WO2023245800A1 - 一种抗隐球菌荚膜多糖单克隆抗体及其应用 - Google Patents

一种抗隐球菌荚膜多糖单克隆抗体及其应用 Download PDF

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WO2023245800A1
WO2023245800A1 PCT/CN2022/108076 CN2022108076W WO2023245800A1 WO 2023245800 A1 WO2023245800 A1 WO 2023245800A1 CN 2022108076 W CN2022108076 W CN 2022108076W WO 2023245800 A1 WO2023245800 A1 WO 2023245800A1
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monoclonal antibody
capsular polysaccharide
seq
variable region
chain variable
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PCT/CN2022/108076
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French (fr)
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付成华
翟栓柱
魏新宇
盛长忠
粟艳
周泽奇
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丹娜(天津)生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • 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
    • A61P31/10—Antimycotics
    • 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
    • G01N33/56961—Plant cells or fungi
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/375—Assays involving biological materials from specific organisms or of a specific nature from fungi from Basidiomycetes
    • 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

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to an anti-cryptococcal capsular polysaccharide monoclonal antibody and its application.
  • Cryptococcus neoformans is an important opportunistic pathogenic fungus that often invades the meninges, lungs or skin and causes infection in the corresponding parts.
  • Cryptococcal meningitis CNM
  • CNM Cryptococcal meningitis
  • the incidence and mortality of CNM have increased significantly.
  • about 5% to 10% of AIDS patients in the United States develop cryptoencephalon, while in some developing countries, the incidence of cryptoencephalon among AIDS patients is even higher.
  • pulmonary cryptococcosis caused by Cryptococcus neoformans infection of the respiratory system is also increasing year by year. Therefore, there is a need to establish new diagnostic methods for cryptococcal infection.
  • Monoclonal antibodies are highly uniform antibodies produced by a single B cell clone and only target a specific antigenic epitope. They are called monoclonal antibodies. Usually prepared using hybridoma technology. Hybridoma antibody technology is based on cell fusion technology, which fuses sensitized B cells with the ability to secrete specific antibodies and myeloma cells with unlimited reproduction ability into B cell hybrids. tumor. By cultivating a single hybridoma cell with this characteristic into a cell population, a specific antibody, that is, a monoclonal antibody, can be prepared against an antigenic epitope. Currently, the most widely used monoclonal antibodies are mouse monoclonal antibodies, but mouse monoclonal antibodies have problems with weak affinity and poor specificity. Rabbit monoclonal antibody technology has developed rapidly in recent years. Compared with mouse monoclonal antibodies, rabbit monoclonal antibodies have the advantages of recognizing more sites, stronger affinity and specificity.
  • Immunoassay technology is a method that utilizes the specific binding reaction between antigens and antibodies to achieve qualitative or quantitative detection of antigens or antibodies by detecting markers labeled on the reactants. According to the different labeling substances, it is divided into enzyme-linked immunosorbent assay technology (Enzyme-Linked Immunosorbant Assay, ELISA), immunofluorescence detection technology, chemiluminescence immunoassay technology, immune microsphere technology and immunocolloidal gold technology, etc. Among them, ELISA technology has the characteristics of simple operation, high sensitivity and short detection time, and is widely used in clinical testing and scientific research.
  • Antibodies with excellent performance are the basis of immunoassay technology. Only with good antibodies can immunoassay kits with excellent performance be developed. Therefore, providing an anti-cryptococcal capsular polysaccharide monoclonal antibody with high affinity is of great significance for the detection of cryptococci.
  • the object of the present invention is to provide an anti-cryptococcal capsular polysaccharide monoclonal antibody and its application.
  • the monoclonal antibody is a rabbit-derived monoclonal antibody.
  • the antigen recognition site of the monoclonal antibody is It is multiple, has good specificity and high affinity, and solves the problems of the practical application of mouse monoclonal antibodies.
  • the anti-cryptococcal capsular polysaccharide monoclonal antibody has important application value in preparing products for cryptococcal detection.
  • the present invention provides an anti-Cryptococcus capsular polysaccharide monoclonal antibody, which includes a heavy chain variable region and a light chain variable region;
  • the heavy chain variable region includes the heavy chain CDR3 shown in SEQ ID NO.3;
  • the light chain variable region includes the light chain CDR3 shown in SEQ ID NO. 6.
  • the heavy chain variable region also includes the heavy chain CDR1 shown in SEQ ID NO.1 and the heavy chain CDR2 shown in SEQ ID NO.2.
  • the light chain variable region also includes the light chain CDR1 shown in SEQ ID NO.4 and the light chain CDR2 shown in SEQ ID NO.5.
  • SEQ ID NO.1 YYMST.
  • SEQ ID NO.2 YASWTKIHAGGSAYYG.
  • SEQ ID NO.4 QASESVYNNRLS.
  • SEQ ID NO.6 LSGYCFGGYAST.
  • the monoclonal antibody in the present invention has many original recognition sites, good specificity and high affinity; the monoclonal antibody is a rabbit-derived monoclonal antibody and has many antigen recognition sites; the monoclonal antibody does not interact with other carbohydrates. (Including mannan, galactomannan, lipopolysaccharide, peptidoglycan, 1,3- ⁇ -D-glucan and BSA) cross-reactivity and strong specificity; the screened monoclonal antibodies have strong specificity for GXM antigen It has strong binding ability and high affinity, and its titer for GXM antigen reaches 1:1280000 (OD value>0.5).
