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Antibovine mycoplasma and Pasteurella multocida mixed protein spray

A technology of Pasteurella and Mycoplasma bovis, applied in the direction of antibodies, antibacterial drugs, aerosol delivery, etc., can solve problems such as drug resistance, long diagnosis time, and inability to use early medication

Inactive Publication Date: 2015-07-15
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problems that Mycoplasma bovis and Pasteurella multocida anti-bovine capsular serum type A have drug resistance, long diagnosis time, and can not be used early in the process of treating bovine respiratory diseases, and provide A kind of anti-Mycoplasma bovis and Pasteurella mixed protein spray

Method used

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  • Antibovine mycoplasma and Pasteurella multocida mixed protein spray
  • Antibovine mycoplasma and Pasteurella multocida mixed protein spray
  • Antibovine mycoplasma and Pasteurella multocida mixed protein spray

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Md - UF1, Md - Cloning of AP2

[0015] 1. Use the kit produced by Promega (SV Total RNA Isolation) to extract three-day-old larvae and 2- and 3-day-old housefly ( Musca domestica L. )larva （ 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) total RNA, using the kit produced by TaKaRa Company Oligotex TM -dT30 mRNA Purification Kit (From Total RNA) isolates and purifies mRNA;

[0016] 2. Reverse transcription to synthesize the first strand of cDNA

[0017] (1) Take two EP tubes and add the following reagents respectively, see Table 1:

[0018] Table 1 Reaction system

[0019]

[0020] (2) Mix by blowing and blowing, centrifuge briefly, incubate in a PCR machine at 72°C for 3 minutes, and at 42°C for 2 minutes, then centrifuge briefly, add the following reagents, see

[0...

Embodiment 2

[0032] Example 2 Anti-Mycoplasma bovis, Bovine Capsular Serum Type A Pasteurella multocida Protein Md - UF1, Md - AP2 expression and purification

[0033] 1) Put the plasmid Md - UF1-pMD18-T, Md - AP2-pMD18-T, pET-32a(+), via EcoR I. xho I double digestion, the Md - UF1, Md - Insert the AP2 gene into pET-32a(+) to construct a recombinant expression plasmid Md - UF1-pET-32a, Md - AP2-pET-32a;

[0034] 2) Transformed into Escherichia coli BL21 ( DE3 ), induced by IPTG;

[0035] 3) The fusion protein was purified using nickel ion metal chelation affinity chromatography medium (Ni-NTA Agarose).

[0036] Use the eluent containing 500mM imidazole to elute the target protein step by step, so that the protein is detached from the column, and the SDS-PAGE analysis results are shown in image 3 , Figure 4 .

[0037] The protein concentration after Ni-NTA Agarose column purification was measured by a micro-volume ultraviolet spectropho...

Embodiment 3

[0038] Example 3 Md -UF1, Md - Preparation of AP2 mixed protein gel spray

[0039] In this study, according to the environmental characteristics of the nasal cavity, the ion-sensitive instant gel matrix, that is, deacetylated gellan gum, was selected as the coagulation matrix, and the prescribed amount of antibacterial peptide and solvent was added, and stirred together with excipients to make a suspension. The dose contains 0.8mg of mixed protein. The specific composition is shown in Table 3, and the mixing steps are as follows:

[0040]

[0041] Weigh the prescription amount of acetyl gellan gum into the container according to Table 3, add an appropriate amount of deionized water, heat in a water bath at 100°C and stir continuously until it is evenly dispersed, then place it in a refrigerator at 4°C to cool completely for later use, add the prescription Measure the antibacterial protein, mannitol, and ethidium chloride and stir to dissolve them, and fina...

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Abstract

The invention discloses an antibovine mycoplasma and Pasteurella multocida mixed protein spray. Every 100g of the matrix of the spray contains 3-5g of an antibovine mycoplasma-containing protein Md-UF1 and 3-5g of an antibovine Pasteurella multocida protein Md-AP2, and the matrix is a gel matrix. Gene Md-UF1 screened from a suppression subtractive library constructed after inducing housefly larvae by a pathogen through the inventor and expressing an antibovine mycoplasma protein and gene Md-AP2 screened from the library and expressing antibovine capsular serotype A protein are used to construct fusion gene, the fusion gene expresses proteins, and experiment results prove that the spray has an inhibition effect on bovine mycoplasma and bovine Pasteurella multocida. The spray has good effects in the clinical treatment of bovine respiratory diseases induced by bovine mycoplasma and Pasteurella multocida mixed infection. The spray can be used in the early stage of the bovine respiratory disease.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a medicine for resisting bovine mycoplasma disease and bovine capsular serum type A multoccidal pasteurellosis, and an anti-mycoplasma bovis and Pasteurella mixed protein spray. Background technique [0002] In recent years, bovine respiratory disease has become a major disease that endangers my country's animal husbandry industry. The incidence of the disease is as high as 80%, and the mortality rate in a single farm is as high as 60%. Pasteurella multocida type A of capsular serotype of bovine origin ( Pasteurella mutocida, Pm ) and Mycoplasma bovis ( Mycoplasma bovis, M.bovis ) is the main pathogen that causes cattle respiratory diseases in my country, and the diseases caused by it have been prevalent in many provinces of China, seriously restricting the healthy development of animal husbandry in my country. The clinical symptoms of the above two diseases ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/40A61K48/00A61K9/12A61P31/04
Inventor 孔令聪马红霞唐燕高铎刘树明
Owner JILIN AGRICULTURAL UNIV
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