Avian influenza virus NS1 protein B cell epitope polypeptide and applications thereof

A kind of avian influenza virus, antigen epitope technology, applied in the field of molecular immunology

Inactive Publication Date: 2013-10-02
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the identified influenza virus antigen epitopes are derived from human H1N1 and H3N2 virus strains, but the research on the antigen epitopes of H5 subtype avian influenza is basically in the blank, especially no domestic and foreign phages have been used. Related reports on the analysis of H5N1 subtype avian influenza NS1 protein epitopes by display peptide library technology (Akifumi Yamashita, Norihito Kawashita, Ritsuko Kubota-Koketsu, et al. : Implication for human monoclonal antibody recognition[J].Biochemical and Biophysical Research Communications,2010,393:614-618.)

Method used

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  • Avian influenza virus NS1 protein B cell epitope polypeptide and applications thereof
  • Avian influenza virus NS1 protein B cell epitope polypeptide and applications thereof
  • Avian influenza virus NS1 protein B cell epitope polypeptide and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Identification of avian influenza virus NS1 protein B cell antigen epitope polypeptide

[0024] 1. Materials and Methods

[0025] 1.1 Main experimental materials and experimental animals

[0026] SF9 cells and SP2 / 0 cells were preserved by our laboratory; the recombinant plasmid pET-NS1 expressing NS1 of AIV A / duck / Hubei / 2003 (H5N1) was constructed by our laboratory; 6-8 weeks old SPF BALB / c female mice were purchased from the Experimental Animal Center of the Institute.

[0027] 1.2 Main reagents

[0028] Protein purification kits were purchased from Novagen; horseradish peroxidase-labeled goat anti-mouse IgG antibody (HRP-IgG), HAT, HT, PEG2000, and paraffin oil were purchased from Sigma; diaminobenzidine (DAB) chromogenic reagent The kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; the immunoglobulin subclass identification kit was purchased from SouthernBiotech; the transfection reagent Lipofectamine TM 2000 was purchased from ...

Embodiment 2

[0060] Example 2 The use of the avian influenza virus NS1 protein B cell antigen epitope polypeptide of the present invention in the diagnosis or detection of avian influenza virus infection

[0061] Dilute the synthetic polypeptide WNDNTVRVS to 0.1μg / μl, coat ELISA plate according to 100μl / well, use SPF chicken serum as control, bird flu vaccine immunized chicken serum and bird flu virus infected chicken serum as primary antibody, rabbit anti-chicken HRP-IgG As a secondary antibody, indirect ELISA verification was carried out. After OPD color development, the OD value at 490nm was read in a microplate reader. The results showed that the OD value of the synthetic polypeptide after reacting with the infected chicken serum was significantly higher than that of the control group and the immune group. It shows that the synthetic polypeptide has binding characteristics with the serum of infected chickens, and can be used for the diagnosis and detection of avian influenza virus. The ...

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Abstract

The present invention discloses an avian influenza virus NS1 protein B cell epitope polypeptide and applications thereof. According to the present invention, prokaryotic expressed and purified NS1 recombinant protein is adopted to immunize BALB/c mice, a lymphocyte hybridoma technology is adopted to prepare hybridoma cells, and indirect ELISA is adopted to screen to obtain two strains of hybridoma cells capable of stably secreting anti-NS1 protein MAbs, wherein Western blot identification results show that the two strains of the MAbs can identify NS1 recombinant protein; and an oligopeptide WNLNTVRVS corresponding to D9 and obtained by using a phage display technology is basically matched with aa 182-190 of NS1 protein, such that the result prompted that 182WNDNTVRVS190 is alinear epitope of NS1 protein. The present invention further relates to applications of the antigen epitope polypeptide in preparation of drugs for diagnosis or detection of avian influenza virus infections so as to provide a theoretical basis for further establishment of an efficient avian influenza detection method.

Description

technical field [0001] The present invention relates to antigenic epitope polypeptides, especially to avian influenza virus NS1 protein B cell antigenic epitope polypeptides. The present invention also relates to the application of the antigenic epitope polypeptides in preparing medicines for diagnosing or detecting avian influenza virus infection, belonging to the field of molecular immunology . Background technique [0002] Avian influenza (AI) is an infectious disease of poultry caused by type A AI virus (AIV) of the family Orthomyxoviridae. According to different pathogenicity, AIV can be divided into highly pathogenic (HPAIV) and low pathogenic (LPAIV). In recent years, HPAIV has become popular all over the world. [0003] Vaccination is an effective measure to prevent the spread of avian influenza. However, due to the wide-scale use of vaccines, the seropositive rate of immunized chickens is extremely high, which is confused with the seropositive rate caused by the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C12N15/44C12N15/63G01N33/68
Inventor 王秀荣包红梅姜永萍陈化兰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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