  • amino acid sequence of the heavy chain variable region includes the sequence shown in SEQ ID NO. 7.
  • amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO. 8.
  • the anti-cryptococcal capsular polysaccharide monoclonal antibody also includes any one or a combination of at least two of rabbit-derived IgG1, IgG2, IgG3 or IgG4 constant regions, preferably rabbit-derived IgG1 constant region.
  • the present invention selects New Zealand big-eared rabbits as experimental animals to prepare monoclonal antibodies, which overcomes the defect of weak affinity of mouse-derived antibodies; the present invention uses cryptococcal capsular polysaccharide as an antigen for immunization, and the obtained spleen cells are combined with myeloma cells Cell fusion obtains rabbit-derived monoclonal antibodies, which have good stability and strong affinity for cryptococcal capsular polysaccharides; the monoclonal antibodies prepared by the present invention have good specificity and strong affinity for cryptococcal capsulating polysaccharides, but do not It cross-reacts with other sugars such as galactomannan, mannan, and lipopolysaccharide, and can potentially be used in the detection of cryptococcal antigens, the detection and identification of clinical samples of cryptococcal infection, etc.
  • the present invention provides a nucleic acid molecule encoding the anti-cryptococcal capsular polysaccharide monoclonal antibody described in the first aspect.
  • the nucleotide sequence encoding the heavy chain variable region of the anti-cryptococcal capsular polysaccharide monoclonal antibody includes the sequence shown in SEQ ID NO. 9; the nucleotide sequence encoding the anti-cryptococcal capsular polysaccharide monoclonal antibody.
  • the nucleotide sequence of the light chain variable region includes the sequence shown in SEQ ID NO. 10.
  • the present invention provides an expression vector, which includes the nucleic acid molecule described in the second aspect.
  • the present invention provides a host cell containing at least one copy of the expression vector described in the third aspect, or the nucleic acid molecule described in the second aspect is integrated into the genome of the host cell.
  • the host cells include 293T cells or CHO cells.
  • the present invention provides a pharmaceutical composition comprising the anti-cryptococcal capsular polysaccharide monoclonal antibody described in the first aspect.
  • the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
  • the present invention provides a kit for detecting Cryptococcus, which includes the anti-Cryptococcus capsular polysaccharide monoclonal antibody described in the first aspect.
  • the kit for detecting Cryptococcus also includes any one or a combination of at least two of a positive control substance, a negative control substance, an antibody diluent, a chromogenic solution, a stop solution, a blocking solution or a washing solution.
  • the present invention provides the anti-cryptococcal capsular polysaccharide monoclonal antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, or The use of any one or a combination of at least two of the pharmaceutical compositions described in the fifth aspect in the preparation of cryptococcal infection treatment drugs and/or detection products.
  • the present invention has the following beneficial effects:
  • the monoclonal antibody in the present invention has many original recognition sites, good specificity and high affinity; the monoclonal antibody is a rabbit-derived monoclonal antibody and has many antigen recognition sites; the monoclonal antibody does not interact with other sugars (including Mannan, galactomannan, lipopolysaccharide, peptidoglycan, 1,3- ⁇ -D-glucan and BSA) produce cross-reactivity and strong specificity; the screened monoclonal antibodies have strong specificity against GXM antigen The binding ability and affinity are high, and the titer for GXM antigen reaches 1:1280000 (OD value>0.5).
  • Figure 1 is a diagram of the SDS-PAGE electrophoresis results of the monoclonal antibody in Example 6.
  • Figure 2 is the detection result of the affinity activity of the monoclonal antibody in Example 7.
  • FIG. 3 shows the cross-reaction results in Example 8.
  • GXM cryptococcal capsular polysaccharide antigen
  • the specific immunization steps are as follows:
  • the third week To strengthen the immunity, the GXM antigen is diluted to 0.5 mg/mL with physiological saline and fully emulsified with Freund's incomplete adjuvant at 1:1. The immunization dose is 1 mL per animal.
  • the fifth week Boost immunity.
  • the immunization method is the same as the third week.
  • the immunization dose is 1mL per animal.
  • the first serum titer test take 200-500 ⁇ L of ear marginal venous blood from the immunized New Zealand big-eared rabbits, let it stand for 30 minutes, then centrifuge to separate the serum, and measure the antiserum titer.
  • Week 9 Boost immunity, the immunization method is the same as step (3).
  • Week 10 The third serum titer test, the same as step (4).
  • Week 11 Strengthen immunity, the immunization method is the same as step (3).
  • the method for determining antiserum titer is as follows:
  • GXM antigen as the coating antigen.
  • the coating concentration is 100ng/well. Coat at 37°C for 2 hours. Then add blocking solution prepared with 3% BSA and incubate at 37°C for 1 hour. Shake off the liquid and dry it for later use. Then carry out gradient dilution of the prepared antibody serum and add it to the enzyme plate. Add 100 ⁇ L to each well and incubate at 37°C for 1 hour. Then wash it three times with PBST and let it stand for 3 minutes each time.
