Application of MgdE gene in regulating virulence of Mycobacterium bovis

Abstract

The application discloses application of an MgdE gene in regulating virulence of Mycobacterium bovis, a nucleotide sequence of the MgdE gene is shown as SEQ ID NO. 1, an amino acid sequence of a coded protein is shown as SEQ ID NO. 2, the protein coded by the gene is a nuclear regulatory protein, depends on two nuclear localization signal peptides (NLS) to play a nuclear localization function, and then inhibits a host inflammation level, enhances survival and colonization of the bacteria in a host cell, the two NLS are respectively located at positions 108-111 and 300-305 of the amino acid sequence shown as SEQ ID NO. 2. The MgdE gene or the two NLS is modified to obtain a weak strain of Mycobacterium bovis, it is found for the first time that the MgdE gene and the two NLS can be used as important targets for inhibiting Mycobacterium bovis infection. The application has important application value in preparation of Mycobacterium bovis bacillus Calmette-Guerin (BCG), and also makes an important contribution to exploration of a pathogenic mechanism and an immune escape strategy of Mycobacterium bovis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology, and relates to application of MgdE gene in regulating virulence of Mycobacterium bovis, and further relates to a weakly virulent strain of Mycobacterium bovis and a preparation method thereof. BACKGROUND

[0002] Tuberculosis (TB) is a persistent infectious disease caused by Mycobacterium tuberculosis (Mtb). According to the investigation report of the World Health Organization, about 8.2 million new TB patients were diagnosed in 2023, which is the highest number recorded since the World Health Organization began global TB monitoring in 1995, significantly higher than the 7.5 million new cases reported in 2022. The Mycobacterium bovis attenuated strain Bacillus Calmette-Guerin (BCG) is the only licensed and widely used vaccine for preventing human tuberculosis on the market, but its efficacy is limited to young children. Therefore, to overcome the current tuberculosis crisis, better tuberculosis vaccines and innovative strategies are urgently needed.

[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. The host's resistance to Mtb infection requires complex coordination between the body and its immune system, as well as between innate immune cells 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 intracellular environment of the host and manipulate the activity of phagocytes to escape destruction, such as inhibiting phagosome acidification and fusion with lysosomes. Therefore, in-depth study of the pathogenic mechanism and immune escape strategies of Mtb is crucial for the development of new prevention and treatment strategies to curb this serious zoonosis.

[0004] In recent years, targeting host cell nuclei with effector proteins to regulate nuclear processes has become a new research topic for bacterial pathogens. These unique pathogenic effector proteins are called "nuclear modulators", which interact with specific host targets through various mechanisms and reprogram host cells to a niche 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 modulators in Mycobacterium. Therefore, screening and identifying Mtb nuclear modulators and in-depth exploring their mechanism of action in host cell nuclei is a new breakthrough point for studying pathogenic bacteria, which can lay the foundation for developing new vaccines and therapeutic drugs for pathogenic bacteria.

[0005] The present application takes Mycobacterium bovis as the research object, first, the conserved nucleic regulatory protein in Mycobacterium bovis is screened by using bioinformatics analysis technology combined with high content cell imaging system, it is found that the secreted protein MgdE can enter the host cell nucleus; then, taking the strain BCG (ATCC: 35734) as the research object, the influence of MgdE on the virulence of Mycobacterium bovis in the process of infecting the host is identified. 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 enters the host cell nucleus by relying on two nuclear localization signal peptides R 108-111 (R1) and RLRRPR 300-305 (R2); (3) MgdE can significantly inhibit the inflammation level in the host body after entering the host cell nucleus, 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 a new target to develop a new type of anti-tuberculosis vaccine or drug, and play a role in the prevention and treatment of animal and human tuberculosis. SUMMARY

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

[0007] In order to achieve the above purpose, the applicant analyzes the nucleic protein MgdE screened in the early stage, and identifies that the MgdE protein contains two nuclear localization signal peptides (NLS), which are KRIR 108-111 (R1) and RLRRPR 300-305 (R2), respectively. The MgdE protein enters the host cell nucleus with the assistance of R1 and R2. 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.

[0008] Then, the MgdE gene is amplified from the BCG genome and a recombinant plasmid is constructed, the HEK293T cells are transfected with the recombinant plasmid, and the nuclear entry ability of the MgdE protein is detected by qRT-PCR and Western blot experiment, it is confirmed that the protein encoded by the MgdE gene is a nucleic regulatory protein.

