CN111394366B - Mycobacterium bovis BCG low-invasiveness mutant B2855 - Google Patents

Abstract

The invention belongs to the technical field of prevention and control of animal infectious diseases, and relates to a low-invasiveness mutant strain B2855 of Mycobacterium bovis BCG. The mutant strain B2855 contains a mutant gene of Mycobacterium bovis BCG LipQ, and its nucleotide sequence is as shown in SEQ ID NO: 1 As shown, the mutation site is located after the 2757121 site in the genome and after the 564 site of the LipQ gene sequence, which is a new structural gene and functional gene. The mutant strain of the present invention has low invasiveness, high intracellular viability and high growth rate. The mutant strain B2855 was deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 2018865. The isolated mutant gene and mutant strain of the present invention can be expected to be applied in the study of the pathogenic mechanism of Mycobacterium bovis, the study of the immune mechanism and the preparation of vaccines or medicines for preventing bovine tuberculosis.

Description

Mycobacterium bovis BCG vaccine low-invasiveness mutant B2855

Technical Field

The invention belongs to the technical field of infectious disease control of animals, and particularly relates to a mycobacterium bovis bcg vaccine low-invasiveness mutant strain B2855 which contains a mutant gene of mycobacterium bovis bcg vaccine LipQ.

Background

Mycobacterium bovis (Mtb) is a zoonotic intracellular parasitic bacterium, has a very strong ability to evade host immune responses and cause long-term latent infections, and can cause tuberculosis in multiple species, including humans. Since Mycobacterium bovis is 99.95% homologous at the gene level to the main pathogen Mycobacterium tuberculosis (m.tb) causing tuberculosis in humans [1], the infection profiles cross each other, both can infect almost all vertebrates and lead to the spread of the disease between humans and animals.

The only current official vaccine for preventing human tuberculosis is Mycobacterium bovis BCG (m.bovis BCG), which is an attenuated strain obtained after Mycobacterium bovis naturally loses RD1 region encoding virulence factors after 230 passages in a laboratory. BCG was first introduced since 1921 and makes a great contribution to the prevention of tuberculosis in infants, especially tuberculous meningitis, but is unstable in adult protection. Especially for many special groups, especially for the groups with low immunity (such as AIDS patients), BCG can not realize the immune protection function, but accelerates the onset of tuberculosis. To solve this problem, more and more researchers have been working on developing a more safe and effective tuberculosis vaccine, but no vaccine superior to bcg has been developed yet. Therefore, the research and development of BCG which does not influence the protective effect of BCG and is safer aiming at the population with low immunity are very important from different angles.

The virulence of a bacterium depends on the degree of virulence, and in general the more virulent it is, the more virulent it is. Bacterial virulence is usually measured in terms of adhesion, invasion, replication, exotoxin and immunosuppression. The reduction of the toxicity of BCG vaccine by the deletion of virulence related factors is an important research direction for the preparation of safe vaccines.

Invasion is an important step in the bacterial infection of host cells, triggering an immunological response. During mycobacterial infection, bacteria are first drawn into the alveolar space and interact with alveolar macrophages, and then invade host cells through a series of pathways or modes [2 ]. Invasiveness is thus a virulence factor of bacteria, directly affecting the virulence of bacteria and being closely related to the spread of bacteria [3 ]. When a bacterium invades a cell, or the cell phagocytoses the bacterium, the intracellular viability (level of replication in the cell) of the bacterium can be used to map the magnitude of virulence of the bacterium.

Invasion-associated proteins encoded by invasion-associated genes directly affect the invasive potential of bacteria. Therefore, the screening and identification of the invasion related genes are not only beneficial to the research of the interaction mechanism of the mycobacterium tuberculosis and the host cells, but also lay an important foundation for the research and development of novel BCG.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an important mycobacterium bovis low-invasiveness mutant strain (referred to as a mutant strain in the invention) B2888, wherein the related gene of the mutant strain is LipQ, and compared with wild mycobacterium bovis, the mutant strain has obvious low invasiveness, low intracellular viability and high growth speed, and the shown character is related to the toxicity of the inserted inactivated gene.

