WO2016188449A1 - 一种针对cd47的单域抗体 - Google Patents

一种针对cd47的单域抗体 Download PDF

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WO2016188449A1
WO2016188449A1 PCT/CN2016/083469 CN2016083469W WO2016188449A1 WO 2016188449 A1 WO2016188449 A1 WO 2016188449A1 CN 2016083469 W CN2016083469 W CN 2016083469W WO 2016188449 A1 WO2016188449 A1 WO 2016188449A1
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seq
domain antibody
nucleotide sequence
single domain
antibody
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万亚坤
孟红
卞忠华
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Jiangsu Chunshentang Pharmaceutical Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

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  • the invention relates to a single domain antibody against CD47, and specifically designs a camel source single domain antibody against the extracellular segment of CD47 molecule, and belongs to the technical field of nanobodies.
  • CD47 is a membrane glycoprotein widely expressed between multiple species and tissues, which interacts with the inhibitory receptor signal-modulating protein a as a receptor and ligand, and can form a CD47-SIRPa signal complex.
  • the complex has the function of mediating two-way signal regulation and regulating a variety of immune response processes.
  • HSCs hematopoietic stem cells
  • the significance of CD47 expression is to maintain its relative stability in the body.
  • malignant diseases such as leukemia, non-Hodgkin's lymphoma, bladder cancer and breast cancer
  • Tumor cells evade tumor immunity by taking the "Don't Eat Me” signal.
  • Blocking the interaction of CD47 and SIRPa by using an anti-CD47 antibody has the effect of targeted therapy with minimal effect on normal cells.
  • targeted killing of leukemia tumor stem cells can increase the anti-tumor effect to the maximum extent, and provide a new exploration direction for the treatment of leukemia. The importance of individualization and further improvement of efficacy.
  • Nano-antibody technology is the latest and smallest antibody molecule developed by biomedical scientists based on traditional antibodies and using the molecular biology technology combined with the concept of nanoparticle science to develop the latest and smallest antibody molecules, originally by the Belgian scientist Hamers, R. It is isolated from a single heavy chain antibody in camel blood and is therefore also called a camel single-domain antibody. The single domain nature of this antibody gives it some unique properties compared to common antibodies. (1) The molecular weight is small, the structure is stable, the solubility is high, and it can be fixed in order. Nanobodies are the least known antibodies of the molecular weight and are one tenth (15 KD) of common antibodies.
  • Nanobodies Since the hydrophobic residue in the FR2 (frame-work 2) of the Nanobody is substituted with a hydrophilic residue, the water solubility of the Nanobody is increased, and the polymerizability is reduced. The study found that the Nanobody still retains 80% of the binding activity after 1 week of incubation at 37 ° C, indicating that it is easier to use and long-term storage at room temperature. Antigen binding ability can be regained after prolonged placement in an environment above 90 °C. At the same time, Nanobodies also exhibit high stability in strong denaturing agents and extreme pH environments, while ordinary antibodies undergo irreversible polymerization. Because nano-antibodies have a small volume and a regular structure, they are easier to fix and can be ordered and fixed. (2) High affinity and strong specificity.
  • Nanobodies are weakly immunogenic to humans and have good biocompatibility with humans.
  • Nano-antibodies are small in mass and simple in structure, can be encoded by a single gene, and can be expressed in large amounts in microorganisms such as yeast and E. coli by genetic engineering.
  • Nanobodies combine the advantages of traditional antibodies and small molecule drugs, and almost completely overcome the shortcomings of traditional antibody development cycle, low stability and harsh storage conditions.
  • This single-domain antibody with a molecular weight of only 1/10 of the conventional antibody has gradually become an emerging force in the diagnosis and treatment of a new generation of antibodies. Therefore, the application of nano-antibody technology to develop CD47 therapeutic antibody drugs has broad prospects.
