WO2023072315A1 - 抗志贺氏菌的单克隆抗体及其应用 - Google Patents

抗志贺氏菌的单克隆抗体及其应用 Download PDF

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WO2023072315A1
WO2023072315A1 PCT/CN2022/142714 CN2022142714W WO2023072315A1 WO 2023072315 A1 WO2023072315 A1 WO 2023072315A1 CN 2022142714 W CN2022142714 W CN 2022142714W WO 2023072315 A1 WO2023072315 A1 WO 2023072315A1
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seq
antigen
yhdp
antibody
binding portion
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PCT/CN2022/142714
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French (fr)
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赵丽娜
李巍伟
胡芬
王淑青
高爽
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华北理工大学
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Priority claimed from CN202211622912.3A external-priority patent/CN115819570B/zh
Priority claimed from CN202211622922.7A external-priority patent/CN115856296B/zh
Application filed by 华北理工大学 filed Critical 华北理工大学
Publication of WO2023072315A1 publication Critical patent/WO2023072315A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/25—Shigella (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to a monoclonal antibody against Shigella flexneri and its application for treating diseases caused by Shigella flexneri.
  • Shigella flexneri also known as Shigella flexneri, is a non-motile, non-spore-forming, facultative anaerobic Gram-negative bacterium that can cause bacillary dysentery clinically.
  • An important feature of shigellosis mainly manifested as edema, ulceration and inflammation of the colon. About 99% of cases occur in developing countries, with children and primates having higher rates of infection and mortality. posed a significant foodborne threat to public health.
  • Shigella flexneri is mainly transmitted by the fecal-oral route. Other possible modes of transmission may be from ingestion of contaminated food or water and subcutaneous contact with inanimate objects.
  • the infectious dose of Shigella flexneri is very low; only 100-200 bacteria are required to cause a clinical infection.
  • the bacterial cell envelope is the first line of defense and point of contact with the environment and other organisms. Thus, the envelope is critical to the survival and physiology of bacteria and is often a common target for antimicrobial agents.
  • Gram-negative bacteria have a multilayered envelope separated by inner and outer membranes (IM and OM, respectively).
  • OM is a barrier to many antimicrobial agents because of its asymmetric lipid structure, with phospholipids making up the inner leaflet and lipopolysaccharide (LPS) making up the outer leaflet. Since lipid synthesis occurs at the IM, phospholipids and LPS are transported across the cell envelope and assembled asymmetrically at the OM during growth. How phospholipids are transported to the OM remains unknown.
  • YhdP participates in this process through an unknown mechanism.
  • YhdP belongs to the AsmA-like clan and contains domains homologous to lipid transporters.
  • the data show that YhdP and its paralogs TamB and YdbH are redundant but not equivalent in performing essential functions in the cell envelope.
  • AsmA-like paralogs only the combined loss of YhdP, TamB, and YdbH was lethal, and these proteins are required for OM lipid homeostasis, suggesting that they are long-required phospholipid transport required for OM biogenesis protein.
  • Gram-negative bacteria are characterized by having two membranes. The systems required for the biogenesis of the gram-negative outer membrane have been identified, but also for the transport of newly synthesized phospholipids from the inner membrane to the outer membrane, and the YhdP protein is involved in this process.
  • ECA lipopolysaccharide LPS
  • ECA phosphatidylglycerol a surface-exposed ECA chain linked to phosphatidylglycerol
  • cyclic ECA a Circularized soluble ECA molecules found in the periplasm.
  • the present invention aims at the above problems and aims at providing a hybridoma cell line capable of producing monoclonal antibody against YhdP, monoclonal antibody against YhdP, and the preparation method and application of the antibody.
  • the present invention adopts following technical scheme:
  • the embodiment of the present invention discloses a hybridoma cell line capable of producing YhdP monoclonal antibody and a method for preparing the YhdP monoclonal antibody. It includes:
  • Step (2) fusing the myeloma cell SP2/0 with the B lymphocyte of the animal body after immunization to obtain hybridoma cells;
  • Step (3) screening positive clones of specific hybridoma cells, performing cell cloning on the positive clones, screening out hybridoma cells stably secreting YhdP monoclonal antibodies, and performing the screening of positive clones of specific hybridoma cells by ELISA method ;
  • Step (4) obtaining YhdP monoclonal antibody.
