A genetically modified attenuated strain of Mycobacterium bovis, its construction method and application

Abstract

This invention discloses a genetically modified attenuated strain of Mycobacterium bovis, its construction method, and its applications. Utilizing the principle of homologous recombination, the RD1 region of the genome is knocked out and membrane protein genes are inserted to construct a candidate strain for an attenuated live vaccine. This strain can be used as an immunotherapeutic agent for bladder cancer or as an attenuated live vaccine for tuberculosis. This invention is based on the virulent strain of Mycobacterium bovis AF2122 / 97. By genetically modifying it to knock out its RD1 region to reduce its virulence while retaining cell wall antigens (such as MPB70 / 83), and simultaneously inserting antigen fragments related to the deleted region, it enhances its immunogenicity.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a genetically modified attenuated strain of Mycobacterium bovis, its construction method, and its application. Background Technology

[0002] BCG is the only vaccine approved to date for the prevention of tuberculosis. It is made from attenuated Mycobacterium bovis and is highly effective in preventing severe tuberculosis in children, such as miliary tuberculosis and tuberculous meningitis. However, BCG's protective effect against adult pulmonary tuberculosis, the most common and primary source of tuberculosis transmission, is limited and inconsistent. Its protective efficacy decreases with age and it offers almost no protection against the onset of tuberculosis in individuals already infected with latent tuberculosis.

[0003] Gene knockout vaccines are a novel class of biological agents that utilize gene editing technologies (such as CRISPR-Cas9 and homologous recombination) to selectively delete virulence genes in pathogens, constructing live attenuated vaccines. Their core principle is to precisely knock out genes closely related to pathogenicity (such as the mntA gene in Bacillus anthracis and the P52 / P36 / SAP1 genes in Plasmodium), causing the pathogen to lose its core ability to proliferate or cause disease in the host while retaining its immunogenicity, thereby stimulating the host to produce potent and long-lasting immune protection. This strategy overcomes the limitations of traditional live attenuated vaccines that rely on random mutation or passage culture, achieving a rational design of "controllable attenuation and highly effective immunity." Despite its promising prospects, this technology still faces challenges: balancing the degree of attenuation with immunogenicity (excessive attenuation may lead to decreased protective efficacy), optimizing delivery systems (such as the stability of exosome vectors), and advancing clinical trials to verify human safety and long-term efficacy. Future applications will be expanded in the prevention and control of drug-resistant pathogens and emerging infectious diseases through strategies such as multi-gene synergistic knockout, combined adjuvants, or immunomodulatory factors. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a genetically modified attenuated strain of Mycobacterium bovis.

[0005] Another objective of this invention is to provide a method for constructing the above-mentioned genetically modified attenuated strain of Mycobacterium bovis.

[0006] Another object of the present invention is to provide the application of the above-mentioned genetically modified attenuated strain of Mycobacterium bovis.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A genetically modified attenuated strain of Mycobacterium bovis was obtained by knocking out the RD1 region in the genome of Mycobacterium bovis and then inserting the complement antigen sequence into the knockout region to restore its immunogenicity.

[0009] The bovine tuberculosis mycobacterium mentioned is bovine tuberculosis mycobacterium AF2122 / 97.

[0010] The RD1 region was knocked out after the knockout plasmid was transferred into Mycobacterium bovis via phage-mediated knockout.

[0011] The nucleotide sequence of the RD1 region is shown as 435490 to 439888 bp of the genome sequence of Mycobacterium bovis AF2122 / 97 with NC_002945.4 in the NCBI database.

[0012] The complement antigen sequence is obtained by sequentially linking the signal peptide and the M75 fragment.

[0013] The signal peptide is at least one of signal peptide p1, signal peptide p2, and signal peptide p3.

[0014] The nucleotide sequence of the signal peptide p1 is shown in SEQ ID NO.2.

[0015] The nucleotide sequence of the signal peptide p2 is shown in SEQ ID NO.3.

[0016] The nucleotide sequence of the signal peptide p3 is shown in SEQ ID NO.4.