  • the present invention provides a single domain antibody directed against the extracellular domain of CD47, and provides a coding sequence of the single domain antibody, a vector containing the coding sequence, and a host cell.
  • a single domain antibody directed against CD47 which is a single domain antibody directed against the extracellular domain of CD47, the amino acid sequence of which is represented by SEQ ID NO: 9, consisting of four framework regions and three complementarity determining regions, wherein four frameworks
  • the amino acid sequences of the regions are as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, and the amino acid sequences of the three complementarity determining regions are respectively SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.
  • the complementarity determining region is mainly responsible for the recognition of antigens, and the structure of the framework region is relatively stable, mainly supporting the maintenance of protein structure.
  • nucleic acid encoding the above single domain antibody, the nucleotide sequence of which is one of the following sequences:
  • nucleotide sequence of the nucleotide sequence of (1), (2), and (3) is distinguished by the degeneracy of the genetic code.
  • the nucleotide sequence or at least a portion of the sequences of the invention can be expressed by a suitable expression system to yield the corresponding protein or polypeptide.
  • suitable expression systems include bacteria, yeasts, filamentous fungi, animal cells, insect cells, plant cells, or cell-free expression systems.
  • An expression vector comprising the above nucleic acid encoding a single domain antibody against CD47.
  • a host cell comprising the above expression vector.
  • the recipient bacteria of the host cell are preferably Escherichia coli.
  • the use of the single domain antibody against CD47 for detecting CD47 in specific application, using the ability of the single domain antibody against CD47 of the present invention to specifically bind to CD47, using an enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay) ELISA), fluorescence immunoassay (Fluoroimmunoassay, FIA), immunochip method and affinity chromatography were performed.
  • Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay
  • FIA fluorescence immunoassay
  • immunochip method immunochip method and affinity chromatography
  • the present invention first synthesizes a CD47 polypeptide (extracellular segment) and makes it immunogenic, and then couples the CD47 polypeptide molecule to the ELISA plate to display the correct spatial structure of the protein, and the antigen-utilizing phage display technology in this form
  • the immune nanobody gene library (the camelid heavy chain antibody phage display gene library) was screened to obtain the CD47-specific Nanobody gene, which was transferred to E. coli to establish a Nanobody capable of high expression in E. coli. Strain. Needle of the present invention
  • the single domain antibody to CD47 has broad application prospects in the development of CD47 therapeutic antibody drugs.
  • Figure 1 The insertion rate of the CD47 camel single-domain antibody library.
  • the bands from left to right are: the first is the DNA molecular marker, and the other is the PCR product of the VHH insert (single domain antibody gene fragment). It is about 500 bp in size.
  • FIG. 1 Purification map of CD47 single domain antibody, electrophoresis pattern of SDS-PAGE after purification by nickel column resin gel affinity chromatography, the results show that CD47 single domain antibody can achieve purity of more than 90% after the purification process.
  • the instruments, reagents, materials and the like involved in the following examples are conventional instruments, reagents, materials and the like which are available in the prior art unless otherwise specified, and can be obtained by a formal commercial route.
  • the experimental methods, detection methods, and the like involved in the following examples are conventional experimental methods, detection methods, and the like which have been known in the prior art unless otherwise specified.
  • the ligation product was transformed into electroporation competent cell TG1, and the CD47 Nanobody library was constructed and the storage capacity was determined.
  • the storage capacity was 5.85 ⁇ 10 8 CFU.
  • the insertion rate of the constructed library was detected, and 24 monoclonals were randomly selected. PCR analysis of the insert was performed to determine the insertion rate of the VHH fragment, and the result showed that the insertion rate of the constructed library reached 95.8% (Fig. 1).
  • the phage specifically binding to CD47 was eluted with 100 mM TEA (triethylamine), and the E. coli TG1 cells growing in log phase were infected and cultured at 37 ° C for 1 h to produce and purify the phage for the next round of screening. , The same screening process was repeated for 3 to 4 rounds, and the enrichment was gradually obtained.
