protein design. The ultimate goal of protein design is to design proteins with arbitrary specified structures and functions de novo. Whether making existing proteins have new functions or introducing functions into newly designed proteins, it has very important theoretical significance and potential application value for studying the relationship between protein structure and function. Protein function design includes the transformation of protein function and the development of new functions. We developed a protein-function grafting method applicable to non-homologous discontinuous sites and applied it to the grafting of erythropoietin (EPO) function.

EPO是促进红系祖细胞分化、增殖并形成红细胞的主要激素。重组人EPO临床上用于治疗贫血,慢性肾功能衰竭(CRF),HIV感染/ZDU治疗病人,类风湿性关节炎,癌性贫血等症状。最新的研究表明,EPO在神经和大脑发育中具有非常重要的作用。由于避免了过去常采用的输血治疗方法,重组人EPO产生了巨大的社会和经济效益。目前,全世界重组EPO年销售额高达数十亿美元。但是在临床上使用重组EPO的唯一方法是通过静脉或皮下注射,且要多次重复使用才能达到治疗目的。此外,用于生产重组EPO的哺乳动物细胞的培养成本很高,同时表达量不高,因而使用EPO治疗是相当昂贵的。因此,如果能够找到一种易于表达的蛋白质骨架,将EPO的功能位点嫁接到上面,那么就有可能成为重组人EPO的替代品,满足市场需求。EPO is the main hormone that promotes the differentiation, proliferation and formation of erythrocytes from erythroid progenitor cells. Recombinant human EPO is clinically used to treat anemia, chronic renal failure (CRF), HIV infection/ZDU treatment patients, rheumatoid arthritis, cancerous anemia and other symptoms. The latest research shows that EPO plays a very important role in nerve and brain development. Recombinant human EPO has produced enormous social and economic benefits due to the avoidance of blood transfusion therapy, which was commonly used in the past. At present, the annual sales of recombinant EPO around the world are as high as billions of dollars. But the only way to use recombinant EPO clinically is through intravenous or subcutaneous injection, and it needs to be used repeatedly to achieve the therapeutic purpose. In addition, the mammalian cells used to produce recombinant EPO are expensive to culture and have low expression levels, so treatment with EPO is quite expensive. Therefore, if an easy-to-express protein backbone can be found and the functional sites of EPO grafted onto it, it may become a substitute for recombinant human EPO and meet market demand.

PH结构域是一种存在于多种信号蛋白和细胞骨架相关蛋白中的大约由120个氨基酸组成的功能性区域,在许多物种、器官以及不同蛋白中都有分布。由于它们与pleckstrin(血小板蛋白激酶C(PKC)的一个主要底物)中的一段重复序列具有同源性,所以被称为Pleckstrin Homology(PH)结构域。不同蛋白质中的PH结构域在一级结构上同源性并不一定很高,但是通过对其空间结构的研究发现,其肽链主链折叠方式基本相同,三级结构非常保守。关于该结构域本身的功能,目前主要认为其作用是通过与细胞膜结合,从而将同一蛋白其他成分定位在细胞膜上,可能还具有与SH2和SH3结构域类似的介导信号分子间相互作用的功能。由于该结构域的三级结构高度保守,所以非常便于在不改变其三级结构的基础上进行功能改造。The PH domain is a functional region consisting of approximately 120 amino acids that exists in a variety of signaling proteins and cytoskeleton-related proteins, and is distributed in many species, organs, and different proteins. Because of their homology to a repeat sequence in pleckstrin, a major substrate of platelet protein kinase C (PKC), they are called Pleckstrin Homology (PH) domains. The homology of the primary structure of the PH domains in different proteins is not necessarily very high, but through the study of their spatial structure, it is found that the main chain of the peptide chain has basically the same folding method, and the tertiary structure is very conservative. Regarding the function of the domain itself, it is currently believed that its function is to bind to the cell membrane, thereby positioning other components of the same protein on the cell membrane, and may also have the function of mediating the interaction between signaling molecules similar to the SH2 and SH3 domains . Since the tertiary structure of this domain is highly conserved, it is very convenient to carry out functional modification without changing its tertiary structure.

蛋白质与蛋白质的相互作用界面比较大。它们之间的接触面积往往在

Figure C20051008670800041
之间,作用界面涉及20-60个氨基酸残基。这些氨基酸残基所起的作用是不同的。定点突变表明,对结合有重大影响的只有少数几个残基(通常是结合的双方各提供3-4个关键残基),但仅仅考虑起重要作用的氨基酸显然是不够的。我们发展了一套将序列上非连续的蛋白质-蛋白质相互作用区嫁接到非同源骨架蛋白上的切实可行的策略(Biopolymers,54:515-523,2000)。这种方法是在已知配体蛋白和受体蛋白复合物晶体结构的前提下,基于强相互作用残基的Cα、Cβ原子和重要原子的坐标,将骨架蛋白叠加到配体-受体复合物中的配体蛋白上,评估骨架蛋白与受体的互补性并留下互补性好的骨架蛋白。调节骨架蛋白与受体的相对位置,使界面具有合理的堆积密度,并作其它必要的突变。具体算法分为四个步骤:搜索有合适嫁接位点的骨架蛋白;分子叠合及几何互补性评估;叠合构象的取向微调;结合位点的突变、优化及评估。The protein-protein interaction interface is relatively large. The contact area between them is often
Figure C20051008670800041
Between, the interaction interface involves 20-60 amino acid residues. The roles played by these amino acid residues are different. Site-directed mutagenesis has shown that only a few residues have a significant impact on binding (usually 3-4 key residues are provided by each side of the binding), but it is obviously not enough to consider only the amino acids that play an important role. We developed a practical strategy for grafting sequence non-contiguous protein-protein interaction domains onto non-homologous scaffold proteins (Biopolymers, 54:515-523, 2000). This method is based on the coordinates of the C α , C β atoms and important atoms of the strongly interacting residues on the premise that the crystal structure of the ligand protein and receptor protein complex is known, and the skeleton protein is superimposed on the ligand-receptor protein complex. On the ligand protein in the receptor complex, the complementarity of the scaffold protein to the receptor is evaluated and the good complementarity protein is left. Adjust the relative position of the skeleton protein and the receptor, make the interface have a reasonable packing density, and make other necessary mutations. The specific algorithm is divided into four steps: search for a backbone protein with a suitable grafting site; evaluation of molecular alignment and geometric complementarity; fine-tuning of the orientation of the alignment conformation; mutation, optimization, and evaluation of the binding site.

利用上述方法可以将所需的蛋白功能位点嫁接到合适的蛋白质骨架上,从而获得更便于应用的新的功能蛋白质。Using the above method, the required protein functional sites can be grafted onto the appropriate protein backbone, so as to obtain new functional proteins that are more convenient for application.

