Application of Rv1815 as target spot in preparation of anti-tubercle bacillus preparation and construction method of gene knockout vector and gene knockout mutant of Rv1815

Abstract

The invention provides application of Rv1815 serving as a target spot to preparation of an anti-tubercle bacillus preparation and a construction method for constructing a gene knockout vector and a gene knockout mutant of the Rv1815, belongs to the technical field of biological medicine, and provides application of the Rv1815 serving as the target spot to preparation of the anti-tubercle bacillus preparation. It is proved that the Rv1815 can be applied to preparation of a preparation for inhibiting mycobacterium tuberculosis and has a good effect, a new target spot for treating tuberculosis is found, an important theoretical basis is provided for development of a novel anti-tuberculosis preparation, an important theoretical reference is provided for prevention and clinical treatment of mycobacterium tuberculosis infection, and important practical significance is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to the application of Rv1815 as a target in the preparation of an anti-tuberculosis preparation and a method for constructing a gene knockout vector and a gene knockout mutant thereof. Background Art

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

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

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

[0005] Therefore, there is no relevant functional study on the effect of Rv1815 on Mycobacterium tuberculosis. Summary of the invention

[0006] The main purpose of the present invention is to provide an application of Rv1815 as a target in the preparation of anti-tuberculosis preparations and a method for constructing its gene knockout vector and gene knockout mutant, aiming to study the effect of Rv1815 on anti-tuberculosis preparations and provide an important theoretical basis for the development of new anti-tuberculosis preparations.

[0007] To achieve the above object, the present invention provides the use of Rv1815 as a target in the preparation of an anti-tuberculosis preparation.

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

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

[0010] The present invention also provides an application of a biological material in inhibiting the expression of the Rv1815 gene to reduce the content of the Rv1815 protein in Mycobacterium tuberculosis.

[0011] In some embodiments, the preparation includes any one of a protein encoded by Rv1815, a gene knockout vector, and a gene knockout mutant.

[0012] In some embodiments, the preparation contains excipients, and the excipients include at least one of a solubilizer, a lubricant, and an emulsifier.

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

[0016] Obtain multiple primer pairs to amplify the upper and lower arm sequences of the Rv1815 gene respectively, to obtain an amplified upper arm fragment and an amplified lower arm fragment;

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

[0018] In some embodiments, the plurality of primers include 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] The present invention provides a method for constructing an Rv1815 gene knockout mutant. The constructed Rv1815 gene knockout vector is introduced into a target cell, and a marker gene is used for screening to obtain the Rv1815 gene knockout mutant after verification.

[0028] The present invention has confirmed through functional experiments on Rv1815 that Rv1815 can be used in the preparation of preparations for regulating tuberculosis, and has discovered a new target for the treatment of tuberculosis. This provides an important theoretical basis for the development of new anti-tuberculosis preparations and an important theoretical reference for the prevention and clinical treatment of tuberculosis infection, and has important practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 It is the electrophoresis diagram of the left and right arm DNA fragments amplified by PCR;

[0031] Figure 2 Schematic diagram of primer design for gene knockout and verification;

[0032] Figure 3 This is the electrophoresis diagram of the PCR-verified Rv1815 gene knockout strain;

[0033] Figure 4 This is a diagram of the morphological observation and determination results of wild-type H37Rv and Rv1815 knockout bacteria;

[0034] Figure 5 The scanning electron micrographs and morphological determination results of wild-type H37Rv and Rv1815 knockout bacteria are shown;

[0035] Figure 6This is a graph showing the results of the intracellular survival ability assay of macrophages infected with wild-type H37Rv and Rv1815 knockout bacteria;

[0036] Figure 7 This is the transcriptome sequencing and qPCR verification results of macrophages infected with wild-type H37Rv and Rv1815 knockout bacteria.

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

[0038] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

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

[0040] Therefore, there is no relevant functional study on the effect of Rv1815 on Mycobacterium tuberculosis.

[0041] In view of this, Rv1815 is used as a target in the preparation of anti-tuberculosis preparations.

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

[0043] The present invention has confirmed through functional experiments on Rv1815 in Mycobacterium tuberculosis that Rv1815 in Mycobacterium tuberculosis can be used in the preparation of anti-tuberculosis preparations, and has discovered a new target for the treatment of tuberculosis. This provides an important theoretical basis for the development of new anti-tuberculosis preparations and an important theoretical reference for the prevention and clinical treatment of tuberculosis infection, and has important practical significance.

