Monoclonal antibody of mycobacterium tuberculosis secreted protein 64 and its application

Abstract

The application discloses a kind of simultaneously identifying wild type (W-MPT64) and 63bp deletion mutant (D-MPT64) Mycobacterium tuberculosis secreted protein 64 monoclonal antibody.The antibody heavy chain and light chain variable region amino acid sequence as shown in SEQ ID NO.1 and SEQ ID NO.5.The Mycobacterium tuberculosis MPT64 double antibody sandwich enzyme-linked immunosorbent assay and fluorescence immunoassay method established using the monoclonal antibody reach 1.5625 pg / mL, significantly higher than the minimum detection limit of prior art, can be used for qualitative and quantitative detection of Mycobacterium tuberculosis secreted protein 64.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical immunodetection technology, and particularly relates to a double-recognizing monoclonal antibody for simultaneously recognizing wild-type Mycobacterium tuberculosis secreted protein 64 (W-MPT64) and 63bp deletion mutant Mycobacterium tuberculosis secreted protein 64 (D-MPT64) and application thereof in immunodetection. BACKGROUND

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB). For the diagnosis of tuberculosis, the internationally recognized gold standard is positive culture of MTB in sputum, bronchoalveolar lavage fluid or other body fluids, but MTB culture needs at least 3-4 weeks to obtain results, and the positive rate is low. Acid-fast staining can be used to identify mycobacteria, but it cannot distinguish non-tuberculous mycobacteria (NTM) infection with similar clinical symptoms. Although tuberculin skin test is simple to operate, its sensitivity and specificity are not high, and it cannot distinguish between BCG vaccination and MTB or NTM infection. The nucleic acid molecular detection method has high sensitivity and specificity, but the operation is complex and requires special equipment, which cannot be popularized and used in primary medical institutions. Tuberculosis diagnosis methods based on immunology principles include three categories. The first category takes the antibodies produced by humoral immunity as the detection target, and the positive rate of existing reagents cannot meet the clinical demand and cannot distinguish between current infection and past infection. The second category is the gamma-interferon release test based on cellular immunity, which also cannot distinguish between current infection and past infection. The third category takes MTB-specific antigen as the detection target. After the body is infected with MTB, MTB-specific antigen appears earlier than antibody, which is direct evidence of active MTB infection, and can avoid false negative of humoral immunity or cellular immunity detection due to low immune response of patients.

[0003] The existing MTB antigen detection reagent mainly takes the Mycobacterium tuberculosis secreted protein 64 (MPT64) as the detection target. MPT64 is also called MPB64, which is encoded by the Rv1980c (mpt64) gene in the differential coding region RD2, has a full length of 228 amino acids, and has a molecular weight of about 24 kD. The advantages of MPT64 as the detection target of MTB antigen mainly lie in the following three aspects. Firstly, MPT64 is one of the main proteins secreted during the growth and reproduction of MTB, accounts for 8% of the total secreted proteins, and is relatively easy to detect. Secondly, MPT64 exists in actively replicating bacterial cells and is secreted in large quantities in the early culture period, and is a specific marker for early infection and active infection of MTB. Thirdly, MPT64 is only secreted in MTB and is not secreted in NTM, and is a specific marker of MTB. However, in clinical detection, some culture-positive samples still show negative results in MPT64 antigen detection, causing missed detection and delayed treatment. The reason for the false negative result of MPT64 antigen detection is that MPT64 protein has polymorphism, including 21 amino acids deletion (63 bp deletion) caused by 197-259 nucleotide deletion mutation of MPT64 protein gene, single amino acid sense or nonsense mutation caused by single nucleotide mutation, frame shift mutation caused by single amino acid insertion or deletion, and amino acid sequence change caused by IS6110 fragment insertion. According to the literature reports, among all the mutations, the 63 bp deletion mutation accounts for more than 90%, which is the main reason for the negative result of MPT64 antigen detection. The 63 bp deletion mutation causes the change of the tertiary structure of MPT64 protein, the defect of α-helix and the increase of the degree of curling of adjacent β-fragments, so that the monoclonal antibody specific to wild type MPT64 protein cannot be recognized, and finally the false negative result of the existing reagent MPT64 antigen detection is caused.

[0004] In view of the above problems, the present application prepares and screens a double-recognizing monoclonal antibody capable of recognizing wild type MPT64 (W-MPT64) and 63 bp deletion MPT64 (D-MPT64) at the same time, realizes the simultaneous detection of the two kinds of MPT64 proteins, avoids missed detection, reduces the false negative rate of the existing reagent, and improves the detection rate. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a monoclonal antibody capable of recognizing wild type MPT64 (W-MPT64) and 63 bp deletion mutant MPT64 (D-MPT64) at the same time, which is prepared by using mouse hybridoma technology (in the present patent application, the antibody can also be called W-MPT64 and D-MPT64 double-recognizing monoclonal antibody or double-recognizing monoclonal antibody), which can be used for qualitative and quantitative detection of Mycobacterium tuberculosis MPT64 protein, and preparation of detection reagent for detecting Mycobacterium tuberculosis infection.

[0006] Therefore, the first aspect of the present application relates to a monoclonal antibody or an antigen-binding fragment thereof capable of simultaneously recognizing wild-type MPT64 (W-MPT64) and 63 bp deletion mutant MPT64 (D-MPT64), comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprising a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein,

[0007] the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO. 2;

[0008] the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID NO. 3;

[0009] the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO. 4;

[0010] the amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO. 6;

[0011] the amino acid sequence of the light chain CDR2 is LVS;

[0012] the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO. 7.

[0013] Further, the present application also relates to the above-mentioned monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 5.