[0017] The M75 fragment is obtained by sequentially linking the sequences of membrane protein Rv1508c (106-547) and membrane protein Rv3888c (85-341); preferably, it is sequentially linked by (GGGGS). n The sequence is obtained by concatenating the sequences; more preferably, (GGGGS). n n=1 in the sequence.

[0018] The nucleotide sequence of the M75 fragment is shown in SEQ ID NO.1.

[0019] A method for constructing a genetically modified attenuated strain of Mycobacterium bovis includes the following steps:

[0020] (1) Design primers to amplify the left and right arms of the RD1 region, and ligate them with the linearized p0004s plasmid to obtain the p0004s-AES plasmid;

[0021] (2) After digesting phAE159 and p0004s-AES plasmids with enzymes, they were ligated, transformed into Escherichia coli, screened and sequenced to verify positive clones, namely phAE159-AES phage particles.

[0022] (3) PhAE159-AES phage particles were transferred into Mycobacterium smegmatis, and after culture, phage plaques were picked and added to fresh Mycobacterium smegmatis for culture. After filtration, high-titer phages were obtained.

[0023] (4) High-titer bacteriophages were mixed with Mycobacterium bovis, and after culturing, screening, and sequencing verification, Mycobacterium bovis with the RD1 region knocked out was obtained;

[0024] (5) Amplify the nucleotide sequence fragments of the signal peptide and M75, link them sequentially to the linearized pMV361 plasmid, transform them into E. coli, and obtain positive clones after screening and sequencing verification, namely pMV361-M75 plasmid.

[0025] (6) The pMV361-M75 plasmid was transferred into Mycobacterium bovis with the RD1 region knocked out. After culturing, screening and sequencing verification, a genetically modified attenuated Mycobacterium bovis strain was obtained.

[0026] The bovine tuberculosis mycobacterium mentioned is bovine tuberculosis mycobacterium AF2122 / 97.

[0027] The aforementioned Mycobacterium smegmatis is Mycobacterium smegmatis mc 2 155.

[0028] The signal peptide is at least one of signal peptide p1, signal peptide p2, and signal peptide p3.

[0029] The nucleotide sequence of the signal peptide p1 is shown in SEQ ID NO.2.

[0030] The nucleotide sequence of the signal peptide p2 is shown in SEQ ID NO.3.

[0031] The nucleotide sequence of the signal peptide p3 is shown in SEQ ID NO.4.

[0032] The M75 fragment is obtained by sequentially linking the nucleotide sequences of membrane protein Rv1508c (106-547) and membrane protein Rv3888c (85-341); preferably, it is sequentially linked by (GGGGS). n The sequence is obtained by concatenating the sequences; more preferably, (GGGGS). n n=1 in the sequence.

[0033] The nucleotide sequence of the M75 fragment is shown in SEQ ID NO.1.

[0034] A genetically modified attenuated strain of Mycobacterium bovis was constructed using the method described above.

[0035] A live attenuated tuberculosis vaccine, prepared by including the above-mentioned genetically modified attenuated strain of Mycobacterium bovis.

[0036] The live attenuated tuberculosis vaccine is prepared by diluting the bacterial cells obtained from culturing the above-mentioned genetically modified attenuated Mycobacterium bovis strain to 0.1-1 mg / mL.

[0037] The above-mentioned genetically modified attenuated strains of Mycobacterium bovis were used in the preparation of tuberculosis vaccines.

[0038] The above-mentioned genetically modified attenuated strain of Mycobacterium bovis was used in the preparation of drugs for treating bladder cancer.

[0039] The present invention has the following advantages and effects compared with the prior art:

[0040] Mycobacterium tuberculosis membrane proteins play a dual role in infection and immunity: they are both major targets for host immune recognition and key tools for bacterial escape and survival. As potent immunogens, they are recognized by host antigen-presenting cells (APCs), activating CD4+ / CD8+ T cell-mediated cellular immunity. This invention utilizes homologous recombination to modify the genome, achieving RD1 region gene knockout and membrane protein gene insertion to construct a live attenuated vaccine candidate strain. This strain could serve as an immunotherapeutic agent for bladder cancer treatment or a live attenuated vaccine for tuberculosis.

