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Duck plague virus cyst membrane gI protein polyclonal antibody as well as preparation method and application thereof

A polyclonal antibody, duck plague virus technology, applied in the fields of biotechnology and animal immunology, can solve problems such as lag, and achieve the effects of high specificity, high titer and simple operation

Inactive Publication Date: 2011-09-07
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In comparison, research on the function of the gI protein of duck plague virus is relatively lagging behind, and there are no related reports so far

Method used

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  • Duck plague virus cyst membrane gI protein polyclonal antibody as well as preparation method and application thereof
  • Duck plague virus cyst membrane gI protein polyclonal antibody as well as preparation method and application thereof
  • Duck plague virus cyst membrane gI protein polyclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Preparation of duck plague virus envelope gI protein

[0034] 1. Proliferation and Culture of Duck Plague Virus

[0035] Inoculate the CHv strain of duck plague virus into the newly grown dense monolayer duck embryo fibroblasts (DEF), discard the virus solution after adsorption at 37°C for 60 minutes, and then add calf serum with a volume fraction of 2% and 100IU / mL double Anti-constituent MEM was maintained in nutrient solution, followed by incubation at 37°C for 48 hours.

[0036] 2. DNA extraction

[0037] 1) Select DEF (100mL cell bottle) with 60%-70% cytopathic effect (CPE) after infection with DPV seed virus;

[0038] 2) Pour off the cell culture medium, add 500 μL of cell lysate, and at the same time add proteinase K (10 mg / mL) to a final concentration of 200 μg / mL, mix gently, and incubate at 37°C for 10 minutes;

[0039] 3) Pour the cell suspension into an EP centrifuge tube, wash the lysate remaining in the cell bottle with 500 μL of saturated phenol, an...

Embodiment 2

[0082] Example 2 Preparation of Polyclonal Antibody Based on Recombinant Envelope gI Protein of Duck Plague Virus

[0083] 1. Preparation of recombinant protein vaccine

[0084] Mix paraffin oil and lanolin in a ratio of 3:1, heat to dissolve, and sterilize at high temperature to make incomplete Freund's adjuvant, and add 1 mg of BCG to each milliliter of incomplete adjuvant to make complete Freund's adjuvant. Before use, mix Freund's complete adjuvant or incomplete adjuvant with equal volumes of the antigen liquid, ultrasonically emulsify in an ice bath, and check the emulsification effect. The oil droplets are emulsified completely.

[0085] 2. The immunization procedure is as follows

[0086]

[0087] The day before the immunization, blood was drawn from the rabbits, and the serum was separated as a negative control. After the fourth immunization, the carotid artery was bled, and the blood was collected. Place at 37°C for 30 minutes, and freeze at 4°C overnight. T...

Embodiment 3

[0106] Example 3 Application of polyclonal antibody based on duck plague virus envelope gI protein in the preparation of duck plague virus and duck plague virus envelope gI protein indirect immunofluorescence detection reagent

[0107] The application of the polyclonal antibody of DPV gI protein in the indirect immunofluorescence method, the steps are as follows:

[0108] 1) Prepare duck embryo fibroblast fly sheets by conventional methods, inoculate DPV after DEF grows into typical spindle cells 24 hours later, and harvest cell fly sheets 24 hours after inoculation;

[0109] 2) Wash the residual nutrient solution on the surface with sterilized pre-cooled PBS;

[0110] 3) Fix with 4% paraformaldehyde at room temperature, wash with sterilized PBS for 3×10 minutes;

[0111] 4) Permeabilize with PBS with a mass fraction of 0.2% Triton for 15 minutes, wash with 0.1% Tween-20 PBS (PBST) (same as above);

[0112] 5) Block with PBST containing 5% bovine serum albumin (BSA) at 37°...

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Abstract

The invention relates to the field of biological technology and animal immunology, in particular to a duck plague virus cyst membrane gI protein polyclonal antibody. The polyclonal antibody is obtained from a duck plague virus cyst membrane gI protein immune animal which has a DNA sequence shown as SEQ ID No.1 and a polypeptide sequence shown as SEQ ID No.2; the polyclonal antibody can be used for preparing a duck plague virus detecting reagent; and the polyclonal antibody has high antibody titer and high specificity, can meet the duck plague virus and duck plague virus cyst membrane gI protein-associated detecting test and fills the research blank of duck plague virus cyst membrane gI protein polyclonal antibody.

Description

technical field [0001] The invention relates to the fields of biotechnology and animal immunology, in particular to a polyclonal antibody of duck plague virus envelope gI protein and a preparation method and application thereof. [0002] Background technique [0003] Duck plague (Duck plague, DP), also known as duck viral enteritis (Duck Viral Enteritis, DVE), is caused by duck plague virus (Duck plague virus, DPV). , febrile and septic infectious diseases are one of the main infectious diseases that endanger waterfowl farming in the world. The pathogen DPV is a double-stranded DNA herpes virus. In the eighth virus classification report in 2005, it was classified as an unclassified virus in the subfamily a herpesviridae. Herpes virus is mainly composed of four parts: core, capsid, tegument and envelope. The diameter of the virus particle is about 150 nm, and the double-stranded DNA and protein are wound to form the core of the virus. Eleven glycoproteins of Herpes simple...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/569
Inventor 程安春李丽娟汪铭书陈孝跃
Owner SICHUAN AGRI UNIV
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