A monoclonal antibody against Rv0340 that can improve drug resistance in Mycobacterium tuberculosis and its application

Abstract

This invention provides a monoclonal antibody 4D5 against Rv0340 that can improve drug resistance in Mycobacterium tuberculosis. The heavy chain variable region of antibody 4D5 has the following amino acid sequences: CDR-H1 (SEQ ID No. 1), CDR-H2 (SEQ ID No. 2), and CDR-H3 (SEQ ID No. 3); and the light chain variable region of anti-Rv0340 monoclonal antibody 4D5 has the following amino acid sequences: CDR-L1 (SEQ ID No. 4), CDR-L2 (SEQ ID No. 5), and CDR-L3 (SEQ ID No. 6). This anti-Rv0340 monoclonal antibody 4D5 was prepared using Rv0340 protein as an antigen, and the effect of the anti-Rv0340 monoclonal antibody on the in vitro growth of Mtb in the presence of anti-tuberculosis drugs was investigated. The results showed that antibody 4D5 could significantly increase the sensitivity of Mtb to isoniazid; and when antibody 4D5 was used in combination with isoniazid, the survival rate of Mtb decreased by 26.58%. This invention provides a new monoclonal antibody drug for specifically improving the sensitivity of Mtb to isoniazid, and has the prospect of application as a monoclonal antibody drug to improve the drug resistance of Mycobacterium tuberculosis.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more specifically, to an anti-Rv0340 monoclonal antibody that can improve drug resistance in Mycobacterium tuberculosis and its application. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) infection. It can affect multiple organs and cause systemic disease.

[0003] Currently, there are four main classes of first-line antibacterial drugs for treating tuberculosis (TB): isoniazid (INH), rifampicin (RFP), pyrazinamide (PZA), and ethambutol (EMB). Their widespread and standardized use once effectively controlled the global TB epidemic. However, with the continuous mutation of Mycobacterium tuberculosis, drug-resistant strains, especially multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains, have emerged, seriously threatening global TB prevention and control efforts, making the situation extremely challenging. MDR-TB refers to Mycobacterium tuberculosis strains resistant to at least two first-line anti-TB drugs—INH and RFP; XDR-TB refers to multidrug-resistant Mycobacterium tuberculosis strains also resistant to at least three of the six second-line drugs. Overcoming drug resistance in Mycobacterium tuberculosis and increasing its sensitivity to clinical drugs has become an urgent task.

[0004] Reducing drug resistance can be achieved through two main approaches. First, optimizing drug combinations can improve treatment success rates and prevent the further spread of antibiotic resistance. Chinese invention patent CN103357016A discloses a pharmaceutical composition for treating drug-resistant tuberculosis. This composition comprises a combination of nano-chitosan artemisinin for treating drug-resistant tuberculosis and an anti-tuberculosis drug, including the following active ingredients: an anti-tuberculosis drug, nano-chitosan artemisinin, and three nano-chitosan artemisinin derivatives or artemisinin and its derivatives. This composition can inhibit and kill drug-resistant tuberculosis bacteria, showing outstanding efficacy in treating drug-resistant tuberculosis, but its specificity is relatively weak. Second, by identifying key mutant genes mediating drug resistance in Mycobacterium tuberculosis and designing novel molecular drugs targeting these genes, the sensitivity of bacteria to existing anti-tuberculosis drugs can be improved. This approach offers better drug targeting, but the number of key mutant genes and mutations related to drug resistance in Mycobacterium tuberculosis is vast, and the design of suitable drugs to increase the sensitivity of tuberculosis drugs remains a critical challenge. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned technical problems and provide an anti-Rv0340 monoclonal antibody that can improve the drug resistance of Mycobacterium tuberculosis. This antibody can increase the sensitivity to tuberculosis drugs. Another purpose of this invention is to provide the application of the anti-Rv0340 monoclonal antibody.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A monoclonal antibody 4D5 against Rv0340 that can improve drug resistance in Mycobacterium tuberculosis, wherein the CDR-H1 of the heavy chain variable region of the anti-Rv0340 monoclonal antibody 4D5 has the amino acid sequence shown in SEQ ID No. 1, the CDR-H2 of the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 2, and the CDR-H3 of the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 3; the CDR-L1 of the light chain variable region of the anti-Rv0340 monoclonal antibody 4D5 has the amino acid sequence shown in SEQ ID No. 4, the CDR-L2 of the light chain variable region has the amino acid sequence shown in SEQ ID No. 5, and the CDR-L3 of the light chain variable region has the amino acid sequence shown in SEQ ID No. 6.

