CN120966730B - A genetically modified attenuated strain of Mycobacterium bovis, its construction method and application - Google Patents

Abstract

The invention discloses a genetically modified mycobacterium bovis attenuated strain, a construction method and application thereof. The homologous recombination principle is utilized to modify, so that gene knockout and membrane protein gene insertion of a genome RD1 region are realized, and thus, attenuated live vaccine candidate strains are constructed. As immunotherapeutic agent for the treatment of bladder cancer or as live attenuated tuberculosis vaccine. The invention is based on the bovine tuberculosis mycobacterium virulent strain AF2122/97, the RD1 region is knocked out by genetic modification to reduce the toxicity, but cell wall antigens (such as MPB 70/83) are reserved, and meanwhile, the relevant antigen fragments of the insertion deletion region are supplemented back to enhance the immunogenicity.

Description

Genetically modified mycobacterium bovis attenuated strain and construction method and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a genetically modified mycobacterium bovis attenuated strain, a construction method and application thereof.

Background

BCG vaccine is the only vaccine approved to date for preventing tuberculosis, is prepared from attenuated mycobacterium bovis, and has remarkable effect in preventing severe tuberculosis of children (such as castanopsis glaucomatous tuberculosis and tubercular meningitis). However, BCG has limited and unstable protective effects against adult tuberculosis, which is the most common and major source of tuberculosis transmission, its protective efficacy decreases with increasing age of the inoculator, and has little preventive effect against the onset of disease in people who have already been infected with latent tuberculosis.

The gene knockout vaccine is a novel biological agent for constructing attenuated live vaccines by utilizing gene editing technology (such as CRISPR-Cas9, homologous recombination and the like) to directionally delete virulence genes of pathogens. The core principle is that the pathogen loses the core ability of proliferation or pathogenicity in a host body by precisely knocking out genes closely related to pathogenicity of the pathogen (such as mntA genes of bacillus anthracis, P52/P36/SAP1 genes of plasmodium and the like), and meanwhile, the immunogenicity of the pathogen is kept, so that the host is stimulated to produce strong and durable immune protection. The strategy breaks through the limitation that the traditional attenuated vaccine depends on random mutation or subculture, and realizes rational design of 'attenuation controllable and immunity efficient'. Despite its broad prospects, this technology still faces challenges in balancing the degree of attenuation with immunogenicity (excessive attenuation may lead to reduced protective power), optimizing delivery systems (e.g., exosome carrier stability), and advancing clinical trials to verify human safety and long-lasting efficacy. In the future, the application of the recombinant strain in drug-resistant pathogens and prevention and control of new infectious diseases are expanded through strategies such as polygenic collaborative knockout, combined adjuvant or immune regulation factor and the like.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provides a genetically modified mycobacterium bovis attenuated strain.

Another object of the present invention is to provide a method for constructing the above genetically modified Mycobacterium bovis attenuated strain.

It is a further object of the present invention to provide the use of the genetically modified Mycobacterium bovis attenuated strain as described above.

The aim of the invention is achieved by the following technical scheme:

A genetically modified mycobacterium bovis attenuated strain is obtained by knocking out RD1 region in mycobacterium bovis genome, and inserting a complement antigen sequence into the knocked-out deletion region to restore immunogenicity.

The mycobacterium bovis is mycobacterium bovis AF2122/97.

The RD1 region is knocked out after phage-mediated transfer of the knocked-out plasmid into Mycobacterium bovis.

The nucleotide sequence of the RD1 region is shown as 435490 to 439888 bp of the bovine mycobacterium tuberculosis AF2122/97 genome sequence with NCBI database number NC_ 002945.4.

The anaplerotic antigen sequence is obtained by sequentially connecting a signal peptide and an M75 fragment.

The signal peptide is at least one of signal peptide p1, signal peptide p2 and signal peptide p 3.

The nucleotide sequence of the signal peptide p1 is shown as SEQ ID NO. 2.

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

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

The M75 fragment is obtained by sequentially connecting sequences of the membrane protein Rv1508c (106-547) and the membrane protein Rv3888c (85-341), preferably is obtained by sequentially connecting sequences of (GGGGS) _n in series, and more preferably is obtained by connecting n=1 in the sequence of (GGGGS) _n.

The nucleotide sequence of the M75 fragment is shown as SEQ ID NO. 1.

