Preparation and application of egg yolk antibody containing florfenicol drug resistance gene protein

A yolk antibody and anti-florfenicol technology, which is applied in the field of biological products and livestock and poultry disease prevention and control, can solve problems not involved in the important application of florfenicol-resistant proteins, achieve good immunogenicity, improve sensitivity, The effect of easy operation

Inactive Publication Date: 2013-12-04
武汉市畜牧兽医科学研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, the literature does not involve the preparation of florfenicol-resistant protein (FloR protein) egg yolk antibody and its important application in animal disease prevention and control

Method used

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  • Preparation and application of egg yolk antibody containing florfenicol drug resistance gene protein
  • Preparation and application of egg yolk antibody containing florfenicol drug resistance gene protein
  • Preparation and application of egg yolk antibody containing florfenicol drug resistance gene protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Cloning and expression of partial gene fragments of floR gene

[0038] 1.1 Main materials, reagents and preparation

[0039] Escherichia coli BL(DE3), plasmid vector pET-28a(+), protein marker, rabbit anti-HIS antibody (i.e. primary antibody) and goat anti-rabbit IgG HRP-labeled antibody (i.e. secondary antibody) were purchased from Wuhan Sanying Biotechnology Ltd.

[0040] Riemerella anatipestifer (RA-1): preserved by the Wuhan Institute of Animal Husbandry and Veterinary Sciences, and submitted to China. Wuhan. Wuhan University China Type Culture Collection Center for preservation on September 10, 2008. The preservation number is CCTCC NO : M208128; The present invention utilizes the Kirby-Barer method (Xu Shuyun, Bian Rulian, Chen Xiu. Pharmacological Experimental Methodology, the third edition. Beijing: People's Health Publishing House, 2001) to measure the inhibition zone of florfenicol to the bacteria is 7.5 mm, judged to be florfenicol-resistant strai...

Embodiment 2

[0073] The preparation of embodiment 2 immunogen

[0074] 2.1 Main materials, reagents and preparation

[0075] Freund's complete adjuvant and Freund's incomplete adjuvant were products of Sigma, USA.

[0076] LB broth: tryptone 10g, yeast extract 5g, NaCl 10g, dilute to 1000mL with distilled water, adjust the pH value to pH7.0, sterilize by autoclaving at 121°C for 20min, store at 4°C for later use. If kanamycin-resistant LB broth is prepared, when the temperature of the culture medium drops to about 50°C, add kanamycin to make the final concentration 60 μg / mL, and store at 4°C for later use.

[0077] Phosphate buffered saline (PBS): Weigh 8g of NaCl, 0.2g of KCl, NaCl 2 HPO 4 1.44g, KH 2 PO 4 0.24g, dissolved in ddH 2 O800mL, adjust the pH to 7.4, and dilute to 1000mL with double distilled water.

[0078] Inclusion body washing solution: weigh 8g of NaCl, 0.2g of KCl, NaCl 2 HPO 4 1.44g, Triton-X 10mL, dilute to 1000mL with double distilled water.

[0079] Inclu...

Embodiment 3

[0093] Example 3 Preparation of Antibodies and Measurement of Biological Activity

[0094] 3.1 Main materials, reagents and preparation

[0095] Riemerella anatipestifer: preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M208128.

[0096] Escherichia coli (ATCC25922) was donated by the Veterinary Pharmacology Laboratory of Huazhong Agricultural University (purchased in Beijing, China Veterinary Drug Control Institute).

[0097] Florfenicol was donated by Hubei Zhongmu Anda Pharmaceutical Co., Ltd.

[0098] The mouse anti-chicken IgG enzyme-labeled secondary antibody was a product of Wuhan Sanying Biotechnology Co., Ltd.

[0099] Phosphate buffered saline (PBS): Weigh 8g of NaCl, 0.2g of KCl, NaCl 2 HPO 4 1.44g, KH 2 PO 4 0.24g, dissolved in ddH 2 In O800mL, adjust the pH to 7.4, and set the volume to 1000mL.

[0100] Coating solution: NaHCO 3 2.93g; Na 2 CO 3 1.95g; Dissolve in 900mL distilled water, adjust pH to 9.6...

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Abstract

The invention relates to preparation and application of an egg yolk antibody containing florfenicol drug resistance gene protein (FloR). Riemerella anatipestifer (CCTCC No: M208128) resistant to florfenicol is separated from a duck dies clinically; PCR is adopted to amplify a section of the drug resistance gene (floR); the section of the drug resistance gene is cloned to an expression plasmid pET28a (+) for transfecting host bacteria BL21 (DE3). The recombination strain is subject to induction expression, and fusion protein containing the target gene section and having a HIS label is prepared; the fusion protein exists in the form of an inclusion body; the inclusion body is extracted for the preparation of an immunogen; the immunogen is used for immunizing a laying hen, so as to obtain the egg yolk antibody capable of improving the sensitivity of the drug resistance strain to florfenicol; the egg yolk antibody is used in combination with florfenicol to remarkably improve the treatment effect on a duck infected with a drug-resistant riemerella anatipestifer strain ; as other strains resistant to florfenicol are also mediated through a floR gene, the egg yolk antibody can be applied to the veterinary clinical treatment on livestock and poultry infected with strains resistant to florfenicol.

Description

technical field [0001] The invention belongs to the technical field of prevention and control of biological products and livestock and poultry diseases. It specifically relates to an egg yolk antibody against florfenicol drug resistance gene protein (FloR), its preparation method and application. Background technique [0002] Florfenicol is a broad-spectrum, high-efficiency, animal-specific antibacterial drug. China also approved the drug in 1999, and it has been widely used to treat bacterial diseases in cattle, pigs, chickens, ducks and fish. However, with the increase of drug use, the clinical problem of bacterial resistance to this drug has become increasingly prominent, and the rate of drug resistance has increased year by year. In recent years, due to the widespread use of the drug in duck farms, the problem of drug resistance of Riemerella anatipestifer and pathogenic Escherichia coli to it is very serious. [0003] Bacterial resistance to florfenicol is mainly med...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N1/21C07K14/195C07K16/12C07K16/02A61K31/165A61P31/04C12R1/19
Inventor 叶胜强钱运国冉志平陈洁王丽霞喻婷邓兵杨宇周华童新红龚萍
Owner 武汉市畜牧兽医科学研究所
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