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Mycoplasma bovis gene mutation strain having reduced adhesion capacity and adhesion protein

A technology of Mycoplasma bovis and mutant strains, which can be applied in genetic engineering, plant genetic improvement, and microbial-based methods, and can solve problems such as unclear effects

Active Publication Date: 2019-06-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, some adhesion-related proteins such as α-enolase (Song et al., 2012), VSPs (Sachse et al., 2000) and NADH Oxidase (Zhao et al., 2017) have been reported, but most of the functional studies of these proteins are based on recombinant proteins. The role of M. bovis in the actual adhesion process is unclear

Method used

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  • Mycoplasma bovis gene mutation strain having reduced adhesion capacity and adhesion protein
  • Mycoplasma bovis gene mutation strain having reduced adhesion capacity and adhesion protein
  • Mycoplasma bovis gene mutation strain having reduced adhesion capacity and adhesion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Screening and identification of adhesion-deficient mutants of Mycoplasma bovis

[0044] 1. Preliminary screening of adhesion-deficient mutants of Mycoplasma bovis

[0045] (1) Cultivation and enumeration of mutant strains of Mycoplasma bovis: The mutant library of Mycoplasma bovis was constructed by the Ruminant Pathogen Division of the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University where the applicant is located, and stored at -80°C. The M. bovis mutants with different ORFs inserted into the transposon were inoculated with liquid medium (10.5 g of PPLO powder, 0.5 g of sodium pyruvate powder, 2.5 g of yeast, and 440 mL of ddH 2 Dilute to volume in O and sterilize at 121°C for 18 minutes, add 50 mL of 10% horse serum, 5 mL of 10×MEM, 1 mL of 400,000 units / mL penicillin solution, 500 μL of phenol red growth indicator) to recover, place at 37°C, 5 %CO 2 Cultivate in the incubator for 36 hours, that is, after reaching the l...

Embodiment 2

[0050] Embodiment 2: Expression of Mycoplasma bovis Mbov0503 protein

[0051] 1. Cloning and expression of Mycoplasma bovis Mbov_0503 gene

[0052] Because E. coli is to the preference of codon, in the present invention, the codon UGA of tryptophan of encoding tryptophan in the Mycoplasma bovis genome is used as terminator in Escherichia coli, therefore, when expressing Mycoplasma bovis gene with E. coli, need to mycoplasma gene Mutations were performed so that the codon UGA was mutated to the codon UGG for expression of tryptophan in E. coli. In order to express the Mbov_0503 gene of Mycoplasma bovis, the applicant mutated the corresponding 5 codons of the gene using self-designed PCR primers. The specific steps are: using the Mbov_0503 gene in Mycoplasma bovis HB0801 (GenBank accession number CP002058) as a template 6 fragments of the mutated Mbov_0503 gene were respectively amplified using 6 pairs of primers designed as follows (numbers: 0503a1 / 0503a2, 0503b1 / 0503b2, 0503c...

Embodiment 3

[0092] Example 3: Detection of Adhesion Ability of Recombinant Protein rMbov0503

[0093] 1. Adhesion detection of recombinant protein rMbov0503 to EBL cells

[0094] (1) Shop 1×10 5 EBL cells were placed in a 24-well plate at 37°C, 5% CO 2 Cell adherent culture under the condition;

[0095] (2) Interact 10 μg of Mbov0503 with EBL cells for 1 hour, with a reaction volume of 200 μL, so that the protein can fully contact with the cells, and set up the following controls: only MEM basal medium (Hyclone) was added as a blank control; rMbov0503 was treated with anti-Mbov0503 The polyclonal antibody was incubated at 37°C for 1h; rMbov0503 was incubated with mouse negative serum at 37°C for 1h;

[0096] (3) Sealing: wash the non-adhered protein with the open PBS gently, wash 5 times, add PBS to wash in different directions each time, add 5% skimmed milk to seal at room temperature for 2 hours, wash 3 times with PBS ;

[0097] (4) Fixation and permeabilization treatment: Add 1 mL...

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Abstract

The invention belongs to the technical field of prevention and treatment of animal borne diseases, and relates to a mycoplasma bovis gene mutation strain having reduced adhesion capacity and adhesionprotein. The protein gene Mbov_0503 is cloned from a mycoplasma bovis HB0801 genome. According to the partiality properties of escherichia coli to codons, the Mbov_0503 gene is modified, and mycoplasma bovis tryptophan codons UGA are mutated into codons UGG for coding tryptophan in the escherichia coli, so that recombination protein Mbov0503 is obtained. The nucleotide sequence of the protein geneis shown as SEQID NO:13, and the coded protein sequence is shown as SEQID NO:14. The mutation strain is the adhesion defect strain screened from a mutant library. Compared with wild strains, the mutation stain has the advantages that the adhesion capacity to host EBL cells, the cross-membrane transmission capacity to MDBK cells and destructivity to tight connection between cells of the mutation strain are notably reduced. The mycoplasma bovis gene mutation strain can be used for prevention and treatment of mycoplasma bovis diseases.

Description

technical field [0001] The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and in particular relates to the mycoplasma bovis Mbov_0503 gene deletion mutant strain T4.4 and the function-unknown protein Mbov0503 encoded by the gene. The mutation site is located after the 586830 site of the M. bovis genome and after the 313 site of the Mbov_0503 gene. Compared with the wild strain, the mutant strain has a significant defect in the ability to adhere to host cells, and the ability to destroy the tight junction between cells and the ability to spread across the membrane are significantly reduced. The recombinant protein rMbov0503 can adhere to host cells and cell membrane proteins. The mutant strain and protein are expected to have application prospects in the fields of Mycoplasma bovis pathogenic mechanism and immune prevention and control. Background technique [0002] Mycoplasma bovis (M.bovis) mainly causes bovine bronchopn...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/31C12N1/21C07K14/30C12Q1/02C12R1/35C12R1/19
CPCC07K14/30C12Q1/025G01N2333/30
Inventor 郭爱珍朱习芳董亚旗李茜茜陈颖钰胡长敏陈焕春
Owner HUAZHONG AGRI UNIV
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