Application of MgdE gene in regulating virulence of mycobacterium bovis

Abstract

The invention discloses application of an MgdE gene in regulation and control of virulence of mycobacterium bovis, the nucleotide sequence of the MgdE gene is shown as SEQ ID NO.1, the amino acid sequence of encoded protein is shown as SEQ ID NO.2, the protein encoded by the gene is a nuclear regulatory protein, the nuclear localization function is achieved by depending on two nuclear localization signal peptides (NLS), and the nucleotide sequence of the MgdE gene is shown as SEQ ID NO.1. The two NLSs are respectively located at the 108th-111th site and the 300th-305th site of the amino acid sequence shown in SEQ ID NO.2, so that the level of host inflammation is inhibited, the survival and colonization of bacteria in host cells are enhanced. The mycobacterium bovis attenuated strain is obtained by modifying the MgdE gene or the two NLSs of the MgdE gene, and it is found for the first time that the MgdE gene and the two NLSs of the MgdE gene can serve as important targets for inhibiting mycobacterium bovis infection. The invention has important application value in preparation of bovine mycobacterium bacillus calmette guerin vaccine, and also makes important contribution to exploration of pathogenic mechanism and immune escape strategy of bovine mycobacterium.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, relates to the application of MgdE gene in regulating the virulence of Mycobacterium bovis, and also relates to a Mycobacterium bovis attenuated strain and a preparation method thereof. Background Art

[0002] Tuberculosis (TB) is a persistent infectious disease caused by Mycobacterium tuberculosis (Mtb). According to a survey report by the World Health Organization, there will be approximately 8.2 million new TB patients diagnosed in 2023, the highest number recorded by the WHO since the start of global TB surveillance in 1995, significantly higher than the 7.5 million new cases reported in 2022. The Bacillus Calmette-Guérin (BCG) vaccine, a weakly virulent strain of Mycobacterium bovis, is the only licensed and widely used vaccine on the market to prevent human tuberculosis, but its effectiveness is limited to young children. Therefore, better TB vaccines and innovative strategies are urgently needed to overcome the current TB crisis.

[0003] As a typical intracellular pathogen, Mtb enters the lungs after being inhaled into the respiratory system and invades macrophages and dendritic cells in the alveoli. Host resistance to Mtb infection requires complex coordination between the body and its immune system and between innate and adaptive immune cells. For example, innate immune receptors on macrophages and dendritic cells can recognize Mtb, thereby initiating phagocytosis and inflammatory responses. However, Mtb has developed strategies to adapt to the host cell environment during its long evolution and manipulate the activity of phagocytes to escape destruction, such as inhibiting the acidification of phagosomes and fusion with lysosomes. Therefore, in-depth research on the pathogenic mechanism and immune escape strategy of Mtb is crucial for developing new prevention and treatment options to curb this serious zoonosis.

[0004] In recent years, it has become an emerging research topic that bacterial pathogens target effector proteins to the host cell nucleus to regulate nuclear processes. These unique pathogen effector proteins are called "nuclear regulatory proteins". They interact with specific host targets through various mechanisms and reprogram host cells into niches suitable for pathogen proliferation. These mechanisms include: chromatin dynamics, histone modification, DNA methylation, RNA splicing, DNA replication, cell cycle and cell signaling pathways. However, little is known about the mechanism of action of nuclear regulatory proteins in mycobacteria. Therefore, screening and identifying Mtb nuclear regulatory proteins and in-depth exploration of their mechanism of action in the host cell nucleus is a new breakthrough in the study of pathogens, which can lay the foundation for the development of new vaccines and therapeutic drugs for pathogens.

[0005] The present invention uses Mycobacterium bovis as the research object. First, the conserved nuclear regulatory proteins in mycobacteria are screened by using bioinformatics analysis technology combined with a high-content cell imaging system, and it is found that the secretory protein MgdE can enter the host cell nucleus; then, the strain BCG (ATCC: 35734) is used as the research object to identify the effect of MgdE on the virulence of Mycobacterium bovis during the host infection process. In this process, we found that: (1) the hypothetical protein MgdE has the function of entering the nucleus and can significantly enhance the virulence of bacteria; (2) the virulence factor MgdE depends on RRR 108-111 (R1) and RLRRPR 300-305 (R2) Two nuclear localization signal peptides enter the host cell nucleus; (3) After MgdE enters the host cell nucleus, it can significantly inhibit the level of inflammation in the host and enhance the survival and colonization of Mycobacterium bovis in the host cell. Therefore, MgdE and its nuclear localization signal peptide are expected to be used as new targets to develop new anti-tuberculosis vaccines or drugs, and play a role in the prevention and treatment of tuberculosis in animals and humans. Summary of the invention

[0006] The purpose of the present invention is to provide an application of the MgdE gene in regulating the virulence of Mycobacterium bovis, and also to provide a Mycobacterium bovis attenuated strain and a preparation method thereof. The present invention utilizes genetic engineering technology to recombinant Mycobacterium bovis, and the obtained attenuated strain has the advantages of good immunogenicity and high production safety.

[0007] To achieve the above objectives, the applicant analyzed the nuclear import protein MgdE screened in the early stage and identified that the MgdE protein contains two nuclear localization signal peptides (NLS), namely KRIR 108-111 (R1) and RLRRPR 300-305 (R2), MgdE protein enters the host cell nucleus with the assistance of R1 and R2. The nucleotide sequence of MgdE gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0008] Next, the MgdE gene was amplified from the BCG genome and a recombinant plasmid was constructed. The recombinant plasmid was used to transfect HEK293T cells, and the nuclear import ability of the MgdE protein was detected by qRT-PCR and Western blot experiments, confirming that the protein encoded by the MgdE gene is a nuclear regulatory protein.

