Application of a target Rv0087 against Mycobacterium tuberculosis

Abstract

The present application relates to the field of biotechnology, and in particular to an application of Rv0087, a target against Mycobacterium tuberculosis. Specifically, it is at least one of the following applications of a substance that inhibits the activity of the Rv0087 protein and / or a substance that reduces the content of the Rv0087 protein: 1) Application in the preparation of products for the treatment or auxiliary treatment of tuberculosis; 2) Application in the preparation of products for the prevention or inhibition of Mycobacterium tuberculosis infection. The Rv0087 protein secreted by Mycobacterium tuberculosis can interact with the host target protein PKCγ, thereby promoting the intracellular survival of Mycobacterium tuberculosis. Therefore, this not only provides a new target for clinical tuberculosis treatment and anti-tuberculosis drug development, but also provides guidance for the research and development of anti-tuberculosis drugs targeting the Rv0087 domain.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and in particular relates to an application of a target Rv0087 against Mycobacterium tuberculosis. Background Art

[0002] Tuberculosis is an ancient chronic infectious disease caused by infection with Mycobacterium tuberculosis. New vaccines and new drugs to overcome drug resistance are urgently needed. In-depth elucidation of the virulence factors of pathogens and their interaction mechanisms with the host is a prerequisite for the development of effective drugs / vaccines to prevent and treat pathogen infection and transmission. However, the current understanding of the functions and mechanisms of action of nearly 4,000 proteins encoded by Mycobacterium tuberculosis is very limited, and no Mycobacterium tuberculosis virulence factors similar to tetanus exotoxin have been found to play a decisive role. Although existing studies have shown that many substances in Mycobacterium tuberculosis, such as bacterial lipids, bacterial substances such as PDIM and various secretory proteins such as ESAT-6, Rv0222, PtpA, etc., are important pathogenic substances / virulence factors of Mycobacterium tuberculosis, they play an indispensable role in the infection / pathogenesis of Mycobacterium tuberculosis, and can interact with the host through different mechanisms to affect cell necrosis / inhibit host immunity, thereby promoting the occurrence and development of tuberculosis. But overall, our understanding of the virulence factors of Mycobacterium tuberculosis and its host interaction mechanisms is still very limited, which limits the development of effective tuberculosis vaccines and therapeutic drugs. Summary of the invention

[0003] The purpose of this application is to provide an application of Rv0087, an anti-Mycobacterium tuberculosis target, to solve the lack of new anti-Mycobacterium tuberculosis drug targets and to better cope with the technical problems of tuberculosis treatment.

[0004] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0005] The present application provides at least one of the following uses of a substance for inhibiting the activity of Rv0087 protein and / or a substance for reducing the content of Rv0087 protein, wherein the amino acid sequence of the Rv0087 protein is shown in SEQ ID No. 1:

[0006] 1) Application in the preparation of products for the treatment or auxiliary treatment of tuberculosis;

[0007] 2) Use in the preparation of products for preventing or inhibiting Mycobacterium tuberculosis infection.

[0008] The present application experimentally discovered that the Rv0087 protein secreted by Mycobacterium tuberculosis can interact with the host target protein PKCγ (i.e., protein kinase Cγ) to inhibit the activation of the NF-κB innate immune signaling pathway, thereby promoting the intracellular survival of Mycobacterium tuberculosis. Therefore, substances that inhibit the activity of the Rv0087 protein and / or reduce the content of the Rv0087 protein can treat or assist in the treatment of tuberculosis and prevent or inhibit Mycobacterium tuberculosis infection, which not only provides new targets for clinical tuberculosis treatment and anti-tuberculosis drug development, but also provides guidance for the research and development of anti-tuberculosis drugs targeting the Rv0087 domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0010] Figure 1 is the result of immunoblotting to detect the secretion expression of Rv0087 protein; among them, a is the result of anti-FLAG tag antibody to detect the secretion expression of H37Rv strain cultured in Sutong medium and overexpressing FLAG- Rv0087 Protein expression results in whole cell lysates (W) and supernatant (S) of the strain; b is the detection of H37Rv strain and Δ in Sutong medium by anti-Rv0087 antibody Rv0087 Protein expression results in the strain culture supernatant (S);