[0014] Still further, the present application also relates to the above-mentioned monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody or a humanized antibody, and these antibodies or antigen-binding fragments are capable of simultaneously recognizing W-MPT64 and D-MPT64 due to the retention of the variable regions of the light chain and the heavy chain, or only the retention of the variable region of the heavy chain.

[0015] The second aspect of the present application relates to a nucleic acid molecule comprising a nucleic acid encoding the above-mentioned monoclonal antibody or antigen-binding fragment thereof.

[0016] The third aspect of the present application relates to an expression vector comprising the above-mentioned nucleic acid molecule, and the expression vector is capable of expressing the above-mentioned monoclonal antibody or antigen-binding fragment thereof.

[0017] The fourth aspect of the present application relates to a recombinant comprising the above-mentioned nucleic acid molecule or the above-mentioned expression vector, which can produce the above-mentioned monoclonal antibody or antigen-binding fragment thereof, and further, which can be a mammalian cell recombinant, a bacterial recombinant or a yeast recombinant.

[0018] The fifth aspect of the present application relates to a monoclonal antibody mouse hybridoma cell strain secreting the above-mentioned double-recognizing monoclonal antibody which can recognize both wild-type MPT64 (W-MPT64) and 63 bp deletion mutant MPT64 (D-MPT64). Further, the monoclonal antibody mouse hybridoma cell strain is the monoclonal antibody mouse hybridoma cell strain 6112 with the accession number of CGMCC No. 46599.

[0019] The sixth aspect of the present application relates to the use of the above-mentioned monoclonal antibody or antigen-binding fragment thereof in the preparation of a kit for detecting Mycobacterium tuberculosis MPT64 protein.

[0020] The seventh aspect of the present application relates to a kit for detecting Mycobacterium tuberculosis MPT64 protein, which comprises the above-mentioned monoclonal antibody or antigen-binding fragment thereof for recognizing both W-MPT64 and D-MPT64. Further, the kit is a double-antibody sandwich type kit, in which the monoclonal antibody or antigen-binding fragment thereof is used as a capture antibody or a detection antibody. Further, the kit is an enzyme-linked immunosorbent assay kit or a fluorescent immunochromatography kit.

[0021] Biological material preservation instructions

[0022] The monoclonal antibody mouse hybridoma cell strain 6112 of the present application has been preserved in the China General Microbiological Culture Collection Center (CGMCC), and the accession number is CGMCC No. 46599, the preservation date is October 15, 2025, and the classification name is monoclonal antibody mouse hybridoma cell strain. The address of the China General Microbiological Culture Collection Center is No. 1, Yihuangyuan, Beichen West Road, Chaoyang District, Beijing, China, with a postal code of 100101. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a SDS-PAGE electrophoresis map showing the expression of Mycobacterium tuberculosis W-MPT64 protein and D-MPT64 protein in prokaryotes, in which M is a molecular weight marker; 1 is W-MPT64 protein expressed by the recombinant expression vector pBV-W-MPT64; and 2 is D-MPT64 protein expressed by the recombinant expression vector pGEX-D-MPT64.

[0024] Figure 2Figure is the minimum detection limit result chart of the double antibody sandwich enzyme-linked immunoassay method of Mycobacterium tuberculosis MPT64 protein. Wherein A is a double recognition monoclonal antibody 6112 as a capture antibody, and a HRP labeled rabbit anti MPT64 protein polyclonal antibody as a detection antibody group, B is a rabbit anti MPT64 protein polyclonal antibody as a capture antibody, and a HRP labeled 6112 monoclonal antibody as a detection antibody group.

[0025] Figure 3 Figure is the subtype identification result chart of the double recognition monoclonal antibody 6112 of W-MPT64 and D-MPT64. DETAILED DESCRIPTION

[0026] The purpose of the present application is to provide a mouse hybridoma cell strain capable of secreting a double recognition monoclonal antibody which can recognize wild type Mycobacterium tuberculosis secreted protein 64 (W-MPT64) and 63bp deletion mutant Mycobacterium tuberculosis secreted protein 64 (D-MPT64) at the same time, prepared by using mouse hybridoma technology. The specific preparation process is as follows: first, W-MPT64 and D-MPT64 are expressed in E. coli. Then, W-MPT64 and D-MPT64 are used as immunogens to cross-immunize BALB / c female mice to produce monoclonal antibodies, and a double recognition monoclonal antibody capable of recognizing W-MPT64 and D-MPT64 at the same time with high affinity is screened and obtained. This antibody is also referred to as monoclonal antibody 6112 which recognizes W-MPT64 and D-MPT64 at the same time, or double recognition monoclonal antibody 6112, or monoclonal antibody 6112 in the present patent application. The mouse hybridoma cell strain secreting the monoclonal antibody is named 6112 or monoclonal antibody mouse hybridoma cell strain 6112 in the present patent application. The present inventors have deposited the monoclonal antibody mouse hybridoma cell strain in China General Microbiological Culture Collection Center on October 15, 2025, with the preservation number of CGMCC No. 46599, and the classification name of monoclonal antibody mouse hybridoma cell strain.

[0027] At the same time, the above-mentioned prokaryotic expressed W-MPT64 and D-MPT64 are used as mixed immunogens to prepare a rabbit anti Mycobacterium tuberculosis MPT64 protein polyclonal antibody, and the detection titer of the polyclonal antibody to W-MPT64 and D-MPT64 is 1:2048000.

