CN111394366A

CN111394366A - Mycobacterium bovis BCG vaccine low-invasiveness mutant B2855

Info

Publication number: CN111394366A
Application number: CN202010153884.XA
Authority: CN
Inventors: 陈颖钰; 师红铃; 郭爱珍; 李倩倩; 郭佳成; 胡长敏; 陈焕春
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-04-16
Filing date: 2020-03-07
Publication date: 2020-07-10
Anticipated expiration: 2040-03-07
Also published as: CN111394366B

Abstract

The invention belongs to the technical field of prevention and control of animal infectious diseases, and relates to a mycobacterium bovis BCG vaccine low-invasiveness mutant B2855, wherein the mutant B2855 contains a mutant gene of mycobacterium bovis BCG vaccine L ipQ, the nucleotide sequence of the mutant gene is shown as SEQ ID NO:1, the mutant site is positioned behind a 2757121 site of a genome and behind a 564 site which is a L ipQ gene sequence, and the mutant is a new structural gene and a new functional gene.

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 low-invasiveness mutant B2855 which contains a mutant gene of mycobacterium bovis bcg L ipQ.

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 are working on developing more safe and effective tuberculosis vaccines, but no vaccine superior to bcg vaccine has been developed yet. Therefore, it is very important to develop bcg vaccine which does not affect the protective effect of bcg vaccine and is safer for the population with low immunity 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 attack the host cell 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 mutant strain relates to L ipQ gene, and has obvious low invasiveness, low intracellular viability and high growth rate compared with wild mycobacterium bovis, and the shown characters are related to the toxicity of the inserted inactivated gene.

In order to achieve the purpose of the invention, the applicant selects a strain B2855 with significantly reduced invasive ability from a mycobacterium bovis BCG gene deletion mutant library in the national emphasis laboratory of agricultural microbiology of Huazhong agriculture university, wherein the mutant strain contains L ipQ gene which encodes a carboxylesterase associated with lipid metabolism, the literature reports that L ipQ has the function of immunosuppression in mycobacterium tuberculosis and can reduce the level of proinflammatory factors [4], but no literature reports exist about the function of mycobacterium bovis.

The specific technical scheme of the invention is as follows:

the M.bovis BCG used by the applicants was Pasteur strain BCG [ American Type Culture Collection (ATCC): 35734 ] presented to professor L uiz Bermudez, Oregon State university, USA the preliminary work of the present invention included the recovery and culture of the deposited M.bovis BCG Pasteur strain.

The invention uses Mycobacterium bovis BCG vaccine Pasteur strain (ATCC: 35734) as wild strain, uses bacteriophage MycoMarT7 with temperature-sensitive property to transform Himar1 transposon into Mycobacterium bovis BCG vaccine, uses kanamycin as resistance screening mark to screen mutant strain, thereby successfully constructing mutant library, the invention screens mutant strain with reduced invasion ability from mutant library by using cell invasion model, obtains BCG mutant strain B2855 (BCG:: Tn L ipQ) with obviously reduced invasion ability, applicant names the mutant strain as Mycobacillus bovis BCG Pasteur B2855, which is delivered to China center for type culture collection of university of Wuhan university at 12 and 6 days in 2018, with preservation number of CCTCC NO: M2018865, inoculates the mutant strain 2855 and Mycobacterium bovis BCG vaccine wild strain screened by the invention into 7H9 culture medium respectively, and carries out intracellular survival ability and growth curve detection, the result shows that the mutant strain has high survival ability and growth speed 2855 and has high growth period.

The mutant strain constructed by the invention relates to gene L ipQ, the gene codes 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-fast performance relative to a wild strain of mycobacterium bovis.

4. The L ipQ gene is one new gene of mycobacterium bovis, and the gene has invasion function, and can reduce the intracellular survival of bacteria and raise the growth speed.

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. The reference numbers indicate: 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 shows the nucleotide sequence of L ipQ gene related to M.bovis BCG mutant B2855 (the nucleotide sequences shown in SEQ ID NO:1 of the sequence Listing are identical).