[0011] Figure 2 The results of immunoblotting detection of Rv0087 protein targeting and binding to host PKCγ and inhibiting its phosphorylation are shown in Figure 1; a is the H37Rv strain, Δ Rv0087 strains and Δ Rv0087 :: Rv0087 a) The phosphorylation results of different PKC subunits when the strain infected THP-1 macrophages for 30 min and 60 min; b) After FLAG-Rv0087 was overexpressed in 293T cells, FLAG magnetic beads were used for immunoprecipitation and the interaction between FLAG-Rv0087 and different PKC protein subunits was detected; c) After FLAG-Rv0087 was overexpressed in 293T cells, anti-PKCγ antibody was used for immunoprecipitation and the interaction between FLAG-Rv0087 and different PKC protein subunits was detected;

[0012] Figure 3 For H37Rv strain, Δ Rv0087 strains and Δ Rv0087 :: Rv0087 Results of the strain's ability to survive in THP-1 macrophages;

[0013] Figure 4 For H37Rv strain, Δ Rv0087 strains and Δ Rv0087 :: Rv0087 The survival results of strains in mice; a is the survival ability of different Mycobacterium tuberculosis strains in mouse lung tissue, b is the survival ability of different Mycobacterium tuberculosis strains in mouse spleen tissue, c is the statistical result of the inflammatory area formed in the lung tissue of mice infected with different Mycobacterium tuberculosis strains, and d is the pathological HE staining result of mouse lung tissue caused by different Mycobacterium tuberculosis infections. DETAILED DESCRIPTION

[0014] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0015] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0016] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0017] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0018] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0019] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0020] The present application found that the increased gene transcription levels of Rv0087 protein of Mycobacterium tuberculosis and host PKCγ protein are related to the survival and proliferation of intracellular Mycobacterium tuberculosis. Rv0087 Through infection experiments with gene-deleted and complemented Mycobacterium tuberculosis strains, we confirmed that the Rv0087 protein significantly increased the proliferation of Mycobacterium tuberculosis in macrophages and mice, thereby promoting the occurrence and development of tuberculosis. In further experimental screening and verification, it was determined that the Rv0087 protein can interact with host PKCγ, thereby promoting the growth of Mycobacterium tuberculosis in macrophages. These research results provide new ideas and possible solutions for updating the understanding of Mycobacterium tuberculosis virulence factors and their mechanisms, and exploring the design of host-directed therapy (HDT) drugs targeting the host-pathogen interaction interface and potential vaccine antigens. Based on this, the technical solution of this application is as follows.

[0021] The application provided in the embodiments of the present application is at least one of the following applications of a substance that inhibits the activity of Rv0087 protein and / or a substance that reduces the content of Rv0087 protein:

[0022] 1) Application in the preparation of products for the treatment or auxiliary treatment of tuberculosis;

[0023] 2) Use in the preparation of products for preventing or inhibiting Mycobacterium tuberculosis infection.

[0024] The embodiments of the present application provide substances that inhibit the activity of Rv0087 protein or substances that reduce the content of Rv0087 protein, which can effectively inhibit the interaction between Mycobacterium tuberculosis and the host, thereby inhibiting the survival of Mycobacterium tuberculosis in cells. Therefore, the above substances can be used to treat tuberculosis or assist in the treatment of tuberculosis, prevent Mycobacterium tuberculosis infection or inhibit Mycobacterium tuberculosis infection.

[0025] Specifically, experiments have shown that the above substances inhibit Mycobacterium tuberculosis infection by at least one of the following 1)-3): 1) reducing the number of Mycobacterium tuberculosis surviving macrophages (such as macrophage THP-1); 2) reducing the load of Mycobacterium tuberculosis in mouse lung or spleen tissue; 3) weakening or improving the pathological inflammatory infiltration of Mycobacterium tuberculosis in mouse lung tissue.

[0026] In some embodiments, the amino acid sequence of the Rv0087 protein is shown in SEQ ID No. 1. Furthermore, the nucleotide sequence of the corresponding gene is shown in SEQ ID No. 2.

