Mycobacterium tuberculosis H37Rv new gene Rv3864c and encoding protein and application thereof

Abstract

The invention relates to a novel coding gene Rv3864c (-4341113-4341511) of mycobacterium tuberculosis H37Rv and a coding protein of the novel coding gene Rv3864c (-4341113-4341511). The Rv3864c gene has high specificity, secretory property and antigenicity, and can be independently or jointly used for clinical rapid screening of latent tuberculosis infected persons and active tuberculosis patients and protecting human bodies from being infected by mycobacterium tuberculosis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to novel genes of Mycobacterium tuberculosis and their applications. Background Technology

[0002] Tuberculosis (TB) is a major global public health problem, and Mycobacterium tuberculosis (Mtb) is the pathogen that causes TB in humans. According to the latest WHO Global TB Report 2023, approximately 7.5 million new cases of active pulmonary TB were diagnosed in 2022. In addition to active TB patients, about one-quarter of the world's population has been infected with Mtb and faces the risk of developing active TB. Therefore, establishing early, rapid, accurate, and inexpensive methods for identifying early Mycobacterium tuberculosis infection and active TB patients is a necessary prerequisite for effectively preventing and controlling the spread of TB.

[0003] Currently, the diagnosis of tuberculosis mainly relies on 1) sputum bacteriological examination and chest X-ray imaging, but these methods suffer from low sensitivity or are time-consuming; 2) immunological diagnostic methods, but due to cross-immune reactions with BCG vaccines or other environmental mycobacteria, a high false-positive rate occurs, especially in BCG-vaccinated individuals. Several kits similar to T-SPOT.TB or kits based on protein chip immunodiagnostic technology exist both domestically and internationally, but due to the lack of effective specific neoantigens for identifying Mycobacterium tuberculosis, existing antigens have low specificity and sensitivity, making it difficult to solve the problem of "positive results not confirming the diagnosis, and negative results not ruling out the possibility of infection." Therefore, the screening of highly specific and sensitive neoantigens for Mycobacterium tuberculosis is a core problem urgently needing to be solved in the field. Any progress will provide original innovative achievements and technical support for the development of clinical diagnostic technology for tuberculosis and the development of kits. Summary of the Invention

[0004] The purpose of this invention is to provide a novel coding gene Rv3864c(-|4341113-4341511|) newly identified for Mycobacterium tuberculosis H37Rv.

[0005] A second objective of the present invention is to provide the protein encoded by the aforementioned gene and the polypeptide contained in said protein.

[0006] A third objective of this invention is to provide applications for the aforementioned novel encoded genes and proteins.

[0007] A fourth object of the present invention is to provide antibodies against the aforementioned proteins or polypeptides and their applications.

[0008] According to one aspect of the present invention, there is an isolated nucleic acid, which is the encoding gene Rv3864c(-|4341113-4341511|) of Mycobacterium tuberculosis H37Rv, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0009] According to another aspect of the present invention, an Rv3864c-encoded protein is encoded by the Mycobacterium tuberculosis H37Rv encoding gene Rv3864c(-|4341113-4341511|), thereby determining the location and complete coding region of the novel gene Rv3864c(-|4341113-4341511|) on the chromosome, the amino acid sequence of which is shown in SEQ ID NO.2. After NCBI-BLASTP analysis, no sequence was found in the database, indicating that the protein is a novel gene-encoded protein with unknown function.

[0010] Based on precise proteomics and proteogenomics results, two high-quality, fully cleaved peptide segments, “LLAAHAGQLAGATAGR” and “TQAGQAGEAAQEVR”, encoding a protein from the novel gene Rv3864c were identified in whole-cell lysate proteins of Mycobacterium tuberculosis, plasma and plasma exosomes of patients with pulmonary tuberculosis, and cerebrospinal fluid and cerebrospinal fluid exosomes of patients with tuberculous meningitis. Figure 1 The peptide was chemically synthesized, and the spectra of the original identified peptide were basically consistent with those of the synthesized peptide by liquid chromatography-mass spectrometry analysis. Figure 2 This verifies the correctness of the new peptides and proteins discovered by proteogenomics.

[0011] Therefore, the present invention provides a method for detecting the presence of Mycobacterium tuberculosis in a sample, which determines the presence of Mycobacterium tuberculosis in the sample by detecting the presence of one or more of the following:

[0012] The Rv3864c encoded protein or a portion thereof as described in this invention, wherein the amino acid sequence of the encoded protein is shown in SEQ ID NO.2;

[0013] The nucleic acid described in this invention is the Mycobacterium tuberculosis H37Rv encoding gene Rv3864c(-|4341113-4341511|), the nucleotide sequence of which is shown in SEQ ID NO.1; or

[0014] A polypeptide whose peptide sequence is included in the encoded protein of the present invention, wherein the amino acid sequence of the polypeptide is selected from one of the sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7.

