The application of Rv1815 as a target in the preparation of anti-tuberculosis agents and the construction methods for its gene knockout vectors and gene knockout mutants.

Abstract

This invention proposes an application of Rv1815 as a target in the preparation of anti-tuberculosis agents, as well as a method for constructing its gene knockout vector and gene knockout mutant, belonging to the field of biomedical technology. Through functional experiments, this invention demonstrates that Rv1815 can be used to prepare agents that inhibit Mycobacterium tuberculosis with good efficacy, discovering a new target for treating tuberculosis. This provides an important theoretical basis for the development of novel anti-tuberculosis agents and offers important theoretical reference for the prevention and clinical treatment of tuberculosis infection, possessing significant practical value.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to the application of Rv1815 as a target in the preparation of anti-tuberculosis agents, and the construction methods for constructing gene knockout vectors and gene knockout mutants. Background Technology

[0002] Mycobacterium tuberculosis is the pathogen that causes tuberculosis. Drug-resistant tuberculosis is a relatively difficult disease to treat because it develops resistance to antibiotics and other drugs, leading to poor treatment outcomes. It is necessary to develop new drugs targeting Mycobacterium tuberculosis to enhance its sensitivity to tuberculosis treatments.

[0003] Serine proteases play a crucial role in bacterial invasion of cells and immune regulation. For example, Neisseria IgA1-specific serine protease can degrade IgG3, enhancing virulence. Vibrio cholerae serine protease can degrade endoglucosidin, thereby inhibiting the interaction between endoglucosidin and Vibrio cholerae. In Campylobacter, the serine protease HtrA participates in virulence by cleaving claudin-8, a tight junction component involved in virulence.

[0004] Mycobacterium tuberculosis adapts to macrophage immune attack and survival through serine proteases. Several serine proteases have been reported in Mycobacterium tuberculosis, such as serine protease Rv3194c, which has been shown to inhibit macrophage migration and enhance intracellular survival, and serine protease Rv3668c, which can affect the expression of host cytokines and regulate apoptosis through Erk-NF-κB. However, Rv1815 is a serine protease in Mycobacterium tuberculosis that has not yet been studied, and it is unknown whether it affects the sensitivity of Mycobacterium tuberculosis to drugs.

[0005] Therefore, there are currently no functional studies on the effects of Rv1815 on Mycobacterium tuberculosis. Summary of the Invention

[0006] The main objective of this invention is to provide an application of Rv1815 as a target in the preparation of anti-tuberculosis agents, as well as a method for constructing its gene knockout vector and gene knockout mutant. The aim is to study the effect of Rv1815 on anti-tuberculosis agents and to provide an important theoretical basis for the development of novel anti-tuberculosis agents.

[0007] To achieve the above objectives, the present invention provides the application of Rv1815 as a target in the preparation of formulations for anti-tuberculosis.

[0008] In some embodiments, the formulation reduces the Rv1815 protein content in Mycobacterium tuberculosis by inhibiting Rv1815 gene expression, thereby inhibiting Mycobacterium tuberculosis.

[0009] In some embodiments, the formulation is used to enhance the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis drugs.

[0010] This invention also provides an application of biomaterials in inhibiting Rv1815 gene expression to reduce the Rv1815 protein content in Mycobacterium tuberculosis.

[0011] In some embodiments, the formulation comprises any one of the following: a protein encoded by Rv1815, a gene knockout vector, and a gene knockout mutant.

[0012] In some embodiments, the formulation contains excipients, including at least one of solubilizers, lubricants, and emulsifiers.

[0013] In some embodiments, the nucleotide sequence of the Rv1815 gene is as shown in SEQ ID NO.1; and / or;

[0014] The amino acid sequence of the protein encoded by the Rv1815 gene is shown in SEQ ID NO.2.

[0015] This invention provides a method for constructing an Rv1815 gene knockout vector, comprising the following steps:

[0016] Multiple primer pairs were obtained to amplify the upper and lower arm sequences of the Rv1815 gene, respectively, to obtain the amplified upper arm fragment and the amplified lower arm fragment.

[0017] The amplified upper arm fragment and the amplified lower arm fragment were inserted into the target vector using homologous recombination to obtain the Rv1815 gene knockout vector.