发明内容 Contents of the invention

本发明的目的是找到具有类似EPO活性的蛋白质突变体,用于治疗贫血、慢性肾功能衰竭等EPO适用症。The purpose of the present invention is to find protein mutants with similar EPO activity for treating EPO applicable diseases such as anemia and chronic renal failure.

本发明的技术方案是利用蛋白质功能嫁接方法寻找能够嫁接EPO功能的合适蛋白质骨架,进行突变体设计、表达及纯化,利用表面等离子体共振技术测定蛋白质突变体与EPO受体的结合活性,并通过细胞实验测定突变体的EPO活性。The technical scheme of the present invention is to use the protein function grafting method to find a suitable protein skeleton capable of grafting EPO functions, carry out mutant design, expression and purification, use surface plasmon resonance technology to measure the binding activity of protein mutants and EPO receptors, and pass Cell experiments were used to measure the EPO activity of the mutants.

按照以上方案进行蛋白质嫁接设计,本发明找到pleckstrin同源性蛋白质结构域(PH结构域)作为嫁接EPO功能的蛋白质骨架。在保持pleckstrin同源性结构域的三级结构不变的情况下,将其氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加可得到具有促红细胞生长因子活性的PH结构域衍生蛋白质。The protein grafting design is carried out according to the above scheme, and the present invention finds the pleckstrin homologous protein domain (PH domain) as the protein skeleton for grafting the EPO function. In the case of keeping the tertiary structure of the pleckstrin homology domain unchanged, its amino acid sequence can be substituted, deleted or added by one or several amino acid residues to obtain a PH domain-derived protein with erythrocyte growth factor activity .

PH结构域是一种存在于多种信号转导蛋白及细胞骨架蛋白中的约由120个氨基酸残基组成的功能性区域,但PH结构域与EPO受体的作用未见报导。文献表明,PH结构域是一种三级结构上非常保守的蛋白(Cell 120:574-576,2005;Biochem Soc Trans.32:707-711,2004)。用关键词PLECKSTRIN HOMOLOGY DOMAIN,搜索PDB数据库(Protein Data Bank),得到43个具有与1mai(大鼠磷脂酶Cδ1蛋白PH结构域)相似结构的含PH结构域的蛋白。通过结构比对发现,所有这些PH结构域的三维结构非常保守。因此,适合于构建本发明的具有促红细胞生长因子活性的蛋白质的PH结构域广泛存在于生物体内。The PH domain is a functional region consisting of about 120 amino acid residues present in various signal transduction proteins and cytoskeleton proteins, but the role of the PH domain and EPO receptors has not been reported. Literature shows that the PH domain is a protein with a very conserved tertiary structure (Cell 120:574-576, 2005; Biochem Soc Trans. 32:707-711, 2004). Using the keywords PLECKSTRIN HOMOLOGY DOMAIN to search the PDB database (Protein Data Bank), 43 proteins containing a PH domain with a structure similar to that of 1mai (rat phospholipase Cδ1 protein PH domain) were obtained. Through structural comparison, it was found that the three-dimensional structures of all these PH domains are very conserved. Therefore, the PH domain suitable for constructing the protein having erythrocyte growth-stimulating factor activity of the present invention widely exists in organisms.

目前,对磷脂酶C PH结构域的研究较为深入,特别是大鼠磷脂酶Cδ1蛋白的PH结构域的晶体结构已经解析出来。通过计算我们发现,在与EPO受体结合的界面上,大鼠磷脂酶Cδ1蛋白的PH结构域上的以下氨基酸残基很重要:18,23,24,41,43,45,46,47,49,50,51,52,56,57,58,59,60,61,62,63,64,65,66,67,68,70,71,72,73,77,80,93,94,101,102,103,104,105,106,119,123,126,127,129,130。这些残基参与了与EPO受体相关残基的直接作用,形成静电力、氢键、范德华力或者疏水堆积等,对两者的结合起到了非常重要的作用。例如根据计算结果突变后的PH结构域上的Asn47能够与EPO受体上的His114形成氢键,Arg49能与EPO受体上的Glu117形成静电作用,Phe63能与EPO受体上的Phe93形成疏水作用,Thr50可能与EPO受体上的Phe93形成疏水作用等等。因此对所有这些残基的突变都有可能改变(提高或者降低)两者的相互作用强度。本发明重点选取了在大鼠的磷脂酶Cδ1蛋白的PH结构域的63,47,49,46等位置上进行氨基酸残基替换,得到了下述具有类似EPO活性的蛋白质突变体:E63F、D47N、K49R、E63F-D47N、E63F-K49R、D47N-K49R、E63F-D47N-K49R和E63F-D47N-K49R-E46A。At present, the research on the PH domain of phospholipase C is more in-depth, especially the crystal structure of the PH domain of rat phospholipase Cδ1 protein has been resolved. We found by calculation that the following amino acid residues on the PH domain of the rat phospholipase Cδ1 protein are important at the interface with the EPO receptor: 18, 23, 24, 41, 43, 45, 46, 47, 49, 50, 51, 52, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 77, 80, 93, 94, 101, 102, 103, 104, 105, 106, 119, 123, 126, 127, 129, 130. These residues participate in the direct interaction with EPO receptor-related residues, forming electrostatic force, hydrogen bond, van der Waals force or hydrophobic stacking, etc., which play a very important role in the combination of the two. For example, according to the calculation results, Asn47 on the mutated PH domain can form a hydrogen bond with His114 on the EPO receptor, Arg49 can form an electrostatic interaction with Glu117 on the EPO receptor, and Phe63 can form a hydrophobic interaction with Phe93 on the EPO receptor , Thr50 may form a hydrophobic interaction with Phe93 on the EPO receptor and so on. Mutations to all of these residues therefore have the potential to alter (increase or decrease) the strength of the interaction between the two. In the present invention, the amino acid residues are replaced at positions 63, 47, 49, and 46 of the PH domain of the rat phospholipase Cδ1 protein, and the following protein mutants with similar EPO activity are obtained: E63F, D47N , K49R, E63F-D47N, E63F-K49R, D47N-K49R, E63F-D47N-K49R, and E63F-D47N-K49R-E46A.

上述63,47,49,46位置还可以突变为其他的氨基酸及其衍生物。除了这四个位置以外,PH结构域上的其它位置也可以进行氨基酸及其衍生物的替代、缺失或添加,只要这些突变加强了PH结构域与EPO受体的相互作用,这样的突变体蛋白就有可能具有类似EPO的活性。The above positions 63, 47, 49 and 46 can also be mutated into other amino acids and their derivatives. In addition to these four positions, other positions on the PH domain can also undergo substitutions, deletions or additions of amino acids and their derivatives, as long as these mutations strengthen the interaction between the PH domain and the EPO receptor, such mutant proteins It is possible to have an activity similar to EPO.