[0028] Next, the inventors established an enzyme-linked immunosorbent assay and a fluorescent immunochromatographic assay for detecting MPT64 protein of Mycobacterium tuberculosis based on the double antibody sandwich principle using the prepared W-MPT64 and D-MPT64 double recognition monoclonal antibody 6112 and rabbit anti-MPT64 protein polyclonal antibody. The minimum detection limit of the two detection methods for the prokaryotic expressed W-MPT64 protein and D-MPT64 protein was 1.5625 pg / mL, and the minimum detection limit of the MPT64 double antibody sandwich fluorescent immunochromatographic assay for the W-MPT64-expressed Mycobacterium tuberculosis culture solution and the D-MPT64-expressed Mycobacterium tuberculosis culture solution was 1x10 3 CFU / mL, which was significantly higher than the minimum detection limit of the existing detection reagent and the minimum detection limit reported in the literature; the positive detection time of the Mycobacterium tuberculosis culture solution was 7 days, which was earlier than the positive detection time of the existing detection reagent and the positive detection time reported in the literature, and could be used for qualitative and quantitative detection of secreted protein 64 of Mycobacterium tuberculosis.

[0029] The inventors amplified and sequenced the gene sequence of the monoclonal antibody 6112 secreted by the above monoclonal antibody mouse hybridoma cell strain CGMCC No. 46599, and then analyzed the immunoglobulin domain sequence of the monoclonal antibody using the public software of the National Center for Biotechnology Information (NCBI) website, and found that the heavy chain variable region has 115 amino acids, and the specific sequence is: EVKVVESGGGLVQPGGSMKLSCAAS GFTFSDAW MDWVRQSPEKGLEWIAE IRNKANDHAT YYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYC TTVPFDC WGQGTTLTVS (SEQ ID NO. 1), and three CDR regions are marked with underlines, wherein CDR1 is located at 26-33 aa, the amino acid sequence is GFTFSDAW (SEQ ID NO. 2); CDR2 is located at 51-60 aa, the amino acid sequence is IRNKANDHAT (SEQ ID NO. 3); and CDR3 is located at 99-105 aa, the amino acid sequence is TTVPFDC (SEQ ID NO. 4). The amino acid sequence of the light chain variable region is 109 amino acids, and the sequence is as follows: DIVLTQSPASLAVSLGQRATISYRAS KSVSTSG YSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTRSEGAPSWKS (SEQ ID NO. 5), the three CDR regions are underlined, wherein CDR1 is located at 27-36 aa, the amino acid sequence is KSVSTSGYSY (SEQ ID NO. 6); CDR2 is located at 54-56 aa, the amino acid sequence is LVS; CDR3 is located at 93-100 aa, the amino acid sequence is QHIRELTR (SEQ ID NO. 7).

[0030] It is well known in the art that although the antibody heavy chain CDR region and the light chain CDR region are important amino acid sequence regions for recognizing and binding to the corresponding antigen, conservative amino acid substitution is a biotechnology means in protein engineering by replacing amino acid residues of the same family with similar properties of the substituents to maintain the functional characteristics of the protein. This method mainly involves directional substitution between amino acids of the same family, thereby ensuring that the binding affinity and specificity of the protein after substitution do not change significantly. In the present patent application, conservative amino acid substitution is the mutual substitution between aromatic amino acids Phe, Trp, Tyr, the mutual substitution between aliphatic amino acids Ala, Gly, Leu, Ile, Val, the mutual substitution between polar amino acids Gln, Asn, the mutual substitution between basic amino acids Lys, Arg, His, the mutual substitution between acidic amino acids Asp, Glu, and the mutual substitution between hydroxyl amino acids Ser, Thr. The conservative amino acid substitution of 1 amino acid in the above heavy chain CDR region amino acid sequence and light chain CDR region amino acid sequence should not change the structure of the protein, so the single amino acid conservative substitution in the above region may still have the characteristics of binding to the corresponding antigen. Therefore, the monoclonal antibody or antigen-binding fragment obtained after conservative substitution of 1 amino acid in the heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 and / or light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 can still recognize W-MPT64 and D-MPT64.

[0031] The skilled person can also use the state of the art to prepare various antibody fragments, i.e. antigen binding fragments, such as but not limited to Fab, Fab', F(ab')2, from the monoclonal antibody of the application described above, which are capable of recognizing both W-MPT64 and D-MPT64. The Fab fragment is the region of an antibody that can bind to an antigen, consisting of one complete light chain and the variable region VH and constant region CH1 domain (Fd fragment) of a heavy chain, with one constant region and one variable region of both light and heavy chains, and a disulfide bond linkage between the light and heavy chains. The antigen binding fragments can be prepared, for example, by using papain to degrade the antibody IgG into two Fab fragments and one Fc fragment. Under the action of pepsin, the antibody IgG is degraded into one F(ab')2 fragment and one Fc fragment, and the F(ab')2 fragment is further reduced to form two Fab' fragments. Because the above-mentioned antigen binding fragments can still bind to the corresponding antigen, they can be used to prepare a kit for detecting Mycobacterium tuberculosis MPT64 protein.

[0032] The skilled person can also use the state of the art to prepare single chain antibodies (scFv) from the monoclonal antibody of the application. Single chain antibodies are antibodies in which the variable region of the heavy chain and the variable region of the light chain are connected by a short peptide linker of several amino acids, and they have only one chain, which is an artificially synthesized antibody. The single chain antibody can also contain only the variable region of the heavy chain. The length and amino acid composition of the short peptide linker are well known in the art, and the short peptide linker that can be used for the monoclonal antibody of the application can be determined by simple repeated experiments. Single chain antibodies can be expressed in, for example, E. coli, by genetic engineering techniques. The single chain antibody of the application thus prepared has the property of recognizing both W-MPT64 and D-MPT64, and therefore can be used to prepare a kit for detecting Mycobacterium tuberculosis MPT64 protein.