In order to achieve the purpose of the invention, the important laboratory ruminant etiology of agricultural microbiology country of Huazhong university of agriculture in China, where the applicant is located, is screened from a mycobacterium bovis BCG gene deletion mutant library by a division to obtain a strain B2855 with remarkably reduced invasive ability, wherein the mutant strain contains a LipQ gene, and the gene codes a carboxylesterase related to lipid metabolism. It is reported in the literature that LipQ has an immunosuppressive effect and is capable of reducing the level of proinflammatory factors in M.tuberculosis [4 ]. However, no document reports the function of Mycobacterium bovis. Through verification, in the process of infecting A549 cells, the invasion capacity of the mutant strain is obviously reduced, and the intracellular survival capacity is improved. In 7H9 medium, the mutant strain grew significantly faster than the wild strain. The relation study of the mutant strain and the mutant gene shows that the phenotype of the mutant strain is related to the virulence of the inactivated gene LipQ. Invasion is an important step in the bacterial realization of infection, and pathogenic virulence can directly affect the level of immune response in the host. Therefore, the successful construction of the mycobacterium bovis bcg B2855 mutant strain and the research on the related functions of the related gene LipQ thereof have important reference values for clarifying the related pathogenic mechanism of the mycobacterium bovis and developing safer and more effective vaccines.

The specific technical scheme of the invention is as follows:

the mycobacterium bovis BCG used by the applicant is pasteur strain BCG [ American Type Culture Collection (ATCC): 35734 to Luiz Bermudez professor, Oregon State university, USA. The earlier work of the present invention involved the recovery and culture of the deposited bcg strains of mycobacterium bovis.

The invention uses a mycobacterium bovis BCG vaccine Pasteur strain (ATCC: 35734) as a wild strain, uses a phage MycoMarT7 with temperature-sensitive property to convert Himar1 transposon into the mycobacterium bovis BCG vaccine, uses kanamycin as a resistance screening mark to screen mutant strains, thereby successfully constructing a mutant library. The invention utilizes a cell invasion model to screen out mutant strains with reduced invasion capacity from a mutant library, obtains BCG mutant strain B2855(BCG:: Tn LipQ) with significantly reduced invasion capacity, and the applicant names the mutant strains as Mycobacterium bovis BCG Pasteur B2855, and sends the mutant strains to the China center for type culture Collection of university of Wuhan, in 2018, 12 and 6 days, wherein the preservation number is CCTCC NO: m2018865. The mutant B2855 and the wild mycobacterium bovis BCG vaccine strain screened by the invention are respectively inoculated in a 7H9 culture medium for intracellular survival ability and growth curve detection, and the result shows that the mutant B2855 has high survival ability and grows rapidly in logarithmic phase.

The mutant strain constructed by the invention relates to a gene LipQ which encodes a carboxylesterase related to lipid metabolism, and the function of the gene in mycobacterium bovis is not reported by relevant documents or databases.

The invention has the following advantages:

1. the B2855 strain is a mutant strain with reduced invasive ability, which is screened from a mycobacterium bovis BCG mutant library by the inventor.

2. The B2855 strain of the present invention has been confirmed by the inventors to exhibit a remarkably high intracellular survival ability relative to a wild strain of mycobacterium bovis.

3. The B2855 strain of the present invention has been demonstrated by the inventors to exhibit significant growth-promoting performance relative to a wild strain of mycobacterium bovis.

4. The LipQ gene related by the invention is a novel gene of mycobacterium bovis, has an invasion function, and can reduce the intracellular survival of bacteria and improve the growth rate.

For a more detailed technical solution, refer to the detailed description.

Drawings

FIG. 1: is the result of high-throughput screening of the mutant bacteria with altered invasion capacity of the mycobacterium bovis bcg vaccine. Description of reference numerals: the results of screening different mutants in the mutant library are shown in FIG. 1, panel A-F, respectively, and only some of the results are shown due to space limitations. The box in panel D of FIG. 1 shows the low invasive potential mutant B2855. Throughout the experiment, "+" indicates P <0.05, "+" indicates P <0.01, and "+" indicates P < 0.001.