[0008] Furthermore, the heavy chain variable region is the amino acid sequence shown in SEQ ID No. 7.

[0009] Furthermore, the light chain variable region is the amino acid sequence shown in SEQ ID No. 8.

[0010] Furthermore, the anti-Rv0340 monoclonal antibody 4D5 is of the IgG1 subtype, and all light chains are κ chains.

[0011] This invention also provides a method for preparing the above-mentioned anti-Rv0340 monoclonal antibody 4D5, which can improve the drug resistance of Mycobacterium tuberculosis: immunizing animals with Rv0340 protein, isolating single B lymphocytes from the animals for myeloma cell fusion, and screening to obtain anti-Rv0340 monoclonal antibody 4D5; wherein the Rv0340 protein is a secreted protein of Mycobacterium tuberculosis, and the amino acid sequence of the Rv0340 protein is the sequence shown in SEQ ID No. 9.

[0012] This invention screened and prepared multiple monoclonal antibodies against the Rv0340 protein. During research on its resistance to Mycobacterium tuberculosis, an anti-Rv0340 monoclonal antibody, 4D5, was unexpectedly discovered. This anti-Rv0340 monoclonal antibody 4D5 can significantly increase the sensitivity of Mtb to isoniazid (INH). Furthermore, when anti-Rv0340 monoclonal antibody 4D5 is used in combination with INH, it can significantly improve the sensitivity of Mtb to the specific drug INH, while having no such effect on other drugs. This demonstrates the potential of monoclonal antibody drugs for improving drug resistance in Mycobacterium tuberculosis.

[0013] Since the anti-Rv0340 monoclonal antibody 4D5 can significantly increase the sensitivity of Mtb to isoniazid INH, the present invention requests protection for the nucleic acid encoding the above-mentioned anti-Rv0340 monoclonal antibody 4D5 that can improve the drug resistance of Mycobacterium tuberculosis, the sequence of which is shown in SEQ ID No. 10.

[0014] Based on this, the present invention provides an expression vector containing the above-mentioned nucleic acid, which can express the nucleic acid in prokaryotic or eukaryotic host cells.

[0015] The present invention provides a host cell that contains and expresses the above-mentioned vector and produces the above-mentioned anti-Rv0340 monoclonal antibody 4D5, which can improve the drug resistance of Mycobacterium tuberculosis.

[0016] The present invention provides a drug that can improve drug resistance of Mycobacterium tuberculosis, comprising the above-mentioned anti-Rv0340 monoclonal antibody 4D5, and a medically acceptable carrier or excipient.

[0017] The present invention provides a drug that can improve drug resistance of Mycobacterium tuberculosis, comprising the above-mentioned composition of anti-Rv0340 monoclonal antibody 4D5 and isoniazid, and a medically acceptable carrier or excipient.

[0018] Preferably, the carrier can be a polymer carrier or a glycosylated ligand; the excipient can be albumin or protein A (Staphylococcus aureus cell wall protein).

[0019] This invention provides the use of the above-mentioned anti-Rv0340 monoclonal antibody 4D5, nucleic acid, vector or host cell in the preparation of drugs for the prevention and treatment of tuberculosis or for improving drug resistance of Mycobacterium tuberculosis.

[0020] Preferably, the preparation of drugs for the prevention and treatment of tuberculosis or for improving drug resistance of Mycobacterium tuberculosis includes anti-Rv0340 monoclonal antibody 4D5 and other immunomodulatory drugs or traditional anti-tuberculosis drugs.

[0021] This anti-Rv0340 monoclonal antibody 4D5 can prevent tuberculosis through passive and active immunization, treat tuberculosis by directly participating in targeted therapy and immunomodulation, be used in combination with traditional anti-tuberculosis drugs, or act on Mycobacterium tuberculosis through different mechanisms, thereby bypassing its drug resistance mechanism and improving the effectiveness of the drug. Therefore, it can be used to prepare such drugs.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) In this invention, multiple monoclonal antibodies were prepared by screening for Rv0340 protein. During the study on its resistance to Mycobacterium tuberculosis, an anti-Rv0340 monoclonal antibody 4D5 was unexpectedly discovered. This anti-Rv0340 monoclonal antibody 4D5 can significantly increase the sensitivity of Mtb to isoniazid INH. Furthermore, when anti-Rv0340 monoclonal antibody 4D5 is used in combination with INH, it can significantly improve the sensitivity of Mtb to the specific drug INH.