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

[0010] The knockout strain and each complementation strain were respectively infected into macrophages to detect the survival ability of M. bovis in host cells. The results showed that compared with the wild-type strain, the survival ability of the knockout strain BCG hyg::MgdE and the complementation strains Comp-MgdE-R1, Comp-MgdE-R2 and Comp-MgdE-R1 / 2 in macrophages was significantly weakened; compared with the knockout strain BCG hyg::MgdE, the survival ability of the complementation strains Comp-MgdE-R1, Comp-MgdE-R2 and Comp-MgdE-R1 / 2 in macrophages was significantly enhanced; compared with the complementation strains Comp-MgdE-R1 and Comp-MgdE-R2, the survival ability of the complementation strain Comp-MgdE-R1 / 2 in macrophages was significantly weakened. The results proved that the MgdE gene can promote the survival and colonization of the pathogenic bacteria in the host, thereby enhancing the virulence of the pathogenic bacteria, and knocking out the MgdE gene can reduce the survival and colonization of the pathogenic bacteria in the host, thereby reducing the virulence, and in this process, the nuclear localization signal peptides KRIR 108-111 (R1) and RLRRPR 300-305 (R2) played an important role.

[0011] In addition, the wild type strain BCG / pLJR965, the MgdE gene knockout strain KoMgdE, the MgdE gene complete complement strain Comp-MgdE, the R1 and R2 double deletion MgdE gene partial complement strain Comp-MgdE-R1 / 2 are respectively infected into mice, and the bacterial survival amount of lung tissue and the inflammatory factor level of spleen are detected. The bacterial survival number result shows that compared with the wild type strain BCG / pLJR965, the survival ability of the knockout strain KoMgdE and the gene partial complement strain Comp-MgdE-R1 / 2 in the mouse body is significantly reduced; compared with the knockout strain KoMgdE, the survival ability of the complement strain Comp-MgdE and Comp-MgdE-R1 / 2 in the mouse lung is obviously enhanced, and the complete complement strain Comp-MgdE is more significant; compared with the complete complement strain Comp-MgdE, the survival ability of the partial complement strain Comp-MgdE-R1 / 2 in the mouse lung is significantly reduced. The inflammatory level detection result shows that in the innate immune stage, compared with the wild type strain BCG / pLJR965 and the complete complement strain Comp-MgdE, the knockout strain KoMgdE and the partial complement strain Comp-MgdE-R1 / 2 can significantly induce the production of inflammatory factors (il1β, il6, il10) in the mouse spleen, indicating that the MgdE gene can significantly inhibit the non-specific immune response produced by the body after antigen stimulation, and the knockout of the MgdE gene or the modification of the NLS can improve the innate immunity of the body to the antigen.

[0012] In summary, the protein encoded by the MgdE gene is a nuclear regulatory protein, which is located in the host cell nucleus, and after entering the nucleus, it inhibits the inflammation level produced by the host and enhances the survival and colonization of the pathogenic bacteria in the host cells, so the MgdE protein is an important virulence factor of Mycobacterium bovis and depends on two NLS to play a role.

[0013] On this basis, the attenuated Mycobacterium bovis provided by the application is a recombinant Mycobacterium bovis with MgdE gene function loss or nuclear localization function loss, which is obtained by modifying the MgdE gene or the two NLS of the MgdE gene.

[0014] The application provides a preparation method of a weak strain of Mycobacterium bovis, which is characterized in that the MgdE gene in the genome of the Mycobacterium bovis is modified by a gene editing technology to make the MgdE gene function deficient, or the two NLSs of the MgdE gene are modified respectively or simultaneously by a gene editing technology to make the nuclear localization function of the NLSs deficient. For those skilled in the art, gene knockout or silencing is a mature technical means, and the gene editing technologies suitable for the application include but are not limited to homologous recombination (HR), clustered regularly interspaced short palindromic repeat-associated nuclease (CRISPR-Cas) technology, TALEN gene editing technology and Cre-loxP recombinase system.