[0033] The skilled in the art can design and synthesize nucleic acid molecules encoding the amino acid sequences of the above monoclonal antibody variable regions which can recognize W-MPT64 and D-MPT64 simultaneously, and can insert the synthesized nucleic acid molecules into nucleic acid vectors to construct expression vectors which can express monoclonal antibodies or antigen-binding fragments thereof which can recognize W-MPT64 and D-MPT64 simultaneously. The skilled in the art can also introduce the synthesized nucleic acid molecules or the constructed expression vectors into organisms such as mammalian cells, bacteria or yeast to obtain mammalian cell recombinants, bacterial recombinants or yeast recombinants, and produce the antibodies or antigen-binding fragments thereof of the present application via expression of the above recombinants. The antibodies or antigen-binding fragments thereof expressed in this way can recognize W-MPT64 and D-MPT64 simultaneously, and therefore the above nucleic acid molecules, expression vectors and mammalian cell recombinants, bacterial recombinants or yeast recombinants are within the scope of protection of the claims of the present application. Moreover, the above techniques are all well-known in the art and can be carried out by the skilled in the art without inventive labor.

[0034] As described above, the antibodies or antigen-binding fragments thereof of the present application can recognize W-MPT64 and D-MPT64 simultaneously, and therefore can be used to prepare a kit for detecting MPT64 protein of Mycobacterium tuberculosis, which can be any kit utilizing the binding reaction between the antibodies or antigen-binding fragments thereof of the present application and MPT64 protein of Mycobacterium tuberculosis, such as but not limited to a double antibody sandwich type kit and the like, and for specific kits, such as but not limited to kits for enzyme-linked immunosorbent assay, chemiluminescence, fluorescent immunochromatography, colloidal gold immunochromatography, immunoblotting, immunohistochemical methods.

[0035] To describe the technical content of the technical solutions, the purposes and effects achieved in detail, the following will be described in combination with specific embodiments.

[0036] Example 1: Prokaryotic expression of Mycobacterium tuberculosis W-MPT64 and D-MPT64 proteins

[0037] Since the wild-type W-MPT64 protein changes its tertiary structure after the 63 bp deletion mutation, resulting in α-helix defects and increased degree of curling of adjacent β-fragments, it cannot be recognized by monoclonal antibodies which specifically bind to wild-type MPT64 protein, and therefore the present application separately prepares prokaryotic expressed wild-type W-MPT64 protein and 63 bp deletion mutant D-MPT64 protein.

[0038] The W-MPT64 protein of Mycobacterium tuberculosis H37Rv strain (GenBank: CAA53143.1) was queried in the GenBank database of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ), which is 228 amino acids in length, and its sequence is MRIKIFMLVTAVVLLCCSGVATAAPKTYCEELKGTDTGQACLIQMSDPAYNTNISLPSYYPDQKSLENYIAQTRDKFLSAATSSTPREAPYELNITSATYQSAIPPRGTQAVVLKVYQNAGGTHPTTTYKAFDWDQAYRKPITYDTLWQADTDPLPVVFPIVQGELSKQTGQQVSIAPNAGLDPVNYQNFAVTNDGVIFFFNPGELLPEAAGPTQVLVHRSAIDSMLA (SEQ ID NO. 8). The 63 bp deletion mutant D-MPT64 is a deletion of amino acids 66-86, which is 207 amino acids in length, and its sequence is MRIKIFMLVTAVVLLCCSGVATAAPKTYCEELKGTDTGQACQIQMSDPAYNINISLPSYYPDQKSREAPYELNITSATYQSAIPPRGTQAVVLKVYQNAGGTHPTTTYKAFDWDQAYRKPITYDTLWQADTDPLPVVFPIVQGELSKQTGQQVSIAPNAGLDPVNYQNFAVTNDGVIFFFNPGELLPEAAGPTQVLVPRSAIDSMLA (SEQ ID NO.9). According to the preference of E. coli genetic code, the optimized nucleotide sequence suitable for the expression system of E. coli was deduced, and the optimized nucleotide sequence of W-MPT64 was ATGCGTATCAAGATCTTCATGCTGGTGACCGCGGTTGTGTTGCTGTGCTGTAGCGGCGTCGCCACGGCGGCACCGAAGACTTATTGCGAAGAGTTAAAAGGTACCGATACTGGCCAGGCTTGCCAAATTCAGATGTCTGACCCAGCGTACAACATCAATATTTCCCTGCCGTCGTATTACCCAGATCAGAAAAGCCTTGAAAACTATATCGCCCAAACGCGCGATAAGTTCCTGAGTGCAGCTACCTCTTCGACTCCGCGTGAAGCGCCATACGAGCTCAATATTACCAGCGCCACCTATCAGAGCGCGATCCCGCCACGCGGTACGCAGGCAGTTGTACTGAAAGTGTACCAAAACGCGTGGCGGTACTCATCCGACCACTACGTATAAAGCGTTTGACTGGGATCAGGCCTATCGTAAGCCGATTACCTACGACACTCTGTGGCAGGCAGATACCGATCCATTGCCGGTTGTCTTCCCAATTGTGCAAGGCGAACTGTCTAAACAGACCGGTCAGCAAGTTTCCATCGCTCCGAATGCGGGCTTAGATCCAGTGAACTATCAGAATTTTGCCGTCACGAACGACGGCGTTATTTTCTTTTTCAACCCGGGTGAACTGCTTCCAGAGGCGGCAGGCCCGACTCAGGTACTGGTGCCGCGCTCGGCTATCGATAGCATGCTCGCT (SEQ ID NO.10); the optimized nucleotide sequence of D-MPT64 is ATGCGTATCAAGATCTTCATGCTGGTGACCGCGGTTGTGTTGCTGTGCTGTAGCGGCGTCGCCACGGCGGCACCGAAGACTTATTGCGAAGAGTTAAAAGGTACCGATACTGGCCAGGCTTGCCAAATTCAGATGTCTGACCCAGCGTACAACATCAATATTTCCCTGCCGTCGTATTACCCAGATCAGAAAAGCCGCGAAGCCCCGTATGAACTTAACATCACGAGTGCAACCTACCAATCTGCTATTCCACCGCGTGGTACTCAGGCGGTTGTACTGAAGGTGTATCAGAATGCCGGCGGTACCCATCCAACCACGACTTACAAAGCGTTCGATTGGGACCAAGCATATCGCAAACCGATCACCTATGATACTCTCTGGCAGGCTGACACGGACCCACTGCCAGTTGTCTTTCCGATTGTGCAGGGCGAGCTGTCGAAGCAAACCGGTCAGCAGGTTAGCATTGCGCCAAACGCCGGCTTGGACCCAGTGAATTACCAAAACTTCGCAGTCACTAATGATGGCGTTATCTTTTTCTTTAACCCAGGTGAACTGTTACCGGAAGCTGCGGGCCCAACCCAGGTACTGGTGCCGCGTAGCGCCATTGACTCTATGCTTGCG (SEQ ID NO. 11). Beijing Qikexing Biotechnology Co., Ltd. was commissioned to synthesize the optimized nucleotide sequences of W-MPT64 and D-MPT64. For the expression of W-MPT64, the pBVIL expression vector was used, and the fusion protein was interleukin IL1, with a molecular weight of about 10 kDa. The sequence of the upstream primer (named W-MPT64-F) used for amplification is GC. CTCGAG ATGCGTATCAAGATCTTC (SEQ ID NO. 12), which contains a Xho I restriction site (indicated by underlining); the sequence of the downstream primer (named W-MPT64-R) is GC TCTAGAAGCGAGCATGCTATCGAT (SEQ ID NO. 13), which contains an Xba I restriction site (underlined). For the expression of D-MPT64, the pGEX-4T-2 expression vector was used, and the fusion protein was glutathione S-transferase (GST), and the molecular weight of the fusion protein was about 26 kDa. The sequence of the upstream primer (named D-MPT64-F) was GC GGATCC ATGCGTATCAAGATCTTC (SEQ ID NO. 14), which contains a BamH I restriction site (underlined); the sequence of the downstream primer (named D-MPT64-R) was GC GAATTC CGCAAGCATAGAGTCAAT (SEQ ID NO. 15), which contains an EcoR I restriction site (underlined). The recombinant expression vectors were constructed using techniques known in the art, and the constructed recombinant expression vectors were named pBV-W-MPT64 and pGEX-D-MPT64, respectively.