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

[0045] In some embodiments, the preparation is used to improve the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis drugs, that is, the preparation includes Rv1815-related biological materials, which are mainly used to improve the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis drugs to achieve the purpose of treating or alleviating tuberculosis.

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

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

[0048] In some embodiments, the preparation contains excipients, and the excipients include at least one of a solubilizer, a lubricant, and an emulsifier.

[0049] It is understandable that the preparation for regulating tuberculosis bacteria also includes excipients. The excipients are substances that have been reasonably evaluated in terms of safety and are included in the pharmaceutical preparation in addition to the active ingredients in the preparation. In addition to excipients, serving as carriers, and improving stability, the excipients also have important functions such as solubilization, solubilization assistance, and sustained-release. They are important ingredients that may affect the quality, safety, and effectiveness of the drug. They may include solubilizers, lubricants, or emulsifiers, or a mixture of any of them.

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

[0051] GTGGTCCGCCTGGTACCACGCGCATTCGCAGCGACGGTCGCCCTATTGGCGGCC

[0052] GGGTTTTCGCCGGCGACCGCCAGTGCCGATCCGGTCTTGGTGTTCCCCGGCATGGAA

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

[0066] Obtain multiple primer pairs to amplify the upper and lower arm sequences of the Rv1815 gene respectively, to obtain an amplified upper arm fragment and an amplified lower arm fragment;

[0067] The amplified upper arm fragment and the amplified lower arm fragment are inserted into a target vector by homologous recombination method to obtain a knockout vector of the Rv1815 gene in Mycobacterium tuberculosis.

[0068] The present invention obtains the Rv1815 gene knockout vector by a homologous recombination method, and the construction method is simple.

[0069] Specifically, the plurality of primers include 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] The above primers have good specificity and can efficiently obtain the success rate of the Rv1815 gene knockout vector.

[0079] The present invention provides a method for constructing an Rv1815 gene knockout mutant. The Rv1815 gene knockout vector constructed above in Mycobacterium tuberculosis is introduced into target cells, and marker gene screening is used to obtain the Rv1815 gene knockout mutant in Mycobacterium tuberculosis after verification.

[0080] The present invention also provides an anti-tuberculosis preparation, which comprises inhibiting the expression of Rv1815 gene to reduce the content of Rv1815 protein in Mycobacterium tuberculosis.

[0081] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used 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] OADC in the following embodiments refers to OADC enrichment solution, purchased from BD; ADC refers to ADC enrichment solution, purchased from BD, Mycobacterium smegmatis is derived from laboratory storage, and Mycobacterium tuberculosis H37Rv is derived from standard bacteria preserved in the laboratory. For details, see Mycobacterium tuberculosis H37Rv in patent application number CN202410531146.2

[0087] Example 1

[0088] 1 Construction of knockout mutant of Mycobacterium tuberculosis H37Rv strain Rv1815

[0089] 1.1 Mycobacterium culture and preservation

[0090] (1) As needed, Mycobacterium smegmatis 2 155) 7H9 liquid medium or 7H10 solid medium can be used. For liquid culture, the culture can be static or shaken. For Mycobacterium tuberculosis H37Rv, 7H9+OADC / ADC liquid medium or 7H10+OADC solid medium can be used. For liquid culture, the culture can be static.

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

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

[0093] 1.2 Construction of p0004s-AES plasmid

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

[0095] The nucleotide sequence of the left arm DNA fragment (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 the figure, Figure 11 and 2 are the DNA fragments of the left arm; 3 and 4 are the DNA fragments of the right arm; M is a DNA marker, wherein 1 and 2 are the results of amplifying the genomic DNA of the wild-type strain (WT strain) using the primer pair LRP / LRP; 3 and 4 are the results of amplifying the genomic DNA of the wild-type strain (WT strain) using the primer pair RFP / RRP. Figure 1 It can be seen that the left and right arm homologous fragments of Rv1815 were successfully amplified, and the fragment sizes were 797bp and 837bp respectively.