[0024] (2) This invention provides a new monoclonal antibody drug that specifically improves the sensitivity of Mtb to INH, and has the application prospect of monoclonal antibody drugs that improve the drug resistance of Mycobacterium tuberculosis. Attached Figure Description

[0025] Figure 1 The image shows the SDS-PAGE electrophoresis results of the purified anti-Rv0340 monoclonal antibodies prepared. Figure 1 In the figure, 2E5, 2E9, 3D5, 4C4, 4D5, 4D9, 4E4, 4F9, 5B12, 12C7, 20E5, and 20H11 represent 12 monoclonal antibodies against Rv0340. The electrophoresis symbols are: M-Marker; 1-full-length IgG monoclonal antibody; 2-heavy chain and light chain.

[0026] Figure 2 The subclasses, subtypes, and light chains of the 12 anti-Rv0340 monoclonal antibodies prepared are shown. Figure 2 In the diagram, A represents the subclass or subtype of each monoclonal antibody; B represents the type of light chain of each monoclonal antibody.

[0027] Figure 3 The binding activity of the prepared anti-Rv0340 monoclonal antibody is shown.

[0028] Figure 4 The prepared anti-Rv0340 monoclonal antibody was shown to affect the survival rate of mycobacteria in isoniazid.

[0029] Figure 5 The prepared anti-Rv0340 monoclonal antibody showed that it reduced the resistance of mycobacteria to isoniazid.

[0030] Figure 6This demonstrates the effect of the prepared anti-Rv0340 monoclonal antibody on the growth of mycobacteria in vitro.

[0031] Figure 7 This demonstrates the effect of the prepared anti-Rv0340 monoclonal antibody on resistance to other anti-tuberculosis drugs; Figure 7 In the diagram, A represents the drug rifampin, and B represents the drug ethambutol. Detailed Implementation

[0032] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention. Example 1: Expression and purification of Rv0340 protein

[0033] 1. Construction of recombinant plasmid pET30a-Rv0340

[0034] (1) Obtaining the target gene

[0035] The CDS sequence of the Rv0340 protein was found on Mycobrowser. Upstream and downstream primers were designed and synthesized (Table 1). The primer sequences are SEQ ID No. 19 and SEQ ID No. 20, respectively. The designed primers were synthesized by Genewiz Biotechnology Co., Ltd. The Rv0340 target gene was amplified by PCR using H37Rv whole-genome DNA as a template, and the target gene was recovered using an agarose gel extraction kit.

[0036] Table 1. Primers for amplifying the Rv0340 fragment.

[0037]

[0038] (2) Construction of recombinant prokaryotic expression vector

[0039] The optimized pET-30a-trx vector and the Rv0340 target gene were digested with XhoI enzyme, and the target fragment was recovered and ligated by homologous recombination to construct the pET-Rv0340 recombinant vector.

[0040] (3) Obtain the strain containing the recombinant expression plasmid and the recombinant expression plasmid.

[0041] The recombinant plasmids obtained above were transformed into E. coli Top10 competent cells, and selected using solid LB medium containing ampicillin. Single clones were picked, cultured, and a small amount of plasmid was extracted. Preliminary identification was performed by double enzyme digestion. The sequence of the target gene was verified by sequencing. A large quantity of correctly sequenced recombinant plasmids were extracted, and some plasmids and the bacterial strain were preserved.

[0042] 2. Expression and purification of Rv0340 protein

[0043] (1) Expression of recombinant protein Rv0340

[0044] The pET-Rv0340 plasmid was transformed into competent BL21 cells, and the cells were expanded in LB culture at OD. 600 When the value reaches 0.6-1.0, add 1mM IPTG to induce protein expression. The induction conditions are 25℃ for 12h and 250rpm.

[0045] (2) Purification of Rv0340 protein

[0046] Bacterial cells were collected, lysed, and purified by nickel affinity chromatography followed by elution with 100 mM imidazole. The column buffer was collected, dialyzed against PBS, and then treated with thrombin to remove the trx tag from the Rv0340-trx protein. The protein was then purified again by nickel affinity chromatography to obtain pure Rv0340 protein with a purity exceeding 90%. The amino acid sequence is shown in SEQ ID No. 9.