[0041] This invention is based on the highly virulent strain of Mycobacterium bovis AF2122 / 97. By genetically modifying it to knock out its RD1 region to reduce its virulence, but retaining cell wall antigens (such as MPB70 / 83), and simultaneously reintroducing related antigen fragments into the deleted region to enhance its immunogenicity. Attached Figure Description

[0042] Figure 1 This is the plasmid map of the complementation vector pMV361-M75-p1 constructed in Example 4;

[0043] Figure 2 These are photographs of samples cultured from the strains in Example 5; from left to right, they are BCG, M. bovis-ΔRD1, M. bovis-ΔRD1::M75p1, M. bovis-ΔRD1::M75p2, and M. bovis-ΔRD1::M75p3.

[0044] Figure 3 These are the experimental results of immunogenicity assessment in Example 6; where A to C are the results of ELISA detection of IgG, IgG1, and IgG2a, respectively, and D is the result of ELISPOT detection of IFN-γ;

[0045] Figure 4 This is a schematic diagram of the experimental procedure in Example 7;

[0046] Figure 5These are photographs of the internal organs of the guinea pig after dissection in Example 7; from top to bottom, they are BCG, M.bovis-ΔRD1, M.bovis-ΔRD1::M75p1, M.bovis-ΔRD1::M75p2, and M.bovis-ΔRD1::M75p3.

[0047] Figure 6 This is a graph showing the statistical results of bacterial load in the major organs of the guinea pig after dissection in Example 7, with the unit being CFU (Log 10); where A is the bacterial load in the lungs and B is the bacterial load in the spleen;

[0048] Figure 7 These are the results of quantitative analysis of the histopathological lesion index of the major organs after guinea pig dissection in Example 7; where A is the pathological index of the liver, B is the pathological index of the spleen, and C is the pathological index of the lungs. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0050] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0051] All biological materials used in this invention were purchased from publicly available commercial channels. Among them, Mycobacterium bovis AF2122 / 97 was purchased from Qifa Biotechnology, and Mycobacterium smegmatis mc2155, phAE159, p0004s-AES, and pMV361 were all purchased from Jingnuo Biotechnology.

[0052] Example 1: Construction of the shuttle carrier

[0053] 1.1 Construction of p0004s-AES plasmid

[0054] Using the virulent strain of Mycobacterium bovis AF2122 / 97 as a template, two pairs of primers targeting the left and right arms of the RD1 region were designed. The left and right arms were amplified by Hi-Fi DNA Polymerase PCR. The target DNA fragments were recovered using a DNA gel recovery kit and digested with AdeI restriction endonuclease. The DNA fragments after gel recovery and digestion were then used for future reference.

[0055] RD1 left and right arm primers:

[0056] LFP-B:TTTTTTTTCACAAAGTGATTGCGCACCACCAGCTCTC;

[0057] LRP-B: TTTTTTTTCACTTCGTGTGAACGTTGCGCGGTGAGTGTGT;

[0058] RFP-B: TTTTTTTTCACAGAGTG AGGTGTCCAATACATCGGTGAC;

[0059] RRP-B: TTTTTTTTCACCTTGTG AGTGGCTCACCGGCCTGCGCATC;

[0060] The p0004s plasmid was extracted using a plasmid extraction kit and digested with Van91I restriction endonuclease. The digested DNA fragment was then recovered from the gel and kept for later use.

[0061] The DNA fragments obtained in the above two steps were ligated using T4 DNA Ligase and transformed into E. coli DH5α competent cells. The cells were plated on LB agar plates containing 150 μg / mL hygromycin and cultured overnight. Single colonies were picked and inoculated into LB liquid medium containing 150 μg / mL hygromycin and cultured at 37°C. Plasmids were extracted, and positive clones were confirmed by sequencing and stored for later use.