[0100] Then, the target DNA fragment was recovered using the Omega gel recovery purification kit. The specific steps of gel recovery were carried out according to the operating manual of the kit. The recovered left and right arm DNA fragments were digested with Bgl I restriction endonuclease, and then the target DNA fragments after digestion were recovered using a quick recovery kit for standby use. At the same time, the p0004s plasmid was extracted using the Tiangen plasmid extraction kit and digested with Van91I restriction endonuclease. After digestion, the target DNA fragment was recovered using a gel recovery 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. The method was referred to the molecular cloning experiment guide. The positive clones were screened and sequenced to verify whether the plasmid was correct or not, and the p0004s-AES positive plasmid was obtained and stored for standby use.

[0101] 1.3 Construction of phAE159-AES plasmid

[0102] The Omega plasmid kit was used to extract the phAE159 and p0004s-AES positive plasmids. The obtained plasmids were digested with Pac I and the target fragments were recovered. The digestion system was referred to the reagent instructions. After the digestion reaction system was completed, the Omega company's rapid purification kit was used to recover the digestion products. The two linearized plasmid fragments were ligated using T4 DNA ligase. The reaction system used a packaging kit (EPICENTRE Biotechnologies, MP5120) to assist in transforming E. coli HB101 competent cells. The method was referred to the kit instructions. The single colony grown on the hygromycin resistance plate was picked and placed in LB + 75μg / mL hygromycin B liquid medium and cultured at 37°C. The plasmid was extracted and digested with Pac I restriction endonuclease for identification to obtain the positive phAE159-AES plasmid.

[0103] 1.4 Phage preparation

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

[0105] Pick fresh M.smegmatis mc 2 155 single colonies were inoculated into 5 mL 7H9 liquid culture medium and cultured at 37°C with shaking until the logarithmic growth phase; the culture was inoculated into 100 mL 7H9 culture medium at a ratio of 1:100 and cultured at 37°C overnight until OD600 reached about 0.6; the culture was placed on ice for 0.5 h, and then centrifuged at 4°C, 5000 rpm for 10 min to collect the bacteria; the bacteria were washed at least twice with 10% sterile glycerol that had been pre-ice-bathed, and finally 10 mL of pre-cooled 10% glycerol was added, the bacteria were blown evenly, and 200 μL were dispensed into each tube and stored at -80°C for later use.

[0106] (2) Electroporation of phAE159-AES into M. smegmatis mc 2 Obtaining plaques (phage) in 155

[0107] Take the positive phAE159-AES plasmid and add it to M.smegmatis mc 2 155 electroporation competent state was mixed, and electroporation was performed using a 2 mm electroporation cup Bio-rad electroporator (electroporation parameters: voltage 2.5 kV, resistance 1000 Ω, capacitance 25 μF). After electroporation, H9 medium was added, and cultured in a 37°C incubator overnight. The bacterial solution was mixed with Top Agar and inoculated on a 7H10 solid plate. After culturing at 30°C for 3 days, phage plaques were screened.

[0108] (3) Amplification of high titer phage

[0109] Pick the empty plaque containing phage from the plate into MP buffer and incubate at 4℃ overnight. Mix the phage-containing liquid with freshly cultured M.smegmatis mc 2 155 bacteria were mixed, then mixed with Top Agar and plated. After the plate was incubated at 30℃ for 3 days, MP buffer was added to the plate with plaques and placed at 4℃ overnight. High titer phages were collected and filtered with a 0.22μm sterile filter and stored in a 4℃ refrigerator for later use.

[0110] 1.5 Phage lysate transfection of the knockout strain

[0111] The high-titer phage lysate was mixed with Mycobacterium tuberculosis H37Rv grown to the logarithmic phase (pre-washed with MP buffer), incubated at 37°C overnight, centrifuged and discarded the supernatant, and then 7H9 liquid culture medium was added. After incubation at 37°C overnight, the supernatant was centrifuged and collected, and the cells were coated on plates (7H10+OADC+75μg / mL hygromycin B). After culturing at 37°C for 4 weeks, the genome was extracted and PCR was used to verify whether the gene was successfully knocked out.

