CN111635880A

CN111635880A - Mycoplasma bovis VspX gene mutant strain and construction method and application thereof

Info

Publication number: CN111635880A
Application number: CN202010372788.4A
Authority: CN
Inventors: 郭爱珍; 陈曦; 刘东明; 朱习芳; 张慧; 路豆昆; 陈颖钰; 胡长敏; 陈建国; 陈焕春
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-05-06
Filing date: 2020-05-06
Publication date: 2020-09-08
Anticipated expiration: 2040-05-06
Also published as: CN111635880B

Abstract

The invention discloses a Mycoplasma bovis VspX gene mutant, which is named as Mycoplasma bovis (Mycoplasma bovis) T8.101 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M20191038, and the VspX gene has a nucleotide sequence shown as SEQ ID NO: 1. The invention also discloses a construction method of the mutant strain and application of the mutant strain in the field of mycoplasma bovis pathogenicity mechanism and immune control. The mutant strain does not express the protein coded by the mutant gene, and the growth curve, the colony morphology and the size of the mutant strain have no obvious difference from those of a wild strain. But the binding capacity to bovine pulmonary epithelial cell line and fibronectin is obviously reduced, and the strain has the potential of being applied to the field of research of bovine mycoplasma pathogenic mechanisms and development of preventive drugs as a virulence attenuated strain.

Description

Mycoplasma bovis VspX gene mutant strain and construction method and application thereof

Technical Field

The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and particularly relates to a Mycoplasma bovis VspX gene mutant strain, a construction method of the strain, and application of the strain in the fields of mycoplasma bovis pathogenicity mechanism and immune prevention and control.

Background

Mycoplasma bovis (m.bovis) is one of the important pathogens causing bovine respiratory diseases, resulting in various clinical conditions, mainly including bronchopneumonia, mastitis, arthritis, genital tract inflammation, tenosynovitis, and the like. Because the pathogenesis of the traditional Chinese medicine is unclear, the traditional Chinese medicine still lacks effective vaccines clinically at present, has poor antibiotic treatment effect and long treatment course, is easy to generate drug resistance, causes difficult prevention and control of the disease, and causes huge economic loss to beef cattle and dairy cow industries.

Although mycoplasma is the smallest genome among known genomes, there is a lack of effective genetic manipulation techniques available for mycoplasma, thus leaving annotation of its gene function long behind other bacteria. The reasons mainly include: the codon UGA of mycoplasma bovis encoding tryptophan is considered a stop codon in the heterologous host and, therefore, the full-length protein cannot be expressed in the exogenous expression system; secondly, homologous recombination efficiency is very low in some mycoplasma, and site-directed mutagenesis of genes is limited; in addition, many mycoplasma lack plasmids themselves that can be used for genetic manipulation, necessitating the introduction of foreign plasmids.

At present, transposon-based mutagenesis is a very valuable genetic tool for mycoplasma. The Tn4001 transposon has the size of 4.7kb, is derived from staphylococcus aureus, has the functional advantages of easy phenotype screening, multiple insertion sites, easy operation and the like, and is particularly suitable for mycoplasma gene operation; new advantages such as unique modification sites, increased resistance screening range or reduced transposition probability are added to the modified Tn4001 mod, min-Tn4001, mini-Tn4001-SGM and the like. These transposons are now widely used in the construction of pools of random mutants of various mycoplasmas. No report on the construction of a mycoplasma bovis random insertion mutant library by using a min-Tn4001 transposon is found at home. The construction of a mycoplasma bovis saturated transposition mutant library can draw a whole gene map and quickly decode the function of each gene.