[0062] 1.2 Construction of phAE159-AES shuttle plasmid

[0063] Positive plasmids phAE159 and p0004s-AES were extracted using a plasmid extraction kit. All obtained plasmids were digested with PacI and the target fragments were recovered. The two linearized plasmid fragments were ligated using T4 DNA Ligase. The resulting fragments were then transformed into E. coli HB101 competent cells using a phage packaging kit. The cells were plated on 150 μg / mL LB agar plates and cultured overnight. Single colonies were picked and inoculated into 150 μg / mL LB broth and cultured at 37°C. Plasmids were extracted, and positive clones were sequenced and stored for later use.

[0064] Example 2 Preparation of bacteriophage

[0065] 2.1 Mycobacterium smegmatis mc 2 155 Preparation of Electrocompetent Cells

[0066] Select fresh Mycobacterium smegma mc 2155 single colonies were inoculated into 5 mL of complete medium and incubated statically at 37°C until the logarithmic growth phase (OD 0.5–1.0, approximately 1–2 days). The culture was then inoculated into 100 mL of complete medium at a ratio of 1:100 and incubated overnight at 37°C until the OD600 reached approximately 0.6. After incubating the culture on ice for 0.5–1 h, the cells were collected by centrifugation at 5000 rpm for 10 min at 4°C. The cells were washed at least twice with 10% sterile glycerol in an ice bath, and finally 10 mL of pre-cooled 10% glycerol was added. After mixing the cells, 200 μL of each tube was dispensed and stored at -80°C for later use.

[0067] 2.2 Phage amplification

[0068] Take 2 μg of the phAE159-AES shuttle plasmid prepared in Example 1 and add it to Mycobacterium smegma mc 2 Mix the 155g of the bacterial culture with the competent cells and transform them using a Bio-rad electroporator with a 2mm electroporation cup (electroporation parameters: voltage 2.5 kV, resistance 1000Ω, capacitance 25 μF). After electroporation, add 7H9 complete medium and incubate overnight at 37°C. Then, mix the bacterial culture with an appropriate amount of topagar and plate it onto mycobacterial solid medium. After incubating at 30°C for 2–3 days, screen for phage plaques.

[0069] Pick empty plaques containing bacteriophages from the plate and add them to MP buffer. Incubate overnight at 4°C. Then, mix this bacteriophage-containing liquid with an appropriate amount of freshly cultured Mycobacterium smegma. 2 After mixing 155 bacteria, mix with an appropriate amount of Top Agar and plate. Incubate the plates at 30°C for 2–3 days. Add an appropriate amount of MP buffer to the plates with plaques and incubate overnight at 4°C. Collect and filter the high-titer phages using a 0.22µm sterile filter, and store at 4°C for later use.

[0070] Example 3 Construction of RD1 knockout strain

[0071] A suitable amount of high-titer phage lysis buffer was mixed with *Mycobacterium bovis* AF2122 / 97 (pre-washed with MP buffer) grown to the logarithmic phase. The mixture was incubated overnight at 37°C in the dark, then centrifuged and the supernatant was discarded. An appropriate amount of 7H10 complete medium was added, and the mixture was incubated overnight at 37°C. The bacterial cells were then collected, centrifuged, and the supernatant was discarded. The cells were spread onto mycobacterial solid medium (75 μg / mL hygromycin) and cultured at 37°C for 4–6 weeks. Single clones were picked, and whole-genome DNA was extracted after culturing. The target band was recovered by PCR amplification, and sequencing verification confirmed that the RD1 region had been knocked out. The positive clone strain was named *M. bovis* ΔRD1 strain.

[0072] Example 4 Construction of complement strain

[0073] After knocking out the RD1 region, the virulence of Mycobacterium tuberculosis decreased significantly, but its immunogenicity also decreased. In this experiment, we designed a complement sequence to insert into the deleted region to restore its immunogenicity.

