Avian influenza hemagglutinin, epitope peptide, monoclonal antibodies for resisting avian influenza hemagglutinin and epitope peptide, and applications of monoclonal antibodies

A monoclonal antibody, bird flu technology, applied in the direction of viral peptides, applications, antiviral agents, etc.

Inactive Publication Date: 2012-12-05
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There is no research on the epitope peptide sequence of avian influenza virus in mainland China

Method used

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  • Avian influenza hemagglutinin, epitope peptide, monoclonal antibodies for resisting avian influenza hemagglutinin and epitope peptide, and applications of monoclonal antibodies
  • Avian influenza hemagglutinin, epitope peptide, monoclonal antibodies for resisting avian influenza hemagglutinin and epitope peptide, and applications of monoclonal antibodies
  • Avian influenza hemagglutinin, epitope peptide, monoclonal antibodies for resisting avian influenza hemagglutinin and epitope peptide, and applications of monoclonal antibodies

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The specific procedures of expression, purification and identification of recombinant HA gene and recombinant HA protein are obtained by PCR.

[0049] In this study, the recombinant HA protein was expressed by the baculovirus eukaryotic protein expression system. The HA protein expressed by this system has a natural conformation and is active. Its main process is as figure 1 shown.

[0050] 1. PCR amplification to obtain recombinant HA gene fragments

[0051] 1.1 Template preparation:

[0052] 1.1.1 Isolation and cultivation of highly pathogenic avian influenza H5N1 virus

[0053] 1.1.2 Extraction of total virus RNA, extraction of plasmid digestion and identification

[0054] 1.1.3 Reverse transcription to synthesize the first strand of cDNA

[0055] 1.1.4 HA fragment PCR, recovery, ligation into pCR-Blunt II-Topo vector, as a template

[0056] 1.2 PCR amplification: (the amplification ranges from 17-532aa in length of amino acid residues of HA)

[0057] 1.2.1 P...

Embodiment 2

[0106] Embodiment 2: Preparation of monoclonal antibody

[0107] 1. Immunization of animals:

[0108] Clean-grade female Balb / c mice (purchased from the Institute of Animals, Chinese Academy of Medical Sciences) aged 6-8 weeks were selected as immunized objects. According to the monoclonal antibody preparation method in the book "Contemporary Immunology Technology and Application" (Editor-in-Chief Badenian) (Beijing Medical University, China Peking Union Medical College United Press, 1998). details as follows:

[0109] Initial immunization Ag 1~50μg / 0.8~1.0ml plus Freund’s complete adjuvant

[0110] Subcutaneous multi-point injection (0.2ml / point)

[0111] ↓After 3 weeks

[0112] The dose of the second immunization is the same as above, plus Freund’s incomplete adjuvant

[0113] Subcutaneous or ip (ip dose should not exceed 0.5ml)

[0114] ↓After 3 weeks

[0115] The dose for the third immunization is the same as above, wi...

Embodiment 3

[0134] Example 3: Identification of Antibody Neutralizing Ability to Viral Cytotoxicity

[0135] 1. Packaging of recombinant virus:

[0136] According to the recombinant WSN virus system (Generation of influenza A viruses entirely from cloned cDNAs, PNAS, 1999, 96: 9345-93 50) used by GABRIELE NEUMANN et al., we used the reverse genetic operating system of 12 plasmids to convert A / Bar- The hemagglutinin (HA) and neuraminidase (NA) of headed Goose / Qinghai / 1 / 05 (H5N1) were constructed into the RNA expression plasmid pHH21, together with A / WSN / 33 (H 1 N 1 ) RNA expression plasmids of the other 6 fragments inside the virus: pHH21-PB2, pHH21-PB1, pHH21-PA, pHH21-NP, pHH21-M, pHH21-NS, and A / PR / 8 / 34(H 1 N 1 ) 3 protein expression plasmids of the virus: pcDNA-PB2, pcDNA-PB1, pcDNA-PA, and A / WSN / 33 NP protein expression plasmid PCAGGS / MCS-NP, etc. A total of 12 plasmids were simultaneously transfected into 293T cells. The 293T cells and their culture medium 3 days after transfecti...

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PUM

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Abstract

The invention discloses an avian influenza hemagglutinin, an epitope peptide, monoclonal antibodies for resisting the avian influenza hemagglutinin and the epitope peptide, and applications of the monoclonal antibodies. The epitope peptide of the invention comprises an amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. The invention further provides the monoclonal antibodies which can be specifically combined with the hemagglutinin protein of the avian influenza virus, and the combination can be blocked by the epitope peptide sequence. The antibodies have important application value in the aspect of specific diagnosis of the avian influenza virus. Because the antibodies provided by the invention specifically aim at the epitope sequence of the avian influenza virus, the cross reactivity of the immune reaction is greatly reduced, and the antibodies are especially suitable for rapid diagnosis of avian influenza diseases.

Description

technical field [0001] The present invention relates to avian influenza virus hemagglutinin, its epitope peptide and anti-monoclonal antibody, and their application in preparation of avian influenza virus vaccine and diagnostic reagent. Background technique [0002] Avian influenza is the viral influenza of poultry. It is an infectious disease with various symptoms from respiratory system to severe systemic sepsis caused by type A influenza virus in poultry. The mortality rate of poultry infection is very high. Normally, such poultry diseases do not have a serious impact on human health. In 1997, when the first human infection case of highly pathogenic avian influenza virus H5N1 occurred in Hong Kong, in 2003 and later in 2005-2006, human infection cases continued to occur all over the world, causing panic in the world. The H5N1 virus has become the focus of attention in the world because of its high mortality rate and the risk of infecting humans. The main method to preve...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K14/11C12N15/44C12N15/63C07K16/10A61K39/145A61P31/16G01N33/569
Inventor 何维李峥舒跃龙马驰崔莲仙
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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