[0015] The embodiment of the application also provides a specific implementation scheme for modifying the MgdE gene or the NLS sequence thereof based on the homologous recombination technology, which comprises the following steps: introducing an exogenous target gene into the genome of the Mycobacterium bovis by using the homologous recombination technology, replacing the MgdE gene at the original site with the DNA fragment of the exogenous target gene through homologous sequence exchange to obtain a Mycobacterium bovis strain in which the MgdE gene is replaced; then, connecting the DNA fragment of the MgdE gene in which the two NLSs are modified respectively or simultaneously with a complementation plasmid by enzyme digestion; and then, electrotransferring the DNA fragment into the Mycobacterium bovis strain in which the MgdE gene is replaced to obtain a MgdE gene complementation strain in which the two NLSs are modified respectively or simultaneously.

[0016] The weak strain of Mycobacterium bovis provided by the application can be used for preparing a Mycobacterium bovis vaccine, and compared with a common Mycobacterium bovis vaccine, the application has the advantages of good immunogenicity and high production safety.

[0017] The application first finds that the MgdE gene and the two nuclear localization signal peptides thereof can be used as important target points for inhibiting Mycobacterium bovis infection, have important application values in the preparation of Mycobacterium bovis bacillus Calmette-Guerin vaccine, and make important contributions to the exploration of the pathogenic mechanism and immune escape strategy of Mycobacterium bovis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 : qRT-PCR experiment for detecting the expression level of the target gene MgdE in HEK293T cells at different transfection time points (A) and Western blot experiment for detecting the content of the target protein MgdE in the nucleus of HEK293T cells at different transfection time points (B).

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

[0020] Figure 3 PCR verification results of knock-out strain BCG hyg::mgdE.

[0021] Figure 4 Sequencing results of DNA fragment of knock-out strain BCG hyg::mgdE amplified by MgdE upstream 360bp sequence forward primer and downstream 360bp reverse primer.

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

[0023] Figure 6 Wild, knock-out, and complemented strain survival number determination in THP-1 macrophages.

[0024] Figure 7 Wild, knock-out, and complemented strain survival number determination in mice.

[0025] Figure 8 Wild, knock-out, and complemented strain expression level determination of spleen inflammatory factors in mice at different time points. DETAILED DESCRIPTION

[0026] The technical solutions of the present application are further described in detail below in combination with specific examples. It should be understood that the following examples are only used to illustrate the present application and are not used to limit the protection scope of the present application. Various modifications or equivalent replacements made by those skilled in the art on the basis of the following examples should also be considered to fall within the protection scope of the present application. The experimental methods not specified in the following examples are generally carried out according to the conventional conditions or reference books such as “Molecular Cloning Laboratory Guide” (New York: Cold Spring Harbor Laboratory, 2017), or according to the methods suggested in the operation manual provided by the manufacturer. The materials not specified in the examples are all common materials known in the art, which can be constructed according to the literature reports or obtained through commercial channels.

[0027] Example 1, construction of MgdE recombinant plasmid and determination of nuclear entry ability

[0028] Firstly, the hypothetical protein MgdE was predicted by using the secreted protein analysis software SignalP-5.0, and it was found that MgdE was a secreted protein with Tat signal peptide ( https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ); then the NLS sequence on MgdE was identified according to the characteristics of the nuclear localization signal sequence (NLS) on the amino acid sequence of the nuclear regulatory protein (a highly conserved short sequence composed of several basic amino acids, usually containing arginine and lysine), and it was found that KRIR 108-111 (R1) and RLRRPR300-305 (R2) can be a NLS sequence which helps MgdE to enter the nucleus.

[0029] In this embodiment, the MgdE gene is cloned into the N-terminal of the pEGFP-N1 vector, transformed into the DH5a competent, and the recombinant expression strain is obtained; then the endotoxin-free plasmid extraction kit is used to obtain the fusion expression plasmid MgdE-pEGFP, which is transfected into the HEK293T cells in a 12-well plate, and the localization of the target protein in the host cells is observed by confocal fluorescence microscopy at 4, 12, 24, 36 and 48 hours after transfection. The specific operation steps are as follows:

[0030] 1. Amplification of the target gene fragment: First, query the MgdE gene information on GenBank and download the gene sequence, use BioEdit software to query the enzyme cutting site inside the MgdE gene, according to the available enzyme cutting sites (NheI and Hind3) on the fluorescent reporter vector EGFP, design the forward primer (MgdE-F) and the reverse primer (MgdE-R) (Table 1) from the front and back of the coding gene, each taking 18-20bp, and amplify the MgdE gene sequence with BCG genome as the template; the PCR amplification system is 50μL, and the reaction conditions and the reaction system are shown in the following tables (Table 2, Table 3).