[0027] Rv0087 amino acid sequence (SEQ ID No. 1):

[0028] VMSASWLRHRVSERGLIATAEQLWADSFRLALVAAHDDGDSLRVVYLFLAGYPDRRVELEYVVPADNPEIRSLAYLSFPAGRFEREMADLYGIRPVGHPKPRRLVRHAHWPDWHPMRTDAGPA PEFTDTGAFPFLAVEGPGVYEIPVGPVHAGLIEPGHFRFSVAGETIVRLKARLWFVHRGIEKLFHGRPATAAVDLAERISGDTSAAHALAHSLAIEDALGIELPHEVHRLRALIVELERLYNH AADLGALANDVGYSLANAHAQRIRENLLRRNAAVTGHRLLRGAIRAGGVALRALPDELAALAVDLAEVATLTLANSVVYDRFAGTAVLHPDDASALGCLGYVARASGLRSDARVEHPTIVL PITEIGAPDGDVLARYTVRRDEFAASAALAQHIVESHTGPIEYAATLHPVGAPSSGIGIVEGWRGTIVHRVEIDVDGRITRAKVVDPSWFNWPALPVAMADTIVPDFPLANKSFNQSYAGNDL.

[0029] Rv0087 Gene sequence (SEQ ID No.2):

[0030]

[0031] In some embodiments, the substance that inhibits the Rv0087 protein includes at least one of a protein, a polypeptide, and a small molecule compound that inhibits the function of the Rv0087 protein.

[0032] In some embodiments, the substance that reduces the content of Rv0087 protein includes a substance that inhibits the synthesis of Rv0087 protein, a substance that promotes the degradation of Rv0087 protein, a substance that knocks down Rv0087 Genetic material, knockout Rv0087 At least one of the genetic materials.

[0033] In some embodiments, knockdown Rv0087 The gene-silencing substance can be any substance that can make the gene encoding Rv0087 protein unable to express, such as silencing Rv0087 Genetic material. For example, including silencing Rv0087 At least one of the miRNA, siRNA, dsRNA, and shRNA of a gene.

[0034] In some embodiments, knockout Rv0087 The genetic material may be in any manner that causes the Mycobacterium tuberculosis cell to not produce Rv0087 A substance that produces a functional protein product of a gene, such as by removing all or part of the coding gene sequence, introducing a frameshift mutation so that no functional protein is produced, removing or changing regulatory components (such as promoter editing) so that the coding gene sequence is not transcribed, or preventing translation by binding to mRNA. Usually, the knockout is performed at the genomic DNA level, so that the offspring of the cell also carry the knockout permanently.

[0035] Specifically, knockout Rv0087 Genetic material can be any substance that can make Rv0087 A substance in which a gene has mutated (the mutation may be in the form of deletion mutation and / or insertion mutation and / or base substitution) and has lost its activity, including at least one of the zinc finger protein ZFN gene editing system, TALENs gene editing system, CRISPR / Cas9 gene editing system, and recombinant plasmid.

[0036] In some embodiments, knockout Rv0087 Gene material can be used to knock out Rv0087 CRISPR / Cas9 gene editing system for knockout Rv0087 The CRISPR / Cas9 gene editing system includes Cas9 nuclease and targeting Rv0087 The sgRNA targeting the gene. The targeting nucleotide sequences of the sgRNA are shown in SEQ ID No.3 and SEQ ID No.4.

[0037] Targeting Rv0087 The target sequence of the sgRNA of the gene is:

[0038] SEQ ID No. 3: atccagatcgccgcgtcgag and SEQ ID No. 4: gtcgaatcacacaccggtcca.

[0039] In some embodiments, the target Rv0087 The recombinant plasmids of the sgRNA of the gene may include pYC1876-Rv0087-sgRNA plasmid, pNHEJ-Cas9sth1 plasmid and pYC1759 plasmid.

[0040] For example, the present application cloned the designed and synthesized Rv0087-sgRNA1 (SEQ ID No. 3) and Rv0087-sgRNA2 (SEQ ID No. 4) sequences into the pYC1876 plasmid (the nucleotide sequence is shown in SEQ ID No. 5), and obtained Rv0087 Gene knockout plasmid pYC1876-Rv0087-sgRNA.