[0074] 4.1 Preparation of electrocompetent cells

[0075] Pick a fresh single colony of M. bovis ΔRD1 and inoculate it into 5 mL of complete medium (containing the corresponding antibiotic), and incubate at 37°C until the logarithmic growth phase (OD 0.5–1.0, about 2–3 weeks). Inoculate the culture at a ratio of 1:100 into 100 mL of complete medium (containing the corresponding antibiotic), and incubate at 37°C until the OD 600 reaches about 0.6. Centrifuge the culture at 5000 rpm for 10 min at room temperature to collect the cells. Wash the cells at least twice with 10% sterile glycerol, and finally add 10 mL (appropriate amount) of 10% glycerol, mix the cells, and then divide into 200 μL tubes and freeze at -80°C for later use.

[0076] 4.2 Construction of the Completion Fragment

[0077] By comparing the protective efficacy and immunogenicity of truncated membrane protein fragments in the missing regions, complement sequences were constructed using membrane proteins Rv1508c (106-547) and Rv3888c (85-341), and analyzed using (GGGGS). n The sequence tandem (n=1 in this example), with a molecular weight of approximately 75 kDa, is named M75. Its nucleotide sequence is shown in SEQ ID NO.1. It was synthesized by a biotechnology company, amplified, and recovered to obtain a fragment for later use.

[0078] In addition, to better express and locate the complement sequence, an endogenous signal peptide from Mycobacterium tuberculosis was used to link to the N-terminus of M75 to construct the complement fragment. Three different signal peptides were constructed in this experiment, as follows:

[0079] Signal peptide p1: MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGA;

[0080] Signal peptide p2: MINVQAKPAAAASLAAIAIAFLAG;

[0081] Signal peptide p3: MKVKNTIAATSFAAAGLAALAVAVSPPAAA.

[0082] Similarly, after being synthesized by a biotechnology company, the fragments are amplified and recovered for later use.

[0083] P1 nucleotide sequence:

[0084] Atgacagacgtgagccgaaagattcgagcttggggacgccgattgatgatcggcacggcagcggctgtagtccttccgggcctggtggggcttgccggcggagcggcaaccgcgggcgcg;

[0085] P2 nucleotide sequence:

[0086] atgatcaacgttcaggccaaaccggccgcagcagcgagcctcgcagccatcgcgattgcgttcttagcgggt;

[0087] P3 nucleotide sequence:

[0088] atgaaggtaaagaacacaattgcggcaaccagtttcgcggcggccggcctggcggctctggcggtggctgtctcaccgccggcggccgca.

[0089] 4.3 Construction of the complement plasmid

[0090] The signal peptide fragment and M75 fragment obtained in section 4.2 were sequentially recombinated with the linearized pMV361 plasmid and transformed into E. coli DH5α competent cells. Single clones were selected for sequencing to verify the successful construction of positive plasmids using the seamless cloning method, resulting in plasmids pMV361-M75-p1, pMV361-M75-p2, and pMV361-M75-p3. The plasmid map of plasmid pMV361-M75-p1 is shown below. Figure 1 As shown.

[0091] 4.4 Electroporation of complement plasmids into competent cells

[0092] Add an appropriate amount of positive plasmid to *M. bovis* ΔRD1 competent cells and mix well. Transform the cells using a 2mm Bio-rad electroporator (electroporation parameters: voltage 2.5 kV, resistance 1000 Ω, capacitance 25 μF). After electroporation, add complete culture medium and incubate overnight at 37°C. Centrifuge, discard the supernatant, and resuspend the cells in approximately 100 μL of the remaining liquid medium. Spread the bacterial suspension parallel to two mycobacterial solid culture media (containing the corresponding antibiotics). Incubate at 37°C for 3–4 weeks. Select single clones and inoculate them into complete culture medium (containing the corresponding antibiotics). Incubate at 37°C for 3–4 weeks, collect the cells, extract the genome, and perform PCR and sequencing verification. The PCR verification primers were JDFP / JDRP (100-200 bp upstream and downstream of the multiple cloning site of the pMV361 vector). Sequencing verification was successful, and strains M. bovis-ΔRD1::M75p1, M. bovis-ΔRD1::M75p2 and M. bovis-ΔRD1::M75p3 were obtained.

[0093] JDFP: TGTCAGTACGCGAAGAACCACGC;

[0094] JDRP:TCGAGCAAGACGTTTCCCCGTTGA.