[0015] The peptides shown in SEQ ID NO.3 and SEQ ID NO.4 are two high-quality, fully cleaved peptides encoding the protein of the novel gene Rv3864c identified above. In addition, peptides such as DGPLLPPLAGR (SEQ ID NO.5), GASVDGCTLR (SEQ ID NO.6), and HELLQLTHGGGHLTHGGQ TGK (SEQ ID NO.7) can be used as potential candidate peptides for the detection and diagnosis of Mycobacterium tuberculosis.

[0016] The samples used for testing can be tissue or body fluid samples from the test subject, with plasma, plasma exosomes, cerebrospinal fluid, and cerebrospinal fluid exosomes being preferred samples.

[0017] In terms of detection methods, antibodies against proteins or epitopes can be used for detection; mass spectrometry can also be used to detect the entire protein or fragments composed of its partial sequences, or peptides of SEQ ID NO.2 to SEQ ID NO.7; these proteins can also be enriched by antibody affinity and then detected by mass spectrometry.

[0018] Those skilled in the art will understand that the detection gene of the present invention can be prepared into various detection reagents as needed, for example, by nucleic acid hybridization (e.g., hybridization assay of gene chip, RNA blotting, dot blot, etc.) and / or by nucleic acid amplification (e.g., PCR, qPCR, RT-PCR, qRT-PCR), mass spectrometry detection of the encoded protein or its enzyme-digested peptides, etc. The detection reagent can also be an antibody, and detection can be performed by immunochemical assays (e.g., ELISA, chemiluminescence, immunofluorescence, etc.).

[0019] In a specific embodiment of the present invention, the above-mentioned detection reagent is used to detect Mycobacterium tuberculosis encoding genes or proteins in subjects who need it. The detection method may include: (1) isolating and / or amplifying DNA or RNA from biological samples obtained from the subject; (2) contacting the isolated and / or amplified DNA or RNA with the detection reagent; (3) detecting the level of H37Rv encoding gene Rv3864c(-|4341113-4341511|) and / or Rv3864c encoding protein; (4) detecting the enzyme-digested peptide sequence of Rv3864c encoding protein; (5) combining with a reference detection reagent if necessary.

[0020] Alternatively, the protein or polypeptide described in this invention can be used as a specific antigen of Mycobacterium tuberculosis to prepare a tuberculosis vaccine against Mycobacterium tuberculosis.

[0021] According to another aspect of the present invention, an antibody is provided, comprising the protein or polypeptide described herein. The antibody is a polyclonal antibody obtained by direct immunization using the Rv3864c-encoded protein, or a monoclonal antibody prepared using a portion of the protein's sequence (e.g., SEQ ID NO. 3-7 sequences) as an epitope peptide.

[0022] Beneficial Effects: This invention provides a novel gene-encoded protein for the accurate diagnosis of early Mycobacterium tuberculosis infection and tuberculosis patients. This protein can effectively distinguish between healthy individuals, latently infected individuals, and patients. The diagnostic method using this protein overcomes the shortcomings of low antigen specificity and sensitivity commonly used in the immunological diagnosis of tuberculosis infection. Furthermore, the sensitivity and specificity of this protein in diagnosing latent tuberculosis infection and active tuberculosis patients are higher than those of the currently used clinical biomarker Rv3874 (also known as CFP-10, Renshaw PS, et al. J. Biol. Chem. 2002, 277:21598-21603). It can be used for the auxiliary diagnosis of early Mycobacterium tuberculosis infection and clinical tuberculosis patients, as well as for large-scale tuberculosis epidemiological surveys. Attached Figure Description

[0023] Figure 1 Mass spectrometry detection of peptide secondary spectra;

[0024] Figure 2 Comparison of the mass spectrum of the synthesized peptide with the mass spectrum of the originally identified peptide;

[0025] Figure 3 Verification diagram of the Rv3864c start codon and complete encoded region;