[0018] In some embodiments, the plurality of primers includes primer LFP, primer LRP, primer RFP, primer RRP, primer LYZFP, primer LYZRP, primer RYZFP, and primer RYZRP; wherein,

[0019] The sequence of the primer LFP is shown in SEQ ID NO.5; and / or;

[0020] The sequence of the primer LRP is shown in SEQ ID NO. 6; and / or;

[0021] The sequence of the primer RFP is shown in SEQ ID NO.7; and / or;

[0022] The sequence of the primer RRP is shown in SEQ ID NO. 8; and / or;

[0023] The sequence of the primer LYZFP is shown in SEQ ID NO.9; and / or;

[0024] The sequence of the primer LYZRP is shown in SEQ ID NO.10; and / or;

[0025] The sequence of the primer RYZFP is shown in SEQ ID NO.11; and / or;

[0026] The sequence of the primer RYZRP is shown in SEQ ID NO.12.

[0027] This invention provides a method for constructing an Rv1815 gene knockout mutant. The Rv1815 gene knockout vector constructed above is introduced into target cells, screened using marker genes, and the Rv1815 gene knockout mutant is obtained after verification.

[0028] This invention, through functional experiments on Rv1815, confirms that Rv1815 can be used in the preparation of agents that regulate Mycobacterium tuberculosis, and discovers a new target for the treatment of tuberculosis. This provides an important theoretical basis for the development of novel anti-tuberculosis agents and provides an important theoretical reference for the prevention and clinical treatment of Mycobacterium tuberculosis infection, and has important practical significance. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 Electrophoresis images of DNA fragments amplified by PCR on the left and right arms;

[0031] Figure 2 A schematic diagram of primer design for gene knockout and validation;

[0032] Figure 3 Electrophoresis image for PCR verification of Rv1815 gene knockout strain;

[0033] Figure 4 Figures showing the morphological observation and measurement results of wild-type H37Rv and Rv1815 knockout bacteria;

[0034] Figure 5 Scanning electron microscope images and morphological determination results of wild-type H37Rv and Rv1815 knockout bacteria;

[0035] Figure 6Figure 1 shows the results of intracellular viability assays of macrophages infected with wild-type H37Rv and Rv1815 knockout bacteria.

[0036] Figure 7 The figure shows the transcriptome sequencing and qPCR validation results of macrophages infected with wild-type H37Rv and Rv1815 knockout bacteria.

[0037] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Mycobacterium tuberculosis adapts to macrophage immune attack and survival through serine proteases. Several serine proteases have been reported in Mycobacterium tuberculosis, such as serine protease Rv3194c, which has been shown to inhibit macrophage migration and enhance intracellular survival, and serine protease Rv3668c, which can affect the expression of host cytokines and regulate apoptosis through Erk-NF-κB. However, Rv1815 is a serine protease in Mycobacterium tuberculosis that has not yet been studied, and it is unknown whether it affects the sensitivity of Mycobacterium tuberculosis to drugs.

[0040] Therefore, there are currently no functional studies on the effects of Rv1815 on Mycobacterium tuberculosis.

[0041] Therefore, Rv1815 is used as a target in the preparation of formulations for anti-tuberculosis.

[0042] It should be noted that Rv1815 is an open reading frame (ORF) in the genome of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB). The Rv1815 gene is located in the genome of this bacterium and encodes a class of proteins that may play an important role in the physiology and pathogenicity of Mycobacterium tuberculosis.

[0043] This invention, through functional experiments on Rv1815 in Mycobacterium tuberculosis, confirms that Rv1815 can be used in the preparation of anti-tuberculosis agents, discovering a new target for the treatment of tuberculosis. This provides an important theoretical basis for the development of novel anti-tuberculosis agents and offers important theoretical reference for the prevention and clinical treatment of tuberculosis infection, and has significant practical implications.

[0044] In any embodiment of the present invention, the prepared formulation reduces the Rv1815 protein content in Mycobacterium tuberculosis by inhibiting the expression of the Rv1815 gene, thereby inhibiting Mycobacterium tuberculosis. The principle of the prepared formulation is to reduce the Rv1815 protein content in Mycobacterium tuberculosis by inhibiting the expression of the Rv1815 gene, thereby achieving the purpose of inhibiting Mycobacterium tuberculosis, and thus can also achieve the treatment or relief of tuberculosis.

[0045] In some embodiments, the formulation is used to improve the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis drugs. That is, the formulation includes Rv1815-related biological materials, which are mainly used to improve the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis drugs, so as to treat or alleviate tuberculosis.

[0046] Furthermore, the biological material includes any one of the following: a protein encoded by Rv1815, a gene knockout vector, and a gene knockout mutant.

[0047] This invention includes not only the protein encoded by Rv1815, gene knockout vectors, or gene knockout mutants, but also a series of biological materials such as recombinant host cells that can reduce the content of Rv1815 protein in Mycobacterium tuberculosis. The protein encoded by Rv1815 can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0048] In some embodiments, the formulation contains excipients, including at least one of solubilizers, lubricants, and emulsifiers.