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

[0032]

[0033] Table 2 PCR amplification system of MgdE gene

[0034]

[0035] Table 3 PCR reaction conditions for amplification of MgdE gene

[0036]

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

[0038] (1) DNA double enzyme digestion system

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

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

[0041]

[0042] (2) 37℃ reaction for 3-4 hours;

[0043] (3) Purify and recover the DNA fragments after enzyme digestion for enzyme ligation reaction.

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

[0045] (1) Enzyme ligation 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 the target gene fragment and the vector

[0048]

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

[0050] 4. Transformation: transform the ligation product into DH5a competent cells, culture at 37°C for 16 hours, pick single transformants, culture in 5 mL LB liquid medium containing 30 μg / mL Kan (kanamycin) for 8 hours, extract plasmid, and obtain recombinant plasmid pEGFP-mgdE;

[0051] 5. Cell transfection: first, plate HEK293T cells in a 12-well plate at a cell number of 2 x 10 5 6 hours before transfection, remove the cell supernatant and add 800 μL Opti-MEM medium; then, during the infection process, configure the DNA-Hieff TransTM liposome nucleic acid transfection reagent complex, wherein 1 μg of DNA diluted with 100 μL of Opti-MEM and 4.5 μL of Hieff TransTM liposome nucleic acid transfection reagent diluted with 100 μL of Opti-MEM are used for each cell in the hole, and after incubation at room temperature for 20 minutes, they are added to the cell hole plate, and 1 mL of DMEM + 10% FBS medium is added 6 hours after transfection, and then the ability of MgdE to enter the host cell nucleus is detected by qRT-PCR and Western blot at 4 hours, 12 hours, 24 hours, 36 hours, and 48 hours after transfection.

[0052] 6. qRT-PCR detection of 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. The Western blot experiment detects the content of the target protein MgdE entering the HEK293T cell nucleus at different transfection times. The cell nucleus protein and cytoplasm protein extraction kit are purchased from Biyun Tian Biological Company. Histone H3 is used as the detection index of the cell nucleus reference protein.

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

[0055]

[0056] The experimental results show that: (1) The qRT-PCR experiment results show that the expression level of the target gene mgdE in the host cell also gradually increases with the increase of the transfection time ( Figure 1 A); (2) The Western blot experiment results show that the accumulation of the target protein MgdE in the cell nucleus also gradually increases with the increase of the transfection time ( Figure 1 B).

[0057] Example Two, Construction of MgdE Gene Knockout Strain

[0058] In this experiment, the DNA homologous recombination technology is used to modify the endogenous gene, so that the exchange occurs between the exogenous DNA and the homologous sequence of the target gene on the receptor genome DNA, the exogenous DNA is integrated into the predetermined position of the target gene, the precise modification and transformation of the target gene are completed, the gene function is lost, and the modified and transformed gene can be stably replicated with the replication of the genome DNA. The main steps of gene knockout are: (1) Construction of knockout vector plasmid, cloning the homologous sequence of the target gene to the vector with a marker gene (usually an antibiotic gene, such as hygromycin hyg); (2) Transformation of the knockout vector plasmid into the receptor bacteria; (3) Screening of the target gene knockout recombinant strain with a selective medium; (4) Identification of the target gene deletion strain.

[0059] The specific implementation steps are as follows:

[0060] 1. Primer design: Query MgdE gene information on GenBank and download gene sequence, use BioEdit software to query enzyme cutting sites in the coding MgdE gene, according to the available enzyme cutting sites on the pMind-LacZ vector (Chen L et al., 2022), determine that the Pac I, Spe I, Hind III and Nhe I enzyme cutting sites on the MgdE gene sequence are available, select the upstream and downstream 600 bp sequences of the target gene sequence, and amplify the MgdE gene upstream about 600 bp fragment (enzyme cutting sites are Pac I and Spe I) (UP), downstream 600 bp fragment (enzyme cutting sites are Hind III and Nhe I) (DN) with BCG genome as template, at the same time construct pMind vector treated by Pac I and Spe I restriction endonuclease, MgdE gene upstream and downstream 600 bp sequence amplification primers are shown in Table 7 below.