A construction method of genetically modified mycobacterium bovis attenuated strain comprises the following steps:

(1) Designing a primer for amplification to obtain left and right arms of an RD1 region, and connecting the left and right arms with a linearized p0004s plasmid to obtain a p0004s-AES plasmid;

(2) Carrying out enzyme digestion on phAE and p0004s-AES plasmids, connecting, transferring into escherichia coli, screening, sequencing and verifying to obtain positive clones, namely phAE-AES phagemid;

(3) Transferring phAE-AES phagemid into mycobacterium smegmatis, culturing, picking phage plaques, adding fresh mycobacterium smegmatis for culturing, and filtering to obtain high-titer phage;

(4) Mixing high-titer phage with mycobacterium bovis, culturing, screening, and sequencing to verify to obtain mycobacterium bovis knocked out RD1 region;

(5) Amplifying the nucleotide sequence fragments of the signal peptide and M75, sequentially connecting with a linearized pMV361 plasmid, transferring into escherichia coli, screening and sequencing to verify to obtain positive cloning, namely the pMV361-M75 plasmid;

(6) Transferring the pMV361-M75 plasmid into mycobacterium bovis which knocks out RD1 region, culturing, screening, sequencing and verifying to obtain the genetically modified mycobacterium bovis attenuated strain.

The mycobacterium bovis is mycobacterium bovis AF2122/97.

The mycobacterium smegmatis is mycobacterium smegmatis mc ² 155,155.

The signal peptide is at least one of signal peptide p1, signal peptide p2 and signal peptide p 3.

The nucleotide sequence of the signal peptide p1 is shown as SEQ ID NO. 2.

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

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

The M75 fragment is obtained by sequentially connecting nucleotide sequences of the membrane protein Rv1508c (106-547) and the membrane protein Rv3888c (85-341), preferably is obtained by sequentially connecting (GGGGS) _n sequences in series, and more preferably, n=1 in the (GGGGS) _n sequence.

The nucleotide sequence of the M75 fragment is shown as SEQ ID NO. 1.

The genetically modified mycobacterium bovis attenuated strain is constructed by the construction method.

A tuberculosis attenuated live vaccine comprises the preparation of the genetically modified mycobacterium bovis attenuated strain.

The tuberculosis attenuated live vaccine is prepared by diluting thalli obtained by culturing the genetically modified mycobacterium bovis attenuated strain to 0.1-1 mg/mL.

The application of the genetically modified mycobacterium bovis attenuated strain in preparing tuberculosis vaccines.

The application of the genetically modified mycobacterium bovis attenuated strain in preparing medicines for treating bladder cancer.

Compared with the prior art, the invention has the following advantages and effects:

The mycobacterium tuberculosis membrane protein has dual roles in infection and immunity, and is not only a main target point of host immune recognition, but also a key tool for bacterial escape and survival. As a strong immunogen, it is recognized by host Antigen Presenting Cells (APCs), activating cd4+/cd8+ T cell-mediated cellular immunity. The invention utilizes the homologous recombination theory to modify, realizes gene knockout and membrane protein gene insertion of the genome RD1 region, thereby constructing attenuated live vaccine candidate strains. As immunotherapeutic agent for the treatment of bladder cancer or as live attenuated tuberculosis vaccine.

The invention is based on the bovine tuberculosis mycobacterium virulent strain AF2122/97, the RD1 region is knocked out by genetic modification to reduce the toxicity, but cell wall antigens (such as MPB 70/83) are reserved, and meanwhile, the relevant antigen fragments of the insertion deletion region are supplemented back to enhance the immunogenicity.

Drawings

FIG. 1 is a plasmid map of the complementation vector pMV361-M75-p1 constructed in example 4;

FIG. 2 is a photograph of a sample of the strain culture in example 5, wherein BCG, M.bovis- ΔRd1: M75p1, M.bovis- ΔRd1:: M75p2, M.bovis- ΔRd1: M75p3 are sequentially from left to right;

FIG. 3 shows the results of the immunogenicity evaluation in example 6, wherein A to C are the results of detecting IgG, igG1 and IgG2a by ELISA method, and D is the result of detecting IFN-gamma by ELISPOT method;

FIG. 4 is a schematic illustration of the experimental procedure in example 7;

FIG. 5 is a photograph record of each viscera of guinea pig dissected in example 7, wherein BCG, M.bovis- ΔRd1:: M75p1, M.bovis- ΔRd1:: M75p2, M.bovis- ΔRd1:: M75p3 are sequentially from top to bottom;

FIG. 6 is a graph showing the statistical result of the biomass of the main organ after the guinea pig is dissected in example 7, wherein the unit is CFU (Log 10), A is the biomass of the lung, and B is the biomass of the spleen;

FIG. 7 is a quantitative analysis result of the histopathological lesion index of the main organ after the guinea pig dissection in example 7, wherein A is the pathological index of the liver, B is the pathological index of the spleen, and C is the pathological index of the lung.