本发明的具有促红细胞生长因子活性的PH结构域衍生蛋白质可以通过以下步骤制备:The PH domain-derived protein with erythrocyte growth-stimulating factor activity of the present invention can be prepared by the following steps:

(1)用PCR的方法扩增得到pleckstrin同源性结构域的基因,并连入合适的表达载体,获得表达pleckstrin同源性结构域的质粒;(1) amplifying the gene of the pleckstrin homology domain by PCR, and connecting it into a suitable expression vector to obtain a plasmid expressing the pleckstrin homology domain;

(2)利用蛋白质功能嫁接方法进行突变体设计;(2) Mutant design by protein function grafting method;

(3)通过引物引入突变,PCR扩增上述表达质粒,得到含有pleckstrin同源性结构域的突变基因的表达质粒;(3) introducing mutations by primers, amplifying the above-mentioned expression plasmids by PCR, and obtaining expression plasmids of mutant genes containing pleckstrin homology domains;

(4)将上述质粒转入原核或真核宿主中,在适宜的条件下表达目的蛋白;(4) Transfer the above-mentioned plasmid into a prokaryotic or eukaryotic host, and express the target protein under suitable conditions;

(5)用适当的方法收集和处理菌体、组织或细胞,得到目的蛋白的粗提物;(5) Collect and process the bacteria, tissue or cells with appropriate methods to obtain the crude extract of the target protein;

(6)用亲和柱、体积排阻柱等方法获得纯化的目的蛋白。(6) Obtain purified target protein by methods such as affinity column and size exclusion column.

本发明利用表面等离子体共振方法测定了PH结构域突变体与EPO受体的结合常数。表面等离子体共振方法是公认的用于测定蛋白质-蛋白质结合常数的通用方法,具有蛋白质用量少、灵敏、结合常数测定准确的特点。The present invention uses the surface plasmon resonance method to measure the binding constant of the PH domain mutant and the EPO receptor. The surface plasmon resonance method is recognized as a general method for the determination of protein-protein binding constants. It has the characteristics of less protein consumption, sensitivity, and accurate determination of binding constants.

同时,本发明利用293T细胞体系,测定了PH结构域突变体激活EPO受体信号通路的作用。实验采用的是PathDetect in vivo signal transduction pathwaytrans-reporting system(购自Stratagene公司),荧光酶活性检测采用的是luciferaseassay system(购自Promega公司).At the same time, the present invention uses the 293T cell system to measure the effect of the PH domain mutant on activating the EPO receptor signaling pathway. The experiment used the PathDetect in vivo signal transduction pathway trans-reporting system (purchased from Stratagene), and the luciferase activity detection used the luciferase assay system (purchased from Promega).

本发明的pleckstrin同源结构域相关突变体有可能作为EPO替代品,用于治疗治疗贫血,慢性肾功能衰竭(CRF),HIV感染/ZDU治疗病人,类风湿性关节炎,癌性贫血以及其它任何采用EPO治疗的症状。该类具有EPO活性的突变体以及其衍生形式蛋白的任何性质的商业化生产和应用在本发明的保护范围之内。The pleckstrin homology domain-related mutants of the present invention may be used as EPO substitutes for the treatment of anemia, chronic renal failure (CRF), HIV infection/ZDU treatment patients, rheumatoid arthritis, cancerous anemia and others Any symptoms treated with EPO. The commercial production and application of any properties of such mutants with EPO activity and their derivative forms fall within the protection scope of the present invention.

具体实施方式: Detailed ways:

为了更清楚地说明本发明,列举以下实施例,但其对本发明的范围无任何限制。In order to illustrate the present invention more clearly, the following examples are cited, but they do not limit the scope of the present invention in any way.

进行实验时,所采用的操作方法和操作步骤以及反应条件等,都是依据本技术领域的普通技术人员熟知的方法设计、实施的。When carrying out the experiment, the operating methods, operating steps and reaction conditions adopted were all designed and implemented according to methods well known to those of ordinary skill in the art.

实施例1:PH结构域突变位点的选择Example 1: Selection of PH domain mutation sites

我们将本实验室发展的蛋白功能嫁接计算策略(Biopolymers,54:515-523,2000)用于EPO表面功能区嫁接的计算。将EPO(Protein Data Bank代码leer)与其受体EPOR相互作用界面的关键残基:48位PHE,147位ASN,150位ARG作为嫁接的关键残基。我们使用基于矢量匹配的嫁接策略将蛋白质关键结合位点嫁接转移到另一个非同源的蛋白质上,成功地找到了用于嫁接EPO功能的候选蛋白骨架PH结构域(Protein Data Bank代码是lmai)。通过我们根据这一策略编写的一个计算机程序计算得到这个骨架的互补性打分是561,关键残基的均方要偏差为0.843埃。需要突变的三个关键残基对应如下:We used the calculation strategy of protein function grafting developed in our laboratory (Biopolymers, 54:515-523, 2000) for the calculation of functional region grafting on the surface of EPO. The key residues at the interaction interface between EPO (Protein Data Bank code leer) and its receptor EPOR: 48-position PHE, 147-position ASN, and 150-position ARG are used as key residues for grafting. We used a grafting strategy based on vector matching to transfer the key binding site of the protein to another non-homologous protein, and successfully found the candidate protein backbone PH domain for grafting EPO function (Protein Data Bank code is lmai) . Calculated by a computer program we wrote according to this strategy, the complementarity score of this backbone is 561, and the mean square deviation of key residues is 0.843 angstroms. The three key residues that need to be mutated correspond to the following:

63位GLU→PHE;47位ASP→ASN;49位LYS→ARG63-bit GLU→PHE; 47-bit ASP→ASN; 49-bit LYS→ARG

同时,该程序建议将46位GLU突变为较小的残基,如GLY。At the same time, the program suggested mutating GLU at position 46 to a smaller residue such as GLY.

上面四个突变之后,互补分数变为571,堆积密度为0.729,埋藏面积约为1610平方埃,疏水互补打分为85。这些数据表明该蛋白经过上述突变后能够与EPO受体形成理想的结合界面。After the above four mutations, the complementary score becomes 571, the packing density is 0.729, the buried area is about 1610 square angstroms, and the hydrophobic complementary score is 85. These data indicate that the protein can form an ideal binding interface with the EPO receptor after the above mutations.

