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Mutant having reduced adhesive ability and with deleted mycoplasma bovis gene

A technology of mycoplasma bovis and gene deletion, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve problems such as unclear effects

Active Publication Date: 2020-06-05
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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  • Mutant having reduced adhesive ability and with deleted mycoplasma bovis gene
  • Mutant having reduced adhesive ability and with deleted mycoplasma bovis gene
  • Mutant having reduced adhesive ability and with deleted mycoplasma bovis gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Screening and identification of adhesion-deficient mutants of Mycoplasma bovis

[0046] 1. Preliminary screening of adhesion-deficient mutants of Mycoplasma bovis

[0047] (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 Mycoplasma bovis mutants with different ORFs inserted into the transposon were inoculated with liquid medium (PPLO powder 10.5g, sodium pyruvate powder 0.5g, yeast 2.5g, and 440mL ddH 2 Diluted to volume and steam sterilized at 121°C for 18 minutes, added 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, placed at 37°C, 5 %CO 2 Cultivate in the incubator for 36 hours, that is, after reaching the log...

Embodiment 2

[0052] Embodiment 2: Expression of Mycoplasma bovis Mbov0503 protein

[0053] 1. Cloning and expression of Mycoplasma bovis Mbov_0503 gene

[0054] 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 , using 6 pairs of primers designed as follows (respectively: 0503a1 / 0503a2, 0503b1 / 0503b2, 0503c1 / 0503c2, 0503d1 / 0503d2, 0503e1 / 0503e2, 0503f1 / 0503f2) to ampli...

Embodiment 3

[0099] Example 3: Detection of Adhesion Ability of Recombinant Protein rMbov0503

[0100] 1. Adhesion detection of recombinant protein rMbov0503 to EBL cells

[0101] (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;

[0102] (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;

[0103] (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 ;

[0104] (4) Fixation and permeabilization: 1 mL of commercial...

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Abstract

The invention belongs to the field of control of animal borne diseases, and relates to a mutant having reduced adhesive ability and with a deleted mycoplasma bovis gene. The protein gene Mbov_0503 isobtained by cloning from a mycoplasma bovis HB0801 genome. According to the partiality of escherichia coli for codons, the Mbov_0503 gene is modified, and mycoplasma bovis tryptophan codon UGA is mutated into a codon UGG for coding tryptophan in the escherichia coli to obtain a recombinant protein Mbov0503. The sequence of the protein gene which is cloned is shown as SEQID NO:13, and the sequenceof the coded protein is shown as SEQID NO:14. The mutant provided by the invention is an adhesion deficient strain screened from a mutant bank. The mutant has adhesive ability for host EBL cells, andcompared with a wild strain, for the mutant disclosed by the invention, the transmembrane transmission capacity for MDBK cells and the destructivity for tight connection between cells are notably reduced. The mutant can be applied to pathopoiesis and control of the mycoplasma bovis.

Description

technical field [0001] The present invention is a divisional application with an application number of 2019101283763 and an application date of February 21, 2019. [0002] The invention belongs to the technical field of animal infectious disease prevention and control, and in particular relates to 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 Mycoplasma bovis genome and after the 313 site of the Mbov_0503 gene. Compared with the wild strain, the mutant strain had a significant defect in the ability to adhere to host cells, and the ability to destroy tight junctions between cells and transmembrane transmission was 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 mecha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12Q1/02C12R1/35
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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