[0047] Example 2: Preparation of monoclonal antibody against Rv0340 protein

[0048] This embodiment also provides a method for preparing anti-Rv0340 monoclonal antibody 4D5: animals are immunized with Rv0340 protein, single B lymphocytes are isolated from the animals for myeloma cell fusion, and the anti-Rv0340 monoclonal antibody is obtained through screening; the Rv0340 protein is a secreted protein of Mycobacterium tuberculosis, and the specific steps are as follows:

[0049] 1. Immunized animals

[0050] Four 6-8 week old female Balb / c mice were selected and administered three immunizations, with one mouse serving as a control. First immunization: Rv0340 protein was diluted with PBS, mixed 1:1 with complete Freund's adjuvant, emulsified, and administered subcutaneously at 30 μg / mouse. Second immunization: 10-14 days after the first immunization, Rv0340 protein was diluted with PBS, mixed 1:1 with incomplete Freund's adjuvant, emulsified, and administered subcutaneously at 30 μg / mouse. Third immunization: 10-14 days after the second immunization, Rv0340 protein was diluted with PBS, mixed 1:1 with incomplete Freund's adjuvant, emulsified, and administered subcutaneously at 30 μg / mouse. Three days after each immunization, orbital blood was collected from the mice, and antibody titers were measured using ELISA. Mice with the highest titers were selected for cell fusion. Enhanced immunity: Two days before cell fusion, mice were injected intraperitoneally with Rv0340 protein, 30 μg / mouse. Two days later, the spleens of the mice were harvested for fusion.

[0051] 2. Preparation of mouse peritoneal macrophages

[0052] Feeder cells were prepared 24 hours before cell fusion. Mice were euthanized by dislocation and disinfected with 70% alcohol. An abdominal incision was made, the incision was opened, and the abdominal wall was cleaned with 70% alcohol. The abdominal wall was lifted with forceps, and 4.5-5.5 ml of DMEM culture medium was injected intraperitoneally. The abdomen was massaged, and 4-5 ml of fluid was aspirated. The cells were centrifuged at 1000 rpm for 7 minutes, and the supernatant was discarded. The cell culture medium was resuspended to 10⁻⁶. 5 / ml cells, seed the cells in a 96-well plate, 100μl / well.

[0053] 3. Preparation of mouse spleen cells

[0054] Three days after booster immunization, mice were enucleated, blood was collected, and they were euthanized by cervical dislocation. The cells were then disinfected by soaking in 75% alcohol for 3 minutes. An abdominal incision was made, tearing open the abdominal wall to fully expose it. The peritoneum was cut open, and the spleen was removed using forceps. The spleen was ground using a 100-mesh cell filter while simultaneously washing with basal RPMI-1640 medium. It was then ground again using a 40-mesh cell filter while washing with basal medium. The filtrate was collected, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. 1 ml of RBC was added for 1-2 minutes to break down the cells, followed by the addition of an equal volume of basal medium to stop the process. The mixture was then centrifuged at 200 RCF for 7 minutes, the supernatant was removed, and the cells were diluted with 20 ml of complete medium for cell counting.

[0055] 4. Preparation of syngeneic myeloma cells SP2 / 0

[0056] Pre-culture SP2 / 0 cells in good growth condition in T75 cell culture flasks. The cells can be detached by tapping the back of the culture flask. After collection, centrifuge at 1200 rpm for 6 min, remove the supernatant, dilute with complete culture medium, and count the cells.

[0057] 5. PEG cell fusion

[0058] After mixing spleen cells with SP2 / 0 cells at a 4:1 ratio, centrifuge at 1500 rpm for 7 min, remove the supernatant, and homogenize to a paste-like consistency. Place the mixture in a 37°C water bath. Once the temperature is maintained, add 1 ml of polyethylene glycol uniformly over 1 min while stirring, then let it stand for 45 s. Next, add 1 ml of basal culture medium uniformly over 1 min while stirring. Then, add 5 ml of basal culture medium over 2 min while stirring, and repeat this process. Finally, fill the well with basal culture medium, centrifuge at 1000 rpm for 7 min, remove the supernatant, homogenize to a paste-like consistency, add 50 ml of selection medium containing HAT, and incubate at 37°C for 30-60 min. Finally, add the mixture to 96-well plates from step b at a rate of 30,000-50,000 cells / well.