  • step (4) said obtaining YhdP monoclonal antibody comprises:
  • the hybridoma cells stably secreting the YhdP monoclonal antibody are cultured in vitro, and the culture fluid is separated and purified to obtain the YhdP monoclonal antibody.
  • the embodiment of the present invention also provides that the above-mentioned YhdP monoclonal antibody is used as an auxiliary antibiotic in the treatment of Shigella flexneri infection.
  • the type of the monoclonal antibody of the present invention is identified as IgG2b by ELISA method.
  • the embodiments of the present invention disclose an isolated antibody or an antigen-binding portion thereof, which binds to a protein YhdP of Shigella flexneri, wherein the antibody or an antigen-binding portion thereof comprises as shown in SEQ ID NO: 1 HCDR1 as shown, HCDR2 as shown in SEQ ID NO:2 and the heavy chain variable region of HCDR3 as shown in SEQ ID NO:3 and LCDR1 as shown in SEQ ID NO:4, as shown in SEQ ID NO:5
  • SEQ ID NO:6 The light chain variable region of LCDR2 shown and LCDR3 shown as SEQ ID NO:6.
  • the heavy chain variable region (VH) amino acid sequence of the isolated antibody, or antigen-binding portion thereof is SEQ ID NO: 7 and the light chain variable region (VL) amino acid sequence is SEQ ID NO: 8.
  • the isolated antibody, or antigen-binding portion thereof comprises a heavy chain set forth in SEQ ID NO:9 and a light chain set forth in SEQ ID NO:10.
  • a vector comprising a nucleic acid molecule capable of encoding the above isolated antibody or antigen-binding portion thereof.
  • a cell comprising the above-mentioned vector.
  • a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding portion thereof.
  • the pharmaceutical composition containing YhdP monoclonal antibody is used for treating Shigella infection, and the antibody is used in combination with antibiotics to enhance the effect of antibiotics and avoid the problem of antibiotic resistance.
  • the present invention uses recombinant YhdP protein as immunogen to immunize Balb/c mice, and adopts classic cell fusion technology to obtain hybridoma cells stably secreting YhdP monoclonal antibody.
  • the YhdP monoclonal antibody secreted by it can be used to assist antibiotics in treating Shigella flexneri infection.
  • Figure 1 The effect of YhdP antibody and combined antibiotics on inhibiting sf301 replication
  • the invention provides an anti-Shigella flexneri YhdP antibody, its preparation method, and the application of using the YhdP antibody to treat Shigella flexneri infection.
  • the present invention provides a method for producing an anti-S. flexneri YhdP antibody, comprising the steps of: - fusing an antibody that produces mouse spleen cells previously immunized with a whole cell lysate of S. flexneri with myeloma B cells ;
  • Hybridoma cell lines are cultivated to produce monoclonal antibodies, and the antibody-producing mouse splenocytes are fused with myeloma B cells; monoclonal antibodies with high specificity and affinity for Shigella flexneri are selected.
  • the YhdP antibody produced was further sequenced to find that the produced antibody had a heavy chain comprising the region shown in SEQ ID NO: 9: and a light chain comprising the region shown in SEQ ID NO: 10 or a part thereof.
  • Shigella flexneri 2a 301 strain was isolated from stool samples of patients with diarrhea in Changping District, Beijing, and named S. flexneri 2a str. 301 (Sf301 for short). This strain has been used as the standard strain of Shigella flexneri 2a serotype in my country, provided by the Institute of Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention.
  • the harvested Sf2a301 was resuspended in sterile water, treated with lysozyme at a final concentration of 20 g/L, added with lysate and lysed by shaking, and then extracted DNA.
  • the YhdP gene was amplified by PCR reaction.
  • the reaction conditions were: pre-denaturation at 94°C for 3 minutes; 30s at 94°C, 30s at 55°C, 30s at 72°C; extension at 72°C for 3 minutes, and a total of 30 cycles.
  • the amplified product was correctly sequenced and cloned into pcDNA3.1.