[0049] It is understood that the preparation of the formulation for regulating Mycobacterium tuberculosis also includes excipients. These excipients are substances, other than the active ingredients in the formulation, that have undergone reasonable safety assessments and are included in the pharmaceutical formulation. In addition to being excipients, acting as carriers, and improving stability, these excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of the drug and may include solubilizers, lubricants, or emulsifiers, or mixtures of any of these.

[0050] In some embodiments, the nucleotide sequence of the Rv1815 gene is shown in SEQ ID NO.1, and the specific sequence is as follows:

[0051] GTGGTCCGCCTGGTACCACGCGCATTCGCAGCGACGGTCGCCCTATTGGCGGCC

[0052] GGGTTTTCGCCGGCGACCGCCAGTGCCGATCCGGTCTTGGTGTTTCCCCGGCATGGAA

[0053] ATCCGTCAGGACAACCACGTCTGCACCCTGGGCTACGTCGACCCAGCTCTGAAAATC

[0054] GCGTTTACCGCGGGGCATTGTCGGGGGCGGGGGAGCGGTCACCAGCCGGGACTACAA

[0055] GGTTATCGGCCATCTCAGGGCCATCCGGGACACACACCCAGCGGCTCCACCGTGGC

[0056] CACGCACGAGTTGATCGCCGACTACGAGGCGATTGTGCTGGCTGACGACGTCACGGC

[0057] AAGCAACATTTTGCCGAGCGGGCGTGCACTGGAATCCAGACCGGGTGTGGTTCTTCA

[0058] CCCGGGCCAAGCGGTCTGCCATTTCGGCGTCAGCACAGGCGAAACCTGTGGGACCG

[0059] TCGAAAGCGTCAACAACGGCTGGTTCACCATGTCCCACGGCGTGCTCAGTGAGAAG

[0060] GGGGATTCGGGGGGGCCCGGTCTACCTGGCCCCCGATGGCGGCCCCGCGCAGATCGTC

[0061] GGGATCTTCAACAGCGTCTGGGGCGGCTTTCCCGCGGCGGTGTCCTGGCGGTCGACG

[0062] TCCGAGCAGGTTCACGCGGATCTCGGCGTGACGCCCCTTGCTTAG.

[0063] The amino acid sequence of the protein encoded by the Rv1815 gene is shown in SEQ ID NO.2, and the specific sequence is as follows:

[0064] MVRLVPRAFAATVALLAAGFSPATASADPVLVFPGMEIRQDNHVCTLGYVDPALKIAFTAGHCRGGGAVTSRDYKVIGHLRAIRDNTPSGSTVATHELIADYEAIVLADDV TASNILPSGRALESRPGVVLHPGQAVCHFGVSTGETCGTVESVNNGWFTMSHGVLSEKGDSGGPVYLAPDGGPAQIVGIFNSVWGGFPAAVSWRSTSEQVHADLGVTPLA.

[0065] This invention provides a method for constructing an Rv1815 gene knockout vector, comprising the following steps:

[0066] Multiple primer pairs were obtained to amplify the upper and lower arm sequences of the Rv1815 gene, respectively, to obtain the amplified upper arm fragment and the amplified lower arm fragment.

[0067] The amplified upper arm fragment and the amplified lower arm fragment were inserted into the target vector using homologous recombination to obtain the Rv1815 gene knockout vector in Mycobacterium tuberculosis.

[0068] This invention obtains the Rv1815 gene knockout vector through homologous recombination, and the construction method is simple.

[0069] Specifically, the plurality of primers includes primer LFP, primer LRP, primer RFP, primer RRP, primer LYZFP, primer LYZRP, primer RYZFP, and primer RYZRP; wherein,

[0070] The sequence of the primer LFP is shown in SEQ ID NO.5; and / or;

[0071] The sequence of the primer LRP is shown in SEQ ID NO. 6; and / or;

[0072] The sequence of the primer RFP is shown in SEQ ID NO.7; and / or;

[0073] The sequence of the primer RRP is shown in SEQ ID NO. 8; and / or;

[0074] The sequence of the primer LYZFP is shown in SEQ ID NO.9; and / or;

[0075] The sequence of the primer LYZRP is shown in SEQ ID NO.10; and / or;

[0076] The sequence of the primer RYZFP is shown in SEQ ID NO.11; and / or;

[0077] The sequence of the primer RYZRP is shown in SEQ ID NO.12.

[0078] Using the primers described above, the Rv1815 gene knockout vector can be obtained with high specificity and high efficiency.