[0041] Furthermore, the pNHEJ-Cas9sth1 plasmid and the pYC1876-Rv0087-sgRNA plasmid were introduced into the wild-type Mycobacterium tuberculosis H37Rv competent cells containing the pYC1759 plasmid (nucleotide sequence as shown in SEQ ID No. 6), and electroporation was performed at room temperature (25-30°C) to obtain Rv0087 Knockout Mycobacterium tuberculosis (Δ Rv0087 ).

[0042] For example, the present application converts the Rv0087 The gene sequence was ligated into the BamH I and Cla I restriction sites of the pYC874_L5 plasmid (nucleotide sequence as shown in SEQ ID No.7) to obtain the pYC874_L5-Rv0087 plasmid. Rv0087 Knockout Mycobacterium tuberculosis (Δ Rv0087 ) was transferred into the pYC874_L5-Rv0087 plasmid to obtain complement Rv0087 Gene Δ Rv0087 strain (Δ Rv0087 :: Rv0087 strains).

[0043] The products corresponding to the applications provided in the embodiments of the present application include substances that inhibit the activity of Rv0087 protein and / or substances that reduce the content of Rv0087 protein. Specifically, the active ingredients of the products provided in the embodiments of the present application include substances that inhibit the activity of Rv0087 protein or substances that reduce the content of Rv0087 protein. The products include at least one of the following: 1) products for treating or assisting in the treatment of tuberculosis; 2) products for preventing or inhibiting infection with Mycobacterium tuberculosis.

[0044] Furthermore, the substance that inhibits the activity of Rv0087 protein includes at least one of proteins, polypeptides and small molecule compounds that inhibit the function of Rv0087 protein. The substance that reduces the content of Rv0087 protein includes substances that inhibit the synthesis of Rv0087 protein, substances that promote the degradation of Rv0087 protein, substances that knock down Rv0087 Genetic material, knockout Rv0087 Further, knocking down Rv0087 Genetic substances include silencing Rv0087 At least one of miRNA, siRNA, dsRNA, and shRNA for a gene; Knockout Rv0087 The gene material includes at least one of zinc finger protein ZFN gene editing system, TALENs gene editing system, CRISPR / Cas9 gene editing system, and recombinant plasmid.

[0045] Specifically, the CRISPR / Cas9 gene editing system or the recombinant plasmid contains a targeting Rv0087 The sgRNA of the gene, the targeting nucleotide sequence of the sgRNA is shown in SEQ ID No.3 and SEQ ID No.4.

[0046] Specifically, the above-mentioned product may be a drug or an inhibitor.

[0047] In some embodiments, the Mycobacterium tuberculosis is specifically Mycobacterium tuberculosis H37Rv.

[0048] In the present application, a knockout system coupled with CRISPR-Cas9 system-mediated genome cleavage and non-homologous end-joining (NHEJ) system was used to construct a targeted knockout. Rv0087 Knockout strain Δ Rv0087 as well as Rv0087 Gene complement strain Δ Rv0087 :: Rv0087Rv0087 was detected by Rv0087 antibody, which confirmed that Rv0087 was a secreted effector protein. Rv0087 protein was further overexpressed in 293T cells, and then the host proteins interacting with Rv0087 were identified by immunoprecipitation and Western Blotting experiments. It was confirmed that Rv0087 interacted with proteins such as PKCγ in eukaryotic cells, thereby inhibiting the activation of PKCγ phosphorylation host innate immune signaling pathways. Finally, macrophages and mice were used to confirm that Rv0087 was a virulence factor of Mycobacterium tuberculosis. Therefore, Rv0087 can be used as a potential new target for anti-tuberculosis drugs. Inhibitors or drugs targeting Rv0087 can effectively inhibit the interaction between Mycobacterium tuberculosis and the host, thereby inhibiting the survival of Mycobacterium tuberculosis in cells. Therefore, the embodiments of the present application help to better understand the immunological pathogenesis of tuberculosis, not only provide new targets for clinical tuberculosis treatment and anti-tuberculosis drug development, but also provide guidance for the research and development of anti-tuberculosis drugs such as chemical small molecule preparations and polypeptide antagonists targeting Rv0087.

[0049] The pLVX-Flag plasmid in the following example is described in the document “Xu Y, Cheng S, Zeng H, et al. ARF GTPases activate Salmonella effector SopF to ADP-ribosylate host V-ATPase and inhibit endomembrane damage-induced autophagy[J]. Nature Structural&Molecular Biology, 2022, 29(1): 67-77.”.