[0039] The correctly sequenced pBV-W-MPT64 recombinant expression plasmid was transformed into E. coli HB101, and the identified positive clones were cultured at 37°C overnight, then transferred to fresh LB medium and cultured at 37°C for 2-3 hours to an OD600nm of 0.4-0.6, and then transferred to a 42°C water bath for induction of expression for at least 4 hours. The bacterial cells were collected, the inclusion bodies were extracted, and protein purification was performed, and the purified protein was identified by SDS-PAGE electrophoresis. The correctly sequenced pGEX-D-MPT64 recombinant expression plasmid was transformed into E. coli BL21, and the identified positive clones were cultured at 37°C overnight, and the next day, 250 mL of fresh LB liquid medium was inoculated, and the temperature was adjusted to 16°C, and after 30 minutes, 150 μl of 1 mol / L IPTG induction solution was added, and induction was performed at 16°C for 12-14 hours. The bacterial cells were collected, the inclusion bodies were extracted, and protein purification was performed, and the purified protein was identified by SDS-PAGE electrophoresis. The results are shown in Figure 1, wherein 1 is the W-MPT64 protein expressed by the recombinant expression vector pBV-W-MPT64, and the molecular weight is about 35 kDa; and 2 is the D-MPT64 protein expressed by the recombinant expression vector pGEX-D-MPT64, and the molecular weight is about 48 kDa. Figure 1

[0040] Example 2: Preparation and screening of W-MPT64 and D-MPT64 double- recognition monoclonal antibodies

[0041] ​In order to obtain a double-recognizing monoclonal antibody capable of simultaneously recognizing W-MPT64 and D-MPT64, 6-8 week old BALB / c female mice were used. W-MPT64 was used as the immunogen for the first immunization, 100 μg / antigen plus an equal amount of complete Freund's adjuvant, emulsified and injected subcutaneously on the back and intraperitoneally. The second immunization was performed 4 weeks later, using D-MPT64 as the immunogen, 60 μg / antigen plus an equal amount of incomplete Freund's adjuvant. Eight weeks later, the third immunization was performed, using 30 μg / antigen of W-MPT64 and 30 μg / antigen of D-MPT64 plus incomplete Freund's adjuvant to emulsify and immunize the mice. One week after the third immunization, the mouse with the highest serum titer was selected for intraperitoneal booster immunization, with the same dose as the third immunization. Three days later, the spleen cells were collected and fused with SP20 myeloma cells according to the conventional operation. When the fused cells covered about 60% of the well bottom, the cell culture supernatant was collected and screened for double-recognizing monoclonal antibodies capable of simultaneously recognizing W-MPT64 and D-MPT64 by indirect enzyme-linked immunoassay. The specific method is as follows: dilute W-MPT64 and D-MPT64 in carbonate buffer, both at a concentration of 2.0 μg / ml, coat 100 μl of W-MPT64 or D-MPT64 per well at 4°C overnight; wash the plate twice with washing solution; add 150 μl / well blocking solution and incubate at room temperature for 6 hours; wash the plate 5 times with washing solution. Add 100 μl of sample diluent to each well, then add 10 μl of cell culture supernatant, shake and incubate at room temperature for 15 min, and discard the liquid; wash the plate 5 times with washing solution, add 100 μl / well of HRP-labeled goat anti-mouse IgG antibody, shake and incubate at room temperature for 15 min. Wash the plate 5 times. Add 50 μL of TMB color developing solution A and B to each well, and develop color at room temperature for 15 minutes in the dark. Add 50 μL of 2 M H2SO4 to each well to stop the reaction, and measure the absorbance of each well at 450 nm using an enzyme-labeled instrument within 10 minutes after termination. Select the positive clones that show strong positive reactions in both W-MPT64-coated wells and D-MPT64-coated wells as the double-recognizing monoclonal antibody positive clones for W-MPT64 and D-MPT64, and obtain three positive clones, 1134, 6112 and 6261. The three positive clone mouse hybridoma cell lines were cultured in 1640 culture medium containing 10% fetal bovine serum. After 10 days, the cells were collected, resuspended in normal saline, and each mouse was injected intraperitoneally with 1×10 6