  • HRP-labeled goat anti-rabbit IgG as the Enzyme-labeled antibody, diluted 1:5000, add 100 ⁇ L to each well, incubate at 37°C for 1 hour, then wash three times with PBST and let stand for 40 seconds each time. Add 100 ⁇ L of TMB to each well and let stand, incubate at 37°C for 15 min to develop color, then add 50 ⁇ L of stop solution to each well to end the reaction, and detect the OD 450 value on a microplate reader.
  • the titer of the prepared rabbit antiserum is tested. If the titer is qualified, the spleens of qualified New Zealand big-eared rabbits are used for cell fusion to prepare monoclonal hybridoma cell lines.
  • the specific preparation method is as follows:
  • the immunized New Zealand big-eared rabbits were sacrificed, and the spleens were removed under aseptic conditions. Wash once with cell culture medium, grind, pass through a stainless steel mesh, wash twice with cell culture medium, and centrifuge to obtain cells.
  • the obtained hybridoma cells were screened using the ELISA method to find polyclonal antibody cell lines with up to 10 wells targeting the cryptococcal capsular polysaccharide antigen.
  • the cell lines can produce specific monoclonal antibodies with high affinity against the respective antigens.
  • the cell fusion step there are two parental cells and three randomly fused cells in the culture medium.
  • the successfully fused hybridoma cells must be separated from the numerous cells.
  • B lymphocytes cannot survive in vitro for a long time. Only myeloma cells and their own fused cells need to be removed. Therefore, the fused cells need to be cultured in HAT culture medium to selectively retain hybridoma cells.
  • the growth of the cells can be observed on the 5th day after fusion.
  • the indirect ELISA method can be used to detect the cell culture supernatant and screen positive hybridoma cell lines for cloning culture.
  • the positive hybridoma cells were cloned and cultured using the limiting dilution method.
  • the positive hybridoma cells with the strongest detection result titer were expanded until the cell positivity rate reached 100% to determine the strain.
  • Use ELISA to measure the titer of the culture supernatant of the established hybridoma cell line, and freeze the expanded cultured monoclonal hybridoma cell line in liquid nitrogen.
  • RNA is then precipitated from the aqueous layer using isopropyl alcohol.
  • DNA was precipitated from the organic layer using ethanol.
  • Proteins were precipitated from the phenol-ethanol supernatant using isopropanol precipitation. Wash the precipitated RNA to remove impurities and resuspend it for later use.
  • RNA as the template
  • tRNA as the primer
  • a cDNA single strand complementary to the RNA template is synthesized, which forms an RNA-cDNA hybrid with the RNA template. body.
  • the RNA strand is hydrolyzed, and the second DNA strand is synthesized using cDNA as a template. At this point, total RNA was reverse transcribed into cDNA.
  • Antibody fragments of heavy and light chains were amplified according to the standard operating procedure of Rapid Amplification of cDNA Ends (RACE).
  • RACE Rapid Amplification of cDNA Ends
  • the amplified antibody fragments were individually cloned into standard cloning vectors. Perform colony PCR to screen for clones with the correct size insert.
  • the antibody sequence is obtained.
  • the nucleotide sequence of the antibody is as follows:
  • the nucleotide sequence encoding the heavy chain variable region of the anti-cryptococcal capsular polysaccharide monoclonal antibody includes the sequence shown in SEQ ID NO. 9; the light chain encoding the anti-cryptococcal capsular polysaccharide monoclonal antibody may The nucleotide sequence of the variable region includes the sequence shown in SEQ ID NO.10.
  • Cell culture medium harvest Centrifuge to collect the cell culture medium supernatant, and use Protein A Beads for antibody purification;
  • Example 7 Using ELISA method to detect the affinity activity (titer) of monoclonal antibodies against Cryptococcus capsular polysaccharide (GXM)
  • affinity activity detection the steps for detecting affinity activity are as follows:
  • Example 8 Using ELISA method to detect the specificity of monoclonal antibodies
  • the specificity of monoclonal antibodies is detected through cross-reaction.
  • the steps of cross-reaction are as follows:
  • the enzyme plate was coated with capsular polysaccharide, mannan, galactomannan, lipopolysaccharide, peptidoglycan, 1,3- ⁇ -D-glucan and BSA respectively, and the coating amount per well was 50ng/ hole; dilute the monoclonal antibody to 10ng/mL, add it to each microplate, add 100 ⁇ L to each well, and incubate at 37°C for 1 hour; after washing, add 100 ⁇ L of secondary antibody (HRP-labeled goat anti-rabbit IgG) to each well, and incubate at 37°C. Incubate for 0.5h; add TMB after washing, incubate at 37°C for 15min, and stop reading.
  • the cross-reaction results are shown in Figure 3. The results show that the obtained monoclonal antibody does not cross-react with other sugars and has strong specificity.
  • the present invention provides an anti-cryptococcal capsular polysaccharide monoclonal antibody.
  • the monoclonal antibody is a rabbit-derived monoclonal antibody with multiple antigen recognition sites, good specificity, and high affinity. It solves the problem of mouse-derived monoclonal antibodies. There are problems in the practical application of antibodies.
  • the anti-cryptococcal capsular polysaccharide monoclonal antibody has important application value in preparing products for cryptococcal detection.