Detailed Description

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

Unless specific test conditions are noted in the following embodiments, conventional test conditions or test conditions recommended by the reagent company are generally followed. The materials, reagents and the like used are those obtained commercially unless otherwise specified.

The biological materials used in the invention are all purchased from public commercial sources, wherein mycobacterium bovis AF2122/97 is purchased from an initiator, and mycobacterium smegmatis mc2155, phAE159, p0004s-AES and pMV361 are all purchased from Jinuo organism.

EXAMPLE 1 construction of shuttle vector

1.1 Construction of p0004s-AES plasmid

Two pairs of left and right arm primers aiming at RD1 region are respectively designed by taking bovine mycobacterium tuberculosis virulent strain AF2122/97 as a template, hi-Fi DNA Polymerase high-fidelity polymerase PCR is used for amplifying the left and right arms, a DNA gel recovery kit is used for recovering a target DNA fragment, then an AdeI restriction enzyme is used for enzyme digestion, and the DNA fragment after gel recovery enzyme digestion is used for standby.

RD1 left and right arm primers:

LFP-B:TTTTTTTTCACAAAGTGATTGCGCACCACCAGCTCTC;

LRP-B:TTTTTTTTCACTTCGTGTGAACGTTGCGCGGTGAGTGTGT;

RFP-B:TTTTTTTTCACAGAGTG AGGTGTCCAATACATCGGTGAC;

RRP-B:TTTTTTTTCACCTTGTG AGTGGCTCACCGGCCTGCGCATC;

The p0004s plasmid is extracted by using a plasmid extraction kit, and is digested by Van91I restriction enzyme, and the target DNA fragment after the digestion is recovered for standby.

The DNA fragments obtained in the above two steps were ligated using T4 DNA LIGASE to transform E.coli DH 5. Alpha. Competent cells, plated on hygromycin 150. Mu.g/mL LB plates and cultured overnight. The monoclonal was selected and inoculated into 150. Mu.g/mLLB of hygromycin liquid medium and cultured at 37 ℃. Extracting plasmid, sequencing to confirm positive clone and storing for standby.

1.2 Construction of phAE-AES shuttle plasmid

PhAE159 and p0004s-AES positive plasmids were extracted with a plasmid extraction kit, and the obtained plasmids were digested with PacI and the desired fragment was recovered. Two linearized plasmid fragments were ligated using T4 DNA LIGASE, and the fragments obtained after ligation were used to aid in transforming e.coli HB101 competent cells using phage in vitro packaging kit, plated on hygromycin 150ug/mL LB plates and cultured overnight. The monoclonal is selected and inoculated in 150ug/mL LB liquid medium of hygromycin, and cultured at 37 ℃. Extracting plasmid, sequencing to confirm positive clone and storing for standby.

Example 2 preparation of phages

2.1 Preparation of Mycobacterium smegmatis mc ² electric transduction competent cells

Selecting fresh Mycobacterium smegmatis mc ² 155 5 single colony, inoculating the single colony into 5mL complete culture medium, standing and culturing at 37 ℃ until the logarithmic phase (OD 0.5-1.0, about 1-2 days), inoculating the culture into 100 mL complete culture medium at a ratio of 1:100, culturing overnight at 37 ℃ until the OD600 is about 0.6, ice-bathing the culture for 0.5-1h, centrifuging at 5000 rpm for 10min to collect thalli, washing the thalli at least twice with 10% sterile glycerol in the ice-bath, adding 10% glycerol precooled by 10 mL, blowing uniformly, and freezing and storing 200 mu L of each tube at-80 ℃ for later use.

2.2 Phage amplification

Mu.g of phAE-AES shuttle plasmid prepared in example 1 was added to the electric transformation competence of Mycobacterium smegmatis mc ² and mixed well, and was subjected to electric shock transformation (electric shock parameters: voltage 2.5 kV, resistance 1000. OMEGA., capacitance 25. Mu.F) using a 2mm electric-rotating cup Bio-rad electric-rotating instrument, after electric shock, 7H9 complete medium was added, after incubation in a 37℃incubator overnight, bacterial solution was mixed well with an appropriate amount of top agar and plated with Mycobacterium solid medium. After culturing for 2-3 days at 30 ℃, phage plaques are screened.