[0026] Figure 4 Hypermatrix phylogenetic tree of 209 species in the Mycobacterium family (arrows indicate the locations Mycobacterium tuberculosis ATCC 27294). T , Mycobacterium bovisATCC 19210 T , Mycobacterium shinjukuenseDSM 45663 T , Mycobacterium kansasiiATCC 12478 T , Mycobacterium intracellulareATCC 13950 T ,MycolicibacterterraeATCC 15755 T , MycolicibacillusparakoreensisDSM 45575 T, MycolicibacteriumsmegmatisATCC 19420 T and MycobacteroidesabscessusATCC 19977 T );

[0027] Figure 5 Comparison of gene sequence homology of Rv3864c(-|4341113-4341511|) in mycobacteria other than Mycobacterium tuberculosis;

[0028] Figure 6 Comparison of protein sequence homology of Rv3864c(-|4341113-4341511|) in mycobacteria other than Mycobacterium tuberculosis;

[0029] Figure 7 : Homology map of gene sequences of Rv3864c(-|4341113-4341511|) and 8 other strains with different phylogenetic relationships;

[0030] Figure 8 Homology map of protein sequences of Rv3864c(-|4341113-4341511|) with 8 other strains with different phylogenetic relationships.

[0031] Figure 9 The protein encoded by Rv3864c(-|4341113-4341511|) was found in purified protein, whole-cell protein of Mycobacterium tuberculosis H37Rv, and Mycobacterium smegmatis MC. 2 Western blot results of whole-cell proteins (pro, purified protein; H37Rv, H37Rv whole-cell protein; MC) 2 155, MC 2 155 whole-cell protein);

[0032] Figure 10 Graphs showing the protein content of plasma and plasma exosomes in patients with pulmonary tuberculosis and cerebrospinal fluid and cerebrospinal fluid exosomes in patients with tuberculous meningitis (plasma, plasma; plasma exosomes, plasma exosomes; CSF, cerebrospinal fluid; CSF exosomes, cerebrospinal fluid exosomes).

[0033] Figure 11 Abundance ranking of the protein encoded by Rv3864c (-|4341113-4341511|) in healthy individuals, individuals with latent tuberculosis infection, and patients with pulmonary tuberculosis (Healthy, healthy individuals; Latent, latent tuberculosis infection; Patient, pulmonary tuberculosis).

[0034] Figure 12Abundance expression plot of the protein encoded by Rv3864c(-|4341113-4341511|) in healthy individuals, individuals with latent tuberculosis infection, and patients with pulmonary tuberculosis (H, healthy individuals; L, individuals with latent tuberculosis infection; P, patients with pulmonary tuberculosis);

[0035] Figure 13 ROC curves of the protein encoded by Rv3864c(-|4341113-4341511|) in differentiating between tuberculosis-infected individuals and healthy individuals (H, healthy individuals; L, latent tuberculosis-infected individuals; P, pulmonary tuberculosis patients);

[0036] Figure 14 Abundance plot of the protein encoded by Rv3864c(-|4341113-4341511|) in pulmonary tuberculosis patients with different copy numbers of Mycobacterium tuberculosis (H, high copy; M, medium copy; L, low copy; EL, very low copy). Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, but this does not limit the scope of the claims. All reagents used in this invention are commercially available.

[0038] Example 1: Discovery and Validation of the Novel Encoding Gene Rv3864c for Mycobacterium tuberculosis H37Rv

[0039] Deep-coverage proteomics was used to study the Mycobacterium tuberculosis H37Rv strain. Using pAnno software (http: / / www.pfind.org / software / pAnno / index.html), developed in collaboration with the Institute of Computing Technology, Chinese Academy of Sciences, the whole genome (NC_000962.3) of Mycobacterium tuberculosis H37Rv published on NCBI was translated according to the rule of translating one amino acid every three consecutive nucleotides on the positive and complementary strands. A total of 141,851 open reading frames (ORFs) were obtained using a terminator-to-terminator translation pattern, and a six-ORF database was constructed.

[0040] This database was used to search for H37Rv deep-coverage proteomic data. Two high-quality peptides encoded by H37Rv were identified between the negative-chain terminator and terminator 4341119-4341845: “LLAAHAGQLAGATAGR” and “TQAGQAGEAAQEVR”. Figure 1 The spectra of the two peptide segments are of good quality, with continuous matching of b / y daughter ions and low impurity peak signals, making the results reliable.

[0041] To further verify the authenticity of these two peptides, we artificially synthesized peptides according to the amino acid sequences “LLAAHAGQLAGATAGR” and “TQAGQAGEAAQEVR”. Liquid chromatography-mass spectrometry (LC-MS) analysis yielded the mass spectra of the synthesized peptides, which were largely consistent with the mass spectra of the new peptides obtained from large-scale identification using deep-coverage proteomics, with similarity (cosin values) of 0.99 and 0.97, respectively. Figure 2 This indicates that we have used proteogenomics to identify a protein restriction peptide encoded by an unannotated and undiscovered gene corresponding to a DNA sequence from Mycobacterium tuberculosis.