[0095] Example 5: Strain culture and preparation and validation of attenuated live vaccine

[0096] 5.1 Experimental Methods

[0097] (1) Resuscitation and culture of strains: Take one strain, thaw it quickly in a 37°C water bath, inoculate it onto Sutong potato slant culture medium, and incubate it at 37°C for 3 to 5 weeks.

[0098] (2) Harvesting: After the bacteria have matured, collect the bacterial film or bacterial blocks from the surface or inside of the culture medium.

[0099] (3) Physical treatment: Disperse the bacterial blocks by grinding or dilution to form a uniform bacterial suspension.

[0100] (4) Dilution and packaging: Dilution and packaging into 0.5 mg / mL bacterial suspension to obtain live attenuated vaccine.

[0101] (5) Attenuated live vaccines are repackaged or freeze-dried.

[0102] 5.2 Experimental Results

[0103] Take 2 tubes each of the cultures of different strains cultured for 4 weeks (with the same inoculum size, cultured in medium tubes), add 4 mL of dilute Sauton solution, take a sterilized cotton swab, insert it into the tube, dip the cotton swab in the dilute Sauton solution, wash down the bacterial lawn on the slant of each culture medium, and dissolve it in Sauton liquid medium. Suck out the bacterial liquid, add it to a sterilized grinder, and grind it slowly until there are no small particles. Use a sterile pipette to suck out the bacterial liquid in the grinder and store it in a 5 mL centrifuge tube. Take 1 mL of the bacterial liquid into a pre-weighed centrifuge tube, weigh it to calculate the cell weight (mg / mL), measure OD600, and perform viable cell counting.

[0104] Table 1 Results of strain culture

[0105]

[0106] The experimental results are as Figure 2 shown in and Table 1. It can be seen that the growth rate of the complemented strain M.bovis-ΔRD1::M75p1 is similar to that of the original strain, and even slightly improved; while the growth of M.bovis-ΔRD1::M75p2 and M.bovis-ΔRD1::M75p3 is inhibited to a certain extent, among which M.bovis-ΔRD1::M75p2 is most significantly affected, with obvious decreases in cell weight and OD value, proving that the production efficiency will be affected after the complementation scheme of this strain.

[0107] Example 6 Evaluation of the immunogenicity of the live attenuated vaccine

[0108] 6.1 Experimental materials

[0109] Select SPF-grade BalB / c female mice, weighing 20 - 25 g, 30 in number. From the Guangdong Provincial Center for Medical Laboratory Animals. Animal certificate number: No.44007200123277, license number: SYXK (Guangdong) 2022 - 0275.

[0110] Divide the above mice into:

[0111] a. BCG (Bacillus Calmette-Guérin) group; [[ID=2B]]

[0112] b. M.bovis -ΔRD1 (RD1 knockout) group;

[0113] c. M.bovis -ΔRD1::M75p1 (M75p1 complementation) group;

[0114] d. M.bovis -ΔRD1::M75p2 (M75p2 complementation) group;

[0115] e. M.bovis -ΔRD1::M75p3 (M75p3 complementation) group;

[0116] f. Saline group.

[0117] 6.2 Immunogenicity evaluation experiment

[0118] (1)Immunization

[0119] Take BalB / c mice, disinfect the back with 75% alcohol cotton. After disinfection, inject 0.1 mL of live bacteria vaccine subcutaneously at the disinfected site. Immunize twice at an interval of 4 weeks, and collect blood 3 weeks after immunization.

[0120] (2)Blood collection

[0121] Before the primary immunization and before sacrificing the mice, collect blood by orbital blood collection method respectively and collect the blood. Let it stand at 37 °C for 2 h, centrifuge at 4000 rpm for 10 min, separate the serum, and store it in a -20 °C refrigerator after sub-packaging for later use.

[0122] (3)Detection of blood antibody level

[0123] Use ELISA method to detect the titer of serum specific antibodies. Dilute the lysate of each strain to � μg / mL with coating buffer as antigen, add 100 μL per well, incubate the plate overnight at 4 °C, and detect the antibody levels of IgG, IgG1, and IgG2a.