Plaques containing phage on the plates were picked up and incubated overnight at 4℃with a suitable amount of freshly cultured Mycobacterium smegmatis mc ² strain, then mixed with a suitable amount of Top Agar and plated. The plates are placed at 30 ℃ for 2-3 days of culture. An appropriate amount of MP buffer was added to the plaque plates and allowed to stand at 4℃overnight. High titer phage were collected and filtered through a 0.22um sterile filter and stored in a 4 ℃ refrigerator for later use.

EXAMPLE 3 construction of RD1 knockout Strain

An appropriate amount of high titer phage lysate was mixed with Mycobacterium bovis AF2122/97 (pre-washed with MP buffer) grown to log phase, centrifuged to discard supernatant after incubation at 37℃overnight in the dark, then supplemented with an appropriate amount of 7H10 complete medium, incubated at 37℃overnight, centrifuged to discard supernatant to collect the cells coated with Mycobacterium solid medium (hygromycin 75. Mu.g/mL), and cultured at 37℃for 4-6 weeks. And (3) selecting a monoclonal, culturing, extracting whole genome DNA, and recovering a target band by PCR amplification, wherein sequencing verification proves that the RD1 region is knocked out, and the positive clone strain is named as M.bovis delta RD1 strain.

EXAMPLE 4 construction of the anaplerotic Strain

After the RD1 region is knocked out, the toxicity of the mycobacterium tuberculosis is obviously reduced, but the immunogenicity of the mycobacterium tuberculosis is also reduced, and the immunogenicity of the mycobacterium tuberculosis is recovered by designing a complement sequence to insert the deletion region.

4.1 Preparation of electrotransformation competent cells

Selecting fresh M.bovis DeltaRD 1 single colony, inoculating the single colony into 5mL complete culture medium (containing corresponding resistance), standing and culturing at 37 ℃ to logarithmic phase (OD 0.5-1.0, 2-3 Zhou Zuo), inoculating the culture into 100 mL complete culture medium (containing corresponding resistance) in a ratio of 1:100, standing and culturing at 37 ℃ to OD 600-0.6, centrifuging the culture at room temperature at 5000rpm for 10 min to collect thalli, washing the thalli at least twice with 10% sterile glycerol, adding 10mL (proper amount) of 10% glycerol, blowing the thalli uniformly, and freezing and storing 200 mu L of the thalli per tube at-80 ℃ for later use.

4.2 Construction of a patch segment

The protective power and immunogenicity of the deletion region membrane protein truncated fragment are compared, a complementary sequence is constructed by selecting membrane protein Rv1508c (106-547) and membrane protein Rv3888c (85-341), and the fragment is obtained for standby after serial connection of (GGGGS) _n sequences (n=1 in the embodiment), the molecular weight is about 75kDa, the molecular weight is named M75, the nucleotide sequence is shown as SEQ ID NO.1, and the fragment is synthesized by biological company, amplified and recovered.

In addition, for better expression and localization of the anaplerotic sequence, the anaplerotic fragment was constructed using the endogenous signal peptide of mycobacterium tuberculosis linked to the N-terminus of M75, and 3 different signal peptides were simultaneously constructed in this experiment, as follows:

signal peptide p1: MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGA;

Signal peptide p2: MINVQAKPAAAASLAAIAIAFLAG;

signal peptide p3: MKVKNTIAATSFAAAGLAALAVAVSPPAAA.

Also synthesized by biological company, amplified and recovered to obtain fragments for later use.

P1 nucleotide sequence:

Atgacagacgtgagccgaaagattcgagcttggggacgccgattgatgatcggcacggcagcggctgtagtccttccgggcctggtggggcttgccggcggagcggcaaccgcgggcgcg;

p2 nucleotide sequence:

atgatcaacgttcaggccaaaccggccgcagcagcgagcctcgcagccatcgcgattgcgttcttagcgggt;

p3 nucleotide sequence:

atgaaggtaaagaacacaattgcggcaaccagtttcgcggcggccggcctggcggctctggcggtggctgtctcaccgccggcggccgca。

4.3 construction of the make-up plasmid

And (3) recombining and connecting the signal peptide fragment and the M75 fragment obtained in 4.2 with a linearized pMV361 plasmid in sequence, transforming E.coli DH5 alpha competent cells, and carrying out monoclonal sequencing to verify that a seamless cloning method is adopted to construct positive plasmids successfully, so as to obtain plasmids pMV361-M75-p1, pMV361-M75-p2 and pMV361-M75-p3, wherein a plasmid map of the plasmids pMV361-M75-p1 is shown in figure 1.

