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Bovine mycoplasma hypothetical protein MbovP732 and application thereof

A technology of Mycoplasma bovis, hypothetical protein, applied in application, immunoglobulin, antibacterial immunoglobulin, etc., can solve the problems of deficiency, increased serum antibody, and insufficient detection accuracy.

Inactive Publication Date: 2019-11-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since animals infected with M. bovis wild virus and immunized attenuated vaccines can both cause an increase in serum antibodies, and currently there is no effective serological method to distinguish between natural M. bovis infection and vaccine-immune populations, it is difficult to judge whether the attenuated vaccine is successful in clinical practice.
The Virus Division of the State Key Laboratory of Agricultural Microbiology, where the applicant works, found that the differential protein MbovP730 protein of strong and weak strains of Mycoplasma bovis has strong immunogenicity and reactogenicity through comparative genomics and immunoproteology, and successfully synthesized the MbovP730 protein As a molecular target for the differential diagnosis of Mycoplasma bovis wild virus infection and vaccine immunity, the protein applied for an invention patent on March 11, 2016, and was disclosed in the patent literature of CN107176977A, but the MbovP730 protein still has defects such as insufficient detection accuracy

Method used

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  • Bovine mycoplasma hypothetical protein MbovP732 and application thereof
  • Bovine mycoplasma hypothetical protein MbovP732 and application thereof
  • Bovine mycoplasma hypothetical protein MbovP732 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Mycoplasma bovis MbovP732 protein clone expression and purification

[0034] 1.1 Cloning and expression of Mycoplasma bovis Mbov_0732 gene

[0035] Due to the codon preference of Escherichia coli, the codon UGA of Mycoplasma bovis encoding tryptophan in the present invention is used as a terminator in Escherichia coli, therefore, when expressing the Mycoplasma bovis gene with Escherichia coli, the Mycoplasma bovis gene needs to be mutated , to mutate the codon UGA to the codon UGG for expression of tryptophan in E. coli.

[0036]The present invention clones and modifies the Mbov_0732 gene sequence in Mycoplasma bovis HB0801 (genome GenBank accession number is CP002058) according to the codon preference of Escherichia coli and enables the gene to be expressed efficiently in Escherichia coli, and then artificially synthesizes the optimized Mbov_0732 gene The complete sequence of the sequence is 990bp in length (see the sequence shown in the 1-990 bases in t...

Embodiment 2

[0053] Example 2: Verification of reactogenicity of Mycoplasma bovis His-rMbovP732 protein

[0054] Western blot method was used to identify the reactogenicity of His-rMbovP732 protein. The main steps are: perform SDS-PAGE electrophoresis on the purified recombinant protein His-rMbovP732, transfer the protein on the gel to a PVDF membrane, wash the membrane, and put the membrane into In 5% skimmed milk diluted with TBST, block at room temperature for 3 hours; after fully washing, put the PVDF membrane into 100-fold diluted M. test), incubated overnight at 4°C; after fully washing, put the PVDF membrane into 5000 times diluted horseradish peroxidase-labeled goat anti-bovine IgG (purchased from Southern Biotech Company), at room temperature for 1h; after washing three times with TBST, mix A good ECL luminescence solution (purchased from Thermo Company, USA) was evenly added dropwise on the membrane, and the signal was detected by a Kodak Image Station chemiluminescence detector....

Embodiment 3

[0055] Example 3: Verification of differential expression of Mycoplasma bovis His-rMbovP732 protein between strong and weak strains

[0056] 3.1 Preparation of polyclonal antibody

[0057] Mix the prepared His-rMbovP732 protein with complete Freund's adjuvant (purchased from Sigma, USA) at a ratio of 1:1, fully emulsify, and immunize three six-week-old Balb / c female mice to prepare multiple antiserum (experimental animal The use license number is SYXK (E) 2015-0084; the experimental animal ethics approval number is HZAUMO-2018-026). Immunization procedure: before immunization, blood was collected from the tip of the mouse tail, and the collected serum was negative serum. For the first immunization, the amount of His-rMbovP732 protein was 100 μg / rat, mixed with complete Freund's adjuvant at a volume ratio of 1:1, fully emulsified, and injected subcutaneously at multiple points on the back of the neck. Two weeks after the first immunization, an equal dose of protein was emulsi...

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Abstract

The invention discloses a bovine mycoplasma hypothetical protein MbovP732 and application thereof. The bovine mycoplasma hypothetical protein MbovP732 has an amino acid sequence as shown in the sequence table SEQ ID NO:1, and a gene encoding the protein has a nucleotide sequence as shown in the sequence table SEQ ID NO:2. The bovine mycoplasma hypothetical protein MbovP732 has reactogenicity and immunogenicity, can specifically react with bovine mycoplasma wild strains, and cannot react with vaccine strains, therefore, the bovine mycoplasma hypothetical protein MbovP732 can be used for distinguishing infection of the bovine mycoplasma wild strains from immunization of the vaccine strains.

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 hypothetical protein MbovP732 of Mycoplasma bovis and its application. Background technique [0002] Mycoplasma bovis (M.bovis) is an important pathogenic pathogen of cattle in the genus Mycoplasma. It can cause various diseases such as mastitis, pneumonia, arthritis and keratoconjunctivitis in cattle, and it is also the cause of bovine respiratory disease syndrome. The main pathogen of symptoms. In 1961, it was isolated for the first time from a sick cow suffering from severe mastitis in the United States, and then spread in various countries through animal migration, causing huge economic losses to the cattle industry. [0003] In my country, in 2008, a respiratory disease broke out in beef cattle imported from other places in Hubei Province. The disease occurred about two weeks after the cattle were introduced, mani...

Claims

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Application Information

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IPC IPC(8): C07K14/30C12N15/31C12N1/21C07K16/12G01N33/569C12R1/19
CPCC07K14/30C07K16/1253G01N33/56933G01N2333/30G01N2469/10
Inventor 郭爱珍聂晶晶陈颖钰杨莉赵刚胡长敏陈曦陈焕春
Owner HUAZHONG AGRI UNIV
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