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Abstract

本发明提供了一种抗隐球菌荚膜多糖单克隆抗体及其应用，所述抗隐球菌荚膜多糖单克隆抗体包括重链可变区和轻链可变区，所述重链可变区包括SEQ ID NO.3所示的重链CDR3，所述轻链可变区包括SEQ ID NO.6所示的轻链CDR3。所述单克隆抗体为兔源单克隆抗体，所述单克隆抗体的抗原识别位点多、特异性好、亲和力高，解决了鼠源单克隆抗体在实际应用方面的问题，所述抗隐球菌荚膜多糖单克隆抗体在制备隐球菌检测的产品中具有重要应用价值。

Description

一种抗隐球菌荚膜多糖单克隆抗体及其应用 技术领域
本发明属于生物技术领域，具体涉及一种抗隐球菌荚膜多糖单克隆抗体及其应用。
背景技术
新型隐球菌是一种重要的条件致病性真菌，常侵犯脑膜、肺或皮肤，引起相应部位感染。新型隐球菌脑膜炎(CNM)是中枢神经系统真菌感染最常见的类型。近年来，CNM的发病率和病死率明显增加。据统计，在美国大约有5％～10％的AIDS患者发生隐脑，而在一些发展中国家，AIDS患者隐脑的发病率更高。同时，新型隐球菌感染呼吸系统引起的肺新型隐球菌病也呈逐年增多的趋势。因此，需要建立新的隐球菌感染诊断方法。
单克隆抗体是由单一B细胞克隆产生的高度均一、仅针对某一特定抗原表位的抗体，称为单克隆抗体。通常采用杂交瘤技术来制备，杂交瘤(hybridoma)抗体技术是在细胞融合技术的基础上，将具有分泌特异性抗体能力的致敏B细胞和具有无限繁殖能力的骨髓瘤细胞融合为B细胞杂交瘤。用具备这种特性的单个杂交瘤细胞培养成细胞群，可制备针对一种抗原表位的特异性抗体即单克隆抗体。目前，使用最广泛的单克隆抗体是鼠源单克隆抗体，但鼠源单克隆抗体存在亲和力弱、特异性差的问题。近些年兔单克隆抗体技术发展迅速，与鼠单克隆抗体相比，兔单克隆抗体具有识别更多位点、更强的亲和性和特异性等优点。
免疫检测技术是利用抗原抗体间能特异性结合反应，通过检测标记在反应物上的标记物，对抗原或抗体实现定性或定量检测的方法。根据标记物质的不 同，分为酶联免疫分析技术(Enzyme-Linked Immunosorbant Assay，ELISA)、免疫荧光检测技术、化学发光免疫分析技术、免疫微球技术和免疫胶体金技术等。其中，ELISA技术具有操作简单、灵敏度高、检测时间短的特点，在临床检测和科学研究中被广泛使用。
性能优良的抗体是免疫检测技术的基础，只有具有好的抗体，才能研发出性能优良的免疫检测试剂盒。因此，提供一种亲和能力高的抗隐球菌荚膜多糖单克隆抗体对于检测隐球菌检测具有重要意义。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种抗隐球菌荚膜多糖单克隆抗体及其应用，所述单克隆抗体为兔源单克隆抗体，所述单克隆抗体抗原识别位点多、特异性好、亲和力高，解决了鼠源单克隆抗体在实际应用方面的问题，所述抗隐球菌荚膜多糖单克隆抗体在制备隐球菌检测的产品中具有重要应用价值。
为达到此发明目的，本发明采用以下技术方案：
第一方面，本发明提供了一种抗隐球菌荚膜多糖单克隆抗体，所述抗隐球菌荚膜多糖单克隆抗体包括重链可变区和轻链可变区；
所述重链可变区包括SEQ ID NO.3所示的重链CDR3；
所述轻链可变区包括SEQ ID NO.6所示的轻链CDR3。
优选地，所述重链可变区还包括SEQ ID NO.1所示的重链CDR1和SEQ ID NO.2所示的重链CDR2。
优选地，所述轻链可变区还包括SEQ ID NO.4所示的轻链CDR1和SEQ ID NO.5所示的轻链CDR2。
SEQ ID NO.1：YYMST。
SEQ ID NO.2：YASWTKIHAGGSAYYG。
SEQ ID NO.3：EYLNNFGL。
SEQ ID NO.4：QASESVYNNRLS。
SEQ ID NO.5：DAASKLS。
SEQ ID NO.6：LSGYCFGGYAST。
本发明中所述单克隆抗体抗的原识别位点多、特异性好、亲和力高；所述单克隆抗体为兔源单克隆抗体，抗原识别位点多；所述单克隆抗体不与其他糖(包括甘露聚糖、半乳甘露聚糖、脂多糖、肽聚糖、1,3-β-D-葡聚糖和BSA)产生交叉反应，特异性强；筛选的单克隆抗体对GXM抗原具有较强的结合能力，亲和力高，对GXM抗原的效价达到1:1280000(OD值＞0.5)。
优选地，所述重链可变区的氨基酸序列包括SEQ ID NO.7所示的序列。
优选地，所述轻链可变区的氨基酸序列包括SEQ ID NO.8所示的序列。
SEQ ID NO.7：
Figure PCTCN2022108076-appb-000001