  • TEA triethylamine
  • a crude antibody was obtained by an infiltration method, and the antibody was transferred to an antigen-coated ELISA plate and allowed to stand at room temperature for 1 hour.
  • mouse anti-HA tag antibody anti-mouse anti-HA antibody, purchased from Beijing Kangwei Century Biotechnology Co., Ltd.
  • the positive cloned bacteria were shaken in a LA liquid containing 100 ug/mL to extract a plasmid and perform sequencing.
  • each clone was analyzed according to the sequence alignment software Vector NTI, and the strains with the same CDR3 sequence were regarded as the same clone, and the strains with different sequences were regarded as different clones, and the amino acid sequence of the VHH chain of the antibody was SEQ ID.
  • NO: 9 is composed of 4 framework regions and 3 complementarity determining regions, wherein the amino acid sequences of the four framework regions are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID, respectively.
  • the amino acid sequences of the three complementarity determining regions are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.
  • the plasmids of the different clones obtained by the above sequencing analysis were electrotransformed into Escherichia coli WK6, and coated on LA+2% Gl ⁇ cose (ie containing ampicillin and glucose) culture plates, and cultured at 37 ° C overnight;

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Abstract

本发明提供了一种针对CD47胞外区的单域抗体，其氨基酸序列如SEQ ID NO:9所示。本发明还提供了编码该抗体的核酸，含有该核酸的表达载体，含有该表达载体的宿主细胞。

Description

一种针对CD47的单域抗体 技术领域
本发明涉及一种针对CD47的单域抗体，具体设计一种针对CD47分子胞外段的驼源单域抗体，属于纳米抗体技术领域。
背景技术