[0042] ​The titer of three W-MPT64 and D-MPT64 double-recognizing monoclonal antibodies was detected by indirect enzyme-linked immunoassay. Specifically, 50 μl of W-MPT64 and D-MPT64 was coated in each well, and the remaining steps were the same as the above-described method. The three monoclonal antibodies were diluted to a concentration of 1 mg / mL with PBS, and then serially diluted to 1:50, 1:200, 1:800, 1:3200, 1:12800, 1:51200 and 1:204800, respectively. The detection results are shown in Table 1. The antibody titer of the double-recognizing monoclonal antibody 6112 was the highest, reaching 1:204800, and was selected for subsequent studies.

[0043] Table 1. Results of W-MPT64 and D-MPT64 double-recognizing monoclonal antibody titer determination (OD450nm)

[0044]

[0045] Example 3: Preparation of rabbit anti-MPT64 protein polyclonal antibody

[0046] W-MPT64 and D-MPT64 prepared in Example 1 were used as immunogens to prepare polyclonal antibodies against MPT64 protein. The specific steps are as follows: one healthy male white rabbit was selected, and 2 mL of blood was collected from the ear vein, and the serum was separated as a negative control. 0.5 mg of W-MPT64 and D-MPT64 prepared in Example 1 were mixed and mixed with 1.0 mL of Freund's complete adjuvant, and the stirrer was thoroughly emulsified into a white oily water emulsion. The rabbit was injected subcutaneously at multiple points on both sides of the spine, and each point was injected with not less than 0.1 mL. Four weeks later, 1.0 mg of W-MPT64 and D-MPT64 mixed protein was mixed with 1.0 mL of Freund's incomplete adjuvant, and the stirrer was thoroughly emulsified for the second immunization; four weeks later, the third boost was performed, and the heart was bled one week later. After the blood clotting and blood clot contraction, centrifugation was performed at 5000 rpm for 15 minutes, and the serum was determined by indirect ELISA. Each well was coated with 100 μl of W-MPT64 or D-MPT64, and the secondary antibody was HRP-labeled goat anti-rabbit secondary antibody, and the remaining steps were the same as the above-described method. The results are shown in Table 2. The pre-immune rabbit serum (1:2000 dilution) was used as a negative control, and the detection titer of the prepared rabbit anti-MPT64 protein polyclonal antibody against W-MPT64 and D-MPT64 reached 1:2048000.

[0047] Table 2. Results of rabbit anti-MPT64 protein polyclonal antibody titer determination (OD450nm)

[0048]

[0049] Example 4: Double antibody sandwich enzyme-linked immunosorbent assay for MPT64 protein of Mycobacterium tuberculosis

[0050] The anti-W-MPT64 and D-MPT64 double recognition monoclonal antibody 6112 and rabbit anti-MPT64 protein polyclonal antibody prepared by the application are used to establish a double antibody sandwich enzyme-linked immunosorbent assay for MPT64 protein of Mycobacterium tuberculosis.

[0051] First, the monoclonal antibody and the polyclonal antibody are labeled with horseradish peroxidase (HRP) respectively, and the specific operation method is as follows: 5 mg of HRP is dissolved in 0.5 mL of deionized water, 1 mL of 0.06 mol / L NaIO4 is added, and the mixture is gently stirred at room temperature for 30 min in the dark. 1 mL of 0.16 mol / L ethylene glycol is added, and the mixture is gently stirred at room temperature for 1 hour, and the oxidation reaction is terminated. The dialysis bag is filled with 1000 mL of 0.01 mol / L carbonate buffer (pH 9.5) at 4°C, and dialysis is performed overnight, and the solution is changed 3 times to obtain aldehyde HRP. 1 mL of carbonate buffer containing 5 mg of antibody is added to 3 mL of aldehyde HRP solution, and the mixture is combined at room temperature in the dark for 2-3 hours, and 5 mg of NaHB4 is added, and the mixture is incubated at 4°C overnight. Then the dialysis bag is filled with 0.01 mol / L PBS (pH 7.2) at 4°C, and dialysis is performed for 24 hours, and the solution is changed 3 times. The mixture is centrifuged at 3000 r / min for 30 min, and the precipitate is removed, and an equal amount of glycerol is added to the supernatant to obtain HRP-labeled antibody, which is stored at low temperature.