SEQ ID NO.8：
Figure PCTCN2022108076-appb-000002
优选地，所述抗隐球菌荚膜多糖单克隆抗体还包括兔源IgG1、IgG2、IgG3或IgG4恒定区中的任意一种或至少两种的组合，优选为兔源IgG1恒定区。
本发明选择新西兰大耳兔为实验动物，制备单克隆抗体，克服了小鼠源抗 体亲和力弱的缺陷；本发明以隐球菌夹膜多糖作为抗原进行免疫，得到的脾脏细胞通过与骨髓瘤细胞进行细胞融合得到兔源单克隆抗体，稳定性好，对隐球菌夹膜多糖具有很强的亲和性；本发明制备的单克隆抗体特异性好，与夹膜多糖具有强亲和性，但是不与半乳甘露聚糖、甘露聚糖、脂多糖等其他糖类产生交叉反应，可潜在地应用于隐球菌的抗原检测、隐球菌感染临床样本的检测与鉴定等。
第二方面，本发明提供一种核酸分子，所述核酸分子编码第一方面所述抗隐球菌荚膜多糖单克隆抗体。
优选地，编码所述抗隐球菌荚膜多糖单克隆抗体的重链可变区的核苷酸序列包括SEQ ID NO.9所示的序列；编码所述抗隐球菌荚膜多糖单克隆抗体的轻链可变区的核苷酸序列包括SEQ ID NO.10所示的序列。
SEQ ID NO.9：
Figure PCTCN2022108076-appb-000003
SEQ ID NO.10：
Figure PCTCN2022108076-appb-000004
第三方面，本发明提供一种表达载体，所述表达载体包括第二方面所述的核酸分子。
第四方面，本发明提供一种宿主细胞，所述宿主细胞中含有至少一个拷贝的第三方面所述的表达载体，或所述宿主细胞的基因组中整合有第二方面所述的核酸分子。
优选地，所述宿主细胞包括293T细胞或CHO细胞。
第五方面，本发明提供一种药物组合物，所述药物组合物包括第一方面所述的抗隐球菌荚膜多糖单克隆抗体。
优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第六方面，本发明提供一种检测隐球菌的试剂盒，所述检测隐球菌的试剂盒包括第一方面所述的抗隐球菌荚膜多糖单克隆抗体。
优选地，所述检测隐球菌的试剂盒还包括阳性对照品、阴性对照品、抗体稀释液、显色液、终止液、封闭液或洗涤液中任意一种或至少两种的组合。
第七方面，本发明提供第一方面所述的抗隐球菌荚膜多糖单克隆抗体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的宿主细胞或第五方面所述的药物组合物中任意一种或至少两种的组合在制备隐球菌感染疾病治疗药物和/或检测产品中的应用。
相对于现有技术，本发明具有以下有益效果：
本发明中所述单克隆抗体抗的原识别位点多、特异性好、亲和力高；所述单克隆抗体为兔源单克隆抗体，抗原识别位点多；单克隆抗体不与其他糖(包括甘露聚糖、半乳甘露聚糖、脂多糖、肽聚糖、1,3-β-D-葡聚糖和BSA)产生交叉反应，特异性强；筛选的单克隆抗体对GXM抗原具有较强的结合能力，亲和 力高，对GXM抗原的效价达到1:1280000(OD值＞0.5)。
附图说明
图1为实施例6中单克隆抗体的SDS-PAGE电泳结果图。
图2为实施例7中单克隆抗体的亲和活性的检测结果。
图3为实施例8中交叉反应的结果。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1抗原制备
隐球菌荚膜多糖抗原(GXM)的制备方法如下所示：
(1)隐球菌的菌体培养及荚膜多糖粗提
将隐球菌接种于沙氏液体培养基(每升培养基含1％蛋白胨、4％D-葡萄糖)中，在30℃摇床于200rpm培养48h左右，当培养液的pH值降至4.2-4.5时停止培养；将所得培养液用121℃高压灭菌锅进行灭菌40min，4℃、16000g离心15min收集菌液上清。
向菌液上清中缓慢加醋酸钙粉末至终浓度为5％，再加冰醋酸调pH值至5.0左右；缓慢向每只烧杯中加入三倍体积95vol％的乙醇，马上会有沉淀产生，4℃静置过夜；4℃、16000g离心15min收集醇沉沉淀，烘干，用去离子水复溶；向复溶液中加适量CTAB处理复溶液，溶液有沉淀析出，4℃、16000g离心15min 收集沉淀。
用20vol％乙醇溶液重悬沉淀，并离心收集沉淀；用1M氯化钠溶液溶解沉淀；用3倍体积无水乙醇醇沉复溶液，有沉淀析出，4℃、16000g离心15min收集沉淀；用70vol％乙醇溶液重悬沉淀，清洗2次；用去离子水复溶沉淀，并进行过滤，得到荚膜多糖粗提物。
(2)对所得荚膜多糖粗提物进一步纯化，纯化的步骤如下所示：
荚膜多糖粗提物→GE高通量分离→凝胶排阻装置优化→梯度洗脱→组分阶段搜集→截留液→透析脱盐→糖浓度测定→浓缩→糖浓度测定→纯度测定→得到隐球菌荚膜多糖抗原纯品。