[0050] The pNHEJ-Cas9sth1 plasmid in the following example is described in the literature (the pNHEJ-Cas9sth1 plasmid is described in the literature “Yan MY, Li SS, Ding XY, et al. A CRISPR-assisted nonhomologous end-joining strategy for efficient genome editing in Mycobacterium tuberculosis[J]. MBio, 2020, 11(1): 10.1128 / mbio. 02364-19.”).

[0051] The following describes the invention in conjunction with specific embodiments.

[0052] Example 1: Identification of secretory expression of Mycobacterium tuberculosis Rv0087 protein

[0053] 1. Δ Rv0087 and Δ Rv0087 :: Rv0087 Construction of strains

[0054] 1. Δ Rv0087 Construction and identification of strains

[0055] 1) Design of sgRNA

[0056] Mycobacterium tuberculosis Rv0087 Gene sequence design based on CRISPR-NHEJ technology for targeted knockout Rv0087 Finally, two sgRNAs were designed, named Rv0087-sgRNA1 and Rv0087-sgRNA2, and their target sequences are as follows:

[0057] Rv0087-sgRNA1: atccagatcgccgcgtcgag;

[0058] Rv0087-sgRNA2:gtcgaatcacacaccggtcca.

[0059] 2) Construction of Rv0087 knockout plasmid

[0060] The Rv0087-sgRNA1 and Rv0087-sgRNA2 sequences designed and synthesized in 1) above were cloned into the pYC1876 plasmid (the nucleotide sequence is shown in SEQ ID No. 5), Rv0087 Gene knockout plasmid pYC1876-Rv0087-sgRNA.

[0061] 3) Rv0087 Obtaining gene knockout Mycobacterium tuberculosis

[0062] 200-500 ng of pNHEJ-Cas9sth1 plasmid and 200-500 ng of pYC1876-Rv0087-sgRNA plasmid were introduced into wild-type Mycobacterium tuberculosis H37Rv competent cells containing pYC1759 plasmid (nucleotide sequence as shown in SEQ ID No. 6), and electroporation was performed at room temperature. Immediately add 1 mL of 7H9 medium (BD, USA, Catalog No.: 271310) containing 10% enrichment solution OADC (BD, USA, Catalog No.: 212351) to the competent bacterial suspension after transformation, and transfer to a 15 mL centrifuge tube, place in a 37 °C incubator for static culture for 48 hours, take all the liquid and spread on a 7H10 plate (BD, USA, Catalog No.: 262710) containing kanamycin, bleomycin and 50 ng / ml tetracycline (anhydrotetracycline, ATc) with OADC added, place in a 37 °C incubator, continue to culture for 21 to 28 days, and after a single colony grows, it is amplified by PCR and sequenced for identification. Rv0087 Knockout Mycobacterium tuberculosis (Δ Rv0087 ).

[0063] 4) Δ Rv0087 Identification

[0064] Remove the Rv0087 7H10 culture plates with single colonies of knockout Mycobacterium tuberculosis were randomly selected and streaked on non-resistant 7H10 culture plates containing OADC. The inoculated plates were placed in a 37°C incubator for further culture for about 14 days. The bacteria on the streaked culture plates were then picked and added to 20 μL of sterile water. After vortex mixing, the bacteria were placed in a 99°C metal bath for 30 min to lyse the bacteria. The lysed bacteria were used as templates for PCR reactions. 1 μL of bacterial lysate was added to the 25 μL PCR reaction system for PCR reaction.

[0065] The mutant strain was identified by PCR amplification using primers (upstream primer: SEQ ID No.8: CGCGGATCCGTGATGAGTGCCAGCTGGCTGCGCC, downstream primer: SEQ ID No.9: CCCATCGATTTAGAGGTCGTTGCCCGCGTAGGAC). The PCR control was a wild-type Mycobacterium tuberculosis strain, and then agarose gel electrophoresis was used for detection and the results were observed using an imaging analysis system and sent for sequencing verification. The sequencing verification results showed that Mycobacterium tuberculosis was successfully knocked out. Rv0087 gene, and the strain was recorded as Δ Rv0087 .