[0059] 6. Screening for positive hybridoma cells

[0060] When the fused cell clones reached 1 / 3 to 1 / 2 of the well bottom, the supernatant of the hybridoma cell culture in a 96-well plate was detected using an indirect ELISA method: Rv0340 protein was diluted to 1 μg / ml with coating buffer, 100 μl per well, and coated with the plate overnight at 4°C. After removing the coating buffer, the plate was blocked with Diluent, 100 μl per well, and incubated at 37°C for 1-2 h. After washing three times with PBST, the liquid was aspirated. Then, 100 μl of the supernatant from the 96-well plate was added to each well, and the plate was incubated at 37°C for 1-2 h. The plate was washed three more times with PBST, and the liquid was aspirated. Then, 100 μl of the secondary antibody (goat anti-mouse IgG-HRP) was added to each well, and the plate was incubated at 37°C for 1-2 h. After washing five times with PBST, the liquid was aspirated. TMB was added to each well under light-protected conditions, 100 μl per well, and ELISA stop solution was added after 10 min. OD was detected using a microplate reader. 450 Cells that tested positive were cloned and expanded, and then cultured in 48-well plates. The first subcloning was performed 3 days later.

[0061] 7. Subcloning of positive hybridoma cells

[0062] Cell counts were performed in the 48-well plates. Hybridoma cells were then seeded into 96-well plates using a limiting dilution method, resulting in approximately 100-200 cells per well. A feeder layer of cells was pre-seeded into the 96-well plates, and the plates were incubated at 37°C with 5% CO2 for 2 weeks. After 2 weeks, the hybridoma cell culture supernatant was analyzed using an indirect ELISA method. Positive wells were selected for at least three more subcloning attempts until hybridoma cell lines capable of secreting specific antibodies and proliferating indefinitely, derived from single-cell clones, were obtained. A total of 12 monoclonal antibodies were obtained.

[0063] 8. Large-scale preparation and purification of monoclonal antibodies

[0064] The obtained 4D5 cell line was expanded and cultured in serum-free hybridoma cell culture medium (SFM), and the cell culture supernatant was collected. Antibody was purified using Protein G affinity chromatography. First, Protein G was prepared and the column was equilibrated with PBS. Then, the cell culture supernatant and Protein G were incubated together overnight at 4°C. The Protein G was then transferred to the chromatography column. The column was washed with PBS, followed by elution with 100 mM glycine-hydrochloric acid solution (pH 2.7). The eluent was collected, dialyzed, and the monoclonal antibody was obtained for later use.

[0065] Example 3: Identification of anti-Rv0340 monoclonal antibody

[0066] 1. Experimental Methods

[0067] (1) SDS-PAGE electrophoresis identification of anti-Rv0340 monoclonal antibody

[0068] The 12 anti-Rv0340 monoclonal antibodies obtained above were subjected to SDS-PAGE electrophoresis. Samples were prepared using reducing and non-reducing loading methods, respectively. After purification, the antibodies were analyzed by SDS-PAGE.

[0069] (2) Detection of anti-Rv0340 monoclonal antibody subclass and subtype

[0070] Antibody subtypes were identified using the Biodragon mouse monoclonal antibody subtype identification kit via indirect ELISA, and 12 monoclonal antibodies were also detected.

[0071] (3) Detection of binding activity of anti-Rv0340 monoclonal antibody

[0072] Binding activity was determined using an indirect ELISA method. Rv0340 protein was diluted to 1 μg / ml with coating buffer, 100 μl per well, and coated onto the microplate overnight at 4°C. After removing the coating buffer, the microplate was blocked with Diluent, 100 μl per well, and incubated at 37°C for 1-2 h. After washing three times with PBST, the liquid was agitated. Twelve anti-Rv0340 monoclonal antibodies were then halved from 10 μg / ml, gradually diluted to a concentration of 39.0625 ng / ml, and added to each well at 100 μl, and incubated at 37°C for 1-2 h. After washing three times with PBST again, the liquid was agitated. Then, 100 μl of secondary antibody (goat anti-mouse IgG-HRP) was added to each well, and incubated at 37°C for 1-2 h. After washing five times with PBST, the liquid was agitated. Add TMB (100 μl per well) under light-protected conditions, and add ELISA stop solution after 10 min. Detect OD using a microplate reader. 450 Value. OD is plotted on the x-axis as antibody concentration. 450 Plot a curve with the OD value as the ordinate, where the curve tends to flatten. 450 The value is taken as 100%, which means that the binding of Rv0340 protein to the monoclonal antibody has reached saturation. The concentration of the monoclonal antibody corresponding to 50% saturation on the curve is the relative affinity of the monoclonal antibody.