实施例2动物免疫
将GXM抗原与弗氏完全佐剂等体积混合至合适体积。充分乳化后对新西兰大耳兔进行皮下多点注射，每只兔的免疫剂量控制在0.1-0.8mg。免疫前3天取新西兰大耳兔的耳血，分离血清做阴性对照。初次免疫后每2周免疫1次，方法与第1次相同。共免疫6次。
挑选适龄，重量在1.5公斤左右的新西兰大耳白兔，在标准动物房中饲养3天，如无异常情况则开始进行免疫。取上述制备的GXM抗原30μg，加入到0.5mL高压灭菌后的生理盐水中，用微型漩涡震荡器充分混匀。然后将其与0.5mL弗氏完全佐剂用注射器相互推拉，充分乳化，对新西兰大耳兔进行背部皮下多点注射免疫。
两周后取GXM抗原30μg，加入到0.5mL灭菌后的生理盐水中，用微型漩涡震荡器充分混匀，然后与0.5mL弗氏不完全佐剂用注射器相互推拉，充分乳化，进行加强免疫。此后每隔一周进行一次加强免疫，共免疫六次。并且从第 三次免疫开始，免疫一周后取新西兰大耳兔耳缘静脉血200-500μL，测定其效价及亲和性。在最后一次免疫之后取脾。
具体免疫步骤如下所示：
(1)第一周：初次免疫，将GXM抗原使用生理盐水稀释为1mg/mL，与弗氏完全佐剂1:l充分乳化，将两种溶液分别装入两个注射器中，不要产生气泡，将两个注射器对接，由慢到快逐渐混合抗原稀释液与佐剂，最终将混合液制成乳白色油包水乳浊液，然后进行背部皮下多点免疫，剂量为每只l mL。
(2)第三周：加强免疫，将GXM抗原使用生理盐水稀释为0.5mg/mL与弗氏不完全佐剂1:1充分乳化，免疫剂量为每只1mL。
(3)第五周：加强免疫，免疫方式与第三周相同，免疫剂量为每只1mL。
(4)第六周：第一次血清效价检测；取免疫后的新西兰大耳兔的耳缘静脉血200-500μL，静置30min后离心分离血清，测定抗血清效价。
(5)第七周：加强免疫，免疫方式同步骤(3)。
(6)第八周：第二次血清效价检测，同步骤(4)。
(7)第九周：加强免疫，免疫方式同步骤(3)。
(8)第十周：第三次血清效价检测，同步骤(4)。
(9)第十一周：加强免疫，免疫方式同步骤(3)。
抗血清效价测定方法如下所示：
以GXM抗原作为包被抗原，包被浓度为100ng/well，37℃包被2h，然后加入3％BSA配制的封闭液，在37℃温育1h，甩掉液体，烘干备用。然后把制备的抗体血清进行梯度稀释，加入到酶标板中，每孔加入100μL，37℃温育1h， 然后用PBST洗涤三次，每次静置3min，然后以HRP标记的羊抗兔IgG作为酶标抗体，1:5000稀释，每孔加入100μL，37℃温育1h，然后用PBST洗涤三次，每次静置40s。每孔加入100μLTMB静置，37℃温育显色15min，然后每孔加入50μL终止液结束反应，于酶标仪检测OD 450值。
实施例3兔单抗制备
对制备的兔抗血清进行效价检测，效价合格的情况下，使用合格的新西兰大耳兔的脾脏分别进行细胞融合，制备单克隆杂交瘤细胞株，具体制备方法如下所示：
(1)制备免疫脾细胞
将免疫完成的新西兰大耳兔处死，无菌条件下取脾脏。用细胞培养液洗1次后研碎，过不锈钢筛网，用细胞培养液洗2次，离心得到细胞。
(2)细胞融合
取对数期的SP2/0骨髓瘤细胞，与得到的脾细胞混合，用不含胎牛血清的细胞培养液洗一次后离心、弃上清，加入聚乙二醇溶液后37℃恒温90s左右，用不含胎牛血清的细胞培养液终止反应后离心，用含20％胎牛血清的HAT选择培养液重悬，将细胞加到96孔板内，放入细胞培养箱内培养，培养温度为37℃，CO 2含量为5.0％。
(3)细胞单克隆化与筛选
将96孔板内生长状态良好的细胞用细胞培养液稀释至1-3个细胞/mL，加入到96孔板内，放入细胞培养箱内培养，培养温度为37℃，CO 2含量为5.0％。对每个细胞株分别编号，选取培养液上清呈阳性的细胞株扩大培养。最终得到杂交瘤细胞株。
(4)筛选杂交瘤细胞
对所获得的杂交瘤细胞采用ELISA方法使用进行筛选，寻找到最多10孔针对隐球菌荚膜多糖抗原的多克隆抗体细胞株，细胞株能产生针对各自抗原高亲和力的特异性单克隆抗体。
细胞融合步骤之后，培养基里存在两种亲本细胞和三种随机融合的细胞，要想得到能分泌目的抗体的杂交瘤细胞株，就必须把成功融合的杂交瘤细胞从众多细胞中分离出来。B淋巴细胞不能在体外长期生存，需要除去的只有骨髓瘤细胞和其自身融合细胞，因此需要将融合后的细胞通过HAT培养基培养，选择性的保留杂交瘤细胞。
融合后第5天可观察细胞的生长情况，第10-14天，即可采用间接ELISA法检测细胞培养上清并筛选阳性杂交瘤细胞株，进行克隆化培养。采用有限稀释法对阳性杂交瘤细胞进行克隆化培养。将检测结果效价最强的阳性杂交瘤细胞扩大化至细胞阳性率达100％时定株。用ELISA测量定株的杂交瘤细胞株培养上清的效价，将扩大化培养的单克隆杂交瘤细胞株冻存于液氮中。