[0066] 2. Δ Rv0087 :: Rv0087 Construction and identification of strains

[0067] 1) Construction of pYC874_L5-Rv0087 plasmid

[0068] Will Rv0087 The gene sequence (shown in SEQ ID No. 2) was ligated into the BamH I and Cla I restriction sites of the pYC874_L5 plasmid (the nucleotide sequence is shown in SEQ ID No. 7) to obtain the pYC874_L5-Rv0087 plasmid.

[0069] 2) Δ Rv0087 :: Rv0087 Acquisition of strains

[0070] Using electroporation in Δ Rv0087 Transfer into pYC874_L5-Rv0087 plasmid to obtain complement Rv0087 Gene Δ Rv0087 strain (Δ Rv0087 :: Rv0087 strains).

[0071] 3) Δ Rv0087 :: Rv0087 Identification of strains

[0072] Pick and Cover Rv0087 Gene Δ Rv0087 The single clone was amplified by PCR and sequenced using primer sequences (upstream primer: SEQ ID No. 8: CGCGGATCCGTGATGAGTGCCAGCTGGCTGCGCC, downstream primer: SEQ ID No. 9: CCCATCGATTTAGAGGTCGTTGCCCGCGTAGGAC) and the Rv0087 The strain with successful gene complementation is recorded as Δ Rv0087 :: Rv0087 strains.

[0073] 2. Identification of secretory expression of Rv0087 in different Mycobacterium tuberculosis strains

[0074] Sutong medium (Shanghai Ruichu Biotechnology Co., Ltd., catalog number T2000) was used to culture wild-type Mycobacterium tuberculosis H37Rv strain, H37Rv:Flag -Rv0087 strain, Δ Rv0087 strains and Δ Rv0087 :: Rv0087After 4 weeks of culture, the bacterial liquid was collected and centrifuged at 4500 rpm for 10 min to collect the supernatant, which was then sterilized by filtration through a 0.22 μm filter to obtain the culture supernatant; the remaining bacterial liquid was centrifuged at 12000 rpm for 10 min to obtain the bacterial cells. Anti-Flag tag antibody (MBL product, catalog number: M185-3L) and Rv0087 polyclonal antibody (Wuhan Abotek Biotechnology Co., Ltd.) were used to detect secretory proteins and bacterial proteins in the culture supernatants of different Mycobacterium tuberculosis.

[0075] The results are as follows Figure 1 The results of anti-Flag tag antibody detection showed that the Flag tag could be detected in H37Rv:Flag-Rv0087 bacteria (W) and culture supernatant (S), but the Flag tag could not be detected in H37Rv culture supernatant ( Figure 1 Similarly, the results of anti-Rv0087 antibody detection showed that in H37Rv and Δ Rv0087 :: Rv0087 Rv0087 protein expression was detected in the culture supernatant, but in Δ Rv0087 No Rv0087 protein expression could be detected in the culture supernatant ( Figure 1 The above results indicate that Rv0087 is a secretory protein expressed by Mycobacterium tuberculosis.

[0076] Example 2: Rv0087 protein targets and binds to host PKCγ protein and regulates its phosphorylation modification

[0077] Using H37Rv strain, Δ Rv0087 strains and Δ Rv0087 :: Rv0087 The strains were used to infect THP-1 macrophages, and cell samples were collected at 30 and 60 minutes after infection. Anti-P-PKCα / β antibody (Abclonal, Catalog No.: AP0191), anti-P-PKCγ(T514) antibody (Abclonal, Catalog No.: AP0613), anti-P-PKCγ(S655) antibody (GeneTex, Catalog No.: GTX25796), and anti-P-PKCγ(S674) antibody (GeneTex, Catalog No.: GTX25797) were used to detect the phosphorylation modification of classical protein kinase C (PKC). 2 μg of pLvx-Flag-Rv0087 plasmid (pLvx-Flag-Rv0087 plasmid is the plasmid of SEQ ID No. 2) was transfected into 293T cells. Rv0087The plasmid obtained by connecting the gene sequence to the XhoI multiple cloning site of the pLvx-Flag plasmid) was collected 48 hours after transfection, and the cells were lysed with immunoprecipitation lysis buffer, and Flag magnetic beads were added to enrich the Flag-Rv0087 protein and its interacting host proteins for immunoblotting identification. Similarly, 293T cells transfected with the pLvx-Flag-Rv0087 plasmid were collected and lysed, and anti-PKCγ antibodies (Abclonal, Cat. No.: A7922) were added for immunoprecipitation, and then the interaction between Rv0087 and PKCγ was verified by immunoblotting. In the above immunoprecipitations, 293T cells transfected with the pLvx-Flag empty vector were used as controls (Vector).

