Mycoplasma bovis secretory protein MbovP0145 and application thereof

A technology of mycoplasma bovis and protein, which is applied in the application field of mycoplasma bovis secretory protein MbovP0145, its antibody, and the preparation of mycoplasma bovis detection kit, which can solve the problems of inapplicable clinical batches, rapid and early detection, time-consuming and labor-intensive, and easy pollution And other issues

Active Publication Date: 2020-09-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current gold standard for diagnosing mycoplasma bovis is pathogen isolation. This method is time-consuming and labor-intensive and requires professional laboratories and professio

Method used

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  • Mycoplasma bovis secretory protein MbovP0145 and application thereof
  • Mycoplasma bovis secretory protein MbovP0145 and application thereof
  • Mycoplasma bovis secretory protein MbovP0145 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Cloning and expression of Mycoplasma bovis Mbov_0145 gene

[0021] 1.1 Cloning of Mycoplasma bovis Mbov_0145 gene

[0022] Due to the codon preference of Escherichia coli, the codon UGA encoding tryptophan in Mycoplasma bovis is used as a terminator in Escherichia coli, therefore, when expressing the Mycoplasma bovis gene with Escherichia coli, the mycoplasma gene needs to be mutated so that The codon UGA was mutated to the codon UGG for expression of tryptophan in E. coli.

[0023] The applicant isolated a local strain of Mycoplasma bovis from diseased lung tissue of sick cattle in June 2008, and named it Mycoplasma bovis HB0801 (Mycoplasma bovis HB0801), which has been disclosed in the patent literature of CN 102220263A.

[0024] According to the codon preference of Escherichia coli, the Mbov_0145 gene in Mycoplasma bovis HB0801 (genome GenBank accession number is CP002058) is modified by codons. , 1284, 1359 synonymous mutations of 11 codons occurred,...

Embodiment 2

[0039] Example 2: Identification of the secretion properties of the Mycoplasma bovis protein MbovP0145

[0040] 2.1 Preparation and titer detection of mouse polyclonal antibody against recombinant Mycoplasma bovis rMbovP0145 protein

[0041] Using the rMbovP0145 protein prepared above to immunize BALB / C mice, the immunization procedure is as follows:

[0042] (1) For the first immunization, the dose of Mycoplasma bovis rMbovP0145 antigen was 100 μg / mouse, and Freund's complete adjuvant was added to subcutaneously inject multiple points on the back of the neck, 0.2 mL / mouse.

[0043] (2) Two weeks later, the second immunization, the dose of Mycoplasma bovis rMbovP0145 antigen was 100 μg / mouse, and Freund's incomplete adjuvant was added to subcutaneously inject multiple points on the back of the neck, 0.2 mL / mouse.

[0044] (3) Four weeks later, for the third immunization, the dose of Mycoplasma bovis rMbovP0145 antigen was 100 μg / mouse, and Freund's incomplete adjuvant was add...

Embodiment 3

[0052] Example 3: Identification of antigenicity of Mycoplasma bovis protein MbovP0145

[0053] The antigenicity identification of rMbovP0145 protein was carried out by western blot method. The main steps were as follows: SDS-PAGE electrophoresis was performed on the purified recombinant protein rMbovP0145, and the protein on the gel was transferred to PVDF membrane. Positive serum and bovine negative serum were used as the primary antibody for incubation, and HRP-labeled goat anti-bovine IgG was used as the secondary antibody for western blot verification.

[0054] The results show that MbovP0145 can react with the positive serum of Mycoplasma bovis, and there is a band at the corresponding protein size of 55kDa ( Figure 4 A), consistent with the expected molecular weight of MbovP0145, but no obvious reaction with the negative serum of Mycoplasma bovis ( Figure 4 B).

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Abstract

The invention discloses a protein encoded by a mycoplasma bovis Mbov-0145 gene. The mycoplasma bovis Mbov-0145 gene has a nucleotide sequence as shown in SEQ ID NO: 1. The invention also discloses anantibody of the gene, and an application of the protein and the antibody in preparation of a mycoplasma bovis detection kit, and belongs to the technical field of prevention and treatment of animal infectious diseases. The protein can generate specific reaction with positive serum of naturally infected and artificially infected cattle of a mycoplasma bovis virulence strain M.bovis HB0801, and canidentify animals infected with wild virus after vaccine immunization. The protein is expected to be used as a molecular target for diagnosis reagent and vaccine of mycoplasma bovis infection and drugresearch and development, and plays an important role in prevention and control of mycoplasma bovis.

Description

technical field [0001] The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and in particular relates to a mycoplasma bovis secretory protein MbovP0145, an antibody thereof, and the application of the protein and the antibody in preparing a detection kit for mycoplasma bovis. Background technique [0002] Mycoplasma bovis is an important pathogen that causes bovine respiratory disease syndrome. It often breaks out after transportation stress, causing bovine pneumonia, mastitis, and secondary arthritis, abortion and infertility. It is an important threat to the world's cattle industry today. One of the common infectious diseases. With the rapid development of domestic and international trade in the cattle industry, mycoplasma bovis has spread globally. Mycoplasma bovis pneumonia outbreak in beef cattle was first reported in my country in 2008, the morbidity rate was more than 80%, and the fatality rate was more than 10%, whi...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/30C07K16/12G01N33/68G01N33/569C12R1/35
CPCC07K14/30C07K16/1253G01N33/56933G01N33/6854G01N2469/20
Inventor 郭爱珍张慧胡古月路豆昆赵刚张怡秋陈颖钰胡长敏陈曦陈建国
Owner HUAZHONG AGRI UNIV
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