[0042] Using published Mycobacterium tuberculosis ribosomal sequencing data (E-MTAB-8039 and E-MTAB-8335) downloaded from the ArrayExpress database, the start codon of this coding region was identified, thus determining that the coding region is the negative strand 4341113-4341511. Figure 3 The complete open reading frame DNA sequence was obtained, as shown in SEQ ID NO.1.

[0043] The new coding gene sequence begins with ATG, totaling 396 bp, and encodes 131 amino acids, with a theoretical molecular weight of 13.42 kDa. Based on its location on the chromosome and the naming conventions for Mycobacterium tuberculosis genes, it has been named the Rv3864c gene.

[0044] ATGGCACAGACCTGCTGGCGACTCGGTGTCCGATCAAAGGACGGTCCCCTCCTGCCCCC

[0045] GTTGGCTGGCCGCGGTGCCAGCGTCGATGGGTGCACGCTCCGCCTCGGCCACGCCTTCC

[0046] TCGTCTTCCTTCTTGTCGCTCACGAGGGTGGCGTGCTGCTGGCCTCCTTGCTGGGCCTGC

[0047] GACGAGATCAGTTGCGCTTGTTGGCTGCCCATGCTGGCCAGTTGGCTGGGGCCACCGCC

[0048] GGCCGCCACGAGCTGTTGCAGCTGACCCATGGTGGCGGTCACCTGACCCACGGTGGGC

[0049] AAACTGGCAAAGCTGAGCAGATTGCCAAAACCCAAGCTGGGCAGGCCGGCGAAGCCG

[0050] CCCAAGAAGTCAGGCAAAGCGGCCAGCTCAGTCCAGGTGGGTAA

[0051] (SEQ ID NO.1)

[0052] The amino acid sequence of the theoretically encoded product of this gene is shown in SEQ ID NO.2:

[0053] MAQTCWRLGVRSKDGPLLPLPLAGRGASVDGCTLRLGHAFLVFLLVAHEGGVLLASLLGLR

[0054] RDQLR LLAAHAGQLAGATAGR HELLQLTHGGGHLTHGGQ TGKAEQIAK TQAGQAGEAAQEVR QSGQLSPGG(SEQ ID NO.2)

[0055] The peptide sequences identified by proteogenomics techniques are shown in SEQ ID NO.3 and SEQ ID NO.4:

[0056] LLAAHAGQLAGATAGR(SEQ ID NO.3)

[0057] TQAGQAGEAAQEVR(SEQ ID NO.4)

[0058] The protein sequence encoded by the Rv3864c gene was entered into a database, and a re-search of proteomic data was conducted. Restricting peptide lengths to 7 amino acids or more, three additional peptides were identified: DGPLLPPLAGR, GASVDGCTLR, and HELLQLTHGGGHLTHGGQ TGK. These peptides cover almost all amino acid residues from the N-terminus to the C-terminus of the encoded protein, excluding artificially restricted peptide lengths, indicating that this high-abundance protein may be a high-abundance protein. The re-search for these additional peptides was due to the reduced sensitivity of mass spectrometry searches caused by database expansion in proteogenomics research. This not only increases the reliability of the identification of the new Rv3864c gene, but also suggests that peptides such as DGPLLPPLAGR (SEQ ID NO. 5), GASVDGCTLR (SEQ ID NO. 6), and HELLQLTHGGGHLTHGGQ TGK (SEQ ID NO. 7) can be considered as potential candidate peptides for Mycobacterium tuberculosis detection and diagnosis.

[0059] Example 2: Development of a rabbit polyclonal antibody against the novel encoding gene Rv3864c of Mycobacterium tuberculosis H37Rv

[0060] 1) Antibody customization

[0061] 1. The protein encoded by Rv3864c was expressed and purified in mammalian cells with a purity of over 90%;

[0062] 2. The recombinant protein was mixed with complete Freund's adjuvant (500 μg / rabbit), and New Zealand white rabbits weighing 2 kg were selected for immunization, with two rabbits immunized at a time; a booster immunization was performed at an interval of about 2 weeks, with the recombinant Rv3864c encoded protein mixed with incomplete Freund's adjuvant (450 μg / rabbit) for booster immunization; a total of 5 immunizations were performed.