[0078] The results are as follows Figure 2 The results of immunoblotting showed that: Rv0087 :: Rv0087 Compared with macrophages infected with strains at 30 min and 60 min, Δ Rv0087 Infection with the strain significantly reduced the phosphorylation level of PKCγ, but did not affect the phosphorylation of PKCα or PKCβ ( Figure 2 The results of immunoprecipitation (IP: FLAG) using anti-FLAG tag antibody showed that Rv0087 interacted with classical protein kinase C (cPKC) isoforms (PKCα, β, and γ), but not with novel (ε and δ) or atypical (λ and ι) isoforms or other reported transcription factors ( Figure 2 The results of immunoprecipitation using anti-PKCγ antibody (IP: PKCγ) showed that endogenous PKCγ and Rv0087 were significantly enriched in the immunoprecipitation fraction of HEK293T cells expressing Flag-Rv0087, further confirming the interaction between Rv0087 and PKCγ ( Figure 2 (as shown in c).

[0079] The above results indicate that Rv0087 is involved in regulating the activation of PKCγ and that there is an interaction between Rv0087 and PKCγ.

[0080] Example 3, Δ Rv0087 The survival ability of the strain in macrophages was significantly reduced

[0081] The wild-type Mycobacterium tuberculosis H37Rv strain, Δ Rv0087 strains and Δ Rv0087 :: Rv0087The strain was infected with macrophage THP-1 according to the infection method in Example 2. At 6 hours, 3 days, 4 days and 5 days after infection, the cells were collected and lysed by adding 0.1% SDS. After pipetting and mixing, the lysate was diluted 10 times and coated on a 7H10 culture plate. After further culture in a 37°C incubator for 2-3 weeks, the colony count (CFU) of Mycobacterium tuberculosis was calculated.

[0082] The results are as follows Figure 3 The results showed that 3 days after infection, the average number of wild-type Mycobacterium tuberculosis H37Rv bacteria surviving in macrophages was 1.28×10 6 ;Δ Rv0087 The average number of bacteria surviving in macrophages of the strains cultured for the same time was only 6.27×10 5 ;Δ Rv0087 :: Rv0087 The average number of bacteria surviving in macrophages was 9.53×10 5 From the above results, it can be seen that Δ Rv0087 :: Rv0087 The number of bacteria surviving in macrophages was higher than that of Δ Rv0087 The average number of strains increased by 3.26×10 5 ;Δ Rv0087 The strains were compared with wild-type Mycobacterium tuberculosis H37Rv strain and Δ Rv0087 :: Rv0087 The survival rates of the strains in macrophages were reduced by 51% and 34%, respectively.

[0083] On day 4 of infection, the average number of wild-type Mycobacterium tuberculosis H37Rv bacteria surviving in macrophages was 1.39×10 6 ;Δ Rv0087 The average number of bacteria surviving in macrophages of the strains cultured for the same period of time was only 8.1×10 5 ;Δ Rv0087 :: Rv0087 The average number of bacteria surviving in macrophages was 1.21×10 6 From the above results, it can be seen that Δ Rv0087 :: Rv0087 The number of bacteria surviving in macrophages was higher than that of Δ Rv0087 The average number of strains increased by 4.0×10 5 ;Δ Rv0087 The strains were compared with wild-type Mycobacterium tuberculosis H37Rv strain and Δ Rv0087 :: Rv0087 The survival rates of the strains in macrophages were reduced by 41% and 33%, respectively.