[0009] Furthermore, the exogenous hyg resistance gene was introduced into the genome of Mycobacterium bovis using homologous recombination technology, and the DNA fragment of the exogenous gene replaced the MgdE gene at the original site through homologous sequence exchange, and the recombinant BCG hyg::MgdE in which the MgdE gene was replaced was successfully constructed. At the same time, the MgdE gene sequence was subjected to point mutation using overlap extension PCR to obtain truncated mutant fragments MgdE-R1, MgdE-R2, and MgdE-R1 / 2 in which the two NLS of the MgdE gene were modified separately or simultaneously, and the truncated mutant fragments were fused and connected with the linearized complementation plasmid pLJR965 vector to obtain the MgdE gene complementation plasmid and transform the successfully constructed MgdE gene knockout strain BCG hyg::MgdE, and finally the MgdE gene partial complementation strain Comp-MgdE-R1 (KRIR) in which the two NLS were modified separately or simultaneously was successfully constructed. 108-111 Deletion), Comp-MgdE-R2(RLRRPR 300 -305 Deletion), Comp-MgdE-R1 / 2(KRIR 108-111 and RLRRPR 300-305 double deletion).

[0010] The knockout strain and each complemented strain were used to infect macrophages, and the survival ability of Mycobacterium bovis in host cells was detected. The results showed that compared with the wild-type strain, the survival ability of the knockout strain BCG hyg::MgdE and the complemented strains Comp-MgdE-R1, Comp-MgdE-R2, and Comp-MgdE-R1 / 2 in macrophages was significantly weakened; compared with the knockout strain BCGhyg::MgdE, the survival ability of the complemented strains Comp-MgdE-R1, Comp-MgdE-R2, and Comp-MgdE-R1 / 2 in macrophages was significantly enhanced; compared with the complemented strains Comp-MgdE-R1 and Comp-MgdE-R2, the survival ability of the complemented strain Comp-MgdE-R1 / 2 in macrophages was significantly weakened. The results show that the MgdE gene can promote the survival and colonization of pathogens in the host, thereby enhancing the virulence of pathogens, while knocking out the MgdE gene can reduce the survival and colonization of pathogens in the host, thereby reducing virulence, and in this process, the nuclear localization signal peptide KRIR 108-111 (R1) and RLRRPR 300-305 (R2) played an important role.

[0011] In addition, mice were infected with wild-type strain BCG / pLJR965, MgdE gene knockout strain KoMgdE, MgdE gene complete complement strain Comp-MgdE, R1 and R2 double deletion MgdE gene partial complement strain Comp-MgdE-R1 / 2, and the bacterial survival in lung tissue and inflammatory factor levels in spleen were detected. The results of bacterial survival showed that compared with wild-type strain BCG / pLJR965, the survival ability of knockout strain KoMgdE and gene partial complement strain Comp-MgdE-R1 / 2 in mice was significantly reduced; compared with knockout strain KoMgdE, the survival ability of complement strains Comp-MgdE and Comp-MgdE-R1 / 2 in mouse lungs was significantly enhanced, and the complete complement strain Comp-MgdE was more significant; compared with the complete complement strain Comp-MgdE, the survival ability of the partial complement strain Comp-MgdE-R1 / 2 in mouse lungs was significantly reduced. The results of inflammation level detection showed that in the innate immunity stage, compared with the wild-type strain BCG / pLJR965 and the complete complemented strain Comp-MgdE, the knockout strain KoMgdE and the partial complemented strain Comp-MgdE-R1 / 2 could significantly induce the production of inflammatory factors (il1β, il6, il10) in the spleen of mice, indicating that the MgdE gene can significantly inhibit the nonspecific immune response produced by the body after antigen stimulation, and knocking out the MgdE gene or modifying the NLS can enhance the body's innate immunity to antigens.

[0012] In summary, the protein encoded by the MgdE gene is a nuclear regulatory protein that is localized in the host cell nucleus. After entering the cell nucleus, it inhibits the level of inflammation produced by the host and enhances the survival and colonization of pathogens in the host cells. Therefore, the MgdE protein is an important virulence factor of Mycobacterium bovis and depends on two NLSs to function.

[0013] On this basis, the attenuated strain of Mycobacterium bovis provided by the present invention is a recombinant Mycobacterium bovis with MgdE gene function deficiency or nuclear localization function deficiency obtained by modifying the MgdE gene or the two NLSs of the MgdE gene.

[0014] The preparation method of the attenuated strain of Mycobacterium bovis provided by the present invention is to modify the MgdE gene in the Mycobacterium bovis genome by gene editing technology to make the MgdE gene function lost, or to modify the two NLSs of the MgdE gene separately or simultaneously by gene editing technology to make the nuclear localization function of the NLS lost. For those skilled in the art, gene knockout or silencing belongs to mature technical means, and the gene editing technology applicable to the present invention includes but is not limited to homologous recombination technology (homologous recombination, HR), clustered regularly interspaced short palindromic repeats-associated nuclease (CRISPR-Cas) technology, TALEN gene editing technology and Cre-loxP recombinase system, etc.

[0015] The embodiments of the present invention also provide a specific implementation scheme for modifying the MgdE gene or its NLS sequence based on homologous recombination technology, and the steps are as follows: using homologous recombination technology, an exogenous target gene is introduced into the Mycobacterium bovis genome, and through homologous sequence exchange, the DNA fragment of the exogenous target gene replaces the MgdE gene at the original site to obtain a Mycobacterium bovis strain in which the MgdE gene is replaced; then, the MgdE gene DNA fragment in which the two NLSs are modified separately or simultaneously is enzyme-cut and ligated with a complementing plasmid; and then, it is electroporated into the Mycobacterium bovis strain in which the MgdE gene is replaced to obtain a MgdE gene complementing strain in which the two NLSs are modified separately or simultaneously.