[0079] This invention provides a method for constructing an Rv1815 gene knockout mutant. The Rv1815 gene knockout vector of Mycobacterium tuberculosis constructed above is introduced into target cells, and the Rv1815 gene knockout mutant of Mycobacterium tuberculosis is obtained after screening using marker genes and verification.

[0080] The present invention also provides an anti-tuberculosis agent comprising inhibiting Rv1815 gene expression to reduce the Rv1815 protein content in Mycobacterium tuberculosis.

[0081] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0082] The p0004s plasmid used in the following examples was purchased from Shanghai Jingnuo Biotechnology Co., Ltd.

[0083] The primer sequences used in the following examples are shown in Table 1.

[0084] Table 1 Primer sequences

[0085]

[0086] In the following examples, OADC refers to OADC enrichment broth, purchased from BD Biosciences; ADC refers to ADC enrichment broth, purchased from BD Biosciences; Mycobacterium smegmatis was obtained from laboratory preservation; and Mycobacterium tuberculosis H37Rv was obtained from laboratory-preserved standard bacteria. For details, see also Mycobacterium tuberculosis H37Rv in patent application number CN202410531146.2.

[0087] Example 1

[0088] Construction of the Rv1815 knockout mutant of Mycobacterium tuberculosis H37Rv strain

[0089] 1.1 Mycobacterial Culture Methods and Preservation

[0090] (1) As needed, Mycobacterium smegmatis mc 2 155) 7H9 liquid medium or 7H10 solid medium can be used. Liquid culture can be carried out statically or with shaking on a shaker. Mycobacterium tuberculosis H37Rv can be cultured using 7H9+OADC / ADC liquid medium or 7H10+OADC solid medium. Liquid culture should be carried out statically.

[0091] (2) The final concentration of hygromycin B resistance for Mycobacterium tuberculosis H37Rv knockout strain was 75 μg / mL.

[0092] (3) For liquid culture strains that have grown to the logarithmic phase, short-term storage (1 month) can be done at room temperature or 4°C. For long-term storage (1-2 years), add 15-20% glycerol and freeze at -80°C. After 1-2 years of freezing, pay attention to the revival of the strain.

[0093] 1.2 p0004s-AES plasmid construction

[0094] DNA fragments of the left and right arms of the Rv1815 gene in Mycobacterium tuberculosis H37Rv were amplified using Phusion high-fidelity DNA polymerase from Thermo Scientific. The left arm (797bp) and the right arm (837bp) were amplified using primer pairs LFP / LRP and RFP / RRP, respectively.

[0095] Nucleotide sequence of the DNA fragment in the left arm (SEQ ID NO.3):

[0096] TGCCGCAATCTATGCCTACCTTGCCCCCTGGCAGCTGTCCGCCCACCGGTGGTACACCTGGGTGATCGCGATCGTTGGTGTCGATCTGCTGTACTACTCCTATCACCGCATCGCCCACCGAGTTCGGCTGATCTGGGCTACCCACCAGGCGCATCACTCCAGCGAATACTTCAACTTCGCCACCGCGCTGCGCCAGAAGTGGAACAACAGCGGCGAGATTCTCATGTGGGTTCCGCTGCCACTGATGGGGCTTCCCCCTTGGATGGTGTTCTGCAGTTGGTCGCTGAACTTGATCTACCAGTTCTGGGTGCACACCGAGCGGATCGACAGGCTGCCGCGGTGGTTCGAATTCGTCTTCAATACCCCGTCGCACCACCGGGTCCACCACGGAATGGACCCGGTGTATCTGGACAAGAACTATGGCGGCATCCTCATCATCTGGGACCGCCTGTTCGGTAGCTTTCAGCCGGAGCTATTCCGACCGCATTATGGCCTGACCAAGCGGGTCGACACGTTCAACATCTGGAAGCTGCAGACCCGCGAGTACGTGGCGATCGTGCGTGACTGGCGGTCGGCAACACGTCTGCGGGATCGGCTGGGCTACGTCTTCGGACCGCCGGGCTGGGAACCGCGCACCATCGATAAATCCAATGCCGCCGCCTCCCTGGTCACGTCTCGGTAACGTCGCGACCCGACATTGCGAAAGTATTACCGTCGGGTTTTGGTACGCCTTAGC CGTAACCGGCGGCGGGCGATGCGCTTGGCCCCGACGGATGGGAGTTCAAGGTGGTCCGCCT。

[0097] Nucleotide sequence of the right arm DNA fragment (SEQ ID NO.4):