CD47是一种广泛表达于多个物种和各个组织之间的膜糖蛋白，其与抑制性受体信号调节蛋白a互为受体和配体，并可以形成CD47-SIRPa信号复合体，该信号复合物具有介导双向信号调节并调控多种免疫反应进程的作用。在正常造血干细胞(hematopoietic stem cells,HSCs)上，CD47表达的意义在于保持其在机体内的相对稳定。在白血病、非霍奇金淋巴瘤、膀胱癌和乳腺癌等恶性疾病的研究中，发现肿瘤细胞存在CD47的升高，且CD47的高表达提示临床预后不良；一些类型的肿瘤干细胞(cancer stem cells,CDCs)中存在CD47的过表达。肿瘤细胞通过借此“别吃我”信号，逃避了肿瘤免疫。通过使用抗CD47抗体阻断CD47和SIRPa的相互作用具有靶向性治疗的效果，对正常细胞的影响非常小。在白血病的治疗中，可在现有的经典化疗的基础上联合CD47抗体针对白血病肿瘤干细胞进行靶向杀伤，可在最大限度上增加抗肿瘤疗效，为白血病的治疗提供新的探索方向，对治疗的个体化和进一步提高疗效具有什么重要的意义。
纳米抗体技术，是生物医学科学家在传统抗体的基础上，运用分子生物学技术结合纳米粒子科学的概念进行的抗体工程革命，从而研发出的最新和最小的抗体分子，最初由比利时科学家Hamers，R从骆驼血液中的单独重链抗体中分离得到，因此也叫做驼源单域抗体。该种抗体的单域性质使其较普通抗体具有一些独特的性质。(1)分子量小，结构稳定，溶解性高，可有序固定。纳米抗体是已知分子量最小的抗体，为普通抗体的十分之一(15KD)。由于纳米抗体的FR2(frame-work 2)中的疏水残基被亲水残基取代，使纳米抗体水溶性增加，聚合性减少。研究发现，在37℃孵育1周后纳米抗体仍保持80％的结合活性，说明其在室温下更易于使用和长时间保存。在高于90℃的环境中长期放置后仍能重新获得抗原结合能力。同时，纳米抗体在强变性剂和极度pH值环境下也表现出高度的稳定性，而普通的抗体则发生不可逆的聚合。由于纳米抗体拥有很小的体积且结构规整，所以固定起来更加容易，可实现有序固定。(2)亲和力高，特异性强。相对于普通抗体的凹形或平面的抗原结合面，其拥有较长的CDR3(complementarity-determining region 3)，可形成稳定、暴露的凸形结构的结合面。这个凸形结构可以深入抗原内部，更有效的与抗原表位形成的凹形拓扑结构结合，不 仅提高了纳米抗体的抗原特异性和亲和力，同时识别许多普通抗体无法识别的抗原。甚至，当蛋白紧密包裹隐藏了普通抗体识别位点时，纳米抗体也可对其进行表位识别。(3)免疫原性弱，穿透性强。纳米抗体对人体的免疫原性弱，与人的生物相容性好。免疫原性与分子的大小、化学结构有关，相对分子量越小免疫原性越小。研究证明，动物实验中纳米抗体未引起任何体液和细胞免疫应答。同时，纳米抗体的组织穿透能力强，可进入致密的组织，多余未结合的纳米抗体能够被快速清除，有利于疾病的诊断及治疗。(4)成本低廉，可规模化生产。纳米抗体相对于分子质量小，且结构简单，能被单个基因编码，利用基因工程可在酵母菌、E.coli等微生物中大量表达。
纳米抗体因其特殊的结构性质，兼具了传统抗体和小分子药物的优势，几乎完美地克服了传统抗体的开发周期长，稳定性低，保存条件苛刻等缺陷。这种分子量仅为常规抗体1/10的驼源单域抗体逐渐成为新一代抗体诊断及治疗中的新兴力量。因此应用纳米抗体技术研发CD47治疗性抗体药物具有广阔的前景。
发明内容
针对上述现有技术，本发明提供了一种针对CD47胞外段的单域抗体，同时提供了该单域抗体的编码序列，含有该编码序列的载体、宿主细胞。
本发明是通过以下技术方案实现的：
一种针对CD47的单域抗体，为针对CD47胞外段的单域抗体，其氨基酸序列如SEQ ID NO：9所示，由4个框架区和3个互补决定区组成，其中，4个框架区的氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示，3个互补决定区的氨基酸序列分别如SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7所示。互补决定区主要负责抗原的识别，框架区结构相对稳定，主要起着支撑维持蛋白质结构的作用。
一种编码上述单域抗体的核酸，其核苷酸序列为以下序列之一：
(1)如SEQ ID NO.8所示的核苷酸序列；
(2)SEQ ID NO.8所示的核苷酸序列添加、取代、缺失或插入一个或若干个核苷酸的序列；
(3)在严格条件下，与(1)或(2)的核苷酸序列杂交的核苷酸序列；
(4)由于遗传密码子的简并性区别于(1)、(2)、(3)的核苷酸序列的核苷酸序列。
Figure PCTCN2016083469-appb-000001