FIG. 2: the invention discloses an analysis chart for quantitative detection of invasion capacity of a mycobacterium bovis BCG mutant B2855 infected with A549 cells. "x" indicates P < 0.001. Compared with a wild strain of the mycobacterium bovis, the invasion capacity of the B2855 is obviously reduced.

FIG. 3: the nucleotide sequence of LipQ gene related to the mycobacterium bovis BCG mutant B2855 (the nucleotide sequences shown in the sequence table SEQ ID NO:1 are consistent).

FIG. 4: the nucleotide sequence of gene LipQ related to the mycobacterium bovis BCG mutant B2855 (the nucleotide sequences shown in the sequence table SEQ ID NO:1 are consistent). Wherein transposon insertion inactivation occurs after the 564 th site of the gene, i.e., the 2757121 th site of the genome.

FIG. 5: the invention discloses a quantitative detection and analysis graph of intracellular survival capacity of a mycobacterium bovis BCG mutant B2855 infected with A549 cells. Description of reference numerals: "+" indicates P <0.05 and "+" indicates P < 0.01. The intracellular viability of the B2855 strain is obviously higher than that of a wild strain 12h and 24h after infection.

FIG. 6: the growth curve analysis chart of the mycobacterium bovis BCG in the 7H9 culture medium is shown. Description of reference numerals: "+" indicates P <0.05 and "+" indicates P < 0.01. During infection 12-24d, the growth rate of the B2855 mutant strain is significantly higher than that of the wild Mycobacterium bovis strain.

Detailed Description

Description of sequence listing:

SEQ ID NO 1 is the nucleotide sequence of the isolated Mycobacterium bovis mutant gene LipQ gene of the present invention.

Example 1: screening and identification of low-invasiveness mutant of mycobacterium bovis bcg

1.1 high throughput screening of low invasiveness mutants of M.bovis

Clones in the M.bovis BCG mutant library were individually transferred to 7H9 liquid medium (7H9 liquid medium purchased from BD Co.) and cultured, and the M.bovis BCG mutant library was subjected to high-throughput screening using A549 cells (professor Luiz Bermadez, Oregon State university, USA) infection model. A549 cells were cultured in 12-well cell culture plates until they grew into monolayers and reached 2X 10⁵After each well, bacteria were added at a 10:1 infection ratio, 5% CO at 37 ℃%₂After 1H in the incubator, gentamicin (final concentration 100. mu.g/ml) was added to kill extracellular bacteria, after washing well, cells were lysed with Triton X-100 (Bio-Rad), and intracellular bacteria were collected and plated on 7H11 solid (purchased from BD Co.) plates at 37 ℃ with 5% CO₂The culture was carried out in an incubator for about 15 days. The invasion capacity of 64 mutant strains is obviously changed compared with that of a wild strain BCG through bacterial counting, wherein 28 strains show reduced invasion capacity, and 36 strains show enhanced invasion capacity (figure 1).

1.2 quantitative detection and analysis of low-invasiveness mutants of M.bovis BCG

To verify the results in step 1.1, the applicant further carried out quantitative analysis of the 28 mutants with reduced invasive capacity. A549 cells were prepared at 2X 10⁵Spreading the cells/well on a 12-well cell culture plate, inoculating the primary-screened mutants into A549 cells one by using an infection ratio of 10:1, and setting wild BCG as a control. Will be protrudedVariants with A549 cells at 37 ℃ 5% CO₂After the culture box acts for 1 hour, gentamicin (final concentration is 100 mug/ml) is added to kill extracellular bacteria, cells are lysed after full washing, and the colony number of the mutant is quantitatively determined by a colony counting method. The results showed that 10 of the 28 mutants had stable invasive ability, and that the invasive ability was reduced in both experiments. The mutant B2855 of the invention has the advantages that compared with a wild mycobacterium bovis strain, the invasion capacity is reduced by 4.9 times, the difference is very obvious (figure 2), and the function of the mutant is further verified.

