Human avian influenza special target medicine and preparing method thereof

A bird flu, targeted technology, applied in the direction of antiviral agents, antibodies, etc., to achieve the effect of high-efficiency antagonism

Inactive Publication Date: 2007-03-14
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pseudomonas aeruginosa exotoxin A (PE) has a killing effect on cells and has been widely used in tumor therapy,

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Preparation of anti HA / NA ScFv-PE40 antibody fusion protein

[0029] 1. Cloning of HA and NA antibody variable region genes of avian influenza virus

[0030] ①Immunization of BALB / c mice and screening of specific monoclonal antibody hybridoma cell lines

[0031] BALB / c mice were subcutaneously immunized with prepared HA and NA respectively, and the hemagglutination inhibitory antibody titer was greater than 2 7 Or when the ELISA result is positive, aseptically take mouse splenocytes and mouse myeloma cells for fusion, ELISA method to screen positive hybridoma cells, and Western method to detect monoclonal antibody specificity.

[0032] ② Cloning of HA / NA antibody variable region gene

[0033] Select the hybridoma cells that are specifically positive for HA or NA, extract their RNA, and complete the RT reaction with random hexamer primers to obtain cDNA. Use the cDNA of HA and NA monoclonal antibodies as templates, and use the light chain variable region primer LB , L...

Embodiment 2

[0045] Preparation of anti HA-NA BsAb--PE40 Antibody Fusion Protein

[0046] 1. Cloning of HA and NA antibody variable region genes of avian influenza virus

[0047] ①Immunization of BALB / c mice and screening of specific monoclonal antibody hybridoma cell lines

[0048] BALB / c mice were subcutaneously immunized with prepared HA and NA respectively, and the hemagglutination inhibitory antibody titer was greater than 2 7 Or when the ELISA result is positive, aseptically take mouse splenocytes and mouse myeloma cells for fusion, ELISA method to screen positive hybridoma cells, and Western method to detect monoclonal antibody specificity.

[0049] ② Cloning of variable region genes of HA and NA antibodies

[0050] Select hybridoma cells that are specifically positive for HA and NA, extract RNA respectively, complete RT reaction with random hexamer primers, and obtain cDNA, respectively use the cDNA of HA and NA monoclonal antibodies as templates, and use the light chain variable...

Embodiment 3

[0062] Preparation of MIF Targeted Antagonist Using Human Avian Influenza Virus HA / NA Single Chain Antibody as Carrier

[0063] 1. Cloning of HA, NA and MIF antibody variable region genes

[0064] ①Immunization of BALB / c mice and screening of specific monoclonal antibody hybridoma cell lines

[0065] BALB / c mice were subcutaneously immunized with the prepared HA, NA and MIF respectively, and the hemagglutination inhibitory antibody titer was greater than 2 7 Or when the ELISA result is positive, aseptically take mouse splenocytes and mouse myeloma cells for fusion, ELISA method to screen positive hybridoma cells, and Western method to detect monoclonal antibody specificity.

[0066] ② Cloning of HA, NA, and MIF antibody variable region genes

[0067] Select the hybridoma cells that are specifically positive for HA, NA and MIF, extract their RNA, complete RT reaction with random hexamer primers, and obtain cDNA, respectively use the cDNA of HA, NA and MIF monoclonal antibodie...

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PUM

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Abstract

The invention relates to an animal influenza target drug and relative production. Wherein, it uses single colon antibody to select the cross oncocyte seed which can resist animal influenza virus and inflammatory facto, and it can extract different cross oncocytes, via RT-PCT to obtain heavy chain variable area and light chain variable area genes with single antibody; and via SOE-PCT, to connect the VH, VL genes with one short peptide with 15 amino acid, to build the single chain antibody gene of single chain antibody gene, or single chain dual special antibody gene, and inflammatory facto, and connect the single chain antibody gene or single chain dual special antibody gene, via enzyme cutting to connect the single chain antibody gene or bacillus pyocyaneus ectotoxin PE40 gene of inflammatory facto, to build original core represent carrier, transfer Ecoli.BL21 projective virus, then via ferment, extraction, and purification, to obtain target antagonist.

Description

Technical field: [0001] The present invention relates to a series of targeted drugs specific for human avian influenza and its preparation method, in particular, it provides a series of targeted drugs using human avian influenza virus neutralizing single-chain antibody or single-chain bispecific antibody as a carrier and its preparation method The invention is used for emergency prevention and treatment of human avian influenza, and belongs to the technical field of preparation of medicines for prevention and treatment of important zoonotic diseases. Background technique: [0002] According to the data released by the World Health Organization, as of August 23, 2006, 241 people worldwide have been diagnosed with H5N1 subtype HPAIV, causing 141 deaths, and the case fatality rate is as high as 58.5%. Such a high case fatality rate shows that there is still a lack of effective drugs Rescue drugs, once the virus mutates and acquires the ability to spread among humans, it may tri...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P31/16
Inventor 夏咸柱朱平岳玉环王承宇高玉伟侯晓强冯娜杨松涛黄耕王铁成
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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