对杂交瘤细胞上清液进行特异性和亲和力测试，并从中挑选适合配对的杂交瘤细胞；对挑选出的四孔多克隆杂交瘤细胞株进行重组表达得到单克隆抗体；对使用得到的纯化抗体进行夹心法配对，建立隐球菌荚膜多糖抗原的夹心法检测体系，最终分别得到配对最好的单克隆抗体。
实施例4兔单克隆抗体序列测定
(1)从杂交瘤细胞中分离总RNA
将杂交瘤细胞匀浆化后，加入TRIzol，匀浆物可分成透明的上层水相层(含有RNA)、相界面和红色的下层有机层(含有DNA和蛋白质)。然后用异丙醇 从水相层中沉淀出RNA。用乙醇从有机层中沉淀出DNA。利用异丙醇沉淀从酚-乙醇上清液中沉淀出蛋白质。洗涤沉淀的RNA，去除杂质，重悬浮后备用。
(2)将总RNA逆转录成cDNA
以dNTP为底物，以RNA为模板，tRNA为引物，在tRNA3’-末端上，按5’→3’方向，合成一条与RNA模板互补的cDNA单链，它与RNA模板形成RNA-cDNA杂交体。随后又在反转录酶的作用下，水解掉RNA链，再以cDNA为模板合成第二条DNA链。至此，将总RNA逆转录成cDNA。
(3)cDNA末端快速扩增(RACE)
根据cDNA末端快速扩增(RACE)的标准操作程序扩增重链和轻链的抗体片段。将扩增的抗体片段分别克隆到标准克隆载体中。进行菌落PCR以筛选具有正确大小的插入物的克隆。就得到了抗体序列。抗体的核苷酸序列如下所示：
编码所述抗隐球菌荚膜多糖单克隆抗体的重链可变区的核苷酸序列包括SEQ ID NO.9所示的序列；编码所述抗隐球菌荚膜多糖单克隆抗体的轻链可变区的核苷酸序列包括SEQ ID NO.10所示的序列。
SEQ ID NO.9：
Figure PCTCN2022108076-appb-000005
SEQ ID NO.10：
Figure PCTCN2022108076-appb-000006
Figure PCTCN2022108076-appb-000007
实施例5构建单克隆抗体表达载体并进行纯化表达
(1)构建单克隆抗体的表达载体
根据兔单抗序列测定结果，设定合适的酶切位点，分别对重链和轻链进行基因合成；将基因合成产物分别连接到pcDNA3.4表达载体。
(2)单克隆抗体的表达与纯化
单克隆抗体的表达：
(a)转染前的准备：细胞计数及传代处理，保证细胞具备转染条件；
(b)细胞转染：摇匀细胞液后，用移液管无菌吸取50μL细胞悬液，加950μL培养基稀释20倍，用细胞计数板进行细胞计数，活细胞密度约为(4.5-5.5)×10 6cells/mL，细胞活率应为95-99％；
(c)配制转染试剂和质粒混合物，室温孵育20min；
(d)将转染试剂缓慢加入待转染细胞培养液中，摇瓶置37.0±0.2℃，120rpm和8％CO 2的细胞培养摇床中培养；
(e)转染后的第2天(转染后第18至22小时，第1天)，将补料添加到摇瓶中，并在添加过程中轻轻摇动摇瓶。将摇瓶置37.0±0.2℃，120rpm和8％CO 2的细胞培养摇床中培养，继续培养3-5天。
(3)单克隆抗体的纯化
(a)细胞培养液收获：离心收集细胞培养液上清，使用Protein A Beads进行抗体纯化；
(b)纯化完成后进行抗体浓度测定。
实施例6单克隆抗体分子量测定
用SDS-PAGE电泳鉴定单克隆抗体的分子量，每个电泳道加样量为5μg，同时用已知分子量标准系列作为参照(marker)，首先在90V电压下电泳20min，再在140V电压下电泳直到指示剂全部跑出，取下凝胶，用考马斯亮蓝染色，染色后凝胶分析单克隆抗体的分子量。图1为单克隆抗体的SDS-PAGE电泳结果图，从图1可知，单克隆抗体的蛋白重链和轻链主要分布在55kDa和25kDa左右，图1中泳道M 1为marker，泳道1为单克隆抗体。
实施例7采用ELISA方法检测单克隆抗体对隐球菌荚膜多糖(GXM)的亲和活性(效价)
亲和活性检测，亲和活性的检测步骤如下所示：
将GXM抗原用PBS稀释成1ng/μL，以每孔100μL加入到96孔酶标板中，37℃包被2h；弃上清，用0.01M的PBST洗板3次，用PBST配制含3％BSA的封闭液，每孔加入100μL，37℃封闭2h；弃上清，用PBST清洗5次，将纯化浓缩后的抗体进行梯度稀释，浓度从1:1000倍比稀释至1:2560000，加入各孔，每孔加入100μL，37℃温育1h；弃抗体稀释液，用PBST清洗6遍，用1:5000封闭液稀释HRP标记的羊抗兔IgG(二抗)，每孔加入100μL二抗，37℃温育1h；弃二抗稀释液，用PBST清洗6遍，每孔加入100μLTMB，避光37℃放置15min；每孔加入50μL 1M稀硫酸终止反应，在450nm处测定吸光度。
