Recombinant UL55 protein-based duck plague virus antibody detection method

A duck plague virus and antibody detection technology, which is applied in the field of veterinary medicine, can solve problems such as unsatisfactory purification methods, complexity and purity of purification methods, and obstacles to large-scale application of ELISA methods, and achieve good specificity.

Inactive Publication Date: 2011-09-14
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity and unsatisfactory purity of the DPV whole virus purification method, th

Method used

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  • Recombinant UL55 protein-based duck plague virus antibody detection method
  • Recombinant UL55 protein-based duck plague virus antibody detection method
  • Recombinant UL55 protein-based duck plague virus antibody detection method

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0030] Cloning of embodiment 1 duck plague virus UL55 gene, prokaryotic expression and purification of UL55 protein

[0031] 1. Material method

[0032] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental method that does not indicate specific conditions in the preferred embodiment is usually according to conventional conditions, such as described in the Molecular Cloning Experiment Guide (Third Edition, J. Sambrook et al., translated by Huang Peitang, etc., Science Press, 2002) conditions, or as recommended by the manufacturer.

[0033] 1.1 Strains, plasmids and strains

[0034] Plasmid pMD18-T, purchased from Dalian Bao Biological Engineering Co., Ltd.; prokaryotic expression plasmid pET32a(+), product of Novagen; clone host bacteria E.coli DH5a, expression host bacteria E.coli BL21(DE3) and virulent strain of DPV CHv , provided by the Poultry Disease Research Center of Sichu...

Embodiment 2

[0106] Example 2, Establishment and Application of UL55-ELISA Method for Detecting DPV Antibody

[0107] The recombinant UL55 protein that above-mentioned embodiment obtains, anti-DPV duck serum (is the immune duck serum of 14d after attenuated vaccine immunization, and neutralizing titer is 1: 8), anti-DHV (duck hepatitis virus), RA (Riemer anativa ), Salmonella (Salmonella), duck swollen head hemorrhagic disease virus, influenza virus and E.coli (E. Horseradish peroxidase-labeled goat anti-duck IgG) and tetramethylbenzidine (TMB) were purchased from KPL, USA; bovine serum albumin (BSA) was purchased from Sigma, USA.

[0108] 1 Establishment of UL55-ELISA method for detecting DPV antibody

[0109] 1.1 Determination of recombinant UL55 protein coating concentration and serum dilution

[0110] The square array method was used to determine the optimal antigen coating concentration and serum dilution concentration, and the refolded recombinant UL55 protein with a concentration ...

Embodiment 3

[0147] As another example, the detection procedure of the indirect ELISA method is as follows: (1) Preparation of solid-phase antigen: Coat the recombinant UL55 protein on a microtiter plate at a concentration of 0.2 mg / ml, 100 μl / well, and incubate overnight in a humid box at 4°C , wash 3 times with a plate washer, 5 min each time, pat dry; add 100 μl / well blocking solution, seal in a wet box at 37°C for 1 h, wash with a plate washer once, 5 min each time, pat dry; (2) primary antibody binding: Dilute the serum to be tested according to the volume dilution of 1:20, add it to the microtiter plate, incubate at 100 μl / L in a wet box at 37°C for 1 hour, wash the plate washing machine 3 times, 5 minutes each time, and pat on; (3) Secondary antibody binding : Dilute the enzyme-labeled secondary antibody (goat anti-duck IgG-HRP) with the enzyme-labeled secondary antibody diluent at a volume dilution of 1:1000, add 100 μl / well, react at 37°C for 45 minutes, wash with a plate washer 3 ...

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Abstract

The invention relates to the field of animal medicine, in particular to a method for detecting duck plague virus (DPV) antibodies. The method specifically comprises the steps of preparation of solid phase antigens, primary antibody combination, secondary antibody combination, color development, detection, judgment and the like. The method is a purified recombinant UL55 protein-based indirect enzyme-linked immuno sorbent assay (ELISA) method, and has good specificity; the results of detecting the positive serums of duck virus hepatitis viruses (DHV), riemerella anatipestifer (RA), duck escherichia coli (E.coli), duck-derived salmonella, duck swollen head hemorrhagic disease viruses and duck-derived influenza viruses are negative; and the repeated tests in enzyme-labeled plates or between enzyme-labeled plates display that variation coefficients are less than 10 percent, and the method can detect out the positive serums of DPV weak virus vaccine immune ducks, wherein the DPV weak virus vaccine are diluted according to a ratio of 1:6400.

Description

technical field [0001] The invention relates to the field of animal medicine, in particular to a method for establishing and detecting duck plague virus antibodies by using recombinant UL55 prokaryotic expression protein as a coating antigen. Background technique [0002] Duck plague (DP) is a highly fatal infectious disease commonly found in ducks, geese, swans and other waterfowl caused by DPV in the family Herpesviridae. The disease has a high mortality rate and spreads rapidly. Since it first occurred in the Netherlands in 1923, it has spread to many countries and regions in the world, seriously threatening the development of the duck industry. Therefore, it is urgent to establish an effective and reliable monitoring method, so that the health status and immune antibody level of the duck flock can be correctly understood in time. The classic diagnostic method for this disease is the neutralization test, but due to its cumbersome and time-consuming (about 1 to 2 weeks), ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
Inventor 程安春吴英汪铭书陈孝跃
Owner SICHUAN AGRI UNIV
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