[0061] Table 7 List of primers in PCR experiment

[0062]

[0063] 2. Clone the upstream fragment into the multiple cloning site of the pMind vector, then double enzyme cut the pMind-LacZ-UP vector with Hind III and Nhe I restriction endonuclease, connect the downstream fragment, and constitute the complete knockout vector pMind-LacZ-UP-hyg-DN. This vector has the following characteristics: first, it is a suicide vector and cannot replicate autonomously in mycobacteria; second, the successfully constructed knockout strain has three screening markers: (1) with Hyg resistance; (2) the reporter gene LacZ will make the strain blue in X-gal environment; (3) when the recombinant vector double exchanges with the genome, the strain Kan resistance will be lost, that is, Kan is a negative screening marker. The specific operation is as follows:

[0064] (1) Upstream fragment cloning: first, according to the designed mgdE-UP 600 primer sequence, amplify the mgdE-UP 600 fragment with Pac I and Spe I enzyme cutting sites with BCG genome as template; then, use Pac I and Spe I to double enzyme cut the DNA fragment and pMind-LacZ, respectively, to obtain mgdE-UP 600 (Pac I / Spe I) and pMind-LacZ (Pac I / Spe I) DNA fragments (enzyme cutting system is shown in Table 8 below); then, connect pMind-LacZ (Pac I / Spe I) and mgdE-UP 600 (Pac I / Spe I) fragments in a ratio of 1:3, transform; finally, obtain single transformants, expand culture to extract plasmid, and obtain recombinant plasmid pMind-LacZ-UP600 ;

[0065] (2) Downstream fragment ligation: First, according to the designed mgdE-DN 600 primer sequence, the mgdE-DN 600 fragment with Hind III and Nhe I restriction sites was amplified from the BCG genome as a template; then, the DNA fragment and the recombinant plasmid pMind-LacZ-UP 600 obtained in the previous step were double-digested with Hind III and Nhe I, to obtain mgdE-DN 600 (Hind III / Nhe I) and pMind-LacZ-UP 600 (Hind III / Nhe I) DNA fragments (see Table 8 below for the enzyme digestion system); next, the mgdE-DN 600 (Hind III / Nhe I) and pMind-LacZ-UP 600 (Hind III / Nhe I) DNA fragments were ligated at a ratio of 1:3, and transformed; finally, a single transformant was obtained, and the plasmid was extracted by expanding culture, to obtain the recombinant plasmid pMind-LacZ-UP 600 -hyg-DN 600 The plasmid map is shown in Figure 2 .

[0066] Table 8 Enzyme digestion system

[0067]

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

[0069] 4. A white single colony was picked from the plate, cultured in 7H9 medium containing 10% OADC and 50 μg / mL Hyg, and a portion of the activated bacterial liquid was transferred to 7H9 medium containing 30 μg / mL Kan. If the bacteria lost Kan resistance, it was likely to 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 related primers), and amplified the target DNA fragment of the strain that passed the PCR verification, and sent it to a sequencing company (Qingke Biological) for sequencing.

[0071] Table 9 Primer sequence for PCR verification

[0072]

[0073] Experimental results: (1) PCR results show that in wild-type strain BCG / wt, hyg (999 bp) is not amplified, but mgdE (945 bp) can be amplified; in knockout strain BCG hyg: mgdE, hyg (999 bp) can be amplified, but mgdE (945 bp) cannot be amplified; at the same time, when the forward primer is designed with the 600 bp sequence upstream of the target gene mgdE and the reverse primer is designed with the 600 bp sequence downstream, it is found that in wild-type strain BCG / wt, a 2145 DNA fragment can be amplified, and in knockout strain BCG hyg::mgdE, a 2199 bp DNA fragment can be amplified (2), indicating that the hyg gene successfully replaces the mgdE gene on the BCG genome, and the BCG hyg::mgdE knockout strain is successfully constructed. Figure 3

[0074] (2) Further, the 2199 bp DNA fragment amplified in the knockout strain was sent to a biological company for sequencing, and sequencing primers were designed with 360 bp upstream and downstream of the target gene (Table 10), and the results showed that the hyg gene sequence could be successfully measured (3), indicating that the BCG hyg::mgdE knockout strain was successfully constructed. Figure 4