[0084] On the fifth day of infection, the average number of wild-type Mycobacterium tuberculosis H37Rv bacteria surviving in macrophages further increased to 2.18×10 6 ;Δ Rv0087 The average number of bacteria surviving in macrophages of the strains cultured for the same period of time was only 1.01×10 6 ;Δ Rv0087 :: Rv0087 The average number of bacteria surviving in macrophages also increased to 2.16×10 6 From the above results, it can be seen that Δ Rv0087 :: Rv0087 The number of bacteria surviving in macrophages was higher than that of Δ Rv0087 The average number of strains increased by 1.05×10 6 ;Δ Rv0087 The strains were compared with wild-type Mycobacterium tuberculosis H37Rv strain and Δ Rv0087 :: Rv0087 The survival rates of the strains in macrophages were reduced by 54% and 53%, respectively. The above results indicate that Rv0087 plays a key role in promoting the intracellular survival of Mycobacterium tuberculosis.

[0085] Example 4, Δ Rv0087 The survival ability of the strain in mouse lung tissue was significantly reduced

[0086] The wild-type Mycobacterium tuberculosis H37Rv strain, Δ Rv0087 strains and Δ Rv0087 :: Rv0087 The strain was cultured to the logarithmic phase ( AThe bacterial suspension was collected when the CFU / mL was 0.600=0.8, washed with PBST, and then dispersed and filtered with a 5μm filter to obtain single bacteria. Different volumes of bacterial suspensions of the three strains were diluted to the same McFarland turbidimetric value and added to an aerosol generator device (Glas-Col, Inhalation Exposure System, 099C A4224). C57BL / 6 mice aged 4-6 weeks were infected by aerosol inhalation according to the standardized aerosol infection process. Eight mice were infected with each strain so that the initial bacterial infection amount in the lungs of each mouse was about 500-1000 CFU. Three mice in each group were killed on the first day of infection to detect and record whether the mouse lungs were successfully infected. Five mice in each group were killed on the 28th day after infection. The mouse lung tissue was separated and homogenized and coated on a 7H10 culture dish. After further culture in a 37°C incubator for 2-3 weeks, the CFU was counted. At the same time, the remaining lobe of mouse lung tissue was fixed with paraformaldehyde, a tissue fixative, and used for pathological HE staining analysis.

[0087] The results are as follows Figure 4 The results showed that on the first day of infection, wild-type Mycobacterium tuberculosis H37Rv strain, Δ Rv0087 strains and Δ Rv0087 :: Rv0087 There was no difference in bacterial load between strains in mouse lung tissue. Rv0087 Bacterial load of strains in mouse lung tissue log 10 The average value was 4.59, which was higher than that of wild-type Mycobacterium tuberculosis H37Rv and Δ Rv0087 :: Rv0087 Bacterial load of strains in mouse lung tissue log 10 Average values ​​5.79 and 5.18, log 10 The values ​​decreased by about 1.2 and 0.59 respectively ( Figure 4 At the same time, Δ Rv0087 Bacterial load of strains in mouse spleen tissue log 10 The average value was 2.82, which was higher than that of wild-type Mycobacterium tuberculosis H37Rv and Δ Rv0087 :: Rv0087 Bacterial load of strains in mouse lung tissue log 10 The average values ​​were 3.53 and 3.52, which was significantly reduced by about 0.7 log 10 value( Figure 4 (b) and in Δ Rv0087 The pathological inflammatory infiltration in the lung tissue of mice infected with the strain was significantly weakened ( Figure 4 The above results indicate that Rv0087 is a key virulence factor for Mycobacterium tuberculosis infection and tuberculosis in mice.

[0088] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. At least one of the following uses of a substance for reducing the content of Rv0087 protein, wherein the amino acid sequence of the Rv0087 protein is shown in SEQ ID No. 1: 1) Use in the preparation of drugs for the treatment or auxiliary treatment of tuberculosis; 2) Use in the preparation of drugs for preventing or inhibiting Mycobacterium tuberculosis infection; The substance that reduces the content of Rv0087 protein is a knockout Rv0087 Gene material, the knockout Rv0087 Genetic material contains target Rv0087 The CRISPR / Cas9 gene editing system of the sgRNA of the gene, the targeting nucleotide sequence of the sgRNA is shown in SEQ ID No.3 and SEQ ID No.4, the Rv0087 The nucleotide sequence of the gene is shown in SEQ ID No.2.

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