Anti-Mycoplasma bovis and Pasteurella fusion protein md‑UF1‑Md‑AP2

A fusion protein, md-uf1-md-ap2 technology, applied in the direction of DNA / RNA fragments, antibacterial drugs, peptide / protein components, etc., can solve the problems of difficult control and treatment, concurrent infection, and aggravated antibiotic residues

Inactive Publication Date: 2017-12-01
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, only the United States has a commercialized vaccine to prevent Mycoplasma bovis infection, while other countries use antibacterial drugs to treat it. Due to the widespread use and even abuse of antibiotics, the M.bovis The emergence of drug-resistant strains with a high rate of drug resistance, the direct consequences of drug resistance not only bring difficulties to the control and treatment of the disease, but also aggravate the residues of antibiotics in cattle. Therefore, the development of new anti- M.bovis Drugs are imminent
[0006] The symptoms of the above two diseases are similar, and the clinical diagnosis requires measures such as bacterial culture, and it takes a period of time to confirm the diagnosis, and it is often complicated by infection

Method used

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  • Anti-Mycoplasma bovis and Pasteurella fusion protein md‑UF1‑Md‑AP2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of a suppressive subtractive library after induction of housefly larvae by Salmonella and screening of the protein gene against Pasteurella multocida type A from bovine capsular serum

[0029] 1. The source of the induced strain

[0030] The inducer Salmonella chicken origin was isolated from poultry clinically suffering from salmonellosis, and the isolated bacteria were identified by S.S medium and molecular biology methods, and the results proved that it was Salmonella.

[0031] 2. Construction of a suppressive subtraction library after induction of housefly larvae by Salmonella gallinarum

[0032] Select 2 and 3 day-old houseflies ( Musca domestica L. ) larvae (the species is blowfly, the fly eggs were purchased from the Liaoyuan Ecological Breeding Factory in Jilin Province, and incubated by the Pharmacology and Toxicology Laboratory of Jilin Agricultural University). Dip the No. 2 insect needle into the Salmonella gallinarum solution (conc...

Embodiment 2

[0035] Example 2 Construction of a suppressive subtractive library after induction of housefly larvae by Mycoplasma pneumoniae and screening of anti-Mycoplasma bovis protein genes

[0036] 1. Isolation and identification of the induced strain Mycoplasma pneumoniae

[0037] The lung lesions of pigs suffering from pneumonia were collected, isolated and cultured according to the procedure of mycoplasma isolation and culture, colony morphology was observed, and the isolated bacteria were identified by molecular biology methods. The results showed that the colonies of isolated bacteria were observed under a 40 times ordinary optical microscope. The morphology has the typical characteristics of "fried egg", which preliminarily proves that the isolated bacteria are mycoplasma.

[0038]The Mycoplasma pneumoniae 16S rRNA full-length primer was used to identify it (see Table 1), and a fragment of about 1.5 kb was amplified, which was in line with the expected fragment size. After the ge...

Embodiment 3

[0045] Example 3 Fusion gene against Mycoplasma bovis, bovine capsular serum type A Pasteurella multocida Md -UF1- Md -Clone of AP2

[0046] 1. Use the kit (SV Total RNA Isolation) produced by Promega to extract the total RNA of three-day-old housefly larvae induced by Mycoplasma bovis and Salmonella gallinarum respectively, and use the kit produced by TaKaRa Oligotex TM -dT30 mRNA Purification Kit (From Total RNA) isolates and purifies mRNA;

[0047] 2. Reverse transcription to synthesize the first strand of cDNA

[0048] (1) Take two EP tubes and add the following reagents respectively, see Table 1:

[0049] Table 1 Reaction system

[0050]

[0051] (2) Mix by blowing and blowing, centrifuge briefly, incubate in a PCR machine at 72°C for 3 minutes, then at 42°C for 2 minutes, then centrifuge briefly, add the following reagents, see Table 2: Reaction System

[0052]

[0053] (4) Mix well, centrifuge briefly, and incubate in a PCR instrument at ...

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Abstract

The invention discloses an anti-Mycoplasma bovis and Pasteurella fusion protein Md-UF1-Md-AP2 and a gene encoding the fusion protein. It induces housefly larvae with Mycoplasma pneumoniae and constructs an inhibitory subtractive library. In the inhibitory subtractive library The Md-UF1 gene screened out in the chicken-derived Salmonella housefly larvae was used to construct a suppressive subtractive library, and the Md-AP2 gene screened out in the suppressive subtractive library was constructed using the overlap extension and shearing technique. The fusion gene Md-UF1-Md-AP2, the expressed anti-Mycoplasma bovis and Pasteurella fusion protein Md-UF1-Md-AP2, has inhibitory effects on Pasteurella multocida type A and Mycoplasma bovis, The problem of drug resistance of Pasteurella bovis and Mycoplasma bovis has been solved. When the cattle have pneumonia symptoms, early medication can prevent and treat at the same time.

Description

technical field [0001] The invention belongs to the field of biological technology, and has a protein gene related to simultaneously resisting Mycoplasma bovis and bovine capsular serum type A Pasteurella multocida Md -UF1- Md - AP2, anti-Mycoplasma bovis, bovine capsular serum Pasteurella multocida type A protein Md-UF1-Md-AP2. Background technique [0002] Pasteurella multocida type A of capsular serotype of bovine origin ( Pasteurella mutocida, Pm ) and Mycoplasma bovis ( Mycoplasma bovis, M.bovis ) is the main cause of respiratory disease in cattle. It mainly occurs in fattening cattle imported from other places. The common people call it "rotten lung disease". The incidence rate is as high as 80%, and the mortality rate in a single farm is as high as 60%. The occurrence of the disease has seriously restricted the development of the beef cattle breeding industry in Northeast China. The relevant government departments and farmers are eager to develop effective measu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/70C12N1/21C07K19/00A61K38/17A61P31/04
Inventor 马红霞裴志花孔令聪刘树明王梓
Owner JILIN AGRICULTURAL UNIV
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