[0098] CGTCAGCACAGGCGAAACCTGTGGGACCGTCGAAAGCGTCAACAACGGCTGGTTCACCATGTCCCACGGCGTGCTCAGTGAGAAGGGGGATTCGGGGGGCCCGGTCTACCTGGCCCCCGATGGCGGCCCCGCGCAGATCGTCGGGATCTTCAACAGCGTCTGGGGCGGCTTTCCCGCGGCGGTGTCCTGGCGGTCGACGTCCGAGCAGGTTCACGCGGATCTCGGCGTGACGCCCCTTGCTTAGCAAGCACCCCGTTAGCGGCCACCAGGTTGATCGCCGTGTGTTTGCTAGAGCGGTGATCTCGGTTGTGTCAGACTTGCCGCGTGGGCAAACGCCGGGATGCGAGGGAACAGATCGAGGCGAAAATTGTCGAACTCGGCCGTCGCCAGCTGCTGGATCACGGCGCGGCCGGGTTGTCGCTTCGGGCAATTGCCCGCAACCTGGGCATGGTGTCCTCGGCCGTATACCGCTATGTGTCCAGTCGTGATGAGCTGTTGACTTTGCTGCTCGTCGACGCCTACTCCGACCTGGCCGATACCGTGGACCGAGCCCGCGACGACACCGTCGCCGACTCGTGGAGTGACGACGTCATCGCAATCGCTCGAGCGGTGCGCGGTTGGGCAGTCACTAACCCCGCCCGCTGGGCCTTGCTATACGGTAGCCCGGTTCCTGGTTATCACGCGCCGCCTGACCGTACCGCGGGCGTCGCCACCCGCGTGGTCGGAGCGTTCTTCGACGCGATCGCCGCGGGAATCGCCACCGGAGACATCAGGTTAACCGATGACGTTGCGCCGCAGCCGATGTCATCGGACTTCGAAAAGATCCGGCAGGAGTTC。

[0099] After amplifying the target DNA fragment, agarose nucleic acid gel electrophoresis was performed. The electrophoresis gel model used was Plus IIDNA Marker. The electrophoresis results are shown in Figure 1 as follows: Figure 11 and 2: DNA fragments from the left arm; 3 and 4: DNA fragments from the right arm; M: DNA marker. Specifically, 1 and 2 represent the results of amplification of the wild-type strain (WT strain) genomic DNA using primer pair LRP / LRP; 3 and 4 represent the results of amplification of the wild-type strain (WT strain) genomic DNA using primer pair RFP / RRP. Figure 1 The results show that the left and right homologous fragments of Rv1815 were successfully amplified, with fragment sizes of 797bp and 837bp, respectively.

[0100] Next, the target DNA fragment was recovered using an Omega gel extraction and purification kit, following the kit's instruction manual. The recovered DNA fragments from both arms were digested with Bgl I restriction endonuclease, and then the digested target DNA fragment was recovered using a rapid recovery kit for later use. Simultaneously, the p0004s plasmid was extracted using a Tiangen plasmid extraction kit and digested with Van91I restriction endonuclease. The digested target DNA fragment was then recovered using a gel extraction kit. The DNA fragments obtained in the above steps were ligated using T4 DNA ligase, and the reaction system was transformed into E. coli DH5α competent cells, following the method described in Molecular Cloning Laboratory Manual. Positive clones were screened and sequenced to verify plasmid correctness, yielding the p0004s-AES positive plasmid, which was stored for later use.

[0101] 1.3 Construction of phAE159-AES plasmid

[0102] Positive plasmids phAE159 and p0004s-AES were extracted using the Omega plasmid kit. All obtained plasmids were digested with Pac I, and the target fragments were recovered. The digestion system followed the reagent instructions. After digestion, the digestion products were recovered using an Omega rapid purification kit. The two linearized plasmid fragments were ligated using T4 DNA ligase. The reaction system was transformed into E. coli HB101 competent cells using a packaging kit (EPICENTRE Biotechnologies, MP5120), following the kit instructions. Single colonies grown on hygromycin-resistant plates were picked and incubated in LB medium with 75 μg / mL hygromycin B at 37°C. Plasmids were extracted and identified by Pac I restriction endonuclease digestion, yielding the positive phAE159-AES plasmid.

[0103] 1.4 Phage Preparation

[0104] (1) Mycobacterium smegmatis mc 2 Preparation of 155 electrocompetent cells

[0105] Select fresh M. smegmatis mc 2 155 single colonies were inoculated into 5 mL of 7H9 liquid medium and cultured at 37°C with shaking until the logarithmic growth phase. The culture was then inoculated into 100 mL of 7H9 medium at a ratio of 1:100 and cultured overnight at 37°C until the OD600 reached approximately 0.6. The culture was placed on ice for 0.5 h and then centrifuged at 5000 rpm for 10 min at 4°C to collect the cells. The cells were washed at least twice with 10% sterile glycerol in a pre-ice bath, and finally 10 mL of pre-chilled 10% glycerol was added. After mixing the cells, they were aliquoted into 200 μL tubes and stored at -80°C for later use.

