Avian influenza virus NP proteantigen epitope peptide and colloidal gold test paper box thereof

A bird flu virus and antigen epitope technology, applied in instruments, measuring devices, scientific instruments, etc., can solve the problems that surface proteins are prone to mutation and it is difficult to understand the real law of changes

Active Publication Date: 2017-08-18
QINGDAO BOITE BIOPHARM +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason why the epidemic of avian influenza has not been effectively controlled so far is that its surface protein is extremely prone to mutation, making it difficult for people to understand the real law of its change

Method used

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  • Avian influenza virus NP proteantigen epitope peptide and colloidal gold test paper box thereof
  • Avian influenza virus NP proteantigen epitope peptide and colloidal gold test paper box thereof
  • Avian influenza virus NP proteantigen epitope peptide and colloidal gold test paper box thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Identification of linear epitope of avian influenza H9N2 subtype virus

[0031] 1 Materials and methods

[0032] Main experimental materials and experimental animals: SP2 / 0 cells were preserved by the applicant's laboratory; recombinant plasmid Pet32a-NP expressing AIV (H9N2) NP protein was constructed by this laboratory; AIV (H9N2) virus was preserved by this laboratory; SPF chicken embryo Bought from Merial; SPF BALB / c female mice aged 6-8 weeks were purchased from the Experimental Animal Center of Beijing Medical College.

[0033] Main reagents: PEG6000 (Merck), sucrose (Shanghai Sinopharm), Freund's complete adjuvant and Freund's incomplete adjuvant (SIGMA), Rabbit anti mouse-HRP (SIGMA), Rabbit anti mouse-FITC (SIGMA), phage display Peptide Library Kit (NEB)

[0034] Purification and identification of recombinant antigens: The applicant screened avian influenza virus from sick individuals who had been injected with avian influenza virus vaccine but stil...

Embodiment 2

[0053] The preferred of embodiment 2 monoclonal antibody epitope

[0054] 1. The monoclonal antibodies produced by the NP-Mab 1B2 and 2B5 cells were purified separately, and the purified monoclonal antibodies were tested for HI, and the HI titer of 1B2 was higher than that of 2B5.

[0055] Table 3. Detection of NP-Mab antibody titer

[0056]

[0057] 2. Determination of relative affinity constant of monoclonal antibody

[0058] The relative affinity constant of the monoclonal antibody was determined by the thiocyanate elution method, and the specific operation steps were as follows:

[0059] (1) Take the purified H9 subtype avian influenza virus as an antigen-coated and blocked indirect ELISA plate, dilute the monoclonal antibody to be tested to a saturated concentration, add 100 μL to each well, and incubate at 37°C for 1 hour;

[0060] (2) After washing with PBST for 3 times, different concentrations of thiocyanate were eluted, and NaSCN solutions with concentrations of...

Embodiment 3H9

[0066] Embodiment 3H9 subtype avian influenza virus colloidal gold test strip

[0067] 1. Preparation of monoclonal antibody against outer membrane hemagglutinin antigen of H9 subtype avian influenza virus and rabbit anti-mouse IgG antibody

[0068] Preparation of monoclonal antibodies against nucleoprotein antigens of H9 subtype avian influenza virus: (hybridoma cells are prepared and preserved by our laboratory) the selected hybridoma cells are resuscitated and expanded for culture to prepare ascites, which is H9 subtype avian influenza virus antibodies. ELISA to identify the activity and titer of the monoclonal antibody to the H9 subtype avian influenza virus nucleoprotein antigen, and purify it for later use;

[0069] Preparation of rabbit anti-mouse IgG antibody: Use conventional methods to immunize rabbits with purified mouse IgG as the immune antigen, collect venous blood two weeks later, use ELISA method to determine the activity and titer of anti-mouse IgG antibody, a...

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Abstract

The invention relates to an avian influenza virus NP proteantigen epitope peptide and a colloidal gold test paper box thereof. The invention provides an avian influenza H9N2 subtype virus NP protein linear epitope peptide, and the antigen epitope peptide has the amino acid sequence of SEQ ID NO: 1. The antigen epitope peptide provided by the invention is used for preparing a product for detecting avian influenza virus. The avian influenza virus NP protein B cellular antigen epitope peptide is identified for providing a ground for further establishing a method of efficiently detecting avian influenza, and also lays a foundation for studying the structure and function of NP protein.

Description

technical field [0001] The invention belongs to the field of veterinary medicine biotechnology, and in particular relates to a bird flu virus NP protein antigen epitope polypeptide and a colloidal gold test paper box prepared therefrom. Background technique [0002] H9N2 subtype avian influenza virus (AIV) is an infectious disease caused by type A influenza virus in poultry with various symptoms ranging from respiratory system to severe systemic sepsis, belonging to type A influenza virus. In 1966, the first H9N2 subtype avian influenza virus was isolated by HoMee from a turkey suffering from mild respiratory disease. It was not until 1994 that the isolation of H9N2 subtype avian influenza was reported for the first time in my country, and related reports appeared one after another. However, since the pathogenicity and mortality of H9N2 subtype avian influenza virus are not as high as those of H5N1 and H7H1, the infection is often difficult to detect and is not taken seriou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/558G01N33/577
CPCG01N33/558G01N33/56983G01N33/577
Inventor 刘晓婧蒋贻海凌红丽赵明丁江由佳李明举张志东王艳玲武利利周大卫王晓艺杨莉卢香玲王海燕
Owner QINGDAO BOITE BIOPHARM
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