[0073] 2. Experimental Results

[0074] (1) As Figure 1 As shown, under non-denaturing conditions, a protein band with a molecular weight of approximately 180 kDa is visible. When the interchain disulfide bonds are opened with denaturing loading buffer, two bands of 50 kDa and 25 kDa are visible, which are consistent with the molecular weight of the antibody heavy and light chains, and the antibody purity is above 95%.

[0075] (2) The results are as follows Figure 2 As shown, in this invention, the anti-Rv0340 monoclonal antibodies 2E5, 2E9, 3D5, 4C4, 4D5, 12C7, 20E5, and 20H11 are all IgG1 subclasses, while 4D9 and 5B12 are IgG2a subclasses. Monoclonal antibodies 4D9 and 5B12 are IgG2a; monoclonal antibodies 2E5, 2E9, 3D5, 4C4, 4D5, 4E4, 12C7, and 20E5 are κ light chains; and 4D9, 4F9, 5B12, and 20H11 are λ light chains. The sequences of the heavy and light chains of the anti-Rv0340 monoclonal antibody 4D5 are shown in Table 2.

[0076] Table 2. Sequences of the heavy and light chains of the anti-Rv0340 monoclonal antibody 4D5.

[0077]

[0078]

[0079] Note: Heavy chain variable region CDR1 represents CDR-H1; CDR2 represents CDR-H2; CDR3 represents CDR-H3; Light chain variable region CDR1 represents CDR-L1; Light chain variable region CDR2 represents CDR-L2; Light chain variable region CDR3 represents CDR-L3; Total variable region represents the amino acid sequence of the heavy chain or the entire light chain.

[0080] (3) The results are as follows Figure 3 As shown, in this embodiment of the present invention, the anti-Rv0340 monoclonal antibodies 2E9 and 20H11 exhibited the best affinity, while the remaining 10 antibodies showed good affinity. This indicates that all 12 antibodies possess good binding activity to the Rv0340 protein.

[0081] Example 4: Application of anti-Rv0340 monoclonal antibody 4D5 in isoniazid resistance research

[0082] 1. Experimental Methods

[0083] (1) Anti-Rv0340 monoclonal antibody 4D5 reduced the survival rate of mycobacteria in isoniazid (INH).

[0084] Ms_Rv0340 was cultured to the logarithmic growth phase and then diluted to OD. 600 =0.6, add 20 μg / mL isoniazid and 20 μg / mL anti-Rv0340 monoclonal antibody respectively, and incubate at 37℃ for 24 h. Take 10 μL of bacterial culture and add it to 90 μL PBS, then spread it evenly on 7H10 plates and incubate at 37℃ for 3-5 days before counting.

[0085] (2) Anti-Rv0340 monoclonal antibody 4D5 improves the resistance of mycobacteria to isoniazid (INH).

[0086] MS_Rv0340 was cultured to the logarithmic growth phase beforehand, and then diluted to a 0.5 McFarland turbidity standard, with each diluted 1:100 for later use. The bacteria were added to centrifuge tubes containing different concentrations of INH drug and cultured. 1 ml of bacterial culture was added to each sample, and the drug concentration was diluted to 24, 20, 16, 14, 12, 10, 8, 6, 4, and 2 μg / ml. The same procedure was performed on another group, with 20 μg / ml of anti-Rv0340 monoclonal antibody added to each group. All samples were cultured at 37°C, and the OD values ​​were measured after 3 days. 600 Absorbance.

[0087] (3) Effect of anti-Rv0340 monoclonal antibody 4D5 on the growth of mycobacteria

[0088] Equal volumes of Ms_Rv0340 bacterial culture were added to LBT containing 20 μg / mL of anti-Rv0340 monoclonal antibody and cultured. OD was measured every 8 h. 600 To observe its in vitro growth.

[0089] (4) Effects of anti-Rv0340 monoclonal antibody 4D5 on resistance to other anti-tuberculosis drugs

[0090] MS_Rv0340 was cultured to the logarithmic growth phase in advance, then diluted to a 0.5 McFarland turbidity standard. After a 1:100 dilution, the solutions were added to different concentrations of the anti-tuberculosis drugs rifampin and ethambutol. After culturing for 3 days, the growth was observed. The MICs of anti-Rv0340 monoclonal antibody 4D5 against rifampin and ethambutol were detected. The effect of monoclonal antibody 4D5 on the bactericidal effect of other anti-tuberculosis drugs such as rifampin (RFP) and ethambutol (EMB) was investigated.