[0052] The experiment is divided into group A and group B, wherein group A is a double-recognizing monoclonal antibody 6112 against W-MPT64 and D-MPT64 as a capture antibody, and a HRP-labeled rabbit anti-MPT64 protein polyclonal antibody as a detection antibody; group B is a rabbit anti-MPT64 protein polyclonal antibody as a capture antibody, and a HRP-labeled 6112 monoclonal antibody as a detection antibody. The enzyme-linked plate is coated with the double-recognizing monoclonal antibody 6112 against W-MPT64 and D-MPT64 or the rabbit anti-MPT64 protein polyclonal antibody as a capture antibody, and the corresponding HRP-labeled polyclonal antibody or HRP-labeled monoclonal antibody as a detection antibody is detected. The specific steps are as follows: the antibody is coated on the enzyme-linked plate, the antibody concentration is 2.5 μg / mL, 100 μL is added to each well, and it is coated at 4°C overnight, and the plate is washed twice with the washing solution. 110 μL / well of blocking solution is added at 4°C overnight, the liquid is discarded and dried. The prokaryotic expression W-MPT64 and D-MPT64 in Example 1 are gradient-diluted with double-distilled water, and the concentrations are 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 pg / mL, respectively. 100 μL of each is added to the well, incubated at 37°C for 60 min, and the liquid is discarded. The plate is washed 5 times, 100 μL of the corresponding HRP-labeled antibody is added to each well, and it is incubated at 37°C for 45 min. The plate is washed 5 times, dried, 50 μL of TMB color developing liquid A and B is added to each well, and it is colored at room temperature for 15 min. 50 μL of 2 M H2SO4 termination liquid is added to each well to terminate the reaction. The OD450nm value is measured within 10 min by an enzyme-labeled instrument. Each concentration of sample is detected 3 times. The results are shown in Figure 2 As shown in Table 1, the lowest detection limit of W-MPT64 and D-MPT64 in group A reaches 1.5625 pg / mL, which is higher than 6.25 pg / mL in group B, so the double-recognizing monoclonal antibody 6112 against W-MPT64 and D-MPT64 is selected as the capture antibody, and the HRP-labeled rabbit anti-MPT64 protein polyclonal antibody is selected as the detection antibody.

[0053] Example 5: Double-antibody sandwich fluorescence immuno-chromatographic assay for Mycobacterium tuberculosis MPT64 protein

[0054] To establish a sensitive and simple and rapid detection method of MPT64 protein of Mycobacterium tuberculosis, the present application uses the anti-W-MPT64 and D-MPT64 double recognition monoclonal antibody 6112 as the capture antibody labeled with fluorescent microspheres, and uses the rabbit anti-MPT64 protein polyclonal antibody as the detection antibody coated on the nitrocellulose membrane to establish a double antibody sandwich fluorescence immunochromatographic assay. During detection, 100 μL of the serially diluted W-MPT64 and D-MPT64 protein as in Example 4 is vertically added to the detection card at the sample adding position, and is placed at room temperature for 15 min. The dry fluorescence immunoassay instrument AFS-1000 is used for testing. After the sample is added to each sample adding hole of the reagent card, it is transferred to the chromatography area by capillary action. The fluorescent microsphere labeled particles at the detection line and the quality control line form a complex due to the antigen-antibody reaction, and form or do not form the fluorescent microsphere reaction band. Under the action of the excitation light source, the fluorescent substance in the fluorescent microsphere emits a fluorescent signal of a specific wavelength. The fluorescence immunoassay instrument captures the fluorescent signal, and calculates the ratio (T / C) of the fluorescence value of the T line to the fluorescence value of the C line through signal conversion. The amount of MPT64 protein in the sample is positively correlated with the signal intensity of the fluorescent antibody. The three times of the T / C value of the sample diluent is taken as the Cutoff value. The ratio of the T / C value of the sample to the Cutoff, i.e., the S / CO value, is calculated, so as to realize the detection of MPT64 protein. The S / CO value ≥ 1, and the result is positive. The S / CO value < 1, and the result is negative. Each sample of each concentration is repeatedly detected 5 times. The double antibody sandwich fluorescence immunochromatographic assay of the present application uses the W-MPT64 and D-MPT64 double recognition monoclonal antibody 6112 as the capture antibody, and uses the rabbit anti-MPT64 protein polyclonal antibody as the detection antibody. The minimum detection limit of the W-MPT64 and D-MPT64 is 1.5625 pg / mL. The detection time is only 15-20 min, which is significantly shorter than that of the enzyme-linked immunosorbent assay. The results are shown in Table 3.

[0055] Table 3 Minimum detection limit (S / CO value) of Mycobacterium tuberculosis MPT64 protein fluorescence immunochromatographic assay

[0056]

[0057] Example 6: Minimum detection limit of Mycobacterium tuberculosis MPT64 protein by fluorescence immunochromatographic assay

[0058] The fluorescence immunochromatographic assay as described above is used to detect MPT64 protein in Mycobacterium tuberculosis culture. The culture of Mycobacterium tuberculosis standard strain (H37Rv) and clinical 63 bp deletion strain AH03009 is taken into physiological saline and shaken to prepare a physiological saline bacterial suspension with a turbidity of 1 Macleod unit. The bacterial amount is 3×10 8 CFU / mL, which is serially diluted with physiological saline to a bacterial concentration of 1×107 CFU / mL, 1 x 10 6 CFU / mL, 1 x 10 5 CFU / mL, 1 x 10 4 CFU / mL, 1 x 10 3 CFU / mL, 1 x 10 2 CFU / mL, 1 x 10 1 CFU / mL. Respectively take 50 μL bacteria suspension, add 50 μL sample processing liquid mixed, drop to the detection card sample place vertically, placed at room temperature for 15 min, using dry fluorescence immunoassay analyzer AFS-1000 for detection. The results are shown in Table 4, the minimum detection limit of the Mycobacterium tuberculosis MPT64 protein double antibody sandwich fluorescence immunoassay method of the present application for the expression of W-MPT64 Mycobacterium tuberculosis standard strain (H37Rv) and the expression of D-MPT64 clinical 63bp deletion strain AH03009 is 1 x 10 3 CFU / mL, significantly higher than the reported 3~5 x 10 3 CFU / mL (Zhang Y, Cai X, Tan Y. Secreted protein in sputum and culture medium for identification of Mycobacterium tuberculosis complex. Modern hospital, 2011, 11 (2): 17-19).