Figure PCTCN2016083469-appb-000002
本发明的核苷酸序列或至少部分序列可以通过合适的表达系统进行表达以得到相应的蛋白质或多肽。这些表达系统包括细菌，酵母菌，丝状真菌，动物细胞，昆虫细胞，植物细胞，或无细胞表达系统。
一种表达载体，其包含有上述编码针对CD47的单域抗体的核酸。
一种宿主细胞，包含上述表达载体。
所述宿主细胞的受体菌优选大肠杆菌。
所述针对CD47的单域抗体在检测CD47中的应用，具体应用时，利用本发明的针对CD47的单域抗体与CD47特异性结合的能力，采用酶联免疫吸附法(Enzyme-linked immunosorbent assay，ELISA)，荧光免疫法(Fluoroimmunoassay，FIA)，免疫芯片法和亲和层析法等进行检测。
所述针对CD47的单域抗体在制备检测CD47的试剂或试剂盒中的应用；所述检测CD47的试剂或试剂盒中含有针对CD47的单域抗体。
所述针对CD47的单域抗体在制备CD47治疗性抗体药物中的应用。
本发明首先合成CD47多肽(胞外段)，并使其具有免疫原性，然后将CD47多肽分子偶联在酶标板上，展示该蛋白的正确空间结构，以此形式的抗原利用噬菌体展示技术筛选免疫纳米抗体基因库(骆驼重链抗体噬菌体展示基因库)，从而获得了CD47特异性的纳米抗体基因，将此基因转至大肠杆菌中，从而建立了能在大肠杆菌中高效表达的纳米抗体株。本发明的针 对CD47的单域抗体在研发CD47治疗性抗体药物方面具有广阔的应用前景。
附图说明
图1：CD47驼源单域抗体文库插入率检测图，从左到右的条带分别是：第一道为DNA分子标记，其余孔道为VHH插入片段的PCR产物(单域抗体基因片段)，其大小约为500bp。
图2：CD47单域抗体纯化图，经镍柱树脂凝胶亲和层析纯化后的SDS-PAGE的电泳图，结果显示，CD47单域抗体经过该纯化过程，其纯度可达到90％以上。
具体实施方式
下面结合实施例对本发明作进一步的说明。
下述实施例中所涉及的仪器、试剂、材料等，若无特别说明，均为现有技术中已有的常规仪器、试剂、材料等，可通过正规商业途径获得。下述实施例中所涉及的实验方法，检测方法等，若无特别说明，均为现有技术中已有的常规实验方法，检测方法等。
实施例1针对于CD47的纳米抗体文库的构建
(1)首先合成CD47多肽，将1mg CD47与弗氏佐剂等体积混合，免疫一只新疆单峰驼，每周一次，共免疫7次，刺激B细胞表达抗原特异性的纳米抗体；
(2)7次免疫结束后，抽取100mL骆驼外周血，分离淋巴细胞并提取总RNA；
(3)合成cDNA并利用巢式PCR扩增VHH；
(4)利用限制性内切酶PstI及NotI酶切16μg pCMECS3噬菌体展示载体及8μg VHH并连接两个片段；
(5)将连接产物转化至电转感受态细胞TG1中，构建CD47纳米抗体文库并测定库容，库容大小为5.85×108CFU；另外，对构建文库的插入率进行检测，随机挑选24颗单克隆进行插入片段的PCR检测，判断VHH片段的插入率，结果显示出构建文库的插入率达到了95.8％(如图1)。
实施例2针对CD47的纳米抗体筛选过程
(1)将溶解在100mM NaHCO3、pH 8.2中的20μg CD47偶联在NUNC酶标板上，4℃放置过夜；
(2)第二天加入100μL 0.1％BSA，室温封闭2h；
(3)2h后，加入100μL噬菌体(2×1011个免疫骆驼单域抗体噬菌体)，室温作用1h；
(4)用PBS+0.05％Tween-20洗5遍，以洗掉不结合的噬菌体；
(5)用100mM TEA(triethylamine)将与CD47特异性结合的噬菌体洗脱下来，并感染处于对数期生长的大肠杆菌TG1细胞，37℃培养1h，产生并纯化噬菌体用于下一轮的筛选， 相同筛选过程重复3～4轮，逐步得到富集。
实施例3用噬菌体的酶联免疫方法(ELISA)筛选特异性单个阳性克隆
(1)从上述3～4轮筛选后含有噬菌体的细胞培养皿中，挑选96个单菌落并接种于1mL含有100ug/mL的氨苄青霉素的TB培养基(1L TB培养基中含有2.3g KH2PO4，12.52g K2HPO4，12g peptone，24g yeast extract，4mLglycerol)中，生长至对数期后，加终浓度1mM的IPTG，28℃培养过夜。