  • mice (6 weeks old, male) were immunized intramuscularly with 50 ⁇ g of purified target protein YhdP mixed with 50 ⁇ g of Freund's complete adjuvant (Sigma, India). This was followed by two booster immunizations.
  • the antibody reactivity of immunized mouse serum was determined by ELISA, and the mouse with the highest titer value of 1:16000 was selected.
  • Hybridoma cell YhdP-C3-8 producing anti-YhdP antibody was cultured in a 5% CO2 humidified chamber in Eagle medium containing 10% (vol/vol) fetal bovine serum supplemented with 100 U /mL penicillin and 100mg/mL streptomycin. After the cells had grown to a density of 106 cells/ml, they were harvested by centrifugation at 1000 xg for 5 minutes. For total RNA isolation, cells were homogenized in RNA lysis solution (Sigma, USA) containing 5 ⁇ 106 cells/ml, followed by RNA extraction according to the manufacturer's instructions.
  • RNA lysis solution Sigma, USA
  • First-strand cDNA was synthesized from the mRNA template using random hexadeoxyribonucleotide primers.
  • the variable regions of the heavy chain (VH) and light chain (VL) were amplified from the first-strand cDNA with Taq DNA polymerase, and PCR was performed for 30 cycles (1 cycle at 94°C for 1 minute and at 55°C for 1 minute , 1 minute at 72°C).
  • Primers Yh-H-1 and Yh-H-2 were used to amplify VH; primers Yh-L-1 and Yh-L-2 were used to amplify VL.
  • the amplified variable heavy chain (SEQ ID NO:9) and light chain (SEQ ID NO:10) products were sequenced and immunoglobulin homology searches were performed using the Kabat database.
  • a cell model was used to study the pathogenicity of Shigella affected by anti-YhdP antibodies in vitro. Hela cells were infected with Shigella flexneri 2a 301 (Sf301), and the formation of plaques was observed under a high-power microscope to determine the degree to which the pathogenicity of Sf301 was affected by the anti-YhdP antibody.
  • Sf301 Shigella flexneri 2a 301
  • the Hela cells were adjusted to a concentration of 5 ⁇ 10 7 /L, added to a 24-well plate, and cultured overnight in a CO 2 incubator. After the Hela cells grew into a single layer, they were washed twice with 1640 medium without antibiotics.
  • the Sf301 in the logarithmic growth phase was taken out, and serum-free 1640 culture medium was added to the centrifuge tube, and the Sf301 (10 -1 -10 -8 ) was gradually diluted in the centrifuge tube according to a 10-fold gradient.
  • Shigella flexneri 2a strain 301 put the well-growing Hela cell culture plate, add Shigella flexneri 2a strain 301 to each well the next day, and fix the cells infected with Shigella flexneri 2a strain 301 with paraformaldehyde for 6h, 8h, and 12h respectively. After 24 hours, the cells were fixed at room temperature for 20 minutes.
  • a volume of 100 [mu]l of Shigella flexneri 2a strain 301 or S. typhimurium preparations was added to microtiter wells and blocked with 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS). After washing with PBS, the YhdP antibody produced by the mouse was added, and the antibody was serially diluted 10 times, and the plate was incubated at 37° C. for 60 minutes. Wells were washed, then secondary antibody (goat anti-mouse IgG-HRP, 1:1000) was added and incubated at 37°C for 60 minutes.