[0016] The attenuated strain of Mycobacterium bovis provided by the present invention can be used to prepare a Mycobacterium bovis vaccine because it can better induce the body to produce a nonspecific immune response and inhibit the survival and colonization of pathogens in the host. Compared with ordinary Mycobacterium bovis vaccines, the attenuated strain of Mycobacterium bovis provided by the present invention has the advantages of good immunogenicity and high production safety.

[0017] The present invention discovered for the first time that the MgdE gene and its two nuclear localization signal peptides can serve as important targets for inhibiting bovine Mycobacterium infection, and have important application value in the preparation of bovine Mycobacterium BCG vaccine. It also makes an important contribution to exploring the pathogenic mechanism and immune escape strategy of bovine Mycobacterium. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : qRT-PCR experiments were used to detect the expression levels of the target gene MgdE in HEK293T cells at different transfection time points (Figure A), and Western blot experiments were used to detect the content of the target protein MgdE in the nucleus of HEK293T cells at different transfection time points (Figure B).

[0019] Figure 2 : Plasmid map of knockout vector pMind-LacZ-UP600-hyg-DN600.

[0020] Figure 3 : PCR verification results of knockout strain BCG hyg::mgdE.

[0021] Figure 4 : Sequencing results of the DNA fragment of the knockout strain BCGhyg::mgdE amplified by the forward primer of the 360bp upstream sequence and the reverse primer of the 360bp downstream sequence of MgdE.

[0022] Figure 5 :qRT-PCR detection of the expression level of mgdE in the complemented strain.

[0023] Figure 6 : Determination of the survival quantity of wild-type, knockout and complemented strains in THP-1 macrophages.

[0024] Figure 7 : Determination of the survival number of wild-type, knockout and complemented strains in mice.

[0025] Figure 8 : Determination of the expression levels of inflammatory factors in the spleen of mice infected with wild-type, knockout and complemented strains at different time points. DETAILED DESCRIPTION

[0026] The technical scheme of the present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art on the basis of the following examples should also be deemed to fall within the scope of protection of the present invention. The experimental methods for which specific conditions are not indicated in the following examples are usually implemented according to conventional conditions or reference books such as "Molecular Cloning Laboratory Guide" (NewYork: Cold Spring Harbor Laboratory, 2017), or implemented according to the methods recommended in the operating manual provided by the manufacturer. The materials whose sources are not indicated in the examples are all commonly used materials well known in the art, which can be constructed by themselves according to literature reports or obtained through commercial channels.

[0027] Example 1: Construction of MgdE recombinant plasmid and determination of nuclear entry ability

[0028] First, we used the secretory protein analysis software SignalP-5.0 to predict the hypothetical protein MgdE and found that MgdE is a secretory protein with a Tat signal peptide ( https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ); then, based on the characteristics of the nuclear localization signal sequence (NLS) on the amino acid sequence of nuclear regulatory proteins (a highly conserved short sequence consisting of several basic amino acids, usually containing arginine and lysine), the NLS sequence on MgdE was identified and KRIR was found 108-111 (R1) and RLRRPR300-305 (R2) may be the NLS sequence that assists MgdE in nuclear import.

[0029] In this example, the MgdE gene was cloned into the N-terminus of the pEGFP-N1 vector and transformed into the DH5α competent cell to obtain a recombinant expression strain; then, an endotoxin-free plasmid extraction kit was used to obtain the fusion expression plasmid MgdE-pEGFP, which was transfected into HEK293T cells in a 12-well plate, and the localization of the target protein in the host cells was observed by confocal fluorescence microscopy at 4, 12, 24, 36, and 48 hours after transfection. The specific steps are as follows:

[0030] 1. Amplification of target gene fragment: First, query the MgdE gene information on GenBank and download the gene sequence, use BioEdit software to query the restriction sites within the MgdE gene, and design the gene forward primer (MgdE-F) and reverse primer (MgdE-R) (Table 1) from 18-20 bp before and after the coding gene according to the available restriction sites (NheI and Hind3) on the fluorescent reporter vector EGFP. Use the BCG genome as a template to amplify the MgdE gene sequence; PCR amplification system 50 μL, reaction conditions and reaction system are shown in the following table (Table 2, Table 3).

[0031] Table 1 Primer sequences for amplification of MgdE gene fragment

[0032]

[0033] Table 2 PCR amplification system of MgdE gene

[0034]

[0035] Table 3 PCR reaction conditions for amplifying the MgdE gene

[0036]

[0037] 2. Enzyme digestion of MgdE gene fragment and pEGFP vector (reagents ordered from Takara):

[0038] (1) DNA double enzyme digestion system

[0039] The DNA double restriction enzyme digestion system of MgdE gene fragment and pEGFP vector is as follows (Table 4).

[0040] Table 4 DNA double restriction enzyme digestion system of MgdE gene fragment and pEGFP vector

[0041]

[0042] (2) 37°C for 3-4 hours;

[0043] (3) The DNA fragments after enzyme digestion are purified and recovered for enzyme ligation reaction.

[0044] 3. Enzyme connection of target gene fragment and vector (reagents ordered from Takara):

[0045] (1) Enzyme-linked system (5 μL)

[0046] The enzyme ligation system of the target gene fragment and the vector is as follows (Table 5).