[0106] (2) Electro-transfer phAE159-AES to M.smegmatis mc 2 Phage was obtained from 155.

[0107] Take the positive phAE159-AES plasmid and add it to M. smegmatis mc 2 Mix the 155g of the bacterial culture with the competent cells, and then transform them using a Bio-rad electroporator with a 2mm electroporation cup (electroporation parameters: voltage 2.5kV, resistance 1000Ω, capacitance 25μF). After electroporation, add H9 medium and incubate overnight at 37℃. Then, mix the bacterial culture with Top Agar and inoculate it onto 7H10 solid plates. After incubating at 30℃ for 3 days, screen for phage plaques.

[0108] (3) Amplify high-titer phages

[0109] Pick empty plaques containing bacteriophages from the plate and transfer them to MP buffer. Incubate overnight at 4°C. Then, mix this bacteriophage-containing liquid with freshly cultured M. smegmatis mcg. 2 After mixing 155 bacteria, the mixture was then thoroughly mixed with Top Agar and plated. The plates were incubated at 30°C for 3 days. MP buffer was then added to plates with plaques, and the plates were incubated overnight at 4°C. High-titer phages were collected and filtered through a 0.22 μm sterile filter and stored at 4°C for later use.

[0110] 1.5 Phage lysate was used to transfect the strain to be knocked out.

[0111] High-titer phage lysis buffer was mixed with Mycobacterium tuberculosis H37Rv grown to the logarithmic phase (pre-washed with MP buffer), incubated overnight at 37°C, centrifuged and the supernatant was discarded, then 7H9 liquid medium was added, incubated overnight at 37°C, centrifuged and the supernatant was discarded, the bacterial cells were collected and plated (7H10+OADC+75μg / mL hygromycin B), cultured at 37°C for 4 weeks, the genome was extracted, and PCR was used to verify whether the gene knockout was successful.

[0112] For gene knockout validation primers, an upstream validation primer (LYZFP) was designed 100-200 bp upstream of the LFP primer matching sequence, and a downstream validation primer (RYZRP) was designed 100-200 bp downstream of the RRP primer matching sequence. A schematic diagram of the gene knockout and validation primer design is shown below. Figure 2 As shown, where, Figure 2 In the diagram, RV1814 and RV1816 encode a membrane-bound C-5 sterol desaturase, Rv1816 encodes an HTH-type transcription factor, and Rv1815 encodes a serine protease, respectively. PCR verification using primer pairs LYZFP / LYZRP and RYZFP / RYZRP with the knockout strain (named Δ1815) genome (MUT strain) as a template yielded DNA fragments of 1091 bp and 1296 bp, respectively. However, when the control was used with the wild-type strain genome (WT strain) as a template and the same primer pairs were used, no target DNA fragment was amplified. It should be noted that Δ1815 or ΔRv1815 in the attached diagram refers to the Rv1815 knockout strain. The PCR amplification results are as follows... Figure 3 As shown, Figure 3 In the table, 1 shows the PCR identification results of the wild-type strain (WT strain) using primer pair LYZFP / LYZRP; 2 shows the PCR identification results of the Rv1815 gene knockout strain (MUT strain) using primer pair LYZFP / LYZRP; 3 shows the PCR identification results of the wild-type strain using primer pair RYZFP / RYZRP; 4 shows the PCR identification results of the Rv1815 gene knockout strain using primer pair RYZFP / RYZRP; M: DNA Marker, derived from... Figure 3 It can be seen that the Rv1815 gene knockout strain has been successfully constructed.