FIG. 4 shows the nucleotide sequence of L ipQ gene involved in M.bovis BCG mutant B2855 of the present invention (the nucleotide sequence is identical as shown in SEQ ID NO:1 of the sequence Listing). wherein transposon insertion inactivation occurs after the 564 site of the gene, i.e., 2757121 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 Mycobacterium bovis mutant gene L ipQ gene isolated in 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 from the M.bovis BCG mutant pool were individually transferred to 7H9 liquid medium (7H9 liquid medium purchased from BD Co.) and cultured, and the A549 cells (professor L uiz Bermudez, Oregon State university, USA) were used for the inductionThe staining model carries out high-throughput screening on the mycobacterium bovis BCG mutant library, A549 cells are cultured in a 12-hole cell culture plate and grow into a monolayer to reach 2 × 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 bacterial count revealed that 64 mutants showed a significant change in invasive potential compared to the wild strain BCG, with 28 strains showing reduced invasive potential and 36 strains showing enhanced invasive potential (FIG. 1).

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

To verify the results of step 1.1, the applicants further carried out quantitative assay on 28 mutants with reduced invasive potential A549 cells as per 2 × 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. The mutant and A549 cells were incubated at 37 ℃ with 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 mycobacterium bovis BCG B2855 mutant, the junction of a Himar1 transposon and the mycobacterium bovis BCG genome is sequenced, the sequencing result is compared with the whole genomic sequence of the mycobacterium bovis BCG, the result shows that the nucleotide sequence of the gene involved in B2855 is shown in a sequence table SEQ ID NO:1 because L ipQ (figure 3) has the nucleotide sequence, and the insertion site of the transposon is positioned behind a genome 2757121 site, namely behind a L ipQ gene 564 site (figure 4), so the mutant is named as BCG:: Tn L ipQ by the applicant.

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 treated according to 2 × 10⁵Spreading the cells/well on 12-well cell culture plates, inoculating a B2855 mutant strain into A549 cells by using an infection ratio of 10:1, and setting a 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% CO₂Incubate in incubator, at this time record infection 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 grows at day 12 (P) compared to M.bovis wild strain<0.05), 15 days (P)<0.01), 18 days (P)<0.01), 21 days (P)<0.01) and 27 days (P)<0.05) rise by 1.2, respectively,1.2, 1.2 and 1.1 times. 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 involved in M.bovis BCG B2855 is represented by L ipQ.

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 ofMycobacterium 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 Mce3Cpromotes mycobacteria entry into macrophages through activation of beta2integrin-mediated signalling pathway.Cell Microbiol 2018,20；

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

4.Kumar A,Manisha,Sangha1.GK,Shrivastava A,Kaur J:Theimmunosuppressive effects of a novel recombinant LipQ(Rv2485c)protein ofMycobacterium 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. The nucleotide sequence of the mutant gene separated from the mycobacterium bovis BCG is shown as SEQ ID NO. 1.

2. A mutant B2855 of mycobacterium bovis BCG is characterized in that the nucleotide sequence of the mutant gene is shown in SEQ ID NO 1, the mutation site is located behind 2757121 of mycobacterium bovis genome and 564 of L ipQ gene sequence, the mutant B2855 containing the SEQ ID NO 1 sequence is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2018865.

3. The mycobacterium bovis bcg mutant B2855 of claim 2, wherein said mutant has a significantly reduced invasive potential, a significantly enhanced intracellular survival potential, and an increased growth rate.

4. The mycobacterium bovis bcg mutant gene of claim 1, wherein the gene has the functions of increasing the invasive ability of a bacterium, and decreasing the intracellular survival ability and growth rate of a bacterium.

5. The use of the mycobacterium bovis bcg mutant gene of claim 1 in the preparation of a vaccine or medicament for preventing bovine tuberculosis.

6. The use of the mutant B2855 of M.bovis BCG as claimed in claim 2 in the preparation of vaccine or medicament for preventing tuberculosis in cattle.