[0047] Table 5 Enzyme ligation system of target gene fragment and vector

[0048]

[0049] (2) Enzyme incubation at 16°C for 8 hours;

[0050] 4. Transformation: The ligation product was transformed into DH5α competent cells, cultured at 37°C for 16 hours, a single transformant was picked, cultured in 5 mL LB liquid medium containing 30 μg / mL Kan (kanamycin) for 8 hours, and the plasmid was extracted to obtain the recombinant plasmid pEGFP-mgdE;

[0051] 5. Cell transfection: First, HEK293T cells were plated in 12-well plates one day before transfection, with 2×10 cells per well. 5 and cultured in a cell culture incubator at 37°C and 5% CO2 for 12 hours. The cell supernatant was removed half an hour before transfection and 800 μL Opti-MEM culture medium was added. Then, during the infection process, a DNA-Hieff TransTM liposome nucleic acid transfection reagent complex was prepared, wherein each well of cells used 1 μg DNA diluted in 100 μL Opti-MEM and 4.5 μL Hieff TransTM liposome nucleic acid transfection reagent diluted in 100 μL Opti-MEM. The two were co-incubated at room temperature for 20 minutes and then added to the cell well plate. The medium was changed 6 hours after transfection, and 1 mL DMEM+10% FBS culture medium was added. Subsequently, the ability of MgdE to enter the host cell nucleus was detected by qRT-PCR technology and Western blot technology at 4 hours, 12 hours, 24 hours, 36 hours, and 48 hours after transfection, respectively.

[0052] 6.qRT-PCR was used to detect the expression level of the target gene in HEK293T cells at different transfection times.

[0053] The detection primers of the internal reference gene hprt and the target gene mgdE are shown in Table 6. Western blot experiments were performed to detect the content of the target protein MgdE entering the nucleus of HEK293T cells at different transfection times. The nuclear protein and cytoplasmic protein extraction kits were purchased from Bio-Tech Biotechnology Co., Ltd. Histone H3 was used as the detection indicator of the nuclear internal reference protein.

[0054] Table 6 Primer sequences in qRT-PCR experiments

[0055]

[0056] The experimental results showed that: (1) The results of qRT-PCR experiments showed that as the transfection time increased, the expression level of the target gene mgdE in the host cells also gradually increased ( Figure 1 A); (2) Western blot results showed that as the transfection time increased, the accumulation of the target protein MgdE in the cell nucleus gradually increased ( Figure 1 B).

[0057] Example 2: Construction of MgdE gene knockout strain

[0058] This experiment uses DNA homologous recombination technology to modify endogenous genes, so that the exogenous DNA and the target gene homologous sequence on the recipient genomic DNA are exchanged, and the exogenous DNA is integrated into the predetermined site of the target gene to complete the precise modification and transformation of the target gene, resulting in the loss of gene function. The modified and transformed gene can replicate stably with the replication of the genomic DNA. The main steps of gene knockout are: (1) Construction of knockout vector plasmid, cloning the homologous sequence of the target gene into a vector with a marker gene (usually an antibiotic gene, such as hygromycin hyg); (2) Transformation of the knockout vector plasmid into the recipient bacteria; (3) Screening the recombinant strain with target gene knockout using selective culture medium; (4) Identification of the target gene deletion strain.

[0059] The specific implementation steps are as follows:

[0060] 1. Primer design: Query the MgdE gene information on GenBank and download the gene sequence. Use BioEdit software to query the restriction sites inside the MgdE gene. According to the available restriction sites on the pMind-LacZ vector (Chen L et al., 2022), determine that the PacⅠ, SpeⅠ, HindⅢ and NheⅠ restriction sites on the MgdE gene sequence are available. Select 600 bp sequences upstream and downstream of the target gene sequence, and use the BCG genome as a template to amplify the approximately 600 bp upstream fragment of the MgdE gene (the restriction sites are PacⅠ and SpeⅠ) (UP) and the 600 bp downstream fragment (the restriction sites are HindⅢ and NheⅠ) (DN). At the same time, construct the pMind vector treated with PacⅠ and SpeⅠ restriction endonucleases. The primers for amplifying the 600 bp sequences upstream and downstream of the MgdE gene are shown in Table 7 below.

[0061] Table 7 Primer list in PCR experiment

[0062]

[0063] 2. Clone the upstream fragment into the multiple cloning site of the pMind vector, then double-digest the pMind-LacZ-UP vector with HindⅢ and NheⅠ restriction endonucleases, connect the downstream fragments, and construct the complete knockout vector pMind-LacZ-UP-hyg-DN. This vector has the following characteristics: First, it is a suicide vector that cannot replicate autonomously in mycobacteria; second, the successfully constructed knockout strain has three screening markers: (1) Hyg resistance; (2) the reporter gene LacZ will make the strain turn blue in the X-gal environment; (3) when the recombinant vector and the genome undergo double exchange, the strain's Kan resistance will be lost, that is, Kan is a negative screening marker. The specific operations are as follows:

[0064] (1) Upstream fragment cloning: First, according to the designed mgdE-UP 600 Primer sequences, using BCG genome as template, to amplify mgdE-UP with PacI and SpeI restriction sites 600 Then, the DNA fragment and pMind-LacZ were double-digested with PacI and SpeI to obtain mgdE-UP 600 (PacI / SpeI) and pMind-LacZ(PacI / SpeI) DNA fragments (see Table 8 for restriction enzyme digestion system); then, pMind-LacZ(PacI / SpeI) and mgdE-UP 600 The (PacI / SpeI) fragments were ligated and transformed at a ratio of 1:3; finally, a single transformant was obtained, and the plasmid was extracted by amplification culture to obtain the recombinant plasmid pMind-LacZ-UP600 ;

[0065] (2) Downstream fragment connection: First, according to the designed mgdE-DN 600 Primer sequences, using BCG genome as template, to amplify mgdE-DN with HindⅢ and NheⅠ restriction sites 600 Then, the DNA fragment and the recombinant plasmid pMind-LacZ-UP obtained in the previous step were double-digested with HindⅢ and NheⅠ. 600 , and mgdE-DN 600 (HindⅢ / NheⅠ) and pMind-LacZ-UP 600 (HindⅢ / NheⅠ) DNA fragment (enzyme digestion system see Table 8 below); then, mgdE-DN 600 (HindⅢ / NheⅠ) and pMind-LacZ-UP 600 The (HindⅢ / NheⅠ) DNA fragments were ligated and transformed at a ratio of 1:3; finally, a single transformant was obtained, and the plasmid was extracted by amplification culture to obtain the recombinant plasmid pMind-LacZ-UP 600 -hyg-DN 600 , plasmid map see Figure 2 .