[0124] (4)Detection of cellular immune effect

[0125] Sacrifice the mice 8 weeks after the last immunization, aseptically isolate the spleen, isolate splenocytes, and use the ELISPOT method to detect cytokines such as IFN-γ.

[0126] 6.3 Experimental results

[0127] The experimental results are as Figure 3 shown. It can be seen that the guinea pigs in each strain group after immunization all have a certain immune protection effect against Mycobacterium tuberculosis infection. Among them, the protection effect of the M.bovis-ΔRD1::M75p1 group against Mycobacterium tuberculosis infection is better than that of the BCG group.

[0128] Example 7 Evaluation experiment on the protective efficacy of live attenuated vaccine

[0129] 7.1 Experimental materials

[0130] Use SPF-grade Hartley healthy female guinea pigs that have not undergone any tests and weigh more than 300 g, 60 in total. Guangdong Provincial Center for Medical Laboratory Animals. Animal certificate number: No.1112512011`00412353, license number: SCXK(Beijing)2019-0017.

[0131] 7.2 Screening of guinea pig sensitivity

[0132] Guinea pigs were observed in the laboratory for 7 days, their back hair was clipped, and 0.1 mL of 50 IU / mL TB-PPD was injected intradermally. The injection site was observed for redness, swelling and induration at 24 h and 48 g after injection. Guinea pigs with no reaction or very weak reaction were used for sensitization.

[0133] 7.3 Guinea pig sensitization

[0134] After sensitivity screening, guinea pigs were subcutaneously injected with 5.0 × 10³ CFU of attenuated live vaccine (obtained by dilution of the attenuated live vaccine prepared in Example 5) in the groin, and sensitized once a week for a total of 4 times.

[0135] 7.4 Guinea Pig Skin Test

[0136] Two weeks after the final sensitization, guinea pigs sensitized with each vaccine were divided into three groups of five. Three points were selected on each side of the skin on both sides of the back of each guinea pig, and each group received an intradermal injection of 0.1 mL of physiological saline, 50 IU / mL TB-PPD, or 50 IU / mL rEC-TBST, respectively. The size of local skin erythema or induration was observed and recorded 24 hours after injection to observe whether a DTH (delayed-type hypersensitivity) reaction occurred.

[0137] 7.5 Evaluation of Test Results

[0138] 24 hours after the skin test injection, observe the longitudinal and transverse diameters of the local erythema or induration. A positive result is defined as an average erythema or induration reaction (the sum of the longitudinal and transverse diameters divided by 2) of not less than 5 mm, and a negative result is defined as less than 5 mm. Calculate the cumulative average diameter of the erythema or induration reaction for each dilution based on the 24-hour results.

[0139] Table 2 Skin test results

[0140]

[0141] The RD1 region contains the EC gene, while the knockout strain does not contain the EC gene. Therefore, the PPD skin test result was positive after immunization, and the TBST-rEC result was negative. This proves that the RD1 region of the knockout group strains was successfully knocked out without affecting the immunization effect. It also proves that TBST-rEC can effectively distinguish between knockout strain inoculation and wild-type strain infection.

[0142] 7.6 Anatomical Examination

[0143] Thirty-nine days after sensitization, guinea pigs were dissected to examine for pustules on the greater omentum, enlarged mesenteric lymph nodes, and visible tuberculous lesions in the liver and other organs. Furthermore, lesion index scores were calculated for the lungs, spleen, and liver, and serum, lung, liver, and spleen samples were collected from infected guinea pigs. Live bacterial cultures were performed on organ homogenates using a modified Roche method to evaluate the bacterial load in major organs of each group after infection. Hematoxylin-eosin (HE) staining was used for pathological analysis of major organs to assess the impact of the vaccine on the anti-infective protective effect of guinea pigs.