  • BSA bovine serum albumin
  • PBS phosphate buffered saline
  • the partial nucleotide and amino acid sequences involved in the present invention are as follows:
  • HCDR1 GRFTISRDGGG (SEQ ID NO: 1)
  • HCDR2 CKSLGQDT (SEQ ID NO:2)
  • HCDR3 YGDSQSIW (SEQ ID NO: 3)
  • LCDR1 VTVLDKLSRLGAGK (SEQ ID NO: 4)
  • LCDR2 WLVNTR (SEQ ID NO:5)
  • LCDR3 GYGMHWVRQ (SEQ ID NO:6)
  • Yh-L-1 GAAGAGA TTGTTGCAGC TGGACC (SEQ ID NO: 13)
  • Yh-L-2 AATAAG TGATGCCATT ACTAT (SEQ ID NO: 14)

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Abstract

抗志贺氏菌的单克隆抗体，具体提供一种可以结合福氏志贺氏菌的蛋白YhdP的包含特定序列的分离的抗体或其抗原结合部分，该YhdP 单克隆抗体可以用于治疗志贺氏菌感染或橙测食品和临床样品中福氏志贺氏菌。

Description

抗志贺氏菌的单克隆抗体及其应用 技术领域
本发明涉及抗福氏志贺氏菌的单克隆抗体，及其用于治疗福氏志贺氏菌引起的疾病的应用。
背景技术
福氏志贺氏菌也称福氏痢疾杆菌，是一种非能动的，非孢子形成的，兼性厌氧革兰氏阴性菌，临床上可引起细菌性痢疾，肠道的炎症反应是志贺氏菌病的一个重要特征，主要表现为结肠的水肿、溃疡及炎症。大约99％的病例发生在发展中国家，儿童和灵长类动物有较高的感染率和死亡率。对公共卫生造成了重大的食源性威胁。
福氏志贺氏菌主要通过粪便-口服途径传递。其它可能的传播方式可以来自摄取污染的食物或水和与无生命物体的皮下接触。福氏志贺氏菌感染剂量很低；仅需要100-200个细菌就能引起临床感染。但由于近年来，福氏志贺氏菌的耐药性不断增强，给临床治疗带来了较大困难。因此需要进一步研究抗福氏志贺氏菌的感染机制，从而研制预防福氏志贺氏菌的新的抗菌剂。
细菌细胞包膜是与环境和其他生物的第一道防线和接触点。因此，包膜对于细菌的存活和生理学至关重要，并且通常是抗菌剂的常用目标。革兰氏阴性菌具有由内膜和外膜(分别为IM和OM)分隔的多层包膜。OM是许多抗菌剂的屏障，因为它的脂质结构不对称，磷脂组成内小叶，脂多糖(LPS)组成外小叶。由于脂质合成发生在IM处，因此磷脂和LPS在生长过程中通过细胞包膜运输并在OM处不对称组装。磷脂如何转运到OM仍然未知。有研究表明YhdP通过一种未知的机制参与这一过程。YhdP属于AsmA样氏族，包含与脂质转运蛋白同源的结构域。数据显示，YhdP及其旁系同源物TamB和YdbH在细胞包膜中执行基本功能时是多余的，但并不等效。在AsmA样副同源物中，只有YhdP，TamB和YdbH的组合损失是致命的，且这些蛋白质是OM脂质稳态所必需的，并提出它们是OM生物发生所需的长期需要的磷脂转运蛋白。革兰氏阴性菌的特征是具有两个膜。已经确定了革兰氏阴性外膜生物发生所需的系统，但新合成的磷脂从内膜运输到外膜所需的系统，YhdP蛋白与这一过程有关。