[0063] 3. After immunization, rabbit blood was collected to obtain polyclonal antiserum.

[0064] II) Antibody purification

[0065] 1. Ammonium sulfate precipitation

[0066] Centrifuge the antiserum at 8,500 rpm for 20 min. After centrifugation, collect the supernatant, add saturated ammonium sulfate solution while mixing to precipitate the antibody, then centrifuge at 8,500 rpm for 25 min at 4°C, discard the supernatant, and dissolve the precipitate in phosphate-buffered saline (PBS).

[0067] 2. Antibody affinity purification:

[0068] Pre-packed column preparation: Equilibrate the pre-packed column to room temperature, open the bottom cap, allow the liquid inside the column to drain completely, and then rinse the chromatography column with coupling buffer.

[0069] Purified antibody:

[0070] 1) Add coupling buffer to the chromatography column, then add water-dissolved antigen; after antigen binding is complete, wash the chromatography column with coupling buffer, and wash the top cap and bottom cap at the same time.

[0071] 2) Add blocking buffer and invert at 4°C to mix and block. After blocking, add coupling wash buffer and PBS to wash the column sequentially.

[0072] 3) Add the antiserum to the chromatography column and incubate overnight at 4°C. After binding, place the column on a rack to allow the agarose gel to precipitate, and collect the flow-through into a centrifuge tube for storage.

[0073] 4) Wash the chromatography column three times with 0.5M PBS, and then four times with 10x PBS;

[0074] 5) Pre-elute the column with 0.2 mL of elution buffer, then elute with 3 mL of elution buffer;

[0075] 6) Take a dialysis bag of suitable length, boil it in ultrapure water for 20 minutes, then rinse it with ultrapure water before use, or cool it and store it in 20%-50% ethanol at 4°C. Dialyze the antibody for 2-3 hours, then change the dialysate and dialyze overnight. Change the dialysate again the next day, dialyze for 2-3 hours, and then recover and purify the antibody.

[0076] Example 3: Discovery and validation of the specificity of the novel Rv3864c encoding gene identified by proteogenomics in Mycobacterium tuberculosis H37Rv

[0077] To investigate the specificity of Rv3864c in Mycobacterium tuberculosis H37Rv, the genome and proteome sequences of 209 standard strains from the Mycobacteriaceae family were downloaded from the NCBI database, including 85 strains from the genus *Mycobacterium*, 17 strains from the genus *Mycolicibacter*, 7 strains from the genus *Mycobacteroides*, and 3 strains from the genus *Mycolicibacillus*. A super-matrix phylogenetic tree was constructed based on 269 conserved homologous genes. Figure 4 The novel coding gene for Rv3864c and two classic biomarkers for tuberculosis diagnosis, Rv3874 and Rv3875, were determined based on BLASTN (…). Figure 5 ) and BLASTP Figure 6Sequence alignment was performed. The alignment analysis revealed that the Rv3864c gene DNA sequence only shares some homology with 9 of the 209 validly published species of the Mycobacteriumceae family. The highest homology was found in *Mycobacterium persicum*, with a sequence coverage of 72.98% and a homology of 78.50%, for a total homology of only 57.3%. No homologous sequences were found in the remaining 200 species outside the *Mycobacterium tuberculosis* complex. Therefore, the specificity of Rv3864c in the H37Rv genome is 99.5% (i.e., it is absent in 99.5% of the genomes of Mycobacteriumceae). The amino acid sequence of the protein encoded by Rv3864c shows no homology with the proteomes of standard strains from 209 Mycobacterial Families (excluding the Mycobacterium tuberculosis complex), meaning it is absent in the proteomes of all other published species. Therefore, the Rv3864c-encoded protein exhibits 100% specificity in the H37Rv genome (i.e., it is not present in the proteomes of any Mycobacterial Families except the Mycobacterium tuberculosis complex), significantly higher than the known specificity of Rv3874 in the H37Rv genome (90.5%) and proteome (77%), and also higher than the specificity of Rv3875 in the H37Rv genome (94%) and proteome (88.5%). For this reason, we refer to Rv3864c as a highly specific gene and protein for the Mycobacterium tuberculosis complex.