4.4 Electric transformation of the complementing plasmid into competent cells

Proper amount of positive plasmid is added into M.bovis delta RD1 competent cells, mixed evenly, subjected to electric shock transformation (electric shock parameters: voltage 2.5 kV, resistance 1000 omega, capacitance 25 mu F) by using a 2mm electric rotating cup Bio-rad electric rotating instrument, added into complete culture medium after electric shock, cultured overnight in a37 ℃ incubator, centrifuged, the supernatant is discarded, and the residual liquid culture medium is about 100 mu L to resuspend the bacterial liquid in parallel to two mycobacteria solid culture media (containing corresponding resistance). After 3-4 weeks of culture at 37 ℃, the single clone was selected and inoculated into complete medium (containing the corresponding resistance). After culturing for 3-4 weeks at 37 ℃, collecting thalli, extracting genome, and performing PCR and sequencing verification. The PCR verification primer is JDFP/JDRP (100-200 bp upstream and downstream of the multiple cloning site of the pMV361 vector), and the sequencing verification is passed, so that the strain M.bovis-DeltaRD 1:: M75p1, M.bovis-DeltaRD 1:: M75p2 and M.bovis-DeltaRD 1: M75p3 are obtained.

JDFP :TGTCAGTACGCGAAGAACCACGC;

JDRP :TCGAGCAAGACGTTTCCCGTTGA。

EXAMPLE 5 Strain culture and preparation and validation of attenuated live vaccines

5.1 Experimental methods

(1) And (3) resuscitating and culturing the strain, namely taking 1 strain, placing the strain in a 37 ℃ water bath for quick thawing, inoculating the strain to a storax potato slant culture medium, and carrying out stationary culture for 3-5 weeks at the temperature of 37 ℃.

(2) Harvesting, namely collecting bacterial films or bacterial blocks from the surface or the inside of the culture medium after the bacterial cells grow and mature.

(3) And (3) physical treatment, namely dispersing the fungus blocks by a grinding or diluting method to form uniform fungus suspension.

(4) Diluting and subpackaging, namely diluting and subpackaging into 0.5mg/mL bacterial suspension to obtain the attenuated live vaccine.

(5) Split charging or freeze-drying attenuated live vaccine.

5.2 Experimental results

Taking 2 cultures (with consistent inoculum size and medium tube culture) of different strains after 4 weeks of culture, adding 4mL of diluted Sutong solution, taking sterilized cotton swabs, extending into the tube, dipping the diluted Sutong solution with the cotton swabs, washing off the lawn on the upper inclined surface of each culture medium, and dissolving in the Sutong liquid culture medium. Sucking out the bacterial liquid, adding into a sterilized grinder, and grinding slowly until no small particles exist. Using a sterile pipette, the bacterial solution in the mill was aspirated and stored in a 5mL centrifuge tube. 1mL of the bacterial liquid is taken into a weighed centrifuge tube, and the bacterial body weight (mg/mL), OD600 detection and viable bacteria count are calculated by weighing.

TABLE 1 results of Strain culture

As shown in the experimental results of FIG. 2 and Table 1, it can be seen that the growth rate of the anaplerotic strain M.bovis-DeltaRD 1: M75p1 is similar to that of the original strain, even slightly improved, and the growth of the anaplerotic strain M.bovis-DeltaRD 1:: M75p2 and M.bovis-DeltaRD 1: M75p3 are inhibited to a certain extent, wherein the anaplerotic strain M.bovis-DeltaRD 1: M75p2 is affected most significantly, and the weight and OD value of the thalli are reduced obviously, thus proving that the production efficiency is affected after the anaplerotic scheme of the strain.

EXAMPLE 6 evaluation of immunogenicity of live attenuated vaccines

6.1 Experimental materials

SPF-grade BalB/c female mice are selected, the weight is 20-25 g, 30. The medical laboratory animal center in Guangdong province. Animal pass number No.44007200123277, license number SYXK (Yue) 2022-0275.