[0113] 2. Growth curve, observation and determination of plate colony morphology

[0114] Figure 4A in the diagram is a schematic diagram of the Rv1815 protein domain composition; B is the growth curve of wild-type H37Rv and Δ1815 measured at 600 nm wavelength; C is a single colony morphology diagram of wild-type H37Rv and Δ1815 after three weeks of growth on 7H10 plates; D is a statistical graph of bacterial colony diameter in C; E is a colony morphology diagram of wild-type H37Rv and Δ1815 after three weeks of growth on 7H10 plates. Figure 4 In the diagram, A represents the Rv1815 protein domain, which consists of 221 amino acids. Amino acids 1-27 form the signal peptide (SP), and amino acids 55-178 form the trypsin-like serine protease domain (Tryp_SPc). Wild-type Mycobacterium tuberculosis strains H37Rv and ΔRv1815 were inoculated into 7H9 medium and cultured statically at 37°C until the OD reached approximately 1.0 during the logarithmic growth phase. The cultured H37Rv and ΔRv1815 strains were then subinoculated into fresh 7H9 medium to an initial OD of 0.05, and then cultured statically at 37°C. OD was measured on days 7, 10, 14, 18, 21, and 25, and growth curves were plotted as shown below. Figure 4 As shown in Figure B, strain ΔRv1815 (also known as Δ1815 in the figure) exhibits a slower growth rate compared to the wild-type H37Rv strain (WT in the figure). Additionally, 100 μL of bacterial suspension was diluted 10⁶ times and plated onto 7H10 plates. 20 μL of bacterial suspension with an OD of approximately 1.0 was spotted onto 7H10 plates and incubated at 37°C for 3 weeks. The results are as follows. Figure 4 As shown in C and D: ΔRv1815 colonies (0.9 cm in diameter) were found to be smaller than wild-type H37Rv colonies (1.2 cm in diameter). Figure 4 E in the figure shows that after 3 weeks of growth on 7H10 plates, the ΔRv1815 strain exhibited smaller colony morphology compared to the wild-type H37Rv. The above experiment was repeated three times, with three parallel replicates each time.

[0115] 3. Scanning electron microscopy observation and determination of bacterial morphology

[0116] Wild-type Mycobacterium tuberculosis strains H37Rv and ΔRv1815 were inoculated into 7H9 medium and incubated statically at 37°C until the logarithmic growth phase (OD) reached approximately 1.0. 5 mL of bacterial suspension was collected by centrifugation into a 1.5 mL centrifuge tube. The cells were washed once with 1 mL PBS, resuspended in 1 mL of 2.5% glutaraldehyde, and fixed overnight at 4°C. The cells were then sent to the company for electron microscopy observation. The results are as follows: Figure 5 As shown, where, Figure 5In the diagram, A represents scanning electron microscope images of wild-type H37Rv and the knockout bacterium Δ1815 (20,000x magnification); B represents the morphology of individual bacteria of wild-type H37Rv and the knockout bacterium Δ1815 (100,000x magnification); C represents the average length of wild-type H37Rv and the knockout bacterium Δ1815; and D represents the average diameter of wild-type H37Rv and the knockout bacterium Δ1815. Figure 5 A represents wild-type H37Rv and the knockout bacterium ΔRv1815, specifically the morphology of Δ1815 under a scanning electron microscope. It can be observed that some bacteria of the ΔRv1815 strain exhibit a rough and shriveled morphological characteristic. Figure 5 In addition, using Nano Measurer software to measure the length and diameter of bacteria, it was found that there was no significant difference in the length of wild-type H37Rv and ΔRv1815 bacteria. Figure 5 In the middle (C), when the Rv1815 gene was knocked out, the average diameter of the bacteria was significantly reduced (C). Figure 5 (D). The above experiment was repeated three times, with three parallel replicates each time, indicating that Rv1815 plays a role in regulating the morphology of Mycobacterium tuberculosis.

[0117] 4. Assay for tuberculosis macrophage survival

[0118] THP-1 cells were loaded at 2×10 5 Cells were seeded in 24-well plates containing RMPI 1640 medium and cultured at 37°C and 5% CO2. Cells were induced to differentiate into macrophages with 100 ng / mL PMA for 24 hours, then replaced with fresh RMPI 1640 medium and allowed to rest for 24 hours. Wild-type H37Rv and ΔRv1815 strains were infected at an MOI of 10 for 4 hours. The ΔRv1815 strain is shown in the figure. Cells were then washed three times with pre-warmed sterile PBS to remove extracellular bacteria. Cells were cultured in RMPI 1640 medium at 37°C and 5% CO2 for 3 and 5 days, respectively. Cells were lysed with PBS containing 0.1% SDS, and the lysates were cultured in 7H10 medium at 37°C for 3 weeks. Colony numbers (CFU) were then counted. Experimental results are shown below. Figure 6 As shown, compared with the wild-type H37Rv strain, the ΔRv1815 strain had significantly lower intracellular colony counts at 3 and 5 days after macrophage infection. The experiment was repeated three times, with three parallel replicates each time. This indicates that Rv1815 knockout enhances the clearance of Mycobacterium tuberculosis by macrophages, suggesting that Rv1815 plays an important role in the intracellular survival of Mycobacterium tuberculosis in macrophages.