实施例2:PH结构域突变体的构建、表达与纯化Example 2: Construction, expression and purification of PH domain mutants

一、基因扩增与表达载体构建1. Gene amplification and expression vector construction

大鼠(Rat)磷脂酶Cδ1(Phospholipase C Delta 1)的PH结构域(残基1-140;ProteinData Bank代码为1mai)的基因通过引物对1(引物序列如表1所示)以pGST4/PLCδ1质粒(Hitoshi Yagisawa教授赠送并授权;Eur.J.Biochem.265:481-490,1999)为模板,采取聚合酶链式反应(Polymerase Chain Reaction,PCR)扩增。扩增产物经纯化后用NdeI和EcoRI双酶切,酶切产物通过T4DNA连接酶连入经NdeI和EcoRI双酶切的pET-28a载体(购自Novagen公司)中,得到表达载体pETPHD。经DNA测序检验序列正确。The gene of the PH domain (residues 1-140; ProteinData Bank code is 1mai) of rat (Rat) phospholipase Cδ1 (Phospholipase C Delta 1) was converted to pGST4/PLCδ1 by primer pair 1 (primer sequence is shown in Table 1) Plasmid (gifted and authorized by Professor Hitoshi Yagisawa; Eur.J.Biochem.265:481-490, 1999) was used as a template and amplified by Polymerase Chain Reaction (PCR). After purification, the amplified product was double-digested with NdeI and EcoRI, and the digested product was ligated into the pET-28a vector (purchased from Novagen) that had been double-digested with NdeI and EcoRI by T4 DNA ligase to obtain the expression vector pETPHD. The correct sequence was verified by DNA sequencing.

表1.引物设计Table 1. Primer Design

Figure C20051008670800081
Figure C20051008670800081

注:____标志为内切酶位点。CATATG为NdeI位点;GAATTC为EcoRI位点;GGATCC为BamHI位点;TCTAGA为XbaI位点。Note: ____ mark is the endonuclease site. CATATG is NdeI site; GAATTC is EcoRI site; GGATCC is BamHI site; TCTAGA is XbaI site.

二、PH结构域突变体的构建2. Construction of PH domain mutants

1、E63F突变体的构建1. Construction of E63F mutant

以pETPHD为模板,用引物对2(序列见表1)和PfuUltraHigh-Fidelity Polymerase(购自Stratagene公司)采用PCR方法突变。突变后的产物用DpnI酶酶切切断含有甲基化位点的模板载体,得到完整的没有甲基化位点的突变体载体(参见《分子克隆手册》第三版,美国冷泉港实验室出版)。Using pETPHD as a template, primer pair 2 (see Table 1 for the sequence) and PfuUltraHigh-Fidelity Polymerase (purchased from Stratagene) were used to mutate by PCR. The mutated product was digested with DpnI enzyme to cut off the template vector containing the methylation site to obtain a complete mutant vector without the methylation site (see the third edition of "Molecular Cloning Handbook", published by Cold Spring Harbor Laboratory, USA ).

2、E63F-D47N突变体的构建2. Construction of E63F-D47N mutant

方法同上述E63F突变体的构建方法,所用模板为E63F突变体,引物为引物对3(序列见表1)。The method is the same as the above-mentioned construction method of the E63F mutant, the template used is the E63F mutant, and the primer is primer pair 3 (see Table 1 for the sequence).

3、E63F-K49R突变体的构建3. Construction of E63F-K49R mutant

方法同上述E63F突变体的构建方法,所用模板为E63F突变体,引物为引物对4(序列见表1)。The method is the same as the above-mentioned construction method of the E63F mutant, the template used is the E63F mutant, and the primer is primer pair 4 (see Table 1 for the sequence).

4、E63F-D47N-K49R突变体的构建4. Construction of E63F-D47N-K49R mutant

方法同上述E63F突变体的构建方法,所用模板为E63F-K49R突变体,引物为引物对5(序列见表1)。The method is the same as the above-mentioned construction method of the E63F mutant, the template used is the E63F-K49R mutant, and the primer is primer pair 5 (see Table 1 for the sequence).

5、D47N突变体的构建5. Construction of D47N mutant

方法同上述E63F突变体的构建方法,所用模板为pETPHD,引物为引物对3(序列见表1)。The method is the same as the above-mentioned construction method of the E63F mutant, the template used is pETPHD, and the primer is primer pair 3 (see Table 1 for the sequence).

6、K49R突变体的构建6. Construction of K49R mutant

方法同上述E63F突变体的构建方法,所用模板为pETPHD,引物为引物对4(序列见表1)。The method is the same as the above-mentioned construction method of the E63F mutant, the template used is pETPHD, and the primer is primer pair 4 (see Table 1 for the sequence).

7、D47N-K49R突变体的构建7. Construction of D47N-K49R mutant

方法同上述E63F突变体的构建方法,所用模板为K49R突变体,引物为引物对5(序列见表1)。The method is the same as the above-mentioned construction method of the E63F mutant, the template used is the K49R mutant, and the primer is primer pair 5 (see Table 1 for the sequence).

8、E63F-D47N-K49R-E46A突变体的构建8. Construction of E63F-D47N-K49R-E46A mutant

(1)第一步PCR(1) The first step of PCR

模板为E63F-D47N-K49R突变体,反应A的引物为1号正向引物和6号反向引物,反应B的引物为1号反向引物和6号正向引物(引物序列见表1),其他成分相同。A和B的产物经纯化后用于第二步PCR。The template is the E63F-D47N-K49R mutant, the primers of reaction A are forward primer No. 1 and reverse primer No. 6, and the primers of reaction B are reverse primer No. 1 and forward primer No. 6 (see Table 1 for the primer sequence) , the other components are the same. The products of A and B were purified for the second step of PCR.

(2)第二步PCR(2) The second step of PCR

反应A和B的产物等摩尔浓度混合后,用1号正向引物和1号反向引物进行PCR。产物纯化后用于下一步实验。After the products of reactions A and B were mixed at equimolar concentrations, PCR was carried out with forward primer No. 1 and reverse primer No. 1. The product was purified and used in the next experiment.

(3)双酶切并连入载体(3) Double digestion and ligation into the vector

步骤(2)的产物经NdeI和EcoRI双酶切后,用T4DNA连接酶连入经双酶切的pET-28a载体中,得到突变体。The product of step (2) was double digested with NdeI and EcoRI, and then ligated into the double digested pET-28a vector with T4 DNA ligase to obtain a mutant.

上述1-8所有突变体均经过DNA测序确证。All the above-mentioned 1-8 mutants were confirmed by DNA sequencing.