[0078] Based on the phylogenetic tree of mycobacteria, eight standard strains of H37Rv with different phylogenetic distances were selected, including Mycobacterium bovisATCC 19210. T , Mycobacterium shinjukuenseDSM 45663 T , Mycobacterium kansasiiATCC 12478 T , Mycobacterium intracellulareATCC 13950 T ,MycolicibacterterraeATCC 15755 T , MycolicibacillusparakoreensisDSM 45575 T , Mycolicibacterium smegmatisATCC 19420 T and MycobacteroidesabscessusATCC 19977 T Based on the BLASTN and BLASTP results, scatter plots of H37Rv and other strains were constructed respectively. Figure 7 and Figure 8 BLASTN and BLASNP results showed that Rv3864c is homologous to the genes and corresponding encoded proteins of strains within the Mycobacterium tuberculosis complex, but has no homologous sequences with other strains, proving that Rv3864c is a highly specific gene of the Mycobacterium tuberculosis complex and can be used for the differential diagnosis of Mycobacterium tuberculosis complex and other strains.

[0079] To further verify the expression characteristics and specificity of the Rv3864c encoding gene, we cloned, expressed, and purified the new gene Rv3864c expression product, and compared it with Mycobacterium tuberculosis (M. tuberculosis H37Rv) and its close relative Mycobacterium smegmatis MC. 2 155) Whole cell lysate, based on the antibody developed in Example 2 above, was subjected to Western blot with anti-Rv3864c antibody to detect the Rv3864c target protein. The results showed ( Figure 9 A strong expression signal was detected at the theoretical molecular weight of Rv3864c in the purified Rv3864c target protein and the H37Rv whole-cell proteome sample, while a strong signal was detected in the negative control M. smegmatis MC. 2 No expression signal of Rv3864c was detected in any of the 155 strains, verifying the correctness of the complete coding region sequence of Rv3864c that we discovered, validated, and annotated. This also further demonstrates the specificity of the Rv3864c coding gene. This indicates that we can utilize Rv3864c and its encoded protein to further develop technologies such as Western blot, ELISA, and protein microarrays for the identification, diagnosis, and monitoring of treatment efficacy for Mycobacterium tuberculosis, non-tuberculous mycobacteria, or other strains, and even infected samples.

[0080] Example 4: Detection of expression of novel protein Rv3864c in clinical samples from patients with pulmonary tuberculosis and tuberculous meningitis

[0081] To further explore whether the novel, highly specific protein Rv3864c has clinical diagnostic value after Mycobacterium tuberculosis infection in humans, we collected plasma samples from a typical pulmonary tuberculosis patient in Hebei Province, who presented with clinical symptoms, positive T-SPOT and tuberculosis cultures, and abnormal chest X-rays; and cerebrospinal fluid samples from a typical tuberculous meningitis patient in Beijing, who presented with clinical symptoms and a positive GeneXpert test.

[0082] Based on mature laboratory techniques for removing high-abundance proteins from plasma, extracting proteins from cerebrospinal fluid, and extracting exosomes from trace amounts of plasma and cerebrospinal fluid, plasma and plasma exosome protein samples from one patient with pulmonary tuberculosis, and cerebrospinal fluid and cerebrospinal fluid exosome protein samples from one patient with tuberculous meningitis were prepared. These samples were separated using 12% SDS-PAGE gel chromatography. Figure 10 Afterwards, the molecules were excised and digested with trypsin according to their molecular weight, and detected using a Q Exactive HF mass spectrometer in Data Dependent Acquisition (DDA) mode. The liquid chromatography gradient was 120 min, and the B-phase (80% ACN, 0.1% FA) gradients were as follows: 7%-12%, 6 min; 12%-30%, 74 min; 30%-45%, 30 min; 45-95%, 2 min; 95%, 8 min. Primary mass spectrometry parameters: resolution, 120,000; automatic gain control (AGC), 3e6; maximum injection time (MIT), 50 ms. Secondary mass spectrometry parameters: High energy collision-induced dissociation (HCD) fragmentation mode, energy, 35%; AGC, 3e6; MIT, 19 ms.

[0083] The "Human" annotated protein database was downloaded from the UniProt database, and the "Mycobacterium tuberculosis" annotated protein database was downloaded from TubercuList. The "Human", "Mycobacterium tuberculosis" and "Rv3864c" databases were merged into a new database and searched using the pFind search engine. The search parameters were set as follows: (1) Protease, Trypsin and LysC, with a maximum of 2 missed cleavage sites; (2) Peptide length, ≥7 amino acids; (3) Fixed modification, alkylation of cysteine, Carbamidomethyl[C]; (4) Variable modification, methionine oxidation, Oxidation[M]; protein N-terminal acetylation, Acetyl[Protein N-term]; (5) Error, mother ion mass of 20 ppm, daughter ion of 20 ppm; (6) Filtering, target-decoy library strategy; the false discovery rate (FDR) was less than 1% at the spectral, peptide and protein levels.