[0119] 5. Transcriptome sequencing and real-time quantitative PCR

[0120] THP-1 cells at 2×10 5 Cells were seeded in 24-well plates containing RMPI 1640 medium and cultured at 37°C and 5% CO2. Cells were induced to differentiate into macrophages with 100 ng / mL PMA for 24 hours, then replaced with fresh RMPI 1640 medium and allowed to rest for 24 hours. Wild-type H37Rv and Δ1815 strains were infected at MOI=10 for 4 hours, followed by washing three times with pre-warmed sterile PBS to remove extracellular bacteria. Cells were then cultured in RMPI 1640 medium at 37°C and 5% CO2 for 24 hours. RNA was extracted using the Omega Cell RNA Extraction Kit according to the manufacturer's instructions. RNA samples were submitted to Novogene (Beijing) Co., Ltd. for ribosomal RNA removal and sequencing.

[0121] Analysis results as follows Figure 7 As shown, Figure 7 A in the diagram is a volcano plot of differentially expressed genes; B is a qPCR verification result of some differentially expressed genes between strain ΔRv1815 and wild-type strain H37Rv. Figure 7 As shown in Figure A: the horizontal axis represents log2 (FoldChange), and the vertical axis represents -log10 (P-value). Each point represents a gene, and the color is used to distinguish whether genes are differentially expressed. Orange points represent differentially expressed genes, green points represent downregulated genes, and blue points represent genes with no significant difference. Compared with the wild-type H37Rv strain, after infection of THP-1 cells, the ΔRv1815 strain showed significant upregulation of 35 genes (up35) and significant downregulation of 109 genes (down109), especially some chemokine-related genes, including CCL2 (Chemokine (CC motif) ligand 2), CCL8 (Chemokine (CC motif) ligand 8), CXCL10 (CXC chemokine ligand-10), and CXCL9 (CXC chemokine ligand-10). Chemokine ligand-10 (CXC chemokine 9) showed that the deletion of Rv1815 led to a weakened host inflammatory response. Specifically, differentially expressed genes by transcriptome sequencing are shown in Table 2.

[0122] Table 2 Differentially expressed genes from transcriptome sequencing

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] The transcriptome results were validated using qPCR. RNA samples were first reverse transcribed into cDNA using HiScript IIQ RTSuperMix for qPCR (+gDNA wiper) (Novizan Biotechnology). Then, quantitative real-time PCR (qRT-PCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Novizan Biotechnology) on a Roche LightCycler 96 real-time PCR system. The PCR program was as follows: 95℃ 30s, (95℃ 10s; 60℃ 30s) × 40 cycles, 95℃ 10s, 60℃ 60s, 95℃ 15s. The relative expression level of the target gene was normalized to the housekeeping gene GADPH, and the relative gene expression was calculated using the 2-ΔΔCT method. This value reflects the expression difference between the Δ1815 and WT groups, where the WT group is the baseline and Δ1815 represents the difference relative to the WT group. The difference results are shown in […]. Figure 7 As shown in Figure B: The verification results and transcriptome results are consistent, indicating that Rv1815 induces a host inflammatory response during cell infection, suggesting that Rv1815 plays a positive regulatory role in the host immune response. The above experiment was repeated three times, with three parallel replicates each time.

[0129] In summary, experiments using macrophage survival assays, transcriptome sequencing, and real-time quantitative PCR showed that knocking out Rv1815 enhanced the bactericidal ability of macrophages against Mycobacterium tuberculosis. This suggests that inhibiting Rv1815 may help restore the normal immune function of host cells. However, Rv1815 triggers an inflammatory response, which may lead to host tissue damage or immune imbalance. Targeting and inhibiting Rv1815 can reduce immunopathological damage during tuberculosis infection. Therefore, the function of Rv1815 makes it a potential anti-tuberculosis target, paving the way for the preparation or production of future tuberculosis drugs.

[0130] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. The application of the Rv1815 gene knockout vector in the preparation of anti-tuberculosis agents, characterized in that, The nucleotide sequence of the Rv1815 gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The formulation reduces the Rv1815 protein content in Mycobacterium tuberculosis by inhibiting Rv1815 gene expression, thereby inhibiting tuberculosis.

3. The application as described in claim 2, characterized in that, The formulation contains excipients, which include at least one of solubilizers, lubricants, and emulsifiers.

4. The application as described in claim 1, characterized in that, The amino acid sequence of the protein encoded by the Rv1815 gene is shown in SEQ ID NO.

2.

5. The application as described in claim 1, characterized in that, The method for constructing the Rv1815 gene knockout vector includes the following steps: Multiple primer pairs were obtained to amplify the upper and lower arm sequences of the Rv1815 gene, respectively, to obtain the amplified upper arm fragment and the amplified lower arm fragment. The amplified upper arm fragment and the amplified lower arm fragment were inserted into the target vector using homologous recombination to obtain the Rv1815 gene knockout vector in Mycobacterium tuberculosis.

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