[0066] Table 8 Enzyme Digestion Reaction System

[0067]

[0068] 3. The constructed recombinant vector pMind-LacZ-UP600-hyg-DN600 was electroporated into BCG competent cells and screened on 7H10 plates containing 10% OADC, 50 μg / mL Hyg and 50 μg / mL X-gal.

[0069] 4. Pick a single white colony from the plate, culture it in 7H9 medium containing 10% OADC and 50 μg / mL Hyg, and transfer part of the activated bacterial solution to 7H9 medium containing 30 μg / mL Kan. If the bacteria lose Kan resistance, it may be a double-exchange knockout strain.

[0070] 5. We extracted the genome of the successfully constructed MgdE knockout strain, used the wild-type strain genome as a control, performed PCR verification using mgdE gene primers and hyg primers (see Table 9 for relevant primers), and amplified the target DNA fragment from the strain verified to be correct by PCR and sent it to a sequencing company (Qingke Biotechnology) for sequencing.

[0071] Table 9 Primer sequences verified in PCR experiment

[0072]

[0073] Experimental results: (1) PCR results showed that hyg (999 bp) was not amplified in the wild-type strain BCG / wt, but mgdE (945 bp) was amplified; hyg (999 bp) was amplified in the knockout strain BCG hyg::mgdE, but mgdE (945 bp) was not amplified; when the forward primer was designed with the upstream 600 bp sequence of the target gene mgdE, and the reverse primer was designed with the downstream 600 bp sequence for verification, it was found that a DNA fragment of 2145 could be amplified in the wild-type strain BCG / wt, and a DNA fragment of 2199 bp could be amplified in the knockout strain BCG hyg::mgdE ( Figure 3 ), indicating that the hyg gene successfully replaced the mgdE gene on the BCG genome and the BCGhyg::mgdE knockout strain was successfully constructed.

[0074] (2) The 2199 bp DNA fragment amplified from the knockout strain was sent to a biological company for sequencing. Sequencing primers were designed from 360 bp upstream and downstream of the target gene (Table 10). The results showed that the hyg gene sequence could be successfully determined ( Figure 4 ), indicating that the BCG hyg::mgdE knockout strain was successfully constructed.

[0075] Table 10 Knockout strain sequencing primer sequences

[0076]

[0077] Example 3: Construction of complementation strains

[0078] To further explore the relationship between MgdE and nuclear localization signal sequence KRIR 108-111 (R1), RLRRPR 300-305 (R2) Role in the virulence of BCG. We used the pLJR965 vector to construct the following series of recombinant strains:

[0079] (1) BCG / pLJR965: The empty vector pLJR965 plasmid was electroporated into the wild-type strain BCG to obtain the wild-type strain BCG without mutation of the mgdE gene, which was used as a control in the experiment;

[0080] (2) Knockout strain BCG hyg::mgdE / pLJR965 (KoMgdE): The empty vector pLJR965 plasmid was electroporated into the knockout strain BCG hyg::mgdE constructed above to obtain a BCG strain in which the mgdE gene was completely knocked out;

[0081] (3) Complementation strain BCG hyg::mgdE / pLJR965-mgdE (Comp-MgdE): A fusion expression plasmid pLJR965-mgdE was constructed based on the pLJR965 complementation vector, and it was electroporated into the knockout strain BCG hyg::mgdE to obtain a BCG strain with a complete complementation of the mgdE gene;

[0082] (4) Complementation strain BCG hyg::mgdE / pLJR965-mgdE-R1 (Comp-MgdE-R1): KRIR was obtained by gene point mutation technology 108-111 (R1) The MgdE DNA sequence with the amino acid sequence deleted was ligated to the pLJR965 vector to obtain the fusion expression plasmid pLJR965-mgdE-R1; then, it was electroporated into the knockout strain BCG hyg::mgdE to obtain the RLRRPR 300-305 (R2) BCG strain in which the mgdE gene of amino acids was partially complemented;

[0083] (5) Complementation strain BCG hyg::mgdE / pLJR965-mgdE-R2 (Comp-MgdE-R2): RLRRPR obtained by gene point mutation technology 300-305 (R2) The MgdE DNA sequence with the amino acid sequence deleted was ligated to the pLJR965 vector to obtain the fusion expression plasmid pLJR965-mgdE-R2; then, it was electroporated into the knockout strain BCG hyg::mgdE to obtain the KRIR-containing 108-111 (R1) BCG strain in which the mgdE gene of amino acids was partially complemented;

[0084] (6) Complementation strain BCG hyg::mgdE / pLJR965-mgdE-R1 / 2 (Comp-MgdE-R1 / 2): KRIR was obtained by gene point mutation technology 108-111 (R1) and RLRRPR 300-305 (R2) The MgdE DNA sequence with double amino acid sequence deletion was ligated to the pLJR965 vector to obtain the fusion expression plasmid pLJR965-mgdE-R1 / 2; then, it was electroporated into the knockout strain BCG hyg::mgdE to obtain the double deletion KRIR 108-111 (R1) and RLRRPR 300-305 (R2) BCG strain in which the mgdE gene of amino acids was partially complemented.