Silhavy等人证明YhdP可能在磷脂流动的内外膜之间形成疏水通道，Angela M Mitchell等人证明yhdP缺失导致高水平的万古霉素和SDS敏感性，与生长阶段无关。我们分离出yhdP的自发抑制突变体，在ECA生物合成操纵子中具有功能丧失突变。ECA生物合成基因缺失 抑制了yhdP缺失引起的包膜通透性，而与包膜应激反应和与其他生物合成途径的相互作用无关，证明抑制是由去除ECA直接引起的。此外，yhdP缺失改变了细胞ECA水平，并且发现yhdP与ECA生物合成操纵子在系统发育上共同发生。细胞产生三种形式的ECA：ECA脂多糖(LPS)，与LPS核心相连的ECA链；ECA磷脂酰甘油，一种与磷脂酰甘油相连的表面暴露的ECA链；和环状ECA，一种在周质中发现的环化可溶性ECA分子。我们确定，yhdP缺失对包膜通透性的抑制是由环状ECA的损失引起的，尽管yhdP缺失降低了该分子的水平。从野生型细胞中去除环状ECA也会导致OM渗透性的变化。我们的数据表明，循环ECA以YhdP控制的方式维持OM渗透屏障。因此YhdP的缺失以周期性ECA依赖的方式破坏OM通透性屏障，使抗生素等有害分子进入细胞。这种维持包膜通透性屏障的作用是首次描述循环ECA的表型。由于革兰氏阴性包膜通常不透抗生素，因此本申请获得了一株YhdP抗体，了解YhdP维持屏障和排除抗生素的机制可能会改善抗生素的输送，进一步应用于福氏志贺氏菌的治疗。
发明内容
本发明针对上述问题，目的在于提供一种可产生针对YhdP单克隆抗体的杂交瘤细胞株，YhdP单克隆抗体，以及该抗体的制备方法和应用。
本发明采用如下技术方案：
一方面，本发明实施例公开了一种可产生YhdP单克隆抗体的杂交瘤细胞株以及YhdP单克隆抗体的制备方法。其包括：
步骤(1)采用纯化的YhdP重组蛋白作为免疫原免疫动物体Balb/c小鼠；
步骤(2)将骨髓瘤细胞SP2/0与免疫后的所述动物体的B淋巴细胞融合，得到杂交瘤细胞；
步骤(3)筛选特异性杂交瘤细胞阳性克隆，对所述阳性克隆进行细胞克隆化，筛选出稳定分泌YhdP单克隆抗体的杂交瘤细胞，所述筛选特异性杂交瘤细胞阳性克隆通过ELISA法进行；
步骤(4)获得YhdP单克隆抗体。
进一步，步骤(4)中，所述获得YhdP单克隆抗体包括：
体外培养所述稳定分泌YhdP单克隆抗体的杂交瘤细胞，分离纯化培养液，得到YhdP单克隆抗体。
本发明实施例还提供上述的YhdP单克隆抗体应用于辅助抗生素治疗福氏志贺氏菌感染。
ELISA法鉴定本发明的单克隆抗体类型为IgG2b型。
一方面，本发明实施例公开了一种分离的抗体或其抗原结合部分，其结合福氏志贺氏菌的蛋白YhdP，其中，所述抗体或其抗原结合部分包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2和如SEQ ID NO:3所示的HCDR3的重链可变区以及包含如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3的轻链可变区。
在一个实施方式中，分离的抗体或其抗原结合部分的重链可变区(VH)氨基酸序列为SEQ ID NO:7和轻链可变区(VL)氨基酸序列为SEQ ID NO:8。
在一个实施方式中，分离的抗体或其抗原结合部分的包含如SEQ ID NO:9所示的重链和如SEQ ID NO:10所示的轻链。
编码上述分离的抗体或其抗原结合部分的核苷酸。
一种载体，其包含能够编码上述分离的抗体或其抗原结合部分的核酸分子。
一种细胞，其包含上述载体。
一种药物组合物，其包含上述的抗体或其抗原结合部分。
包含YhdP单克隆抗体的药物组合物，用于治疗志贺氏菌的感染，该抗体与抗生素联用，可以增强抗生素的效果，避免抗生素耐药的问题。
所述的分离的抗体或其抗原结合部分在制备用于治疗志贺氏菌感染的药物中的应用。
本发明的有益效果：
本发明应用重组YhdP蛋白为免疫原，免疫Balb/c小鼠，采用经典的细胞融合技术获得稳定分泌YhdP单抗的杂交瘤细胞。其分泌的YhdP单克隆抗体可以用于辅助抗生素治疗福氏志贺氏菌感染。
附图说明
图1 YhdP抗体以及联合抗生素抑制sf301复制的效果
图2免疫荧光检测福氏2a志贺菌301株感染后的定位
图3感染0h、6h、12h、24h、36h、48hWestern Blot监测