[0112] The upstream verification primer LYZFP was designed 100-200bp upstream of the LFP primer matching sequence, and the downstream verification primer RYZRP was designed 100-200bp downstream of the RRP primer matching sequence. The schematic diagram of gene knockout and verification primer design is shown in Figure 2 As shown, Figure 2 RV1814RV1816 in the figure are Rv1814 encoding membrane-bound C-5 sterol desaturase, Rv1816 encoding HTH-type transcriptional regulatory factor, and Rv1815 encoding serine protease. PCR verification using primer pairs LYZFP / LYZRP and RYZFP / RYZRP and using the genome (MUT strain) of the knockout strain (named Δ1815) as a template can amplify DNA fragments of 1091 bp and 1296 bp. When the wild-type strain genome (WT strain) is used as a template in the control, no target DNA fragment is amplified when the same primer pair is used for PCR. It should be noted that Δ1815 or ΔRv1815 in the attached figure are both Rv1815 knockout bacteria. The PCR amplification results are shown in Figure 3 As shown, Figure 3 1 is the result of PCR identification of the wild-type strain (WT strain) using the primer pair LYZFP / LYZRP; 2 is the result of PCR identification of the Rv1815 gene knockout strain (MUT strain) using the primer pair LYZFP / LYZRP; 3 is the result of PCR identification of the wild-type strain using the primer pair RYZFP / RYZRP; 4 is the result of PCR identification of the Rv1815 gene knockout strain using the primer pair RYZFP / RYZRP; M: DNA Marker, represented by Figure 3 It can be seen that the Rv1815 gene knockout strain was successfully constructed.

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

[0114] Figure 4A is a schematic diagram of the composition of the Rv1815 protein domain; B is a growth curve of wild-type H37Rv and Δ1815 measured at a wavelength of 600 nm; C is a single colony morphology of wild-type H37Rv and Δ1815 after three weeks of growth on 7H10 plates; D is a statistical diagram of bacterial colony diameters in C; E is a colony morphology of wild-type H37Rv and Δ1815 after three weeks of growth on 7H10 plates, wherein, Figure 4 The A in the expression is the domain structure of Rv1815 protein, which consists of 221 amino acids, of which 1-27 amino acids are signal peptide (SP), and 55-178 amino acids are trypsin serine protease domain (Tryp_SPc). Wild-type H37Rv and ΔRv1815 strains of Mycobacterium tuberculosis were inoculated into 7H9 medium, cultured at 37°C until the logarithmic OD reached about 1.0, and the cultured H37Rv and ΔRv1815 strains were sub-inoculated into fresh 7H9 medium, respectively, with the initial OD being 0.05, and then cultured at 37°C. The OD was measured on the 7th, 10th, 14th, 18th, 21st and 25th days, and the growth curve was drawn as shown in FIG. Figure 4 As shown in B, the ΔRv1815 strain, also known as Δ1815 in the figure, grows slower than the wild-type H37Rv strain, also known as WT in the figure. In addition, 100 μl of bacterial solution was diluted 106 times and then spread on 7H10 plates. 20 μl of bacterial solution with an OD of about 1.0 was spotted on 7H10 plates and cultured at 37°C for 3 weeks. The results are as follows Figure 4 As shown in C and D: The ΔRv1815 strain colonies (0.9 cm in diameter) were found to be smaller than the wild-type H37Rv colonies (1.2 cm in diameter). Figure 4 E in the figure shows that after being coated on 7H10 plates and grown for 3 weeks, the ΔRv1815 strain showed a 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 bacterial morphology observation and determination

[0116] The wild-type H37Rv and ΔRv1815 strains of Mycobacterium tuberculosis were inoculated into 7H9 medium respectively, and cultured at 37℃ until the logarithmic phase OD was about 1.0. 5mL of bacterial solution was centrifuged and collected into a 1.5mL centrifuge tube. After washing the bacteria once with 1mL PBS, 1mL 2.5% glutaraldehyde was added to resuspend them and fixed at 4℃ overnight. Then they were sent to the company for electron microscopy observation. The results were as follows: Figure 5 As shown, Figure 5A in the figure is a scanning electron microscope image of the wild-type H37Rv and the knockout bacteria Δ1815 (magnified 20,000 times); B is a single bacterial morphology image of the wild-type H37Rv and the knockout bacteria Δ1815 (magnified 100,000 times); C is a statistical graph of the average length of the wild-type H37Rv and the knockout bacteria Δ1815; D is a statistical graph of the average diameter of the wild-type H37Rv and the knockout bacteria Δ1815, Figure 5 A in the figure shows the morphology of wild-type H37Rv and knockout strain ΔRv1815, also known as Δ1815, under a scanning electron microscope. It can be observed that some bacteria of the ΔRv1815 strain have a rough and shrunken surface ( Figure 5 B), and the length and diameter of bacteria were measured using Nano Measurer software, and it was found that there was no significant difference in the length of wild-type H37Rv and ΔRv1815 bacteria ( Figure 5 When the Rv1815 gene was knocked out, the average diameter of the bacteria was significantly reduced ( Figure 5 The above experiment was repeated three times, with three replicates each time, indicating that Rv1815 plays a role in regulating the morphology of tuberculosis bacteria.