[0085] The specific construction method is as follows:

[0086] During the experiment, the vector was constructed using seamless cloning technology, and the point mutation DNA fragment was amplified using overlapping extension PCR technology. The principle of seamless cloning is to design 15-25 homologous bases (homologous arms) at the ends of the vector and primers. Through T5 exonuclease, DNA polymerase, and DNA ligase, the three enzymes function simultaneously to achieve the connection of a single fragment or multiple fragments to the vector. The principle of overlapping extension PCR is to use primers with complementary ends to form a short overlapping chain of the PCR product, and then in the subsequent amplification reaction, the amplified fragments from different sources are overlapped and spliced ​​together by extending the overlapping chain.

[0087] 1. Linearization of pLJR965 vector (For detailed information on pLJR965 vector, see https: / / www.addgene.org / 115163 / )

[0088] According to the sequence information of pLJR965 vector, two pairs of primers were designed and two fragments of pLJR965 vector (pLJR965L, pLJR965R) were amplified using pLJR965 vector as template, and then the linearized pLJR965 vector was obtained by overlap extension PCR (primer information is shown in Table 11 below);

[0089] 2. Construction of mgdE and truncated mutant fragments (mgdE-R1, mgdE-R2, mgdE-R1 / 2)

[0090] (1) Amplification of MgdE gene fragment: First, the DNA sequence of mgdE gene and its upstream 350 bp bases were obtained from GenBank and KEGG databases (www.kegg.jp); then, based on the linearized pLJR965 vector information, mgdE primers (mgdE-F, mgdE-R) containing the homology arms of the pLJR965 vector were designed (primer sequences are shown in Table 11 below); then, PCR amplification was performed using the BCG genome as a template to obtain the MgdE DNA fragment (the 350 bp base sequence upstream of mgdE contains the gene's own promoter, and the target gene needs to carry its own promoter to initiate gene transcription when cloning using the pLJR965 vector);

[0091] (2) Construction of MgdE-R1 gene truncated mutant fragments: First, determine the KRIR 108-111(R1) The base sequence aagcggatccgg on mgdE; then, a sequence of 15-18bp on the left and right fragments (mgdE-R1L, mgdE-R1R) was selected with the sequence as the center to synthesize a sequence as the forward primer (mgdE-R1R-F) for the synthesis of the truncated mutant fragment MgdE-R1R, and after reverse complementation, it was used as the reverse primer (mgdE-R1L-R) for the synthesis of the truncated mutant fragment MgdE-R1L (primer information is shown in Table 11 below); then, using the BCG genome as a template, mgdE-F / mgdE-R1L-R and mgdE-R1R-F / mgdE-R were used as primers, respectively, and KRIR was obtained after amplification. 108-111 (R1) DNA fragments on both sides; finally, the fragments were subjected to overlap extension PCR to obtain the MgdE-R1 truncated mutant fragment (the overlap extension PCR system is shown in Tables 12 and 14 below, and the reaction conditions are shown in Tables 13 and 15 below);

[0092] (3) Construction of mgdE-R2 gene truncated mutant fragments: First, determine the RLRRPR 300-305 (R2) The base sequence on mgdE is cggctgcggcggccccgg; then, 15-18 bp of the left and right fragments and 16 bp of the homology arm of pLJR965LR were selected with this sequence as the center to synthesize a sequence, which was used as the reverse primer (mgdE-R2-R) for synthesizing the truncated mutant fragment MgdE-R2 after reverse complementation (primer information is shown in Table 11 below); then, using the BCG genome as a template and mgdE-F / mgdE-R2R as primers, the truncated mutant fragment of MgdE-R2 was obtained after amplification;

[0093] (4) Construction of truncated mutant fragments of the mgdE-R1 / 2 gene: First, based on the construction of truncated mutant fragments of MgdE-R1 and MgdE-R2, the BCG genome was used as a template and mgdE-R1R-F / mgdE-R2-R were used as primers to amplify the DNA fragment between the R1 and R2 mutant sequences (MgdE-R1 / 2M); then, MgdE-R1 / 2M and the fragment MgdE-R1L were subjected to overlapping extension PCR to obtain the truncated mutant fragment of MgdE-R1 / 2.

[0094] 3. Construction of recombinant plasmids (pLJR965-mgdE, pLJR965-mgdE-R1, pLJR965-mgdE-R2, pLJR965-mgdE-R1 / 2)

[0095] First, the obtained MgdE and truncated mutant fragments were reacted with the linearized pLJR965 vector using the Uniclone One Step Seamless Cloning Kit (Jinsha Biotechnology SC612) (see Table 16 below for the seamless cloning reaction system), placed at 50°C for 30 minutes, and then placed on ice for 2 minutes; then, the reaction product was transformed into DH5α competent cells and spread on LB solid culture medium containing 30 μg / mL Kan, and cultured at 37°C for 16 hours; finally, a single transformant was picked, expanded and cultured on 5 mL of LB liquid culture medium containing 30 μg / mL Kan, and the recombinant plasmid was extracted.

[0096] 4. Electroporation

[0097] The recombinant plasmid was electroporated into BCG hyg::mgdE competent cells and plated on 7H10 medium containing 10% OADC and 30 μg / mL Kan. The cells were cultured at 37°C for 14 days. A single colony was then picked and cultured in 5 mL of 7H9 liquid medium containing 10% OADC and 30 μg / mL Kan. The OD 600 ≈1.0 is used for experiments.