[0144] The experimental procedure for this experiment is as follows: Figure 4 As shown, the experimental results are as follows: Figures 5-7 As shown, the biosafety of guinea pigs was observed by injecting various bacterial strains and BCG into them. Figure 5 The results showed that no visible tuberculosis lesions were found in guinea pigs injected with the various strains and BCG during the experiment. Macroscopic observation of the mesenteric lymph nodes, greater omentum, lungs, liver, spleen, kidneys, stomach, and heart revealed no significant toxic reactions. A few guinea pigs had pustules on the greater omentum and enlarged mesenteric lymph nodes; the spleen, liver, and other organs were completely normal, with no visible changes. This demonstrates the good safety profile of the strains.

[0145] also, Figure 6 and Figure 7 The results showed that each strain significantly reduced the bacterial load in the lungs, liver, and spleen of the guinea pig model, and effectively improved the severity of organ lesions. M. bovis-ΔRD1::M75p1 induced a strong immune response in guinea pigs and effectively prevented MTB infection and proliferation in various organs.

[0146] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A genetically modified attenuated strain of Mycobacterium bovis, characterized in that: It is obtained by knocking out the RD1 region in the genome of Mycobacterium bovis and then inserting the complement antigen sequence into the knocked-out region to restore its immunogenicity. The complement antigen sequence is obtained by sequentially linking the signal peptide p1 and the M75 fragment; The M75 fragment is composed of the sequences of membrane proteins Rv1508c (106-547) and Rv3888c (85-341) in sequence (GGGGS). n The sequence is obtained by concatenating and linking the sequences. The RD1 region was knocked out after the knockout plasmid was transferred into Mycobacterium bovis via phage-mediated transformation. The nucleotide sequence of the RD1 region is shown as 435490 to 439888 bp of the genome sequence of Mycobacterium bovis AF2122 / 97 with NC_002945.4 in the NCBI database; The nucleotide sequence of the signal peptide p1 is shown in SEQ ID NO.2; The bovine tuberculosis mycobacterium mentioned is bovine tuberculosis mycobacterium AF2122 / 97; The M75 fragment (GGGGS) mentioned above n Sequence n=1; The nucleotide sequence of the M75 fragment is shown in SEQ ID NO.

1.

2. A method for constructing a genetically modified attenuated strain of Mycobacterium bovis, characterized in that... Includes the following steps: (1) Design primers to amplify the left and right arms of the RD1 region, and ligate them with the linearized p0004s plasmid to obtain the p0004s-AES plasmid; (2) After digesting phAE159 and p0004s-AES plasmids with enzymes, they were ligated, transformed into Escherichia coli, screened and sequenced to verify positive clones, namely phAE159-AES phage particles. (3) PhAE159-AES phage particles were transferred into Mycobacterium smegmatis, and after culture, phage plaques were picked and added to fresh Mycobacterium smegmatis for culture. After filtration, high-titer phages were obtained. (4) High-titer bacteriophages were mixed with Mycobacterium bovis, and after culturing, screening, and sequencing verification, Mycobacterium bovis with the RD1 region knocked out was obtained; (5) Amplify the nucleotide sequence fragments of the signal peptide and M75, link them sequentially to the linearized pMV361 plasmid, transform them into E. coli, and obtain positive clones after screening and sequencing verification, namely pMV361-M75 plasmid. (6) The pMV361-M75 plasmid was transferred into Mycobacterium bovis with the RD1 region knocked out. After culturing, screening and sequencing verification, a genetically modified attenuated Mycobacterium bovis strain was obtained. The bovine tuberculosis mycobacterium mentioned is bovine tuberculosis mycobacterium AF2122 / 97; The aforementioned Mycobacterium smegmatis is Mycobacterium smegmatis mc 2 155; The signal peptide mentioned is signal peptide p1; The nucleotide sequence of the RD1 region is shown as 435490 to 439888 bp of the genome sequence of Mycobacterium bovis AF2122 / 97 with NC_002945.4 in the NCBI database; The nucleotide sequence of the M75 fragment is shown in SEQ ID NO.1; The nucleotide sequence of the signal peptide p1 is shown in SEQ ID NO.

2.

3. The application of the genetically modified attenuated Mycobacterium bovis strain according to claim 1 in the preparation of tuberculosis vaccine.

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