The Mycoplasma bovis VspX gene is 689bp in length and encodes a VspX protein, which has been shown to have Plasminogen (PLG) binding properties. Therefore, the pMT85 plasmid containing mini-Tn4001 transposon is randomly transferred into mycoplasma bovis under the mediation of PEG, and a VspX gene insertion mutant strain is screened out, and the growth characteristic and the adhesiveness of the mutant strain are preliminarily researched, so that the new characteristics of the mutant strain are determined: the mutant strain had an unaffected growth rate and reduced binding properties to bovine lung epithelial cell line (EBL) and Fibronectin (Fn) compared to the wild type strain. Fn is an extracellular matrix component (ECM) which can be used as a molecular bridge to connect bacterial adhesin and a surface receptor of a host cell, therefore, the protein with Fn binding property plays an important role in the pathogenesis of bacteria, the coding gene is a virulence gene, and the successful construction of the gene mutant strain provides a good genetic operation tool for deeply researching the molecular pathogenesis of mycoplasma bovis. Adhesion of lung epithelial cells is a key step of invasion of lung tissues by m.bovis, and reduction of adhesion suggests possible reduction of virulence, so that the mutant strain also has potential for application in novel vaccine development.

Disclosure of Invention

The invention aims to overcome the technical obstacles in the prior art and provide a mycoplasma bovis VspX gene mutant strain and a construction method and application thereof.

The mutant provided by the invention uses M.bovis HB0801(GenBank accession number is CP002058) as a parent strain, utilizes a polyethylene glycol (PEG) mediated transformation method to transform pMT85 plasmid containing min-Tn4001 transposon into mycoplasma bovis, then utilizes gentamycin as a resistance screening mark to carry out primary screening, secondary screening and transposon stability detection on the mutant strain, successfully constructs a transposon insertion mutant which is inserted into VspX gene and named as M.bovis T8.101, and is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university in Wuhan city, Hubei province in 12 months and 11 months in 2019, and the preservation number is CCTCC NO: M20191038.

The basic idea of the invention is as follows: constructing a mycoplasma bovis random insertion mutant library by using a min-Tn4001 transposon, and screening a VspX gene insertion mutant strain from the mycoplasma bovis mutant library by sequencing; western blot is used for verifying that the mutant gene does not express protein in the mutant strain; then, the growth characteristics of the mutant strain are tested, and T8.101 is similar to the growth curve of the wild strain, and the colony morphology and the colony size of the T8.101 are not obviously different. The experiment of the wild strain and the T8.101 adhering to the EBL cell shows that the T8.101 adhering ability to the cell is obviously lower than that of the wild strain. Finally, ELISA test of the adhesion ability of T8.101 and wild strain as coating antigen to Fn shows that the adhesion ability of T8.101 to Fn is also reduced remarkably compared with wild strain. The protein which is expressed by the mutant strain T8.101 in a deletion way has the characteristic of Fn adhesion, and the reduction of the cell adhesion capacity is realized by weakening the binding force with Fn. The details are shown in the examples.

Drawings

FIG. 1: bovis mutant sequencing results.

FIG. 2: western blot verified the expression of VspX protein in T8.101 and wild strain strains. Description of reference numerals: m represents a chromogenic protein standard; 1 represents the whole mycoprotein of a wild strain; 2 represents T8.101 holomyces protein.

FIG. 3: growth curves of T8.101 and wild strain.

FIG. 4: colony morphology under microscope of T8.101 and wild strain. Description of reference numerals: panel A represents the colony morphology of the wild strain; panel B represents the morphology of the T8.101 colony; bar is 500. mu.m.

FIG. 5: t8.101 was compared with the number of adherent EBL cells of the wild strain.

FIG. 6: t8.101 and the wild strain were compared to Fn adhesion ability.

Detailed Description

The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.

Example 1: construction of Mycoplasma bovis insertion mutant library

The pMT85 plasmid was originally proposed by Bayanowski, university of French, Agrochemical, and contains a mini Tn4001 transposon (mini-Tn Tn4001) into which a gentamicin resistance marker encoded by the aacA-aphD gene was introduced, located between two Inverted Repeats (IR) at the ends of the transposed segment, and a transposase gene (tnPA) located outside the repeats, preventing transposition from occurring again.