Meanwhile, the applicant names the mutant strain as Mycobacterium bovis BCG Pasteur B2855, and sends the mutant strain to the China center for type culture Collection of university of Wuhan, in 2018, 12 and 6 months, wherein the preservation number is CCTCC NO: m2018865.

1.3 identification of mutant genes of B2855 Strain

The conventional CTAB method is utilized to extract the genomic DNA of the B2855 mutant of the mycobacterium bovis BCG, the junction of the Himar1 transposon and the genome of the mycobacterium bovis BCG is sequenced, the sequencing result is compared with the whole genomic sequence of the mycobacterium bovis BCG, the result shows that the gene related to B2855 is LipQ (figure 3), the nucleotide sequence of the gene is shown as the sequence table SEQ ID NO:1, and the insertion site of the transposon is positioned behind the 2757121 site of the genome, namely behind the 564 site of the LipQ gene (figure 4). Therefore, the applicants named this mutant as BCG:: Tn LipQ.

Example 2: detection of intracellular viability of low-invasiveness mutant of mycobacterium bovis bcg

To verify the intracellular viability of the mutants, a549 cells were plated at 2 × 10⁵Spreading the cells/well on 12-well cell culture plates, inoculating the B2855 mutant strain into A549 cells by using an infection ratio of 10:1, and setting the mycobacterium bovis wild strain BCG vaccine as a control. Mutant B2855 and A549 cells were incubated at 37 ℃ with 5% CO₂After 1h in the incubator, the supernatant was discarded, washed thoroughly 3 times with phosphate buffer (PBS, 0.01M, pH 7.4PBS, Hyclone) and then killed with gentamicin (final concentration 100. mu.g/ml), and then placed at 37 ℃ with 5% CO₂When cultured in an incubator, the feeling is recordedDyeing for 0 h. After 12h and 24h of infection, the cells were washed thoroughly with PBS and lysed before the number of mutant colonies was quantified by colony counting. The results show that the intracellular survival capacity of the B2855 mutant strain is obviously improved after 12h and 24h of infection compared with that of the wild strain and is respectively 1.7 times (P)<0.05) and 2.2 (P)<0.01) fold (FIG. 5).

Example 3: mycobacterium bovis BCG vaccine growth curve detection

Taking a wild mycobacterium bovis BCG vaccine strain and the mutant B2855 of the invention according to the volume ratio of 1:100 to inoculate in a 7H9 liquid medium, standing at 37 ℃ and 5% CO₂Continuously culturing in incubator for 27 days, taking appropriate bacterial liquid every 3 days to measure OD₆₃₀Values, results show that the growth rate of the mutant strain in log phase was significantly higher than that of the wild strain (fig. 6). Mutant B2855 was grown at day 12 (P) compared to M.bovis wild-type strain<0.05), 15 days (P)<0.01), 18 days (P)<0.01), 21 days (P)<0.01) and 27 days (P)<0.05) were raised by 1.2, 1.2 and 1.1 times, respectively. The statistical difference was significant. The gene inserted and inactivated by the mutant strain B2855 is suggested to have the function of reducing the growth speed of bacteria.

Compared with a wild mycobacterium bovis strain, the mycobacterium bovis mutant strain B2855 obviously reduces the invasion capacity of host cells. But the intracellular viability is remarkably enhanced due to the fast growth speed. Invasiveness is a virulence related factor of bacteria. It is well known that a reduction in invasiveness is associated with the virulence of the bacterium. The invasion capacity of the mutant strain screened by the invention is obviously reduced, which implies that the toxicity is obviously reduced compared with the wild strain of the mycobacterium bovis, and the high intracellular survival capacity of the mutant strain is beneficial to enhancing the immune response of an organism. Therefore, the method is expected to have wide application value in the preparation of tuberculosis vaccines.

The noun terms describe:

the gene related to M.bovis BCG B2855 is expressed as LipQ.

The mutant B2855 gene of M.bovis BCG is represented by mutant B2855 or B2855.