三、蛋白表达与纯化3. Protein expression and purification

pETPHD载体被转化到大肠杆菌BL21(DE3)pLys(购自Novagen公司)菌株中表达目的蛋白(NH-PHD)。挑取单克隆或者吸取100μL菌种接到50mL含30μg·mL-1硫酸卡那霉素和34μg·mL-1氯霉素的LB液体培养基中,于摇床上37℃,220转/分钟震荡培养过夜。然后以1∶100的比例将过夜培养基转接到1L含同样量抗生素的新鲜LB液体培养基中,于摇床上37℃,220转/分钟震荡培养至OD600为0.6-1.0。然后加入IPTG至终浓度为0.5mM,于30℃,220转/分钟震荡培养4个小时后离心收集细胞(4℃,5000g,15min)。弃去上清,然后用40mL 0.8%的NaCl水溶液重新悬浮细胞,并再次离心沉淀细胞(4℃,4000g,20min)。弃去上清后得到的细胞可以进入下一步纯化实验或者置于-70℃冰箱放置待用。The pETPHD vector was transformed into Escherichia coli BL21(DE3)pLys (purchased from Novagen) strain to express the target protein (NH-PHD). Pick a single clone or pipette 100 μL of strains into 50 mL of LB liquid medium containing 30 μg·mL -1 kanamycin sulfate and 34 μg·mL -1 chloramphenicol, shake on a shaker at 37°C and 220 rpm Incubate overnight. Then the overnight culture medium was transferred to 1 L of fresh LB liquid medium containing the same amount of antibiotics at a ratio of 1:100, and cultured on a shaker at 37°C at 220 rpm until the OD600 was 0.6-1.0. Then IPTG was added to a final concentration of 0.5 mM, and the cells were collected by centrifugation at 30° C., 220 rpm for 4 hours, and then collected by centrifugation (4° C., 5000 g, 15 min). The supernatant was discarded, and then the cells were resuspended with 40 mL of 0.8% NaCl aqueous solution, and the cells were centrifuged again (4° C., 4000 g, 20 min). The cells obtained after discarding the supernatant can be used in the next purification experiment or placed in a -70°C refrigerator for later use.

纯化时,先将上述得到的细胞用裂解液(50mM Na2HP04/NaH2P04,300mM NaCl,10mMImidazole,pH 7.0)悬浮,裂解液用量为5-10mL/g细胞。加入1mM PMSF(Phenylmethylsulphonylfluoride)后,用超声波(3s,5s,300W,90次)重复2-3次裂解细胞至溶液透明无混浊。离心(4℃,40000g,30min)后保留上清。上清用0.22μm滤膜过滤后上Ni-NTA柱纯化。When purifying, first suspend the cells obtained above with lysate (50mM Na2HPO4/NaH2P04, 300mM NaCl, 10mM midazole, pH 7.0), the amount of lysate used is 5-10mL/g cells. After adding 1mM PMSF (Phenylmethylsulphonylfluoride), use ultrasonic waves (3s, 5s, 300W, 90 times) to lyse the cells 2-3 times until the solution is transparent and free of turbidity. The supernatant was retained after centrifugation (4°C, 40000g, 30min). The supernatant was filtered with a 0.22 μm filter membrane and purified on a Ni-NTA column.

细胞裂解液上柱前,先用上述裂解液平衡Ni-NTA柱子。过滤后的细胞裂解液通过FPLC装置,以3mL·min-1的速度上样。上样完毕后,继续用上述裂解液以3mL·min-1速度冲洗柱子,直到基线平稳。然后用冲洗液(50mM Na2HPO4/NaH2PO4,300mM NaCl,20mMImidazole,pH 7.0)同样冲洗柱子至基线平稳。最后用洗脱液(50mM Na2HPO4/NaH2PO4,300mM NaCl,250mM Imidazole,pH 7.0)以3mL·min-1流速洗脱并收集蛋白峰。Before loading the cell lysate onto the column, equilibrate the Ni-NTA column with the above lysate. The filtered cell lysate was loaded through the FPLC device at a rate of 3 mL·min -1 . After loading the sample, continue to wash the column with the above lysate at a speed of 3 mL·min -1 until the baseline is stable. Then wash the column with washing solution (50mM Na2HPO4/NaH2PO4, 300mM NaCl, 20mM midazole, pH 7.0) until the baseline is stable. Finally, elute with eluent (50mM Na2HPO4/NaH2PO4, 300mM NaCl, 250mM Imidazole, pH 7.0) at a flow rate of 3mL·min -1 and collect the protein peak.

将上述得到的蛋白浓缩后,上样到用缓冲液(PBS,pH 7.0)平衡好的HiPrep 16/60Sephacryl S-200凝胶过滤柱(Amersham Biosciences)中,收集目的蛋白峰,加入2mM DTT(dithiothreitol)后保存。After the protein obtained above was concentrated, it was loaded onto a HiPrep 16/60 Sephacryl S-200 gel filtration column (Amersham Biosciences) equilibrated with buffer (PBS, pH 7.0), the target protein peak was collected, and 2mM DTT (dithiothreitol ) and save.

上述各步均保留少量样品,用于12%(w/v)acrylamide gel(Laemmli,1970)检测。最终纯化蛋白(wild type NH-PHD,wtNH-PHD)的纯度为95%以上(SDS-PAGE)。A small amount of sample was reserved in each of the above steps for the detection of 12% (w/v) acrylamide gel (Laemmli, 1970). The purity of the final purified protein (wild type NH-PHD, wtNH-PHD) was above 95% (SDS-PAGE).

表达的到的蛋白在N端比原基因中的PH domain(PHD)蛋白多出一段His-tag序列(MGSSHHHHHHSSGLVPRGSH),主要用途是方便纯化,可以通过凝血酶(Thrombin)切除。经过凝血酶切除N端His-tag后,用HiTrap Benzamidine FF亲和柱(购自AmershamBioSciences公司)除去凝血酶,然后用HiPrep 16/60Sephacryl S-200凝胶过滤柱除去His-tag,得到纯化的PHD蛋白。The expressed protein has an extra His-tag sequence (MGSSHHHHHHSSGLVPRGSH) at the N-terminus than the PH domain (PHD) protein in the original gene. The main purpose is to facilitate purification and can be excised by thrombin. After the N-terminal His-tag was excised by thrombin, the thrombin was removed with a HiTrap Benzamidine FF affinity column (purchased from Amersham BioSciences), and then the His-tag was removed with a HiPrep 16/60 Sephacryl S-200 gel filtration column to obtain purified PHD protein.

其他突变体蛋白的表达纯化采取同样的方法。The same method was used for the expression and purification of other mutant proteins.

实施例3:体外受体结合能力检测Example 3: Detection of receptor binding ability in vitro

wtNH-PHD以及其突变体蛋白与EPOR(EPO受体)的结合能力利用表面等离子体共振技术(SPR)进行测试。实验仪器为Biacore 3000(购自瑞典Uppsala公司),重组人EPO可溶性受体(rhEPO sR)购于R&D Systems。实验采用的缓冲液为HBS-EP(10mM Hepes,150mM NaCl,3.7mM EDTA,pH 7.4,0.005%P20),芯片为CM5芯片。The binding ability of wtNH-PHD and its mutant proteins to EPOR (EPO receptor) was tested by surface plasmon resonance (SPR). The experimental equipment was Biacore 3000 (purchased from Uppsala Company, Sweden), and the recombinant human EPO soluble receptor (rhEPO sR) was purchased from R&D Systems. The buffer used in the experiment was HBS-EP (10mM Hepes, 150mM NaCl, 3.7mM EDTA, pH 7.4, 0.005% P20), and the chip was a CM5 chip.