[0075] Table 10 Sequencing primer sequences of knockout strain

[0076]

[0077] Example Three, Construction of Complementation Strain

[0078] In order to further explore the role of MgdE and nuclear localization signal sequence KRIR 108-111 (R1), RLRRPR 300-305 (R2) in the process of BCG virulence, we used the pLJR965 vector to construct the following series of recombinant strains:

[0079] (1) BCG / pLJR965: In wild-type strain BCG, the empty vector pLJR965 plasmid is electroporated to obtain a wild-type strain BCG in which the mgdE gene is not mutated, which is used as a control in the experiment;

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

[0081] ​​(3) Complementary strain BCG hyg::mgdE / pLJR965-mgdE(Comp-MgdE): Based on the pLJR965 complementation vector, the fusion expression plasmid pLJR965-mgdE was constructed and electroporated into the knockout strain BCG hyg::mgdE, resulting in a BCG strain with the mgdE gene completely complemented.

[0082] (4) Replacement strain BCG hyg::mgdE / pLJR965-mgdE-R1(Comp-MgdE-R1): KRIR was obtained through gene point mutation technology. 108-111 The (R1) amino acid sequence of MgdE DNA was deleted and enzymatically ligated into 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 fusion expression plasmid. 300-305 (R2) The mgdE gene of amino acids was partially replenished in the BCG strain.

[0083] (5) Replacement strain BCG hyg::mgdE / pLJR965-mgdE-R2 (Comp-MgdE-R2): RLRRPR was obtained through gene point mutation technology. 300-305 The (R2) amino acid sequence of MgdE DNA was deleted and enzymatically ligated into 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 plasmid. 108-111 (R1) The mgdE gene of amino acids was partially replenished in the BCG strain.

[0084] (6) Replacement strain BCG hyg::mgdE / pLJR965-mgdE-R1 / 2(Comp-MgdE-R1 / 2): KRIR was obtained through gene point mutation technology. 108-111 (R1) and RLRRPR 300-305 The (R2) amino acid sequence of double-deleted MgdE DNA was enzymatically ligated into 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-deleted KRIR. 108-111 (R1) and RLRRPR 300-305 (R2) The mgdE gene of amino acids was partially replenished in the BCG strain.

[0085] The specific construction method is as follows:

[0086] During the experiment, the construction of the vector used seamless cloning technology, and the amplification of the point mutation DNA fragment used overlap extension PCR technology. The principle of seamless cloning is to design 15-25 homologous bases (homologous arms) at the end of the vector and the end of the primer. Through T5 exonuclease, DNA polymerase, DNA ligase, the three enzymes function at the same time, so as to realize the connection of single fragment or multiple fragments with the vector. The principle of overlap extension PCR is to use primers with complementary ends, so that the PCR product forms a small overlapping chain, and then through the extension of the overlapping chain in the subsequent amplification reaction, the different source amplification fragments are overlapped and spliced.

[0087] 1. pLJR965 vector linearization (pLJR965 vector specific information 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 with pLJR965 vector as template, and then the linearized pLJR965 vector was obtained by overlap extension PCR (primer information see Table 11 below);

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

[0090] (1) MgdE gene fragment amplification: first, obtain the mgdE gene and its upstream 350 bp DNA sequence from GenBank and KEGG database (www.kegg.jp); then, according to the information of linearized pLJR965 vector, design mgdE primers (mgdE-F, mgdE-R) containing pLJR965 vector homologous arms (primer sequences see Table 11 below); then, with BCG genome as template, PCR amplification was carried out to obtain MgdE DNA fragment (the upstream 350 bp sequence of mgdE contains the gene itself promoter, and the target gene needs to carry the promoter to start the transcription of the gene when using pLJR965 vector cloning);

