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Epitope antigens of human infection with H7N9 avian influenza and applications of epitope antigens in immune detection reagent

An immune detection and avian influenza technology, which is applied in the field of preparation and detection of human infection H7N9 avian influenza antibody detection, can solve problems such as affecting the specificity of H7N9 immune detection, incoherent sentences, etc., and achieve stable detection results, good repeatability, and simple and fast technology. Effect

Inactive Publication Date: 2015-07-01
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The main reason is that H7N9 is a new infectious disease, and there is no technical reserve for the development of relevant immune detection reagents. In addition, most people have been infected with seasonal influenza (the sentence is not fluent, and the applicant is recommended to modify it), especially in my country in 2009. Extensive vaccination of influenza A (H1N1) vaccines will still have a large number of anti-influenza virus antibodies in the body, which will greatly affect the specificity of H7N9 immune detection

Method used

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  • Epitope antigens of human infection with H7N9 avian influenza and applications of epitope antigens in immune detection reagent
  • Epitope antigens of human infection with H7N9 avian influenza and applications of epitope antigens in immune detection reagent
  • Epitope antigens of human infection with H7N9 avian influenza and applications of epitope antigens in immune detection reagent

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Embodiment 1

[0037] The selection of embodiment 1 candidate epitope

[0038] We used BioSun bioinformatics software to predict the antigenicity of human infected H7N9 avian influenza virus sequences. The results of HA and NA epitopes are as follows: figure 1 , figure 2 As shown, two main dominant antigen segments are selected for each antigen, and their amino acid sequences are respectively shown in sequence 1-sequence 4 in the sequence listing.

Embodiment 24

[0039] Cloning and expression of 24 kinds of human infection H7N9 avian influenza antigens

[0040] 1. Construction of four kinds of antigen expression plasmids of human infection with H7N9 avian influenza virus

[0041] 1. Synthesis of antigen genes of 14 human-infected H7N9 avian influenza viruses

[0042] According to the amino acid sequences of the above four human-infected H7N9 avian influenza antigens, Shanghai Yingjun Biotechnology Service Co., Ltd. was commissioned to synthesize the genes of the above sequences using the dominant codons of Escherichia coli. The gene sequences are shown in sequences 5-8 in the sequence table.

[0043] 1. Construction of expression plasmids for 24 human-infected H7N9 avian influenza antigens

[0044] 1.2.1 PCR product and expression vector pBVIL1 double digestion

[0045] Take 30 μl of the above synthetic gene product and pBVIL1 expression vector and put them in Eeppendorf centrifuge tubes respectively, add 4 μl of 10×buffer (H), 1 μl ...

Embodiment 3

[0053] Example 3 Establishment and Application of IgG Antibody Detection (Indirect Method) Technology Based on Recombinant Human Infection with H7N9 Avian Influenza Antigen

[0054] 3.1 Establishment of IgG antibody detection (indirect method) technology based on recombinant human infection with H7N9 avian influenza antigen

[0055] Dilute the purified recombinant human infected H7N9 with pH 7.2 phosphate buffer solution to 2.0 μg / ml, 100 μl per well, discard the solution after overnight at 4 °C, coat the ELISA assay plate, rinse with distilled water 3 times, pat dry, and remove each well. Add 1% BSA100μl, room temperature for 2h, discard the solution. After adding 100 μl sample dilute solution to each well, add 10 μl serum of the sample to be tested, 37°C for 30 minutes, discard the solution, wash the plate 5 times with washing solution, discard the solution, pat dry, add horseradish peroxidase-labeled anti-human IgG monoclonal antibody (1:1000) 100μl, incubate at 37°C for 2...

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Abstract

The invention discloses dominant epitope antigens of hemagglutinin (HA) and neuraminidase (NA) of human infection with H7N9 avian influenza, and further discloses the applications of the antigens in an antibody detection reagent of human infection with H7N9 avian influenza. According to the epitope antigens of human infection with H7N9 avian influenza and the applications of epitope antigens in immune detection reagent, bioinformatics technology is employed to analyze the existing HA and NA sequences of H7N9, four antigen sections are screened to be cloned and expressed, the sensitivity and sensitivity of the antigens are screened by serological tests, and the third antigen is finally determined to have the highest diagnostic value so as to be applicable to detect the antibody of human infection with H7N9 avian influenza.

Description

technical field [0001] The present invention relates to human infection H7N9 avian influenza antigen, and also relates to the use of the antigen in the preparation and detection of human infection H7N9 avian influenza antibody detection. Background technique [0002] Although most of the human-infected H7N9 avian influenza viruses discovered in March 2013 were sporadic cases, and there is no definite evidence of human-to-human transmission, it has spread to 10 provinces and cities, with hundreds of people infected and dozens of deaths, causing national health and The epidemic prevention department attaches great importance to it. [0003] Influenza viruses are divided into three types: A (A), B (B), and C (C) according to the antigenic characteristics and differences of the nucleoprotein and matrix protein. Influenza A can be divided into 16 H subtypes (H1-H16) and 9 N subtypes (N1-N9) according to the antigenicity of the outer membrane hemagglutinin (HA) and neuraminidase ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/11G01N33/569
Inventor 修冰水张贺秋张励力陈堃杨锡琴冯晓燕
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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