UL55 recombinant prokaryotic expression protein for detecting DPV antibody and preparation method thereof

A technology of prokaryotic expression and duck plague virus, applied in the field of veterinary medicine, can solve the problems of unsatisfactory complexity and purity, hinder large-scale application, and unavoidable drug release, etc., achieve high added value of products, broad market application prospects, and easy promotion Effect

Active Publication Date: 2014-08-06
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But it has the following disadvantages: one is that it will inevitably cause loose poison during the operation; the other is that the complexity and purity of the preparation of the whole DPV virus as a diagnostic antigen make it difficult to popularize.
It hinders the large-scale application of whole virus as a diagnostic antigen to monitor DPV antibodies

Method used

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  • UL55 recombinant prokaryotic expression protein for detecting DPV antibody and preparation method thereof
  • UL55 recombinant prokaryotic expression protein for detecting DPV antibody and preparation method thereof
  • UL55 recombinant prokaryotic expression protein for detecting DPV antibody and preparation method thereof

Examples

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Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Detection of duck plague virus antibody UL55 recombinant prokaryotic expression detection antigen and preparation method

[0041] 1. Material method

[0042] 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 condition in the preferred embodiment, generally according to conventional conditions, such as Molecular Cloning Experiment Guideline (third edition, J. Sambrook etc., translated by Huang Peitang, etc., Science Press, 2002) described conditions, or as recommended by the manufacturer.

[0043] 1.1 Strains, plasmids and strains

[0044] 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...

Embodiment 2

[0115] Example 2, Establishment and Application of UL55-ELISA Method for Detecting DPV Antibody

[0116] 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.

[0117] 1 Establishment of UL55-ELISA method for detecting DPV antibody

[0118] 1.1 Determination of recombinant UL55 protein coating concentration and serum dilution

[0119] 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 ...

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Abstract

The invention discloses a UL55 recombinant prokaryotic expression protein for detecting a duck plague virus (DPV) antibody and a preparation method thereof. The recombinant prokaryotic expression protein for detecting the DPV antibody is defined by an amino acid sequence table shown as SEQ ID NO.2 and is a prokaryotic expression product of a nucleotide sequence shown as SEQ ID NO.1. The preparation method comprises the following steps of: (1) designing a specific primer for amplifying a DPV-UL55 gene; (2) performing polymerase chain reaction (PCR) amplification to obtain a UL55 gene fragment; (3) cloning, sequencing and identifying a UL55 gene; (4) directionally subcloning the UL55 gene to a pET32a prokaryotic expression vector; (5) performing inducible expression on the UL55 gene in Escherichia coli; (6) purifying a UL55 gene expression protein; and (7) renaturing and detecting a recombinant UL55 protein. The product can be used as a detection antigen for detecting the DPV antibody by indirect enzyme linked immunosorbent assay (ELISA) and a western blot method, and also can be used as a gene engineering subunit vaccine for preventing DPV infection.

Description

technical field [0001] The invention relates to the field of animal medicine, and relates to a genetic engineering product and a preparation method thereof. Specifically, it is a DPV-UL55 gene recombination prokaryotic expression detection antigen protein prepared by genetic engineering technology for detection of duck plague antibody and a preparation method thereof. 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. Since duck plague was first discovered in the Netherlands in the 1920s, it has a history of more than 80 years. The disease is distributed in all duck raising areas in the world, and has caused huge economic losses to the waterfowl breeding industry in various countries in the world. Its prevention and control are directly related to the sustainable and stable development of waterfowl breeding industry. [0003] Clinical and l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/03
Inventor 程安春吴英汪铭书陈孝跃
Owner SICHUAN AGRI UNIV
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