[0091] (2) Construction of MgdE-R1 gene truncated mutant fragment: first, determine the KRIR 108-111(R1) The base sequence of mgdE is aagcggatccgg; then, a sequence of 15-18 bp of the left and right fragments (mgdE-R1L, mgdE-R1R) selected on the basis of the sequence is synthesized as a forward primer (mgdE-R1R-F) of the truncated mutant fragment MgdE-R1R, and the reverse complement is used as a reverse primer (mgdE-R1L-R) of the truncated mutant fragment MgdE-R1L (see Table 11 below for primer information); then, the BCG genome is used as a template, and mgdE-F / mgdE-R1L-R and mgdE-R1R-F / mgdE-R are used as primers to amplify to obtain KRIR 108-111 (R1) The base sequence of mgdE is aagcggatccgg; then, a sequence of 15-18 bp of the left and right fragments (mgdE-R1L, mgdE-R1R) selected on the basis of the sequence is synthesized as a forward primer (mgdE-R1R-F) of the truncated mutant fragment MgdE-R1R, and the reverse complement is used as a reverse primer (mgdE-R1L-R) of the truncated mutant fragment MgdE-R1L (see Table 11 below for primer information); then, the BCG genome is used as a template, and mgdE-F / mgdE-R1L-R and mgdE-R1R-F / mgdE-R are used as primers to amplify to obtain KRIR

[0092] (3) Construction of the truncated mutant fragment of mgdE-R2 gene: first, the RLRRPR 300-305 (R2) The base sequence of mgdE is cggctgcggcggccccgg; then, a sequence of 15-18 bp of the left and right fragments and 16 bp of the homologous arm of pLJR965LR is synthesized, and the reverse complement is used as a reverse primer (mgdE-R2-R) of the truncated mutant fragment MgdE-R2 (see Table 11 below for primer information); then, the BCG genome is used as a template, and mgdE-F / mgdE-R2R is used as a primer to amplify to obtain the truncated mutant fragment MgdE-R2.

[0093] (4) Construction of the truncated mutant fragment of mgdE-R1 / 2 gene: first, on the basis of the construction of the truncated mutant fragments MgdE-R1 and MgdE-R2, the BCG genome is used as a template, and mgdE-R1R-F / mgdE-R2-R is used as a primer to amplify the DNA fragment (MgdE-R1 / 2M) between the R1 and R2 mutant sequences; then, the overlapping extension PCR of MgdE-R1 / 2M and the fragment MgdE-R1L is performed to obtain the truncated mutant fragment 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 linearized pLJR965 vector using Uniclone One Step Seamless Cloning Kit (Jinsa SC612) (see Table 16 below for the reaction system), and then placed at 50°C for 30 minutes and on ice for 2 minutes; then, the reaction product was transformed into DH5a competent cells and plated on LB solid medium containing 30 μg / mL Kan and incubated at 37°C for 16 hours; finally, a single transformant was picked and cultured in 5 mL LB liquid 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 and incubated at 37°C for 14 days, and then a single colony was picked and cultured in 5 mL 7H9 liquid medium containing 10% OADC and 30 μg / mL Kan until the OD 600 ≈1.0 for the experiment.

[0098] 5. Identification

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

[0100] Table 11 List of primers in PCR experiment

[0101]

[0102]

[0103] Overlap extension PCR steps (using MgdE-R1 as an example, the remaining fragment operation steps are the same) Step one:

[0104] Table 12 Overlap extension PCR amplification system 1

[0105]

[0106] Table 13 Overlap extension PCR reaction conditions 1

[0107]

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

[0109] Step two:

[0110] Table 14 Overlap extension PCR reaction conditions 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 sequence list in qRT-PCR experiment

[0118]

[0119] The qRT-PCR experiment results show that the mgdE gene cannot be detected in the knockout strain; the complementation efficiency of MgdE, MgdE-R1, MgdE-R2, and MgdE-R1 / 2 in the complementation strain is 91.9%, 95.1%, 93.6%, and 91.6%, respectively, indicating that the mgdE gene complementation is successful. The error value represents the standard deviation of three biological repeated experiments, and the P value is calculated using the unpaired two-tailed Student's t test in GraphPad Prism 7. The asterisk (*) represents a significant difference between the two groups of data: *, p < 0.1; **, p < 0.01; ***, p < 0.001. Figure 5 ).