[0117] 4. Mycobacterium tuberculosis macrophage survival assay

[0118] THP-1 cells were cultured at 2 × 10 5 Cells / well were seeded in a 24-well plate containing RMPI 1640 medium, cultured at 37°C and 5% CO2, induced with 100 ng / mL PMA for 24 hours to differentiate the cells into macrophages, and then replaced with fresh RMPI 1640 medium for 24 hours. The cells were infected with wild-type H37Rv and ΔRv1815 strains at MOI=10 for 4 hours, where the ΔRv1815 strain is Δ1815 in the figure, and then washed three times with preheated sterile PBS to remove extracellular bacteria, cultured with RPMI 1640 medium at 37°C and 5% CO2 for 3 days and 5 days, and lysed with PBS containing 0.1% SDS. The lysate was cultured in 7H10 medium at 37°C for 3 weeks, and the colony count (CFU numbers) was counted. The experimental results are shown in Figure 2. Figure 6 As shown in the figure, compared with the wild-type H37Rv strain, the number of intracellular colonies of the ΔRv1815 strain was significantly lower than that of the wild-type H37Rv at 3 and 5 days after infection of macrophages. The above experiment was repeated three times, with three parallel replicates each time, which shows that after Rv1815 knockout, the clearance of tuberculosis by macrophages is enhanced, indicating that Rv1815 plays an important role in the intracellular survival of tuberculosis in macrophages.

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

[0120] THP-1 cells were collected at 2×10 5 Cells / well were seeded in 24-well plates containing RMPI 1640 medium, cultured at 37°C and 5% CO2, induced with 100 ng / mL PMA for 24 hours to differentiate cells into macrophages, and then replaced with fresh RMPI1640 medium for 24 hours. Infection of wild-type H37Rv and Δ1815 strains infected cells at MOI = 10 for 4 hours, then washed three times with preheated sterile PBS to remove extracellular bacteria, cultured with RPMI 1640 medium at 37°C and 5% CO2 for 24 hours, and then RNA was extracted using the Omega Cell RNA Extraction Kit according to the manufacturer's instructions, and RNA samples were submitted to Novogene (Beijing) Company for ribosomal RNA removal and sequencing.

[0121] The analysis results are as follows Figure 7 As shown, Figure 7 A is a volcano plot of differentially expressed genes; B is a qPCR validation result of some differentially expressed genes between the ΔRv1815 strain and the wild-type H37Rv strain. Figure 7 As can be seen from A in the figure: the horizontal axis represents log2 (FoldChange), the vertical axis represents -log10 (P value), each point represents a gene, and the color is used to distinguish whether the gene is differentially expressed. The orange points in the figure represent differentially expressed genes, the green points represent down-regulated genes, and the blue points are genes with no significant differences. Compared with the wild-type H37Rv strain, after the ΔRv1815 strain infected THP-1 cells, a total of 35 genes were significantly upregulated (up35) and 109 genes were significantly downregulated (down 109), especially some chemokine-related genes, including CCL2 (Chemokine (CC motif) ligand 2, chemokine 2), CCL8 (Chemokine (CC motif) ligand 8, chemokine 8), CXCL10 (CXC chemokine ligand-10, CXC chemokine 10), CXCL9 (CXC chemokineligand-10, CXC chemokine 9), showing that the absence of Rv1815 leads to a weakening of the host inflammatory response. Specifically, the differentially expressed genes in transcriptome sequencing are shown in Table 2:

[0122] Table 2 Differentially expressed genes by transcriptome sequencing

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] The above gene transcriptome results were verified by qPCR experiments. RNA samples were first reverse transcribed into cDNA using HiScript IIQ RTSuperMix for qPCR (+gDNA wiper) (Novagen Biotech), and then quantitative real-time PCR (qRT-PCR) was performed on Roche's LightCycler 96real-time PCR system using ChamQUniversal SYBR qPCR Master Mix (Novagen Biotech). The PCR program was as follows: 95°C 30s, (95°C 10s; 60°C 30s) × 40 cycles, 95°C 10s, 60°C 60s, 95°C 15s. The relative expression of the target gene was normalized to the housekeeping gene GADPH, and the relative expression of the gene was calculated using the 2-△△CT method. This value reflects the difference in gene expression between the Δ1815 and WT groups, where the WT group is the baseline and Δ1815 is the difference with respect to the WT group. The difference results are shown in Figure 7 As shown in B: The verification results are consistent with the transcriptome results, which indicates that Rv1815 will cause host inflammatory response during cell infection, indicating that Rv1815 has 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, it can be seen from the tuberculosis macrophage survival assay experiment, transcriptome sequencing and real-time fluorescence quantitative PCR experiments that after knocking out Rv1815, the bactericidal ability of macrophages against tuberculosis bacteria is enhanced, indicating that inhibiting Rv1815 may help restore the normal immune function of host cells. Rv1815 triggers an inflammatory response, which may cause host tissue damage or immune response imbalance. Targeted inhibition of Rv1815 can reduce immunopathological damage during tuberculosis infection. Therefore, the function of Rv1815 makes it a potential anti-tuberculosis target, preparing for the preparation or production of tuberculosis drugs in the future.

[0130] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. Application of Rv1815 as a target in the preparation of anti-tuberculosis preparations.

2. The use according to claim 1, characterized in that The preparation reduces the content of Rv1815 protein in Mycobacterium tuberculosis by inhibiting the expression of Rv1815 gene, thereby inhibiting tuberculosis bacteria.

3. The use according to claim 1, characterized in that The preparation is used for improving the sensitivity of mycobacterium tuberculosis to anti-tuberculosis drugs.

4. The use according to claim 1, characterized in that The preparation comprises any one of a protein encoded by Rv1815, a gene knockout vector and a gene knockout mutant.

5. The use according to claim 4, characterized in that The preparation contains auxiliary materials, and the auxiliary materials include at least one of a solubilizer, a lubricant and an emulsifier.

6. The use according to claim 1, characterized in that The nucleotide sequence of the Rv1815 gene is shown in SEQ ID NO.1; and / or; The amino acid sequence of the protein encoded by the Rv1815 gene is shown in SEQ ID NO.

2.

7. A method for constructing a Rv1815 gene knockout vector, characterized in that: The steps include: Obtain multiple primer pairs to amplify the upper and lower arm sequences of the Rv1815 gene respectively, to obtain an amplified upper arm fragment and an amplified lower arm fragment; The amplified upper arm fragment and the amplified lower arm fragment are inserted into a target vector by homologous recombination method to obtain a knockout vector of the Rv1815 gene in Mycobacterium tuberculosis.

8. The method for constructing the Rv1815 gene knockout vector according to claim 7, characterized in that: The plurality of primers include primer LFP, primer LRP, primer RFP, primer RRP, primer LYZFP, primer LYZRP, primer RYZFP and primer RYZRP; wherein, The sequence of the primer LFP is shown in SEQ ID NO.5; and / or; The sequence of the primer LRP is shown in SEQ ID NO.6; and / or; The sequence of the primer RFP is shown in SEQ ID NO.7; and / or; The sequence of the primer RRP is shown in SEQ ID NO.8; and / or; The sequence of the primer LYZFP is shown in SEQ ID NO.9; and / or; The sequence of the primer LYZRP is shown in SEQ ID NO.10; and / or; The sequence of the primer RYZFP is shown in SEQ ID NO.11; and / or; The sequence of the primer RYZRP is shown in SEQ ID NO.

12.

9. A method for constructing a knockout mutant of Rv1815, characterized in that: The Rv1815 gene knockout vector in Mycobacterium tuberculosis constructed according to claim 7 or 8 is introduced into the target cells, and the Rv1815 gene knockout mutant is obtained after screening using the marker gene and verification.

Images