[0084] The database search results revealed that two fully cleaved peptides, “LLAAHAGQLAGATAGR” and “TQAGQAGEAAQEVR”, validated in Example 1, were successfully identified in the plasma and plasma exosomes of patients with pulmonary tuberculosis, and in the cerebrospinal fluid and cerebrospinal fluid exosomes of patients with tuberculous meningitis. This demonstrates that the protein encoded by Rv3864c is present at high abundance in body fluid samples from patients with pulmonary tuberculosis and tuberculous meningitis. Moreover, quantitative results showed that the expression abundance of Rv3864c was significantly higher than that of the classic tuberculosis diagnostic marker Rv3874, indicating that the new protein Rv3864c has clinical diagnostic value for both pulmonary tuberculosis and tuberculous meningitis. Further research can be conducted using the target protein of the new gene Rv3864c or its peptide to develop antibody-based Western blot, ELISA, protein chip, chemiluminescence, and other technologies for the detection, identification, and diagnosis of pulmonary and extrapulmonary tuberculosis caused by Mycobacterium tuberculosis. Mass spectrometry and other technologies can also be used to develop technologies for the detection, identification, and diagnosis of pulmonary and extrapulmonary tuberculosis caused by Mycobacterium tuberculosis caused by the target protein of the new gene Rv3864c or its peptide.

[0085] Example 5: Differential expression detection of novel protein Rv3864c in individuals with different degrees of Mycobacterium tuberculosis infection

[0086] To further evaluate the diagnostic performance of the novel secreted protein in clinical plasma samples, we collected plasma from 10 healthy individuals, 10 individuals with latent tuberculosis infection, and 10 patients with pulmonary tuberculosis from Beijing, Tianjin, and Hebei, and prepared exosomal proteomics samples. After pooling the enzyme-digested peptide fragments from each group, the samples were analyzed using a Q Exactive HF mass spectrometer. The results showed that the mass spectrometry identification of the pooled samples from the 10 patients was consistent with the results of the single patient in Example 3. The novel protein Rv3864c was a medium-to-high abundance protein, and its expression abundance was higher than that of the known marker Rv3874. Comparative analysis of protein quantification among the healthy group, latent group, and patient group revealed that Rv3864c and Rv3874 were not identified in the healthy group, but were identified in both the latent and patient groups, with the expression level in the patient group being significantly higher than that in the latent group. Figure 11 ).

[0087] To further determine the diagnostic efficacy of the novel protein Rv3864c in Mycobacterium tuberculosis infection samples, we re-collected 15 healthy individuals, 18 latently infected individuals, and 19 patients, and performed mass spectrometry detection using data-independent acquisition (DIA) mode. The results showed that both the known Rv3874 and our novel Rv3864c were detectable in all 19 tuberculosis patients, with a false negative rate of 0. In all 18 clinically T-SPOT and PPD-positive latently infected individuals, the existing tuberculosis diagnostic marker Rv3874 was undetectable in 11 latently infected individuals; therefore, the false negative rate of Rv3874-based mass spectrometry detection was as high as 61.1%. However, the novel protein Rv3864c was identified with high confidence not only in patients but also in all latently infected individuals, with a positive rate of 100%, while the false negative rate leading to misdiagnosis was 0. Figure 12 a). Among 15 volunteers considered healthy, Rv3864c was detected in 2 cases, suggesting these two individuals were potential latent infections. Further investigation based on contact history confirmed that these two cases with detectable Rv3864c signals were indeed latent infections, with a false negative and false positive rate of 0%. However, no Rv3874 signals were detected, resulting in a false negative rate of 13.3% in the blind test. Furthermore, the expression abundance of the novel Rv3864c protein was significantly higher than that of Rv3874 (…). Figure 12 (b) The above results mean that the novel Rv3864c protein has higher sensitivity than the currently used clinical tuberculosis diagnostic marker Rv3874 in screening for tuberculosis infection.

[0088] ROC curves further evaluate the diagnostic accuracy of the two proteins, Rv3864c and Rv3874. Figure 13 The results showed that the existing biomarker Rv3874 could distinguish between healthy individuals and patients (AUC = 1.000), and between healthy individuals and latently infected individuals (AUC = 0.694); while the novel protein Rv3864c showed a good diagnostic efficacy in both distinguishing between healthy individuals and patients (AUC = 1.000) and distinguishing between healthy individuals and latently infected individuals (AUC = 1.000). Even in a blinded study of 15 samples, including 2 latently infected individuals, the false negative rate of Rv3874 was 13.3%, while the false negative and false positive rates of Rv3864c were both 0.