[0120] Example five, detection of survival ability of mutant strains in macrophage THP-1

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

[0122] The collected cell samples were gradient diluted (5 -1 , 5 -2 , 5 -3 ) with PBS, and 100 μL was spread in 7H10 culture 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] As shown in Figure 6 , the experimental results show that:

[0124] (1) Compared with the wild-type strain, the survival ability of the knockout strain KoMgdE and the partial complementation strains Comp-MgdE-R1, Comp-MgdE-R2, and Comp-MgdE-R1 / 2 in host cells was significantly decreased. It is shown that MgdE can enhance the survival ability of pathogenic bacteria in the host, and the process depends on the assistance of nuclear localization signal sequence (R1, R2);

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

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

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

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

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

[0130] Example Six, Detection of 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-Rl / 2) were inoculated into 5 mL of 7H9 liquid medium containing 30 μg / mL Kan and cultured. When OD 600 ≈1.0, 1x10 7 cfu / 40 μL was used to infect C57BL / 6 female mice (bacterial infection method: drop nasal treatment; mouse quality: SPF level, 6-8 weeks, 16-18 g), with 6 biological replicates in each group, and the infection time was 2, 14, 21, 28, and 56 days. At different time points of infection, lung and spleen tissues were taken from the mice, and the lung tissue was subjected to bacterial survival number detection, and the spleen sample was subjected to inflammation factor expression level detection.

[0132] Lung bacterial survival number detection: first, the lung sample of the infected mouse was ground; then, 1 mL of PBS was used for gradient dilution (5 -1 , 5 -2 , 5 -3 ) of the sample, and 100 μL was taken and coated on 7H10 medium containing 10% OADC and 30 μg / mL Kan, and cultured at 37°C for 14 days; finally, the survival number of each strain in the lung of each mouse at each infection time point was counted.

[0133] Detection of the level of spleen inflammation: take the spleen sample of the infected mice, extract the tissue RNA, and after reverse transcription into cDNA, detect the expression of spleen inflammatory factors (il1β, il6, il10) by qRT-PCR (see Table 18 for identification of primers, wherein hprt is the intracellular reference gene). Among them, the tissue sample RNA extraction step and the reagents used are the same as Aidlab Biological Company (RN2802), and the RNA reverse transcription operation step is the same as Nuozhan Reverse Transcription Kit (R423-01).

[0134] The results of the number of bacteria survival show 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 is significantly reduced; compared with the knockout strain KoMgdE, the survival ability of the recombinant strains Comp-MgdE and Comp-MgdE-R1 / 2 in the lungs of mice is significantly enhanced, and Comp-MgdE is more significant; compared with the complement strain Comp-MgdE, the survival ability of the partial complement strain Comp-MgdE-R1 / 2 in the lungs of mice is significantly reduced Figure 7 ).

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

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

[0137] Table 18 Primer sequence list in qRT-PCR experiment

[0138]

Claims

1. Use of a weak strain of Mycobacterium bovis for the manufacture of a vaccine against Mycobacterium bovis, characterized in that: The two nuclear localization signal peptides of the MgdE gene of Mycobacterium bovis are simultaneously knocked out by using a gene editing technology, so that the nuclear localization function is lost to obtain an attenuated strain, the nucleotide sequence of the MgdE gene is shown as SEQ ID NO. 1, the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2, the two nuclear localization signal peptides are located at positions 108-111 and 300-305 of the amino acid sequence shown in SEQ ID NO. 2, and the attenuated strain of Mycobacterium bovis can induce a non-specific immune response of the body and inhibit the survival and colonization of pathogenic bacteria in the host.

2. Use of a weak strain of Mycobacterium bovis for the manufacture of a vaccine against Mycobacterium bovis, characterized in that, The preparation method of the attenuated strain of Mycobacterium bovis is as follows: an exogenous target gene is introduced into the genome of Mycobacterium bovis by using a homologous recombination technology, 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 a DNA fragment of the MgdE gene in which the two nuclear localization signal peptides are simultaneously deleted is ligated with a complementation plasmid, and then electroporation is performed on the above-mentioned Mycobacterium bovis strain in which the MgdE gene is replaced to obtain a MgdE gene complementation strain in which the two nuclear localization signal peptides are simultaneously deleted, the nucleotide sequence of the MgdE gene is shown as SEQ ID NO. 1, the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2, the two nuclear localization signal peptides are located at positions 108-111 and 300-305 of the amino acid sequence shown in SEQ ID NO. 2, and the attenuated strain of Mycobacterium bovis can induce a non-specific immune response of the body and inhibit the survival and colonization of pathogenic bacteria in the host.

3. Use according to claim 2, wherein: The exogenous gene of interest is hyg Resistance genes.

4. The use according to claim 2, characterized in that: The complementation plasmid is a pLJR965 plasmid.

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