具体实施方式
本发明提供了一种抗福氏志贺氏菌YhdP抗体，其制备方法，以及通过使用YhdP抗体治疗福氏志贺氏菌感染的用途。
本发明提供了产生抗福氏志贺氏菌YhdP抗体的方法，包括以下步骤：-将预先用福氏志贺氏菌全细胞裂解物免疫的产生小鼠脾细胞的抗体与骨髓瘤B细胞融合；
培养杂交瘤细胞株，产生单克隆抗体，将产生抗体的小鼠脾细胞与骨髓瘤B细胞融合；选择对福氏志贺氏菌显示高特异性和亲和力的单克隆抗体。
进一步对产生YhdP抗体进行测序，以发现产生的抗体具有包含SEQ ID NO:9所示区域的重链：以及包含SEQ ID NO:10所示区域或其一部分的轻链。
在下面的具体实施例中进一步解释本发明，这些具体实施例仅仅是为了举例说明，而不应被解释为限制本发明的范围。
实施例
实施例1-细菌菌株
福氏2a志贺菌301株，从北京市昌平区腹泻患者粪便标本中分离，命名为S.flexneri 2a str.301(简称Sf301)。该菌株一直作为我国福氏2a志贺菌血清型的标准株，由中国疾病预防控制中心传染病预防控制所提供。
实施例2-制备抗福氏志贺氏菌YhdP单克隆抗体
(a)YhdP抗原的制备
收获的Sf2a301重悬于无菌水中，加入质量终浓度20g/L溶菌酶处理，加入裂解液振荡裂解后，提取DNA。以此为模板进行PCR反应扩增YhdP的基因，反应条件为：94℃预变性3min；94℃30s，55℃30s，72℃30s；72℃延伸3min，共进行30个循环。扩增产物测序正确后克隆入pcDNA3.1中。重组质粒转化宿主菌E.coli DH5α中，阳性克隆接种于LB培养液中，加入终浓度为1mmo1/LIPTG进行诱导，诱导后样品进行SDS-PAGE电泳分析。SDS-PAGE结束后电转移至硝酸纤维素膜，抗His单克隆抗体鉴定。表达产物鉴定正确后，将200ml诱导菌离心后收集菌体，超声裂解后，收集上清，采用Invitrogen公司Ni 2+-NTA蛋白纯化试剂盒，对目的蛋白YhdP进行亲和纯化。
(b)小鼠的免疫
用纯化的50μg目的蛋白YhdP混合50μg弗氏完全佐剂(Sigma，India)肌肉免疫BALB/c小鼠(6周龄，雄性)。随后加强免疫两次。
免疫小鼠血清的抗体反应性通过ELISA测定，挑选出最高滴度值为1:16000的小鼠。
(c)通过杂交瘤技术产生单克隆抗体
选择抗体特异性最高的小鼠进行融合反应。
制备饲养层细胞：细胞融合前1-2天制备并饲养巨噬细胞。在96孔板上铺一层饲养细胞层，每孔2×10 4个细胞。然后置于37℃，6％的CO 2培养箱中培养。
无菌取脾细胞，悬浮于5ml HAT培养基中，将1×10 8/ml脾细胞与2×10 7/ml骨髓瘤细胞SP2/0混合，加30ml不完全培养基混匀。1000r/min离心5-10min，将上清尽量吸净，加入预热40℃的50％PEG(PH＝8.0)1ml，边加边轻轻搅拌，然后加入不完全培养基，静置10min，1000r/min离心5min，弃上清，加入HAT培养基重悬，分装96孔板，置于37℃，6％的CO 2培养箱中培养。融合两周后筛选杂交瘤细胞。
通过限制性稀释方法将含有阳性细胞的孔克隆到96孔组织培养板中。ELISA筛选杂交瘤细胞，在间接ELISA中只有8个克隆给出强阳性信号；其中，克隆YhdP-C3-8对YhdP表现出最高的亲和力。选择该克隆以进一步大规模生产抗体。
实施例3-单克隆抗体的测序
(a)mRNA分离
在含有10％(vol/vol)胎牛血清的Eagle培养基中，在5％CO 2湿化室中培养产生抗YhdP抗体的杂交瘤细胞YhdP-C3-8，所述胎牛血清补充有100U/mL青霉素和100mg/mL链霉素。在细胞生长至密度为10 6个细胞/ml后，通过以1000×g离心5分钟将它们离心收获。对于总RNA分离，在含有5×10 6个细胞/ml的RNA裂解溶液(Sigma，USA)中匀浆细胞，然后根据制造商的说明进行RNA提取。