[0089] Furthermore, we collected two blood samples from clinicians specializing in tuberculosis. Neither sample was diagnosed as positive for infection or latent infection using existing technologies. However, we successfully identified the characteristic peptide of the novel protein Rv3864c in both samples, indicating that the novel protein Rv3864c has better diagnostic efficacy.

[0090] The combination of Rv3864c and Rv3874 enables accurate diagnosis of tuberculosis infection. Given the high specificity, high abundance, and differential expression of the novel protein Rv3864c in samples from different infection stages, it can be used for screening individuals with early-stage Mycobacterium tuberculosis infection.

[0091] Example 6: Verification of a positive correlation between the expression of the novel protein Rv3864c and the copy number of Mycobacterium tuberculosis in patients.

[0092] To further validate the detection sensitivity of the novel protein Rv3864c and to examine its value in monitoring the efficacy of tuberculosis treatment, we collected samples from 11 pulmonary tuberculosis patients with different copy numbers of Mycobacterium tuberculosis from a tuberculosis specialist hospital. Based on the GeneXpert detection results, the patients were divided into 3 high copy number (Ct, <16), 2 medium copy number (Ct, 16-22), 4 low copy number (Ct, 22-28), and 2 very low copy number (Ct, >28). Plasma exosomal proteomics samples were prepared from these patients, and mass spectrometry was performed based on DIA mode.

[0093] Quantitative comparison revealed that Rv3864c was unbiasedly identified in patients with different copy number levels of Mycobacterium tuberculosis, while the commonly used tuberculosis marker Rv3874 was detected only in two patients with high copy numbers of Mycobacterium tuberculosis. Figure 14 a) The expression level of Rv3864c decreases with decreasing copy number of Mycobacterium tuberculosis. Figure 14 b), and related to the copy number of Mycobacterium tuberculosis ( Figure 14 c) and clinical sputum smears ( Figure 14 d) showed a significant positive correlation, with correlation coefficients of 0.8285 and 0.8855, respectively. These results confirm that the expression level of Rv3864c is positively correlated with the copy number of Mycobacterium tuberculosis in patients, and can be used for the diagnostic detection of latent tuberculosis infection, early Mycobacterium tuberculosis infection samples, and clinical samples with very low Mycobacterium tuberculosis copy numbers. It can also be used to evaluate the efficacy of anti-tuberculosis drug treatment based on the expression level of Rv3864c.

Claims

1. An isolated protein, the amino acid sequence of which is shown as SEQ ID NO.

2.

2. An isolated nucleic acid, which encodes the protein of claim 1, the nucleic acid being Mycobacterium tuberculosis H37Rv coding gene Rv3864c (-|4341113-4341511|), the nucleotide sequence of which is shown as SEQ ID NO.

1.

3. An isolated polypeptide, the peptide segment sequence of which is contained in the protein of claim 1, the amino acid sequence of which is selected from one of the sequences shown as SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO.

7.

4. A method of detecting the presence or absence of Mycobacterium tuberculosis in a sample, characterized by The method detects whether one or more of the following is present in the sample: the protein or a portion thereof of claim 1; the nucleic acid of claim 2; or the polypeptide of claim 3.

5. The method of claim 4, wherein the sample is selected from a tissue or body fluid sample of a test subject, including but not limited to plasma, plasma exosomes, cerebrospinal fluid and cerebrospinal fluid exosomes.

6. Use of the protein or a portion thereof of claim 1, or the polypeptide of claim 3 in the preparation of a reagent for diagnosing and / or differentiating Mycobacterium tuberculosis.

7. An antibody that binds to the protein of claim 1 or the polypeptide of claim 3.

8. A kit for detecting Mycobacterium tuberculosis, characterized by The kit comprises a detection reagent for detecting and / or differentiating Mycobacterium tuberculosis in a sample, the detection reagent comprising one selected from: a reagent for detecting whether the protein or a portion thereof of claim 1, or the polypeptide of claim 3 is present in the sample; a reagent for detecting whether the nucleic acid of claim 2 is present in the sample.

9. The kit of claim 8, wherein The reagent is the antibody of claim 7.

10. Use of the protein of claim 1 or the polypeptide of claim 3 as an antigen of Mycobacterium tuberculosis in the preparation of a tuberculosis vaccine.

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