[0098] 5. Identification

[0099] The recombinant strain was inoculated into 10 mL of 7H9 liquid medium containing 30 μg / mL Kan and cultured until OD 600 qRT-PCR experiment was performed when ≈1.0 to detect the expression level of mgdE gene in each strain. First, the bacteria were collected in a 50mL sterile centrifuge tube, centrifuged at 6000rpm for 5 minutes, and the supernatant was removed; then, the bacteria were washed twice with PBS, centrifuged at 6000rpm for 5 minutes, and the supernatant was removed; finally, RNA of each sample was extracted with an RNA extraction kit (Aidlab Biological Company, RN2802) and RNA reverse transcription (Novozyme, R423-01) was performed to detect the expression level of mgdE gene in each strain (identification primers are shown in Table 17, where sigA is a bacterial internal reference gene).

[0100] Table 11 Primer list in PCR experiment

[0101]

[0102]

[0103] Overlap extension PCR steps (taking MgdE-R1 as an example, the parameters of the other fragment operation steps are the same) Step 1:

[0104] Table 12 Overlap extension PCR amplification system 1

[0105]

[0106] Table 13 Overlap extension PCR reaction conditions 1

[0107]

[0108] After the reaction is completed, place on ice and add primers and enzymes for subsequent reactions.

[0109] Step 2:

[0110] Table 14 Overlap extension PCR amplification system 2

[0111]

[0112] Table 15 Overlap extension PCR reaction conditions 2

[0113]

[0114] After the reaction, MgdE and truncated mutant fragments (MgdE-R1, MgdE-R2, MgdE-R1 / 2) were obtained.

[0115] Table 16 Seamless cloning reaction system

[0116]

[0117] Table 17 Primer sequences in qRT-PCR experiments

[0118]

[0119] The results of qRT-PCR experiments showed that the mgdE gene could not be detected in the knockout strain; in the complemented strain, the complementation efficiencies of MgdE, MgdE-R1, MgdE-R2, and MgdE-R1 / 2 were 91.9%, 95.1%, 93.6%, and 91.6%, respectively, indicating that the mgdE gene was successfully complemented. The error value represents the standard deviation of three biological replicates, and the P value is calculated using the unpaired two-tailed Student's t-test in GraphPad Prism 7. Asterisks (*) represent significant differences between two groups of data: *, p<0.1; **, p<0.01; ***, p<0.001 ( Figure 5 ).

[0120] Example 5. Detection of the survival ability of mutants in macrophage THP-1

[0121] The above recombinant strains were inoculated into 50 mL of liquid culture medium containing 30 μg / mL Kan 7H9 and cultured until OD 600When the concentration of THP-1 cells was ≈1.0, the cell infection experiment was performed. First, 3 days before the infection experiment, the cultured THP-1 cells were evenly plated in a 24-well plate, with the number of cells in each well being 0.5×10 6 cfu / mL, and 200 ng / mL of PMA was added for 48 hours; then, 500 μL of LPS containing 200 ng / mL was added for 24 hours. Before the infection experiment, the culture medium was removed and 500 μL of 1640+10% FBS culture medium was added; then, the cultured recombinant strain was infected with THP-1 cells at an infection multiplicity of 10:1, and cultured in a cell culture incubator at 37°C and 5% CO2 for 4 hours, and then the supernatant was removed, and 500 μL of 1640+10% FBS culture medium containing double antibodies (penicillin + streptomycin) was added and cultured for 4, 24, 48, and 72 hours respectively; finally, the adherent cells were digested with 0.05% SDS and the infected cell samples were collected.

[0122] The collected cell samples were diluted with PBS (5 -1 , 5 -2 , 5 -3 ), and 100 μL was applied to 7H10 medium containing 10% OADC and 30 μg / mL Kan, and cultured at 37°C for 14 days, and the number of surviving recombinant strains in macrophages at each infection time point was counted.

[0123] like Figure 6 As shown, the experimental results show that:

[0124] (1) Compared with the wild-type strain, the survival ability of the knockout strain KoMgdE and the partially complemented strains Comp-MgdE-R1, Comp-MgdE-R2, and Comp-MgdE-R1 / 2 in host cells was significantly reduced. This indicates that MgdE can enhance the survival ability of pathogens in the host, and this process depends on the assistance of nuclear localization signal sequences (R1, R2);

[0125] (2) Compared with the knockout strain KoMgdE, 24 hours after infection, the survival abilities of the complement strains Comp-MgdE, Comp-MgdE-R1, Comp-MgdE-R2, and Comp-MgdE-R1 / 2 in host cells were significantly enhanced, and Comp-MgdE was the most significant.

[0126] (3) Compared with the complemented strain Comp-MgdE, the survival ability of the complemented strains Comp-MgdE-R1, Comp-MgdE-R2, and Comp-MgdE-R1 / 2 in host cells was significantly weakened 24 hours after infection;

[0127] (4) There was no significant difference in virulence between the recombinant strains Comp-MgdE-R1 and Comp-MgdE-R2.

[0128] (5) Compared with the recombinant strains Comp-MgdE-R1 and Comp-MgdE-R2, the survival ability of the complemented strains Comp-MgdE-R1 / 2 in host cells was significantly weakened 24 hours after infection.

[0129] In summary, in BCG strains, MgdE can significantly enhance the virulence of pathogens and promote the survival and colonization of pathogens in the host, while knocking out MgdE can reduce virulence and thus reduce the survival and colonization of pathogens in the host. In this process, the nuclear localization signal sequence KRIR 108-111 (R1) and RLRRPR 300-305 (R2) played an important role.

