Application of mycoplasma bovis MbovP730 protein in natural infection and vaccine immunity identification

A technology of Mycoplasma bovis, antigenic protein, applied in chemical instruments and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of cattle industry losses, poor effects, long clinical treatment cycles, etc. original effect

Active Publication Date: 2017-09-19
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows researchers to identify different types of mycobacteria without actually testing them or detecting their presence by measuring how well they are able to fight off other harmful organisms like ticks. By comparing these proteins with known ones from previous studies on tick-borne disease patients, we aimed at developing an assay system capable of distinguishing among various forms of meconium infectious diseases (TICs).

Problems solved by technology

This patented technical problem addressed in this patents relates to improving animal husbandry practices while reducing environmental impact on both animals and humans during their transition into production systems or offshore facilities where they may infectious agents like salmonella enterprises. Current methods involve controlling these factors but there remains room for improvement.

Method used

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  • Application of mycoplasma bovis MbovP730 protein in natural infection and vaccine immunity identification
  • Application of mycoplasma bovis MbovP730 protein in natural infection and vaccine immunity identification
  • Application of mycoplasma bovis MbovP730 protein in natural infection and vaccine immunity identification

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Embodiment 1

[0037] Embodiment 1: Cloning expression of Mycoplasma bovis Mbov_730 protein

[0038] 1.1 Cloning and expression of Mycoplasma bovis Mbov_730 gene

[0039] 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 Mbov_730 gene of Mycoplasma bovis with Escherichia coli, the Mycoplasma Mbov_730 gene needs to be mutated, That is, the codon UGA of Mycoplasma bovis was mutated to the codon UGG of tryptophan which can be expressed in Escherichia coli.

[0040] The applicant named the Escherichia coli obtained after mutation as Escherichia coli pET-30-Mbov-730, Escherichia coli, pET-30-Mbov-730, and sent it to China. Wuhan. Wuhan University, China for the preservation of typical cultures on December 18, 2015 Central deposit, deposit number is CCTCC NO: M2015762.

[0041] In this example, PCR primers were used to mutate the corresponding genes. The initial strai...

Embodiment 2

[0078] Embodiment 2: Mycoplasma bovis rMbovP730 protein specific detection

[0079] 2.1 Preparation of polyclonal antibody

[0080] Using the rMbovP730 protein prepared above to immunize BALB / C mice, the immunization procedure is as follows:

[0081] (1) For the first immunization, Mycoplasma bovis rNOX antigen 100 μg / mouse, plus Freund's complete adjuvant, subcutaneously injected multiple points on the back of the neck, 0.2mL / mouse.

[0082] (2) Two weeks later, for the second immunization, the dose of Mycoplasma bovis rMbovP730 antigen was 100 μg / mouse, and Freund's incomplete adjuvant was added to subcutaneously inject multiple points on the back of the neck.

[0083] (3) Four weeks later, for the third immunization, the dose of Mycoplasma bovis rMbovP730 antigen was 100 μg / rat, and Freund's incomplete adjuvant was added to subcutaneously inject multiple points on the back of the neck.

[0084] (4) After 7 days, cut the tip of the tail to collect blood, separate the serum...

Embodiment 3

[0087] Embodiment 3: the application of Mycoplasma bovis MbovP730 protein in distinguishing natural infection of Mycoplasma bovis field strain and immunity of Mycoplasma bovis vaccine strain

[0088] 3.1 Using M.bovis HB0801 whole bacterial protein as the coating antigen to detect serum by indirect ELISA method

[0089] (1) Animal serum sample collection: Mycoplasma bovis wild strain natural infection serum comes from clinical trials of Mycoplasma bovis suspected infected cattle, Mycoplasma bovis vaccine strain Mbov HB0801-150.2 (namely Mycoplasma bovis Mbov HB0801-150.2) (in vitro by M.bovisHB0801 Inoculation of PPLO liquid medium, 3 days as one generation, continuous passage in vitro for 150 generations) The immune serum comes from clinical trials, and the negative serum comes from the blank group of clinical immune experiments.

[0090] (2) Preparation of whole bacterial protein: M.bovis HB0801 on PPLO medium (preparation method: take 10.5g of PPLO powder, 2.5g of yeast pow...

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Abstract

The invention belongs to the technical field of animal infectious disease prevention and curing, and particularly relates to application of mycoplasma bovis MbovP730 protein in natural infection and vaccine immunity identification. According to the codon preference of escherichia coli, the Mbov_730 gene of M.bovis HB0801 is cloned and is modified, and a mycoplasma bovis tryptophan codon UGA is mutated into a tryptophan codon UGG in the escherichia coli so as to express recombinant mycoplasma bovis MbovP730 protein in the escherichia coli. The nucleotide sequence of the MbovP730 protein is disclosed in SEQ ID NO: 13, and the protein can be used as the antigen protein to be applied to the preparation of a kit for identifying mycoplasma bovis wild strain infection and vaccine strain immune identification.

Description

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Claims

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Application Information

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Owner HUAZHONG AGRI UNIV
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