单克隆抗体的亲和活性的检测结果如表1和图2所示，在OD值保持不变的情况下，单克隆抗体的稀释倍数越大，说明抗原抗体的结合能力越强，筛选的单克隆抗体对GXM抗原具有较强的结合能力，对GXM抗原的效价达到 1:1280000(OD值＞0.5)。
表1
稀释倍数 OD值
1:10000 4.134
1:20000 4.122
1:40000 4.086
1:80000 4.057
1:160000 3.785
1:320000 2.996
1:640000 2.108
1:1280000 1.116
实施例8采用ELISA方法检测单克隆抗体的特异性
通过交叉反应检测单克隆抗体的特异性，交叉反应的步骤如下所示：
分别用荚膜多糖、甘露聚糖、半乳甘露聚糖、脂多糖、肽聚糖、1,3-β-D-葡聚糖和BSA包被酶标板，每孔包被量为50ng/孔；将单克隆抗体稀释至10ng/mL，加入到各酶标板中，每孔加入100μL，37℃孵育1h；洗涤后每孔加入100μL二抗(HRP标记的羊抗兔IgG)，37℃孵育0.5h；洗涤后加入TMB，37℃孵育15min，终止读数。交叉反应的结果如图3所示，结果表明所得单克隆抗体不与其他糖产生交叉反应，特异性强。
综上，本发明提供一种了抗隐球菌荚膜多糖单克隆抗体，所述单克隆抗体为兔源单克隆抗体，抗原识别位点多、特异性好、亲和力高，解决了鼠源单克 隆抗体在实际应用方面的问题，所述抗隐球菌荚膜多糖单克隆抗体在制备隐球菌检测的产品中具有重要应用价值。
申请人声明，以上所述仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种抗隐球菌荚膜多糖单克隆抗体，其特征在于，所述抗隐球菌荚膜多糖单克隆抗体包括重链可变区和轻链可变区；
    所述重链可变区包括SEQ ID NO.3所示的重链CDR3；
    所述轻链可变区包括SEQ ID NO.6所示的轻链CDR3。
  2. 根据权利要求1所述的抗隐球菌荚膜多糖单克隆抗体，其特征在于，所述重链可变区还包括SEQ ID NO.1所示的重链CDR1和SEQ ID NO.2所示的重链CDR2；
    所述轻链可变区还包括SEQ ID NO.4所示的轻链CDR1和SEQ ID NO.5所示的轻链CDR2。
  3. 根据权利要求1所述的抗隐球菌荚膜多糖单克隆抗体，其特征在于，所述重链可变区的氨基酸序列包括SEQ ID NO.7所示的序列；
    所述轻链可变区的氨基酸序列包括SEQ ID NO.8所示的序列。
  4. 根据权利要求1中任一项所述的抗隐球菌荚膜多糖单克隆抗体，其特征在于，所述抗隐球菌荚膜多糖单克隆抗体还包括兔源IgG1、IgG2、IgG3或IgG4恒定区中的任意一种或至少两种的组合，优选为兔源IgG1恒定区。
  5. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1-4中任一项所述抗隐球菌荚膜多糖单克隆抗体；
    编码所述抗隐球菌荚膜多糖单克隆抗体的重链可变区的核苷酸序列包括SEQ ID NO.9所示的序列；编码所述抗隐球菌荚膜多糖单克隆抗体的轻链可变区的核苷酸序列包括SEQ ID NO.10所示的序列。
  6. 一种表达载体，其特征在于，所述表达载体包括权利要求5所述的核酸分子。
  7. 一种宿主细胞，其特征在于，所述宿主细胞中含有至少一个拷贝的权利 要求6所述的表达载体，或所述宿主细胞的基因组中整合有权利要求5所述的核酸分子；
    所述宿主细胞包括293F细胞或CHO细胞。
  8. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1-4中任一项所述的抗隐球菌荚膜多糖单克隆抗体；
    所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中任意一种或至少两种的组合。
  9. 一种检测隐球菌的试剂盒，其特征在于，所述检测隐球菌的试剂盒包括权利要求1-4中任一项所述的抗隐球菌荚膜多糖单克隆抗体；
    所述检测隐球菌的试剂盒还包括阳性对照品、阴性对照品、抗体稀释液、显色液、终止液、封闭液或洗涤液中的任意一种或至少两种的组合。
  10. 权利要求1-4中任一项所述的抗隐球菌荚膜多糖单克隆抗体、权利要求5所述的核酸分子、权利要求6所述的表达载体、权利要求7所述的宿主细胞或权利要求8所述的药物组合物中任意一种或至少两种的组合在制备隐球菌感染疾病治疗药物和/或检测产品中的应用。
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