将rhEPO sR溶于pH 3.1的100mM醋酸钠溶液中,得到pH 4的溶液用于固定化。在固定化过程中,流速保持为5μL·min-1。将CM5芯片的第2通道用35μLN-ethyl-N’-(3-diethylaminopropyl)-carbodiimide/N-hydroxysuccinimide(EDC/NHS,1∶1)活化,然后注射45μL rhEPO sR,最后用35μL 1M的ethanolamine-HCl,pH 8.5封闭通道。固定的rhEPO sR为1200RU。用10第μL 10mM Glycine-HCl,pH 2.2以20μL·min-1的流速将通道2洗3遍后,基线稳定。通道1为参照道,处理方式同上,但不注射rhEPO sR。rhEPO sR was dissolved in 100 mM sodium acetate solution at pH 3.1 to obtain a solution at pH 4 for immobilization. During the immobilization process, the flow rate was kept at 5 μL·min -1 . Activate the second channel of the CM5 chip with 35 μL N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS, 1:1), then inject 45 μL rhEPO sR, and finally inject 35 μL 1M ethanolamine- HCl, pH 8.5 blocked the channels. The fixed rhEPO sR was 1200RU. After washing channel 2 three times with 10 μL of 10 mM Glycine-HCl, pH 2.2 at a flow rate of 20 μL·min -1 , the baseline was stable. Channel 1 is the reference channel, and the treatment method is the same as above, but rhEPO sR is not injected.

用HBS-EP将系统冲洗三次后,体系稳定,基线漂移小于1RU·min-1。所有动力学测试均采用50μL·min-1的流速,温度为25℃。每一个分析循环由以下几步组成:(1)1分钟稳定时间;(2)上样150μL;(3)300秒解离时间;(4)采用快速注射方式注射20μL再生缓冲液洗脱结合蛋白;(5)清洗IFC。每个样品均包括至少一个空白对照(零浓度)和5个浓度测试,浓度排列顺序随机,并选取一个浓度重复测试。rhEPO结合的再生缓冲液为10mM Glycine-HCl,pH 2.2,wtNH-PHD及其突变体结合的再生缓冲液为5mM NaOH溶液。得到的动力学数据采用BIAevaluation 4.0处理。After washing the system three times with HBS-EP, the system was stable and the baseline drift was less than 1RU·min -1 . All kinetic tests were performed at a flow rate of 50 μL min -1 at a temperature of 25 °C. Each analysis cycle consists of the following steps: (1) 1 minute stabilization time; (2) 150 μL sample loading; (3) 300 second dissociation time; (4) fast injection of 20 μL regeneration buffer to elute bound proteins ; (5) Clean the IFC. Each sample includes at least one blank control (zero concentration) and 5 concentration tests, the concentrations are arranged in random order, and one concentration is selected to repeat the test. The regeneration buffer combined with rhEPO was 10mM Glycine-HCl, pH 2.2, and the regeneration buffer combined with wtNH-PHD and its mutants was 5mM NaOH solution. The obtained kinetic data were processed with BIAevaluation 4.0.

经SPR测定的蛋白质突变体与EPO受体的结合常数如表2所示。The binding constants of protein mutants and EPO receptors determined by SPR are shown in Table 2.

表2.设计蛋白质与EPO受体结合的离解常数Table 2. Dissociation constants of designed proteins binding to EPO receptors

  蛋白质 protein   K<sub>D</sub>/M K<sub>D</sub>/M   人重组EPO human recombinant EPO   3.2E-10 3.2E-10   PH-E63F-D47N-K49R-E46A突变体 PH-E63F-D47N-K49R-E46A mutant   1.2E-8 1.2E-8   PH-E63F-D47N-K49R突变体 PH-E63F-D47N-K49R mutant   8.9E-9 8.9E-9   PH-E63F突变体 PH-E63F mutant   2.0E-8 2.0E-8   PH-E63F-D47N突变体 PH-E63F-D47N mutant   1.6E-8 1.6E-8   PH-E63F-K49R突变体 PH-E63F-K49R mutant   1.5E-8 1.5E-8   PH-D47N-K49R突变体 PH-D47N-K49R mutant   1.0E-7 1.0E-7

这些动力学数据表明,在体外条件下,野生型的PH结构域不与EPO受体结合,而其上述突变体能够与EPO受体有效结合。These kinetic data indicate that the wild-type PH domain does not bind to the EPO receptor under in vitro conditions, while its above-mentioned mutants can effectively bind to the EPO receptor.

实施例4:体内EPOR结合活性检测Example 4: Detection of EPOR binding activity in vivo

利用293T细胞体系,测定PH结构域突变体激活EPO受体信号通路的作用。实验采用的是PathDetect in vivo signal transduction pathway trans-reporting system(购自Stratagene公司),荧光酶活性检测采用的是luciferase assay system(购自Promega公司).Using the 293T cell system, the effect of the PH domain mutant on activating the EPO receptor signaling pathway was determined. The experiment used the PathDetect in vivo signal transduction pathway trans-reporting system (purchased from Stratagene), and the luciferase activity detection used the luciferase assay system (purchased from Promega).

1、质粒构建1. Plasmid construction

首先在质粒pcDNA3.1(购自Invitrogen公司)的HindIII和KpnI位点间通过PCR引入信号肽序列MSALLILALVGAAVA(SEQ ID NO.18),其碱基序列为5’ATGGACTCGGGTAGGGACTTCCCGACCCTGCAC 3’(SEQ ID NO.17)。得到的质粒标记为pcDNA3.1-SP myc/His A。First, the signal peptide sequence MSALLILLVGAAVA (SEQ ID NO.18) was introduced by PCR between the HindIII and KpnI sites of plasmid pcDNA3.1 (purchased from Invitrogen Company), and its base sequence was 5' ATGGACTCGGGTAGGGACTTCCCGACCCTGCAC 3' (SEQ ID NO.17 ). The resulting plasmid was labeled pcDNA3.1-SP myc/His A.

以pETPHD为模板,以7号引物对为引物(序列见表1),通过PCR扩增出含有BamHI和XbaI位点的PH结构域及其突变体的序列(酶切位点通过引物设计引入)。用BamHI和XbaI双酶切后,用T4DNA连接酶连入用BamHI和XbaI双酶切过的pcDNA3.1-SP myc/HisA载体中,得到pcDNA3.1-SP/PHD载体。其它突变体载体采用相应的模板,用同样的方法构建。Using pETPHD as a template and primer pair No. 7 as primers (see Table 1 for the sequence), the sequences of the PH domain and its mutants containing BamHI and XbaI sites were amplified by PCR (enzyme cleavage sites were introduced by primer design) . After double digestion with BamHI and XbaI, T4 DNA ligase was used to connect into the pcDNA3.1-SP myc/HisA vector cut with BamHI and XbaI to obtain the pcDNA3.1-SP/PHD vector. Other mutant vectors were constructed in the same way using corresponding templates.