[0130] Example 6. Detection of the survival ability of mutant strains in C57BL / 6 mice and detection of spleen inflammation level

[0131] The constructed recombinant strains (BCG / pLJR965, KoMgdE, Comp-MgdE, Comp-MgdE-R1 / 2) were inoculated into 5 mL of 7H9 liquid medium containing 30 μg / mL Kan and cultured until OD 600 ≈1.0, according to 1×10 7 cfu / 40μL were used to infect C57BL / 6 female mice (bacterial infection method: nasal drops; mouse weight: SPF grade, 6-8 weeks, 16-18g), with 6 biological replicates in each group, and the infection time was 2, 14, 21, 28, and 56 days; at different time points after infection, the lung and spleen tissues of the mice were collected, the lung tissues were used to detect the number of bacterial survival, and the spleen samples were used to detect the expression levels of inflammatory factors.

[0132] Detection of the number of surviving bacteria in the lungs: First, the infected mouse lung samples were ground; then, 1 mL of PBS was used to perform gradient dilutions (5 -1 , 5 -2 , 5 -3 ), and 100 μL was applied to 7H10 medium containing 10% OADC and 30 μg / mL Kan, and cultured at 37°C for 14 days; finally, the number of surviving strains in the lungs of each mouse at each infection time point was counted.

[0133] Detection of spleen inflammation level: spleen samples of infected mice were taken, tissue RNA was extracted, and qRT-PCR was performed to detect the expression of spleen inflammatory factors (il1β, il16, il10) (identification primers are shown in Table 18, where hprt is a cell internal reference gene) after reverse transcription into cDNA. Among them, the RNA extraction steps and reagents used in tissue samples were the same as those of Aidlab Biological Company (RN2802), and the RNA reverse transcription operation steps were the same as those of Novozyme reverse transcription kit (R423-01).

[0134] The results of bacterial survival showed that compared with the wild-type strain BCG / pLJR965, the survival ability of the knockout strain KoMgdE and the recombinant strain Comp-MgdE-R1 / 2 in mice was significantly reduced; compared with the knockout strain KoMgdE, the survival ability of the recombinant strains Comp-MgdE and Comp-MgdE-R1 / 2 in the mouse lungs was significantly enhanced, and Comp-MgdE was more significant; compared with the complemented strain Comp-MgdE, the survival ability of the partially complemented strain Comp-MgdE-R1 / 2 in the mouse lungs was significantly decreased ( Figure 7 ).

[0135] The results of the inflammatory level test showed that in the first 28 days of bacterial infection in mice, compared with the wild-type strain BCG / pLJR965 and the complemented strain Comp-MgdE, the knockout strain KoMgdE and the partial complemented strain Comp-MgdE-R1 / 2 could significantly induce the production of inflammatory factors (il1β, il6, il10) in the spleen of mice ( Figure 8 ).

[0136] In summary, during the BCG strain infection of mice, MgdE can significantly enhance the virulence of the pathogen, promote the survival of the pathogen in the host and inhibit the production of inflammatory factors, and the nuclear localization signal sequences R1 and R2 play an important role in this process.

[0137] Table 18 Primer sequences in qRT-PCR experiments

[0138]

Claims

1. Application of the MgdE gene in regulating the virulence of Mycobacterium bovis, the nucleotide sequence of the MgdE gene is shown in SEQ ID NO.1, the amino acid sequence of the encoded protein is shown in SEQ ID NO.2, the protein encoded by the gene is a nuclear regulatory protein, which is localized in the host cell nucleus. After entering the nucleus, it inhibits the host inflammation level and enhances the survival and colonization of Mycobacterium bovis in the host cell. The MgdE gene depends on two NLSs to exert its nuclear localization function, and the two NLSs are respectively located at positions 108-111 and 300-305 of the amino acid sequence shown in SEQ ID NO.

2.

2. A weak strain of Mycobacterium bovis, the MgdE gene of which is modified to result in functional loss, or the two NLSs of the MgdE gene are modified separately or simultaneously to result in nuclear localization function loss, the nucleotide sequence of the MgdE gene is shown in SEQ ID NO.1, the amino acid sequence of the encoded protein is shown in SEQ ID NO.2, and the two NLSs are respectively located at positions 108-111 and positions 300-305 of the amino acid sequence shown in SEQ ID NO.

2.

3. A method for reducing the virulence of Mycobacterium bovis, characterized in that: The MgdE gene in the genome of Mycobacterium bovis is modified using gene editing technology to cause the loss of MgdE gene function, or the two NLSs of the MgdE gene are modified separately or simultaneously using gene editing technology to cause the loss of the nuclear localization function of the NLS. The nucleotide sequence of the MgdE gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.

2. The two NLSs are respectively located at positions 108-111 and positions 300-305 of the amino acid sequence shown in SEQ ID NO.

2.

4. The method for reducing the toxicity of Mycobacterium bovis according to claim 3, characterized in that: Using homologous recombination technology, an exogenous target gene is introduced into the Mycobacterium bovis genome, and the DNA fragment of the exogenous target gene replaces the MgdE gene at the original site through homologous sequence exchange to obtain a Mycobacterium bovis strain with the MgdE gene replaced. Alternatively, a MgdE gene DNA fragment in which two NLSs are modified separately or simultaneously is enzyme-cut and ligated with a complementing plasmid, and then electroporated into the Mycobacterium bovis strain in which the MgdE gene is replaced to obtain a MgdE gene complementing strain in which two NLSs are modified separately or simultaneously.

5. The method for reducing the virulence of Mycobacterium bovis according to claim 4, characterized in that: The exogenous target gene is a hyg resistance gene.

6. The method for reducing the virulence of Mycobacterium bovis according to claim 4, characterized in that: The complementing plasmid is the pLJR965 plasmid.

7. Use of the attenuated strain according to claim 2 in the preparation of a Mycobacterium bovis vaccine.

8. A Mycobacterium bovis vaccine comprising the attenuated strain according to claim 2.

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