[0059] Table 4 Minimum detection limit of Mycobacterium tuberculosis (S / CO value)

[0060]

[0061] Example 7: The earliest detection time of fluorescence immunoassay method for MPT64 protein of Mycobacterium tuberculosis culture

[0062] The fluorescence immunoassay method as described above was used to detect MPT64 protein in Mycobacterium tuberculosis culture to determine the earliest detection time of Mycobacterium tuberculosis. Three clinical active tuberculosis patient sputum samples were cultured for Mycobacterium tuberculosis using BACTEC MGIT 960 rapid culture system, and the experimental operation was strictly in accordance with the system operation manual. The samples were taken on the 3rd, 5th, 7th, 10th, 12th and 15th day of culture for detection, and the operation was the same as described above. The results are shown in Table 5, wherein the first and second samples were detected positive for MPT64 protein on the 7th day, and the third sample was detected positive for MPT64 protein on the 10th day, which was earlier than the average detection time of 12 days reported in the literature (Zhang Y, Cai X, Tan Y. Secreted protein in sputum and culture medium for identification of Mycobacterium tuberculosis complex. Modern hospital, 2011, 11 (2): 17-19).

[0063] Table 5 The earliest detection time of Mycobacterium tuberculosis (S / CO value)

[0064]

[0065] Example 8: Identification of the 6112 subtype of the W-MPT64 and D-MPT64 dual-recognition monoclonal antibody

[0066] The heavy and light chain isotypes of mouse antibodies were identified using the rapid mouse antibody subtype detection card (catalog number THJ-ISO-M8a, batch number 052725) from Antaiji (Beijing) Biotechnology Co., Ltd. 100 μL of supernatant from mouse hybridoma cell line 6112 was added to the sample wells of the rapid mouse antibody subtype detection card, and the results were observed and recorded after standing for 5-10 min. The results are as follows: Figure 3 As shown, the dual recognition monoclonal antibody 6112 for Mycobacterium tuberculosis W-MPT64 and D-MPT64 is mouse IgG1 subtype, and the antibody light chain is Igκ subtype.

[0067] Example 9: Determination of the amino acid sequence of the variable region of W-MPT64 and D-MPT64 dual-recognition monoclonal antibody 6112

[0068] Mouse hybridoma cell line 6112 was cultured, and total RNA was extracted from the hybridoma cells using the Trizol method. After reverse transcription of cDNA, PCR amplification was performed using primers for the Fab fragment of mouse monoclonal antibodies synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequences are available in *Recombinant Antibodies* (Science Press, 2005), edited by Shen Beifen. Amplification conditions were as follows: preheating at 95℃ for 2 min, followed by 30 cycles of 95℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 30 seconds, with a final extension at 72℃ for 5 min. The PCR product was ligated into the pMD18-T vector and transformed into *E. coli* JM109. Positive clones were selected for sequencing. The sequenced data was compared with the mouse-derived monoclonal antibody CDR region sequence using IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) in the NCBI website's BLAST module.

[0069] Sequence analysis revealed that the heavy chain variable region consists of 115 amino acids, with the following sequence: EVKVVESGGGLVQPGGSMKLSCAAS GFTFSDAW MDWVRQSPEKGLEWIAE IRNKANDHAT YYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYC TTVPFDCWGQGTTLTVS (SEQ ID NO. 1), three CDR regions are underlined, wherein CDR1 is located at 26-33 aa, the amino acid sequence is GFTFSDAW (SEQ ID NO. 2); CDR2 is located at 51-60 aa, the amino acid sequence is IRNKANDHAT (SEQ ID NO. 3); CDR3 is located at 99-105 aa, the amino acid sequence is TTVPFDC (SEQ ID NO. 4). The amino acid sequence of the light chain variable region is 109 amino acids, and the sequence is as follows: DIVLTQSPASLAVSLGQRATISYRAS KSVST SGYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEGAPSWKS (SEQ ID NO. 5), three CDR regions are underlined, wherein CDR1 is located at 27-36 aa, the amino acid sequence is KSVSTSGYSY (SEQ ID NO. 6); CDR2 is located at 54-56 aa, the amino acid sequence is LVS; CDR3 is located at 93-100 aa, the amino acid sequence is QHIRELTR (SEQ ID NO. 7).

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that simultaneously recognizes wild-type Mycobacterium tuberculosis secretory protein 64 and 63bp-deletion mutant Mycobacterium tuberculosis secretory protein 64, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprising a light chain CDR1, a light chain CDR2 and a light chain CDR3, characterized in that, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO. 2; the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO. 3; the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO. 4; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO. 6; the amino acid sequence of the light chain CDR2 is LVS; and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.

7. the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

5. It is secreted by the monoclonal antibody mouse hybridoma cell strain 6112 with the accession number of CGMCC No. 46599. The monoclonal antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab') 2 fragment, a single-chain antibody or a humanized antibody. It comprises a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4. It comprises the nucleic acid molecule according to claim 5. It is the monoclonal antibody mouse hybridoma cell strain 6112 with the accession number of CGMCC No. 46599.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that, 8.Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a kit for detecting Mycobacterium tuberculosis secretory protein 64.

3. The monoclonal antibody according to claim 2, characterized in that, It comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The kit is a double antibody sandwich type kit, wherein the monoclonal antibody or antigen-binding fragment thereof is used as a capture antibody or a detection antibody.

5. A nucleic acid molecule, characterized in that, The kit is an enzyme-linked immunosorbent assay kit or a fluorescent immunochromatographic kit.

6. An expression vector, characterized by, ​ 7. A mouse hybridoma cell line secreting monoclonal antibodies recognizing both wild-type Mycobacterium tuberculosis secreted protein 64 and 63 bp deletion mutant Mycobacterium tuberculosis secreted protein 64, characterized in that, ​ ​ 9. A kit for detecting Mycobacterium tuberculosis secreted protein 64, characterized by, ​ 10. The kit of claim 9, characterized in that ​ 11. The kit of claim 10, characterized in that ​

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