Constructing mutant library by using M.bovis HB0801 as parent strain, collecting M.bovis cultured to late logarithmic phase, washing twice with cold DPBS buffer solution, and suspending in 0.1M CaCl₂Incubating in solution on ice for 30 min; the prepared Mycoplasma bovis competent cells were mixed with 3. mu.g of pMT85 plasmid, 10. mu.g of yeast tRNA and 1mL of 50% PEG 8000. After 1min of incubation, the mixture was diluted into 5mL of PPLO medium and incubated at 37 ℃ for 3 h. Mycoplasma bovis was then washed, resuspended in 1mL PPLO medium, and plated onto gentamicin-containing PPLO solid medium. Incubating at 37 ℃ for 3-7 d, selecting single colonies, culturing in 1mL PPLO broth containing gentamicin until logarithmic phase, preserving strains at-80 ℃, and establishing mycoplasma bovis HB0801 mutant library.

Example 2: screening and identification of Mycoplasma bovis VspX gene mutant strain

1. Genome sequencing and screening mutant strain

Mutant genome DNA (gDNA) in a mutant library is extracted by adopting a CTAB method, the DNA is sent to Wuhan Shaozhi Biotechnology Limited to sequence the junction of a transposon sequence and the mutant genome, the sequencing result is compared with a M.bovis HB0801 whole genome sequence, the result shows that a transposon sequence exists in a Vspx gene (the sequence shown in SEQ ID NO: 1), a min-Tn4001 transposon insertion site is positioned behind a genome 548342 site and behind a Vspx gene 384 site (figure 1), and the mutant is named as M.bovis T8.101.

Western blot verification of mutant protein expression

Culturing T8.101 and wild strains in PPLO culture medium for 48h, collecting thallus, splitting, separating whole mycoprotein by SDS-PAGE, and transferring to PVDF membrane; after washing and blocking, a self-made rVspX protein-resistant monoclonal antibody (1:2000) (Chen et al.2017) is used as a primary antibody, a goat anti-mouse IgG-HRP antibody (1:4000) is used as a secondary antibody for incubation, and Western blot analysis is carried out after color development by a chemiluminescence substrate. The results showed that T8.101 did not detect a VspX protein band at 27ku, whereas the wild strain could detect a protein band at this position (fig. 2), indicating that T8.101 mutant VspX protein expression was deficient and that the VspX gene was successfully mutated by transposon insertion.

Example 3: growth characteristics test of Mycoplasma bovis mutant T8.101

The growth curve and colony morphology of the mutant strain were examined by conventional methods.

1. Growth Curve determination

Recovering T8.101 and wild strain, respectively inoculating to PPLO solid culture medium, culturing at 37 deg.C with 5% CO₂Culturing for 72h in an incubator. After 3 monoclonals are picked from each strain, the monoclonals are respectively inoculated in 1mL of PPLO liquid culture medium, the culture is carried out for 96h at the temperature of 37 ℃, bacterial liquid is taken every 12h and coated on a PPLO plate, after the culture is carried out for 72h, the number of colonies is counted under a microscope, and a growth curve of each bacterium is drawn. The results show that the growth rate and growth state of T8.101 and the wild strain are similar and have no obvious difference, 12-24h is in logarithmic phase, 24-60h is in plateau phase, and then the death phase is entered (figure 3)

2. Observation of colony morphology

T8.101 and the wild strain cultured to the logarithmic phase were inoculated to PPLO solid plates, respectively, and after culturing for 72 hours under the same culture conditions, colony morphology was observed under a microscope. The result shows that after 72h of culture, colonies which are visible by naked eyes and have the size of a needle point grow on the two colonies, the colonies under a microscope are circular, have smooth edges and are typical egg-fried shapes, have a central navel, the colony morphology and the size of the two colonies have no obvious difference (figure 4), the growth characteristics of M.bovis are not influenced after the VspX gene is deleted, and the VspX protein does not participate in the growth metabolism of the mycoplasma bovis.