(b)cDNA合成，免疫球蛋白可变区的PCR扩增
用随机的六脱氧核糖核苷酸引物从mRNA模板合成第一链cDNA。重链(VH)和轻链(VL)的可变区用Taq DNA聚合酶从第一链cDNA中扩增，PCR 30个循环(1个循环在94℃为1分钟，在55℃为1分钟，在72℃为1分钟)。引物Yh-H-1和Yh-H-2用于扩增VH；引物 Yh-L-1和Yh-L-2用于扩增VL。对扩增的可变重链(SEQ ID NO:9)和轻链(SEQ ID NO:10)产物进行测序，并使用Kabat数据库进行免疫球蛋白的同源性搜索。
实施例4-抗YhdP的抗体以及联合抗生素治疗效果研究
采用细胞模型研究志贺菌在体外受抗YhdP的抗体影响的致病性。通过福氏2a志贺菌301(Sf301)感染Hela细胞，在高倍镜下观察噬斑的形成，判断Sf301的致病能力受到抗YhdP的抗体影响程度。
将Hela细胞调整浓度为5×10 7/L，加入24孔板，置CO 2培养箱过夜培养，待Hela细胞生长为单层，用不含抗生素的1640培养基洗涤2次。
取出处于对数生长期的Sf301，在离心管中加入无血清1640培养液，按照10倍梯度在离心管中逐级稀释Sf301(10 -1-10 -8)。
将不同稀释度的Sf301混有抗YhdP抗体(1:500稀释)、抗YhdP抗体(1:500稀释)+氨苄青霉素(1％)、氨苄青霉素(1％)的1640培养基，以及对照组Sf301混有无抗体的1640培养基，加入24孔板，每个稀释度3个平行孔，每孔300μl，留一个孔做正常细胞对照，置于37℃培养箱2小时，每30min摇晃一次，使细菌均匀分布，2小时后，弃上清，用PBS洗3次，尽量去除残余液体。
在水浴锅中75℃加热融化2％低熔点琼脂糖(超纯水配制)。待冷却到50℃左右时，与37℃预热的无酚红1640培养基1:1混合(1640中含有20μg/ml庆大霉素、5％小牛血清)，混匀后迅速加入到24孔板中，每孔500μl。
将24孔板在室温孵育半小时，待琼脂糖凝固后再将24孔板翻转过来倒置培养，每天在显微镜下观察细胞病变情况。培养第2-3天时，将24孔板取出加入2ml 4％多聚甲醛，固定过夜，剔除覆盖的琼脂糖，用0.1％的结晶紫染色，对着光线计数空斑数。结果参见图1。
实施例5-免疫荧光检测福氏2a志贺菌301株感染后的定位
将生长状态良好的Hela细胞传孔板中，次日，每孔加入福氏2a志贺菌301株，分别用多聚甲醛固定感染福氏2a志贺菌301株后的细胞6h、8h、12、24h后的细胞，室温固定20min。弃多聚甲醛，用PBS洗3遍，每次5min，加入0.1％Triton-X-100/PBS，室温透化10min，PBS清洗后，加入BSA室温封闭2h，将YhdP单抗以1:200比例稀释，加入培养皿中，孵育1h后，4℃放置过夜，第二天，用PBST清洗3遍，加入1:400山羊抗小鼠IgG/Dylight二抗，室 温避光孵育1h，用PBST清洗2抗，荧光显微镜观察(见图2)。结果显示YhdP单抗可以定位福氏2a志贺菌301株在细胞中的位置。
实施例6-蛋白质印迹分析(Western Blot)
将生长状态良好的Hela细胞传孔板中，次日，每孔加入福氏2a志贺菌301株，感染6h、12h、24h、36h、48h后收获细胞，PBS洗细胞2次，将细胞沉淀中加入100μlPIPA裂解液，轻轻混匀，冰浴裂解30min，离心收集上清，用BCA蛋白定量方法定量细胞裂解液的蛋白浓度，调整蛋白浓度一致，加上样缓冲液制备成蛋白样品，100℃加热变性后，将蛋白样品用10％聚丙烯酰胺凝胶电泳分离，然后电转至硝酸纤维素薄膜上。转模完成后，经脱脂牛奶封闭，加YhdP抗体过夜，然后用PBST洗涤三次，加入抗鼠IgG Dy800二抗，室温下避光孵育40min，PBST洗涤三次，之后用Odyssey进行扫描鉴定。结果显示YhdP抗体能够识别福氏2a志贺菌301株感染后的蛋白裂解物(参见图3)。