The mice were divided into:

a. BCG (BCG) group;

b. group m.bovis- Δrd1 (RD 1 knockout);

c. M75p1 (M75 p1 back-filling) group;

d. M75p2 (M75 p2 back-filling) group;

e. M75p3 (M75 p3 supplement) group;

f. Saline group.

6.2 Immunogenicity evaluation experiment

(1) Immunization

BalB/c mice were treated with 75% alcohol cotton for back disinfection, and the disinfected parts were subcutaneously injected with 0.1 mL live bacteria vaccine 2 times, 4 weeks apart, and blood was collected after 3 weeks of immunization.

(2) Blood collection

Before primary immunization and before mice sacrifice, blood collection and blood collection are respectively carried out through an orbital blood collection method, standing is carried out at 37 ℃ for 2h, centrifugation is carried out at 4000rpm for 10min, serum is separated, and the separated serum is stored in a refrigerator at-20 ℃ for standby.

(3) Blood antibody level detection

ELISA was used to detect serum-specific antibody titers. Lysates of each strain were diluted to 5ug/mL as antigen with coating buffer, incubated at 4℃overnight per well at 100. Mu.L, and assayed for IgG, igG1, and IgG2a antibody levels.

(4) Cell immune effect detection

Mice were sacrificed 8 weeks after the last immunization, spleens were aseptically isolated, spleen cells were isolated, and cytokine IFN-gamma was detected by ELISPOT method, etc.

6.3 Experimental results

As shown in the experimental result in figure 3, it can be seen that each strain group guinea pig after immunization has a certain immune protection effect on mycobacterium tuberculosis infection, wherein the protection effect of M.bovis-delta RD1 in the M75p1 group against mycobacterium tuberculosis infection is better than that of the bacillus calmette-guerin group.

EXAMPLE 7 attenuated live vaccine protective force evaluation experiment

7.1 Experimental materials

Female guinea pigs healthy with SPF grade Hartley and without any trial had a body weight of more than 300g, 60. The medical laboratory animal center in Guangdong province. Animal pass number No.111251201100412353, license number SCXK (Beijing) 2019-0017.

7.2 Guinea pig sensitivity screening

Guinea pigs were observed in the laboratory for 7d, sheared back, injected intradermally with 50IU/mL TB-PPD 0.1mL, and 24h, 48g post injection observed for presence of redness and induration at the injection site, and no or very weak guinea pigs were used for sensitization.

7.3 Guinea pig sensitization

The guinea pig inguinal injection obtained after the sensibility screening was 5.0X103 CFU attenuated live vaccine (obtained after dilution of the attenuated live vaccine prepared in example 5), and the whole was sensitized 1 time per week for 4 times.

7.4 Guinea pig skin test

After 2 weeks of the last sensitization, the guinea pigs sensitized with each vaccine were divided into 3 groups of 5 animals each. 3 spots were selected on each side of the skin on both sides of the back of each guinea pig, and each group was injected intradermally with physiological saline, 50 IU/mL TB-PPD, 50 IU/mL rEC-TBST, 0.1mL each. The local size of the skin halation or induration response was observed and recorded at 24 post-injection, respectively, to see if a DTH (delayed hypersensitivity) response was produced.

7.5 Evaluation of test results

At 24h after skin test injection, the longitudinal diameter and the transverse diameter of local halation or induration are observed, the average halation or induration reaction (the sum of the longitudinal diameter and the transverse diameter divided by 2) is not less than 5mm and is judged to be positive, and is less than 5mm and is judged to be negative, and the diameter cumulative value of the average halation or induration reaction of each dilution is calculated according to the 24-hour result.

TABLE 2 skin test results

The RD1 region has EC genes, and the knocked-out strain does not contain EC genes, so that PPD skin test results are positive after immunization, TBST-rEC results are negative, the knocked-out group strains are proved to be successful in knocking out the RD1 region and have no influence on immune effects, and TBST-rEC can be proved to be capable of effectively identifying knocked-out strain inoculation and wild strain infection.

7.6 Anatomical examination

After 39d sensitization, guinea pigs were dissected and examined for pustules on the greater omentum, enlargement of the mesenteric lymph nodes, and macroscopic tuberculosis lesions of the liver and other organs. In addition, lung, spleen and liver were scored for lesion index, respectively, and guinea pig serum, lung, liver and spleen after infection were collected. The bacterial strain is cultured by adopting an improved Roche culture method to carry out living bacterial culture on the organ homogenate, the bacterial load of each group of main organs after infection is evaluated, and the main organs are subjected to pathological analysis by adopting hematoxylin-eosin staining (HE method) so as to evaluate the influence of the vaccine on the anti-infection protection effect of guinea pigs.