(2)利用渗透法获得粗提抗体，并将抗体转移到经抗原包被的ELISA板中，在室温下放置1h。
(3)用PBST洗去未结合的抗体，加入mouse anti-HA tag antibody(抗鼠抗HA抗体，购自北京康为世纪生物科技有限公司)，在室温下放置1h。
(4)用PBST洗去未结合的抗体，加入anti-mouse alkaline phosphatase conjugate(山羊抗小鼠碱性磷酸酶标记抗体，购自艾美捷科技有限公司)，在室温下放置1h。
(5)用PBST洗去未结合的抗体，加入碱性磷酸酶显色液，置于酶标仪上，在405nm波长，读取吸收值。
(6)当样品孔OD值大于对照孔OD值3倍以上时，判为阳性克隆。
(7)将阳性克隆的菌转摇在含有100ug/mL的LA液体中以便提取质粒并进行测序。
根据序列比对软件Vector NTI分析各个克隆株的基因序列，把CDR3序列相同的株视为同一克隆株，而其序列不同的菌株视为不同克隆株，其抗体的VHH链的氨基酸序列如SEQ ID NO：9所示，由4个框架区和3个互补决定区组成，其中，4个框架区的氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示，3个互补决定区的氨基酸序列分别如SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7所示。
实施例4纳米抗体在宿主菌大肠杆菌中表达、纯化
(1)将上述测序分析所获得不同克隆株的质粒电转化到大肠杆菌WK6中，并将其涂布在LA+2％Glμcose(即含有氨苄青霉素和葡萄糖)培养平板上，37℃培养过夜；
(2)挑选单个菌落接种在5mL含有氨苄青霉素的LB培养液中，37℃摇床培养过夜；
(3)接种1mL的过夜菌种至330mL TB培养液中，37℃摇床培养，培养到OD值达到0.6～0.9时，加入IPTG，28℃摇床培养过夜(约12-16h)；
(4)离心，收菌；
(5)利用渗透法，获得抗体粗提液：用4mL高糖溶液TES处理细胞2h，然后8mL 1/4TES 处理2h；离心后取上清液用于后续纯化；
(6)使用镍柱离子亲和层析的方法纯化单域抗体：蛋白液与镍柱混合反应1h；10×PBS洗涤5次，含20mM咪唑的PBS洗涤2次，含50mM的PBS洗涤1次，1mL含100mM咪唑的PBS洗涤，含500mM咪唑的PBS洗脱3次；收集洗脱的蛋白液，跑胶观察其纯度(图2所示)，并将其超滤至1×PBS中置于-80℃保存。

Claims (8)

  1. 一种针对CD47的单域抗体，其特征在于：其氨基酸序列如SEQ ID NO：9所示，由4个框架区和3个互补决定区组成，其中，4个框架区的氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4所示，3个互补决定区的氨基酸序列分别如SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7所示。
  2. 权利要求1所述的针对CD47的单域抗体在检测CD47中的应用。
  3. 权利要求1所述的针对CD47的单域抗体在制备检测CD47的试剂或试剂盒中的应用。
  4. 权利要求1所述的针对CD47的单域抗体在制备CD47治疗性抗体药物中的应用。
  5. 一种编码权利要求1所述的单域抗体的核酸，其特征在于：其核苷酸序列为以下序列之一：
    (1)如SEQ ID NO.8所示的核苷酸序列；
    (2)SEQ ID NO.8所示的核苷酸序列添加、取代、缺失或插入一个或若干个核苷酸的序列；
    (3)在严格条件下，与(1)或(2)的核苷酸序列杂交的核苷酸序列；
    (4)由于遗传密码子的简并性区别于(1)、(2)、(3)的核苷酸序列的核苷酸序列。
  6. 一种表达载体，其特征在于：其包含有权利要求4所述的核酸。
  7. 一种宿主细胞，其特征在于：其包含有权利要求6所述的表达载体。
  8. 根据权利要求7所述的宿主细胞，其特征在于：所述宿主细胞的受体菌为大肠杆菌。
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