实施例7-间接平板酶联免疫吸附测定
将100μl体积的福氏2a志贺菌301株或鼠伤寒沙门氏菌制剂加入微量滴定孔中，并用5％牛血清白蛋白(BSA)在磷酸盐缓冲盐水(PBS)中封闭。用PBS洗涤后，加入由小鼠产生的YhdP抗体，抗体10倍梯度稀释，将板在37℃孵育60分钟。洗涤孔，然后加入二抗(山羊抗小鼠IgG-HRP，1:1000)并在37℃孵育60分钟。在用PBS再次洗涤之后，向孔中加入100μl TMB/H 2O 2底物，并在室温下孵育约10分钟。结果清楚显示单克隆抗体对福氏志贺氏菌可以特异性识别，但不识别鼠伤寒沙门氏菌，抗体滴度为1:6400。
本发明涉及的部分核苷酸和氨基酸序列如下：
HCDR1：GRFTISRDGGG(SEQ ID NO:1)
HCDR2：CKSLGQDT(SEQ ID NO:2)
HCDR3：YGDSQSIW(SEQ ID NO:3)
LCDR1：VTVLDKLSRLGAGK(SEQ ID NO:4)
LCDR2：WLVNTR(SEQ ID NO:5)
LCDR3：GYGMHWVRQ(SEQ ID NO:6)
重链可变区
Figure PCTCN2022142714-appb-000001
轻链可变区
Figure PCTCN2022142714-appb-000002
重链
Figure PCTCN2022142714-appb-000003
轻链
Figure PCTCN2022142714-appb-000004
Yh-H-1:GTGTGT GCTTGAAGCCAGTG(SEQ ID NO:11)
Yh-H-2:GTCTTGGAGCGGAGTCAACTCC(SEQ ID NO:12)
Yh-L-1:GAAGAGA TTGTTGCAGC TGGACC(SEQ ID NO:13)
Yh-L-2:AATAAG TGATGCCATT ACTAT(SEQ ID NO:14)

Claims (12)

  1. 一种分离的抗体或其抗原结合部分，其结合福氏志贺氏菌的蛋白YhdP，其中，所述抗体或其抗原结合部分包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:2所示的HCDR2和如SEQ ID NO:3所示的HCDR3的重链可变区以及包含如SEQ ID NO:4所示的LCDR1、如SEQ ID NO:5所示的LCDR2和如SEQ ID NO:6所示的LCDR3的轻链可变区。
  2. 如权利要求1所述的分离的抗体或其抗原结合部分，其包含：如所SEQ ID NO:7所示的重链可变区(VH)和如SEQ ID NO:8所示的轻链可变区(VL)。
  3. 如权利要求1或2所述的分离的抗体或其抗原结合部分，其包含：如SEQ ID NO:9所示的重链和如SEQ ID NO:10所示的轻链。
  4. 编码如权利要求1-3任一所述的分离的抗体或其抗原结合部分的核苷酸。
  5. 一种载体，其包含能够编码权利要求1至3中任一项所述的抗体或其抗原结合部分的核酸分子。
  6. 一种细胞，其包含权利要求5所述的载体。
  7. 一种药物组合物，其包含权利要求1-3中任一项所述的抗体或其抗原结合部分。
  8. 如权利要求7所述的药物组合物，其特征在于用于治疗志贺氏菌的感染。
  9. 如权利要求7或8所述的药物组合物，其特征在于与抗生素联用。
  10. 如权利要求1-3任一项所述的分离的抗体或其抗原结合部分在制备用于治疗志贺氏菌感染的药物中的应用。
  11. 如权利要求1-3任一项所述的分离的抗体或其抗原结合部分在制备检测食品和临床样品中福氏志贺氏菌试剂盒中的应用。
  12. 如权利要求11所述的应用，检测方法包括免疫荧光、蛋白质印迹、间接平板ELISA或斑点ELISA。
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