Neutralization molecule of high-pathogenicity avian influenza and preparation method thereof

A bird flu virus, combined with molecular technology, applied in the fields of biotechnology and immunology, can solve problems such as unsatisfactory effect and emergence of drug-resistant strains

Inactive Publication Date: 2015-03-11
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In addition, the characteristic of influenza virus escaping immune surveillance through its genetic drift and recombination has always been a major threat to public health, resulting in the two types of antiviral drugs commonly used in clinical practice are not very effective and resistant to it strains have emerged, so finding new effective treatments is urgently needed

Method used

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  • Neutralization molecule of high-pathogenicity avian influenza and preparation method thereof
  • Neutralization molecule of high-pathogenicity avian influenza and preparation method thereof
  • Neutralization molecule of high-pathogenicity avian influenza and preparation method thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0184] Preparation of HA / NA pseudovirus

[0185] H5 virus includes 10 clades and 5 subclades of clade 2, of which clades 0, 1, 2.1, 2.2, 2.3 and 7 are isolated from humans, and the rest are isolated from birds. For the method of constructing codon-optimized H5 virus and H1HA and N1NA of flag tag and the method of producing influenza HA / NA pseudovirus, refer to the description in the previously published article 34，35

[0186] VSV-G embedded pseudovirus: the pseudovirus embedded in the VSV-G virus envelope protein. For the embedding method, please refer to the method described in the article Vaccine 27:6777-6790 (2009).

[0187]See Table 1 for the source of the original virus strain and its Accession Number of the HA gene used for packaging HA and NA pseudoviruses. HA is obtained by conventional synthetic methods.

[0188] Table 1

[0189]

[0190]

[0191] Neutralization test based on pseudovirus

[0192] The method for screening convalescent serum neutralizing ant...

Embodiment 1

[0227] Example 1. Preparation of human monoclonal antibodies 65C6, 100F4 and 3C11

[0228] Blood samples were obtained from individuals recovering from H5N1 infection for six months. Experiments show that its serum has high neutralizing activity on H5N12.3.4 and 1 branch. Memory B cells were then sorted and seeded into 96 wells containing approximately 30 cells per well, and then treated with Epstein-Barr virus and CpG according to Traggiai et al. 36 Immortalize B cells. The neutralizing activity of the collected supernatant was screened. It was initially observed that the secretion of antibodies by EBV-transfected B cells was not stable. The neutralizing activity of the supernatant decreased significantly after two rounds of subcloning. Thus in subsequent experiments once neutralizing activity wells were found, a round of subcloning was performed on the cells and RNA could be isolated from positive cells. The gene fragments of heavy chain variable region, κ chain variabl...

Embodiment 2

[0242] Example 2. Antigen specificity and affinity experiments of human monoclonal antibodies 65C6, 100F4 and 3C11

[0243] The antigen specificity of human monoclonal antibodies was detected by western blotting. First, HIV-1, HA and NA virus-like particles were subjected to SDS / PAGE electrophoresis and then transferred to PVDF membrane. The reaction is performed and the specificity of the antibody can be analyzed according to the blot. like figure 1 The negative control antibody TG15 shown in b can specifically bind to the envelope protein on HIV-1 viroids but cannot bind to HA and NA on influenza viroids. The immune serum (Immune sera) of the mice used as the positive control can specifically bind to the HA on influenza viroids 0 , HA 1 and HA 2 Binds but cannot bind to envelope proteins on HIV-1 viroids. Antibodies 65C6, 100F4 and 3C11 can specifically bind to HA 0 and HA 1 Binds but not with HA 2 Binds to the envelope protein of HIV-1. This suggests that the epito...

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Abstract

Provided is a binding molecule of highly pathogenic avian influenza virus, and a preparation method therefor. The binding molecule has a good neutralizing effect on avian influenza virus. Also disclosed is a binding site where the binding molecule is bound on hemagglutinin of the avian influenza virus.

Description

technical field [0001] The invention belongs to the field of biotechnology and immunology; more specifically, the invention relates to a highly pathogenic avian influenza neutralizing molecule and a preparation method thereof. Background technique [0002] The highly pathogenic avian influenza H5N1 virus has infected about 500 million birds since 1997, while increasing numbers of people have been infected in Asia, Europe and Africa. Although human infections have been transmitted by birds so far, new virus strains capable of human-to-human transmission may evolve through recombination and evolution of the H5N1 virus. The widespread spread of this new virus combined with the lack of pre-established immunity to the H5N1 virus will cause significant morbidity and mortality in humans. [0003] The main manifestations of infection with highly pathogenic avian influenza H5N1 virus are severe pneumonia, lymphopenia, hyperlymphokineemia, and high viral load in the respiratory tract...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C07K14/11C12N15/13C12N15/63C12N5/10A61K39/42A61P31/16G01N33/569
CPCC07K16/1018C12N2760/16122C07K2317/76A61P31/16
Inventor 周保罗胡红星周伯平
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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