[0091] 2. Experimental Results

[0092] (1) The effect of anti-Rv0340 monoclonal antibody on the survival rate of mycobacteria in isoniazid is as follows: Figure 4 As shown, only the anti-Rv0340 monoclonal antibody 4D5 significantly reduced the survival rate of mycobacteria in isoniazid, with the survival rate decreasing from 49.02% to 22.44% compared to direct culture in isoniazid, suggesting that it may increase the sensitivity of mycobacteria to the drug.

[0093] (2) The results of the reduction of mycobacterial resistance to isoniazid by anti-Rv0340 monoclonal antibody are as follows: Figure 5 As shown, only the anti-Rv0340 monoclonal antibody 4D5 could significantly reduce the resistance of mycobacteria to INH, while the monoclonal antibody 4F9 even promoted the survival of Mycobacterium tuberculosis, and other monoclonal antibodies did not show significant effects.

[0094] (3) The effect of anti-Rv0340 monoclonal antibody on the growth of mycobacteria in vitro is shown in the following results. Figure 6 As shown, the anti-Rv0340 monoclonal antibody 4D5, like other monoclonal antibodies, did not significantly affect the in vitro growth of Mycobacterium smegma and did not possess any antibacterial ability on its own.

[0095] (4) The effects of the prepared anti-Rv0340 monoclonal antibody on resistance to other anti-tuberculosis drugs are as follows: Figure 7 As shown, neither RFP nor EMB has the effect of increasing the sensitivity of mycobacteria to it.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the solutions. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention based on the understanding of the present invention, without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A monoclonal antibody 4D5 against Rv0340 that can improve drug resistance in Mycobacterium tuberculosis, characterized in that, The amino acid sequence of the heavy chain variable region CDR-H1 of the anti-Rv0340 monoclonal antibody 4D5 is shown in SEQ ID No. 1, the amino acid sequence of the heavy chain variable region CDR-H2 is shown in SEQ ID No. 2, and the amino acid sequence of the heavy chain variable region CDR-H3 is shown in SEQ ID No. 3; the amino acid sequence of the light chain variable region CDR-L1 of the anti-Rv0340 monoclonal antibody 4D5 is shown in SEQ ID No. 4, the amino acid sequence of the light chain variable region CDR-L2 is shown in SEQ ID No. 5, and the amino acid sequence of the light chain variable region CDR-L3 is shown in SEQ ID No.

6.

2. The anti-Rv0340 monoclonal antibody 4D5, which can improve drug resistance of Mycobacterium tuberculosis according to claim 1, is characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

7.

3. The anti-Rv0340 monoclonal antibody 4D5, which can improve drug resistance of Mycobacterium tuberculosis according to claim 1, is characterized in that, The amino acid sequence of the variable region of the light chain is shown in SEQ ID No.

8.

4. The anti-Rv0340 monoclonal antibody 4D5, which can improve drug resistance of Mycobacterium tuberculosis according to claim 1, is characterized in that, The anti-Rv0340 monoclonal antibody 4D5 is of the IgG1 subtype, and the light chain is the κ chain.

5. A nucleic acid encoding the anti-Rv0340 monoclonal antibody 4D5 as described in claim 1, which can improve drug resistance in Mycobacterium tuberculosis.

6. An expression carrier, characterized in that, It comprises the nucleic acid of claim 5 and is capable of expressing the nucleic acid in prokaryotic or eukaryotic host cells.

7. A host cell, characterized in that, It contains and can express the vector of claim 6, and produces the anti-Rv0340 monoclonal antibody 4D5 of any of claims 1-4 that can improve drug resistance in Mycobacterium tuberculosis.

8. A drug that can improve drug resistance in Mycobacterium tuberculosis, characterized in that, The composition includes the anti-Rv0340 monoclonal antibody 4D5 as described in any one of claims 1-4 and isoniazid; and a medically acceptable carrier or excipient.

9. The use of the anti-Rv0340 monoclonal antibody 4D5 according to any one of claims 1-4, the nucleic acid according to claim 5, the vector according to claim 6, or the host cell according to claim 7 in the preparation of a drug for enhancing the sensitivity of Mycobacterium tuberculosis to isoniazid.

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