The experimental flow of the experiment is shown in fig. 4, the experimental results are shown in fig. 5-7, and the biological safety in vivo of the guinea pigs is observed by injecting each strain and BCG in the guinea pigs. The results in FIG. 5 show that no macroscopic tuberculosis lesions were found in guinea pigs dissected from each strain and BCG strain injected during the test, and no apparent toxic reactions were found by visual observation of the intestinal lymph nodes, macroreticular membranes, lung, liver, spleen, kidney, stomach and heart. Individual guinea pigs had pus bubbles on their omentum, enlarged mesenteric lymph nodes, and completely normal spleen, liver and other viscera with no macroscopic changes. The strain is proved to have good safety.

In addition, the results of fig. 6 and 7 show that each strain can significantly reduce the amount of lotus in the lung, liver and spleen in a guinea pig model, and can effectively improve the pathological changes of animal organs. M75p1 can induce strong immune response in guinea pigs and effectively prevent MTB infection and proliferation in various organs.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (3)

1. A genetically modified mycobacterium bovis attenuated strain, characterized by:

the method comprises the steps of knocking out RD1 region in bovine tuberculosis mycobacterium genome, and inserting a complement antigen sequence into the knocked-out deletion region to restore immunogenicity;

The anaplerotic antigen sequence is obtained by sequentially connecting signal peptide p1 and M75 fragments;

The M75 fragment is obtained by connecting sequences of a membrane protein Rv1508c (106-547) and a membrane protein Rv3888c (85-341) in series by a (GGGGS) _n sequence;

The RD1 region is knocked out after the knocked-out plasmid is transferred into mycobacterium bovis through phage mediation;

The nucleotide sequence of the RD1 region is shown as 435490 to 439888 bp of the bovine mycobacterium tuberculosis AF2122/97 genome sequence with NCBI database number NC_ 002945.4;

the nucleotide sequence of the signal peptide p1 is shown as SEQ ID NO. 2;

The mycobacterium bovis is mycobacterium bovis AF2122/97;

n=1 of the (GGGGS) _n sequence in the M75 fragment;

The nucleotide sequence of the M75 fragment is shown as SEQ ID NO. 1.

2. The construction method of the genetically modified mycobacterium bovis attenuated strain is characterized by comprising the following steps:

(1) Designing a primer for amplification to obtain left and right arms of an RD1 region, and connecting the left and right arms with a linearized p0004s plasmid to obtain a p0004s-AES plasmid;

(2) Carrying out enzyme digestion on phAE and p0004s-AES plasmids, connecting, transferring into escherichia coli, screening, sequencing and verifying to obtain positive clones, namely phAE-AES phagemid;

(3) Transferring phAE-AES phagemid into mycobacterium smegmatis, culturing, picking phage plaques, adding fresh mycobacterium smegmatis for culturing, and filtering to obtain high-titer phage;

(4) Mixing high-titer phage with mycobacterium bovis, culturing, screening, and sequencing to verify to obtain mycobacterium bovis knocked out RD1 region;

(5) Amplifying the nucleotide sequence fragments of the signal peptide and M75, sequentially connecting with a linearized pMV361 plasmid, transferring into escherichia coli, screening and sequencing to verify to obtain positive cloning, namely the pMV361-M75 plasmid;

(6) Transferring pMV361-M75 plasmid into mycobacterium bovis which knocks out RD1 region, culturing, screening, sequencing and verifying to obtain genetically modified mycobacterium bovis attenuated strain;

The mycobacterium bovis is mycobacterium bovis AF2122/97;

The mycobacterium smegmatis is mycobacterium smegmatis mc ² 155,155;

The signal peptide is a signal peptide p1;

The nucleotide sequence of the RD1 region is shown as 435490 to 439888 bp of the bovine mycobacterium tuberculosis AF2122/97 genome sequence with NCBI database number NC_ 002945.4;

The nucleotide sequence of the M75 fragment is shown as SEQ ID NO. 1;

the nucleotide sequence of the signal peptide p1 is shown as SEQ ID NO. 2.

3. Use of the genetically modified mycobacterium bovis attenuated strain of claim 1 in the preparation of a tuberculosis vaccine.