Example 4: functional verification of mycoplasma bovis mutant strain T8.101

1. Mycoplasma bovis mutant strain T8.101 adhesion EBL cell detection

Bovine lung epithelial cell line (EBL) was seeded in 24-well cell culture plates at 37 ℃ with 5% CO₂Culturing for 20h in incubator, discarding the medium, blocking the cells with 1% BSA-MEM at 37 deg.C for 15min, and mixing T8.101 with wild strain (1 × 10)⁸CFU) was added to the EBL-containing cells (about 2 × 10)⁵One/well) was incubated at 37 ℃ for 30min with shaking. After being fully washed by PBS for four times, a monolayer of cells are digested by 0.25% trypsin, a cell suspension is coated on a PPLO solid plate by a continuous dilution method, the number of colonies is counted under a microscope after being cultured for 72 hours, the number of adhered M.bovis (CFU/hole) of each hole is calculated, and the adhesion capacity of each strain to the cells is judged. The experiment was repeated 5 times, with 6 replicates set up each time. The results showed that the number of T8.101 adhering to EBL cells was significantly lower than the wild strain (p < 0.05) (fig. 5), indicating that deletion of the VspX gene decreased the adhesion ability of the m.bovis mutant.

2. Mycoplasma bovis mutant strain T8.101 adhesion Fn detection

100 μ L of the suspension was added at a concentration of 1 × 10⁸Respectively adding CFU/mL T8.101 and wild strains into a 96-well plate, and centrifuging for 10min at 1000 g; after the skim milk is sealed, incubating the skim milk with bovine-derived Fn (0, 5, 25, 50. mu.g/mL respectively) at room temperature for 1.5 h; then sequentially incubating with rabbit anti-bovine Fn antibody (diluted 1: 500) and goat anti-rabbit IgG-HRP antibody (diluted 1: 6000); measurement of the absorbance at a wavelength of 630nm after color development by TMBThe light value. The experiment was repeated 6 times, 3 replicates each time were set up. The result shows that the amount of Fn bound by the two strains is increased along with the increase of the concentration of the Fn, and the strains are bound with the Fn in a dose-dependent manner; at the same Fn concentration, the wild strain bound Fn more than T8.101, and at Fn concentrations of 50 μ g/mL, the wild strain bound Fn significantly higher than T8.101 (p < 0.05) (fig. 6), indicating that the mutant Fn binding capacity was significantly reduced.

<110> university of agriculture in Huazhong

<120> Mycoplasma bovis VspX gene mutant strain and construction method and application thereof

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Claims

1. A Mycoplasma bovis VspX gene mutant is named as Mycoplasma bovis (Mycoplasma bovis) T8.101 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M20191038, and the VspX gene has a nucleotide sequence shown as SEQ ID NO: 1.

2. The mutant mycoplasma bovis VspX gene strain of claim 1, wherein: the min-Tn4001 transposon is inserted into the VspX gene of the mutant strain.

3. The mutant mycoplasma bovis VspX gene strain of claim 2, wherein: the min-Tn4001 transposon was inserted at a position 548342 after the Mycoplasma bovis genome and at a position 384 after the VspX gene.

4. A method of constructing a mutant VspX gene strain of mycoplasma bovis according to any one of claims 1-3, wherein: the pMT85 plasmid containing the min-Tn4001 transposon was transformed with CaCl in the presence of yeast tRNA and PEG8000₂And (3) screening resistance strains in the prepared mycoplasma bovis competent cells through gentamicin resistance, identifying the resistance strains, and screening to obtain VspX gene mutant strains.

5. A method of constructing a mutant strain of Mycoplasma bovis VspX gene according to claim 4, wherein: the mycoplasma bovis competent cell is a mycoplasma bovis HB0801 cell.

6. Use of the VspX gene mutant strain of mycoplasma bovis according to any one of claims 1-3 for the pathogenesis of mycoplasma bovis and for the preparation of immune control drugs.

7. The use of claim 6, wherein: the immune control drug is a mycoplasma bovis vaccine, and the adhesion of the vaccine to bovine lung epithelial cells and fibronectin is reduced, so that the virulence is weakened.