以pBS-hEPO质粒(Emmanuel Payen赠送;Blood 97:3776-3782,2001)为模板,以8号引物对为引物(序列见表1),PCR扩增出含有BamHI和XbaI酶切位点的人EPO基因序列。用与上面同样的方法得到pcDNA3.1-SP/hEPO载体。Using the pBS-hEPO plasmid (gifted by Emmanuel Payen; Blood 97:3776-3782, 2001) as a template and primer pair No. 8 (see Table 1 for the sequence), PCR amplified the human gene containing the restriction sites BamHI and XbaI. EPO gene sequence. The pcDNA3.1-SP/hEPO vector was obtained in the same manner as above.

2、质粒转染2. Plasmid transfection

2.1质粒单独转染2.1 Plasmid transfection alone

按200μL每孔的量吸取无血清DMEM(Dulbecco’s modified Eagle’s medium)培养基到新的无菌转染管中,然后加入以下四种质粒:STAT5(25ng/孔),鼠EPOR(murineEPOR,250ng/孔),LHRE(100ng/孔),pTK-RL(购自Promega公司1.25ng/孔)。充分混匀后,按600ng/孔的量加入用于检测的质粒,其中阴性对照为pcDNA3.1-SP myc/His A,阳性对照为pcDNA3.1-SP/hEPO。混匀后,按3μL/孔的量加入Tfx-20转染试剂(购自Promega公司),轻轻混匀后静止15分钟。Draw serum-free DMEM (Dulbecco's modified Eagle's medium) medium into a new sterile transfection tube according to the amount of 200 μL per well, and then add the following four plasmids: STAT5 (25ng/well), mouse EPOR (murineEPOR, 250ng/well ), LHRE (100ng/well), pTK-RL (purchased from Promega, 1.25ng/well). After mixing well, add the plasmid used for detection at an amount of 600ng/well, wherein the negative control is pcDNA3.1-SP myc/His A, and the positive control is pcDNA3.1-SP/hEPO. After mixing, Tfx-20 transfection reagent (purchased from Promega) was added in an amount of 3 μL/well, mixed gently and left to stand for 15 minutes.

吸干培养有293T细胞的24孔板中的培养基后,每孔加入上面的混合物200μL。然后将24孔板置于37度,5%二氧化碳的培养箱中培养1小时。再每孔加入含1%双抗(青霉素+链霉素)和10%胎牛血清的DMEM培养基300μL。置于培养箱中继续培养。After the medium in the 24-well plate cultured with 293T cells was blotted dry, 200 μL of the above mixture was added to each well. Then place the 24-well plate in an incubator with 5% carbon dioxide at 37°C for 1 hour. Add 300 μL of DMEM medium containing 1% double antibody (penicillin+streptomycin) and 10% fetal bovine serum to each well. Place in an incubator to continue culturing.

培养24小时后,将孔中的培养基吸干,每孔加入不含血清的DMEM培养基500μL后,继续培养12小时后用于检测。After culturing for 24 hours, the medium in the wells was blotted dry, and 500 μL of serum-free DMEM medium was added to each well, and cultured for 12 hours for detection.

2.2质粒共同转染2.2 Plasmid co-transfection

操作与单独转染相同,但是待测质粒加入量为300ng/孔,同时添加300ngpcDNA3.1-SP/hEPO。阴性对照为600ng pcDNA3.1__sp myc/His A,阳性对照为300ngpcDNA3.1-SP myc/His A和300ng pcDNA3.1-SP/hEPO。The operation is the same as transfection alone, but the amount of the plasmid to be tested is 300ng/well, and 300ngpcDNA3.1-SP/hEPO is added at the same time. Negative control is 600ng pcDNA3.1__sp myc/His A, positive control is 300ngpcDNA3.1-SP myc/His A and 300ng pcDNA3.1-SP/hEPO.

3、活性测试3. Activity test

取出24孔板,吸干培养基,然后用1mL/孔的PBS(pH 7.4)洗一次。吸干PBS后,每孔加入100μL裂解液。轻轻振荡30分钟后测活。Take out the 24-well plate, blot dry the medium, and wash once with 1 mL/well of PBS (pH 7.4). After the PBS was blotted dry, 100 μL of lysate was added to each well. Viability was measured after shaking gently for 30 minutes.

测活采用的仪器是TopCount NXT Microplate Scintillation & Luminescence Counter(购自Packard公司)。测活时,于96孔板中加入30μL/孔底物I和20μL/孔裂解液后检测荧光酶活性;然后加入30μL/孔底物II后检测背景值。The instrument used in measuring activity is TopCount NXT Microplate Scintillation & Luminescence Counter (purchased from Packard Company). When measuring activity, add 30 μL/well substrate I and 20 μL/well lysate to detect luciferase activity in a 96-well plate; then add 30 μL/well substrate II to detect background value.

4、测活结果4. Test results

测活结果表明,在与EPO相当的表达量时,PH结构域突变体蛋白的EPO活性没有明显表现出来。但在与EPO共同转染的实验中,这些突变体表现出了对EPO的不同程度的抑制性或竞争性作用,表明这些突变体在与EPO表达量相当的情况下即可与EPO竞争结合EPO受体,同时预示着通过提高突变体的相对表达量,该实验方法就可能检测得到这些突变体的类似EPO的活性效应。The results of the activity test showed that the EPO activity of the PH domain mutant protein was not obviously displayed when the expression level was equivalent to that of EPO. However, in the co-transfection experiments with EPO, these mutants showed different degrees of inhibitory or competitive effects on EPO, indicating that these mutants can compete with EPO for binding EPO when the expression level of EPO is comparable. receptors, and it also indicates that by increasing the relative expression of the mutants, the experimental method may detect the EPO-like activity effects of these mutants.