The main references:

1.Garnier T,Eiglmeier K,Camus JC,Medina N,Mansoor H,Pryor M,Duthoy S,Grondin S,Lacroix C,Monsempe C,et al:The complete genome sequence of Mycobacterium bovis.Proc Natl Acad Sci U S A 2003,100:7877-7882；

2.Zhang Y,Li J,Li B,Wang J,Liu CH:Mycobacterium tuberculosis Mce3C promotes mycobacteria entry into macrophages through activation of beta2 integrin-mediated signalling pathway.Cell Microbiol 2018,20；

3.Ashiru OT,Pillay M,Sturm AW:Adhesion to and invasion of pulmonary epithelial cells by the F15/LAM4/KZN and Beijing strains of Mycobacterium tuberculosis.J Med Microbiol 2010,59:528-533；

4.Kumar A,Manisha,Sangha1.GK,Shrivastava A,Kaur J:The immunosuppressive effects of a novel recombinant LipQ(Rv2485c)protein of Mycobacterium tuberculosis on human macrophage cell lines.Microb Pathog 2017,107:361-367。

sequence listing

<110> university of agriculture in Huazhong

<120> Mycobacterium bovis BCG vaccine low invasiveness mutant B2855

<141> 2019-04-13

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1266

<212> DNA

<213> Mycobacterium bovis (Mycobacterium bovis)

<220>

<221> gene

<222> (1)..(1266)

<400> 1

atgcacatcg ccagcgtgac ttcgcggtgc tcgcgggccg gcgccgaggc attgcgccag 60

ggagcgcagc tggcggccga cgccagagac acctgccggg ccggcgccct gctgctgcgt 120

ggatcaccgt gcgccatcgg ttgggtcgca ggatggttgt ccgcggagtt tccggcccgc 180

gtcgtcaccg gccacgcgct gtcccgcata tcaccacgct cgatcggccg gtttggcacc 240

agctgggcgg cgcagcgggc ggatcaaatc ctgcacgcgg cgctcgtgga cgctttcggc 300

ccggatttcc gtgacctagt gtggcatccg accggcgaac agtcagaggc tgcccggcgc 360

agcgggctgc tgaacctacc gcacattccc ggaccccatc gccgctatgc ggcccagacc 420

tccgacatcc cgtacgggcc cggcggccgc gagaatctac tcgacatctg gcggcgtccc 480

gatttagcgc ccggccgccg ggctccggtc ctcatccagg tcccgggcgg ggcgtggacc 540

atcaacggca aacgccctca ggcttacccg ttgatgagcc ggatggtgga actcggctgg 600

atctgtgtgt cgatcaacta cagcaagagc ccacggtgca cgtggccggc gcacattgtg 660

gacgtgaaga gggcgattgc gtgggttcgc gagaacatcg ccgactatgg cggcgatccg 720

gatttcatca cgatcaccgg cgggtccgcc ggtgcgcacc tggccgcgtt ggccgcgctt 780

tcggcgaatg atccggcgct gcagccgggc ttcgaaagcg ccgacacggc ggtacaagcc 840

gcggcgccct actacggcgt ctacgacctc accaacgccg agaatatgca cgaaatgatg 900

atgccgttcc tggagcactt cgtgatgcgt agccgctacg tcgacaaccc ggggctgttc 960

aaggcggcgt cgccgatttc gtacgtccac agcgaggctc cgccgttctt cgtgttgcac 1020

ggtgagaagg atccgatggt gccgagcgcg caatctcgcg ccttcagcgc ggcgctgcgc 1080

gacgcgggtg ccgcgacggt gtcctacgct gagctcccca acgcccacca cgcgttcgac 1140

ctcgccgcca cggtccgatc gcggatggtc gccgaagcgg tctcggactt cttgggtgtg 1200

atctacgggc gacggatggg tgctcgcaag ggttcgctgg cgctgtcgtc acctccggcc 1260

agctga 1266

Claims (1)

1. a mutant strain B2855 screened from Mycobacterium bovis BCG, is characterized in that, the nucleotide sequence of the mutant gene of this mutant strain is as shown in SEQ ID NO:1, and its mutation site is in Mycobacterium bovis After the genomic site 2757121, it is located after the 564 site of the LipQ gene sequence; the mutant strain B2855 comprising the sequence of SEQ ID NO: 1 is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 2018865.

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