序列表sequence listing

SEQUENCE LISTINGSEQUENCE LISTING

<110>北京大学<110> Peking University

<120>具有促红细胞生长因子活性的蛋白质及其用途<120> Protein with erythrocyte growth-stimulating factor activity and use thereof

<130>JSP050194<130>JSP050194

<160>18<160>18

<170>PatentIn version 3.1<170>PatentIn version 3.1

<210>1<210>1

<211>28<211>28

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>1<400>1

cactctacat atggactcgg gtagggac                                                  28cactctacat atggactcgg gtagggac 28

<210>2<210>2

<211>32<211>32

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>2<400>2

catgaattct tacttctgcc gctggtccat gg                                             32catgaattct tacttctgcc gctggtccat gg 32

<210>3<210>3

<211>35<211>35

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>3<400>3

ggtcatgagg tccccgttct cgcagctgtt ctcca                                          35ggtcatgagg tccccgttct cgcagctgtt ctcca 35

<210>4<210>4

<211>35<211>35

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>4<400>4

tggagaacag ctgcgagaac ggggacctca tgacc                                          35tggagaacag ctgcgagaac ggggacctca tgacc 35

<210>5<210>5

<211>35<211>35

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>5<400>5

ctacaagcta caggagaact gcaagaccat ctggc                                          35ctacaagcta caggagaact gcaagaccat ctggc 35

<210>6<210>6

<211>35<211>35

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>6<400>6

gccagatggt cttgcagttc tcctgtagct tgtag                                          35gccagatggt cttgcagttc tcctgtagct tgtag 35

<210>7<210>7

<211>27<211>27

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>7<400>7

caggaggact gcaggaccat ctggcag                                                   27caggaggact gcaggaccat ctggcag 27

<210>8<210>8

<211>27<211>27

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>8<400>8

ctgccagatg gtcctgcagt cctcctg                                                   27ctgccagatg gtcctgcagt cctcctg 27

<210>9<210>9

<211>34<211>34

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>9<400>9

ctacaagcta caggagaact gcaggaccat ctgg                                           34ctacaagcta caggagaact gcaggaccat ctgg 34

<210>10<210>10

<211>34<211>34

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>10<400>10

ccagatggtc ctgcagttct cctgtagctt gtag                                           34ccagatggtc ctgcagttct cctgtagctt gtag 34

<210>11<210>11

<211>32<211>32

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>11<400>11

tctacaagct acaggcgaac tgcaggacca tc                                             32tctacaagct acaggcgaac tgcaggacca tc 32

<210>12<210>12

<211>32<211>32

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>12<400>12

gatggtcctg cagttcgcct gtagcttgta ga                                             32gatggtcctg cagttcgcct gtagcttgta ga 32

<210>13<210>13

<211>31<211>31

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>13<400>13

gataggatcc atggactcgg gtagggactt c                                              31gataggatcc atggactcgg gtagggactt c 31

<210>14<210>14

<211>31<211>31

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>14<400>14

gatgttctag acttctgccg ctggtccatg g                                              31gatgttctag acttctgccg ctggtccatg g 31

<210>15<210>15

<211>29<211>29

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>15<400>15

gataggatcc atgggggtgc ac gaatgtc                                                29gataggatcc atgggggtgc ac gaatgtc 29

<210>16<210>16

<211>30<211>30

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>16<400>16

gatgttctag atctgtcccc tgtcctgcag                                                30gatgttctag atctgtcccc tgtcctgcag 30

<210>17<210>17

<211>33<211>33

<212>DNA<212>DNA

<213>人工序列<213> Artificial sequence

<400>17<400>17

atggactcgg gtagggactt cccgaccctg cac                                            33atggactcgg gtagggactt cccgaccctg cac 33

<210>18<210>18

<211>15<211>15

<212>PRT<212>PRT

<213>人工序列<213> Artificial sequence

<400>18<400>18

Met Ser Ala Leu Leu Ile Leu Ala Leu Val G1y Ala Ala Val AlaMet Ser Ala Leu Leu Ile Leu Ala Leu Val G1y Ala Ala Val Ala

1                 5                        10                    151 5 10 15

Claims (7)

1.一种蛋白质,其特征在于:所述蛋白质是在保持pleckstrin同源性结构域的三级结构不变的情况下,将其氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加而得到的具有促红细胞生长因子活性的pleckstrin同源性结构域衍生蛋白。1. A protein, characterized in that: the protein is under the condition of keeping the tertiary structure of the pleckstrin homology domain unchanged, and its amino acid sequence is replaced, deleted or added by one or several amino acid residues The obtained pleckstrin homology domain-derived protein with erythrocyte growth-stimulating factor activity. 2.如权利要求1所述的蛋白质,其特征在于:所述pleckstrin同源性结构域为磷脂酶C的pleckstrin同源性结构域。2. The protein according to claim 1, wherein the pleckstrin homology domain is the pleckstrin homology domain of phospholipase C. 3.如权利要求2所述的蛋白质,其特征在于:所述pleckstrin同源性结构域为磷脂酶Cδ1的pleckstrin同源性结构域。3. The protein according to claim 2, wherein the pleckstrin homology domain is the pleckstrin homology domain of phospholipase Cδ1. 4.如权利要求3所述的蛋白质,其特征在于:所述磷脂酶C来源于大鼠。4. The protein according to claim 3, wherein the phospholipase C is derived from rats. 5.如权利要求4所述的蛋白质,其特征在于,该蛋白质选自:大鼠磷脂酶Cδ1的pleckstrin同源性结构域的E63F、E63F-D47N、E63F-K49R、D47N-K49R、E63F-D47N-K49R、E63F-D47N-K49R-E46A突变体。5. The protein according to claim 4, wherein the protein is selected from the group consisting of: E63F, E63F-D47N, E63F-K49R, D47N-K49R, E63F-D47N of the pleckstrin homology domain of rat phospholipase Cδ1 - K49R, E63F-D47N-K49R-E46A mutant. 6.权利要求1~5中任一权利要求所述蛋白质的制备方法,其特征在于,包括以下步骤:6. The method for preparing the protein according to any one of claims 1 to 5, characterized in that it comprises the following steps: (1)用PCR的方法扩增得到pleckstrin同源性结构域的基因,并连入合适的表达载体,获得表达pleckstrin同源性结构域的质粒;(1) amplifying the gene of the pleckstrin homology domain by PCR, and connecting it into a suitable expression vector to obtain a plasmid expressing the pleckstrin homology domain; (2)利用蛋白质功能嫁接方法进行突变体设计;(2) Mutant design by protein function grafting method; (3)通过引物引入突变,PCR扩增上述表达质粒,得到含有pleckstrin同源性结构域的突变基因的表达质粒;(3) introducing mutations by primers, amplifying the above-mentioned expression plasmids by PCR, and obtaining expression plasmids of mutant genes containing pleckstrin homology domains; (4)将上述质粒转入原核或真核宿主中,在适宜的条件下表达目的蛋白;(4) Transfer the above-mentioned plasmid into a prokaryotic or eukaryotic host, and express the target protein under suitable conditions; (5)用适当的方法收集和处理菌体、组织或细胞,得到目的蛋白的粗提物;(5) Collect and process the bacteria, tissue or cells with appropriate methods to obtain the crude extract of the target protein; (6)用亲和柱、体积排阻柱等方法获得纯化的目的蛋白。(6) Obtain purified target protein by methods such as affinity column and size exclusion column. 7.权利要求1~5中任一权利要求所述的蛋白质在制备促红细胞生成素替代品的药物中的应用。7. The use of the protein according to any one of claims 1 to 5 in the preparation of medicines for erythropoietin substitutes.
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