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ScFv antibody for resisting H5N1 type highly-pathogenic avian influenza and application thereof

A highly pathogenic, H5N1 technology for applications, antibodies, antiviral agents, etc.

Inactive Publication Date: 2011-03-30
NANJING MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It shows that there is indeed a conserved neutralizing epitope on the HA molecule of the H5N1 avian influenza virus, and there is no similar report in China at present

Method used

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  • ScFv antibody for resisting H5N1 type highly-pathogenic avian influenza and application thereof
  • ScFv antibody for resisting H5N1 type highly-pathogenic avian influenza and application thereof
  • ScFv antibody for resisting H5N1 type highly-pathogenic avian influenza and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of fully human immune scFv phage antibody library

[0030] Collect 40 parts of human whole blood immunized with avian influenza virus vaccine and produce antibodies, separate lymphocytes, use Invitrogen’s Trizol to extract total RNA, and use oligo(dT) 20 As primers, cDNA was generated by reverse transcription. Then use specific primer PCR to amplify the heavy and light chain variable region genes respectively. The scFv gene was synthesized by Overlap PCR ( figure 1 ), was connected with the expression vector pComb3XSS cut with the same restriction enzymes to construct the prokaryotic recombinant expression vector of scFv; electrotransformed competent Escherichia coli XL1-Blue, and constructed a capacity of 6.5×10 8 Fully human immunotype scFv antibody library.

[0031] The connection of the constructed scFv gene with the pComb3xSS carrier refers to: the scFv gene after purification and quantification is subjected to double digestion with SfiI e...

Embodiment 2

[0032] Example 2 Screening, expression and purification of anti-H5N1 highly pathogenic avian influenza virus-specific antibodies

[0033] (1) Coat the solid-phase screening ELISA plate with the hemagglutinin protein HA1 expressed by the purified baculovirus-insect cell expression system, 1 μg per well, wash, add blocking solution, wash, add phage antibody library antibody, wash to remove untreated Bound phage antibody; adding trypsin, eluting specifically bound phage antibody, infection multiplication, assisting phage M13K07 superinfection; repeating the above screening steps, a total of three rounds of "adsorption-elution-amplification" enrichment screening;

[0034](2) Dilute the phage obtained from the last round of screening and multiplication, spread on the culture plate and culture overnight, pick 564 single colonies in the cell culture plate, shake and culture overnight; transfer from each well of the first plate Transfer 5 μL of the bacterial solution to the second pla...

Embodiment 3

[0052] Example 3 Identification of fully human scFv antibodies with binding activity and spectral neutralization activity

[0053] The six H5N1 virus strains A / CK / Hongkong / 3-69 (H5N1), A / DK / Beijing / wy12107 (H5N1), A / Goose / Jilin / 514-1 (H5N1), A / Jiangsu / 07-4 (H5N1), A / Goose / ZD / 8-9106 (H5N1), A / Jiangsu / 08-6 (H5N1), A / Jiangsu / 1 / 2007(H5N1) (GenBank: EU434686.1) Provided by the Jiangsu Provincial Center for Disease Control and Prevention. The RNA of 6 virus strains was extracted, the full length of HA was amplified by RT-PC for sequencing, and the evolutionary tree was constructed using MEGA5.0 software, compared with the H5N1 virus strains provided by WHO, and classified into clade.

[0054] The HA proteins of six H5N1 virus strains were sequenced, analyzed using MEGA5.0 software, and a phylogenetic tree was constructed. The results show that A / CK / Hongkong / 3-69 (H5N1) belongs to clade 9, A / Goose / Jilin / 514-1 (H5N1) belongs to clade 2.2, A / Jiangsu / 07-4 (H5N1), A / Goose / ZD / 8-9106...

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Abstract

The invention relates to an scFv antibody for resisting H5N1 type highly-pathogenic avian influenza and an application thereof. A light chain amino acid sequence is shown as SEQIDNo: 1; a heavy chain amino acid sequence is shown as SEQIDNo: 2; a light chain nucleotide sequence is shown as SEQIDNo: 3; and a heavy chain nucleotide sequence is shown as SEQIDNo: 4. The scFv antibody for resisting the H5N1 type highly-pathogenic avian influenza is applied to the preparation of a medicine for resisting the H5N1 type avian influenza virus. Proven by protection tests of embryonated eggs, the 4F5 antibody has a 100% protective rate on prevention of the avian source and human source H5N1 type avian influenza virus infection, and the 4F5 antibody has a 100% protective rate on treatment of the avian source H5N1 type avian influenza virus infection. The highest protective rate on treatment of the human source H5N1 type highly-pathogenic avian influenza virus infection is 62.5%.

Description

technical field [0001] The invention belongs to the technical field of anti-H5N1 highly pathogenic avian influenza antibody, and relates to an anti-H5N1 highly pathogenic avian influenza virus neutralizing scFv antibody 4F5 and its application. Background technique [0002] Avian influenza is a severe infectious disease of poultry caused by type A influenza virus. In the past ten years, highly pathogenic avian influenza (HPAI), represented by the H5N1 subtype, has ravaged the world, not only causing losses to the world's poultry industry, but even more and more people are infected with H5N1 avian influenza Fatal cases, and even a small number of reported human-to-human bird flu. Since there is no antibody against avian influenza virus in the human body, especially the antibody against H5 antigen, human infection with H5N1 type avian influenza virus has a high lethality rate, which has constituted a major threat to human health and world public health security. [0003] The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/16
Inventor 冯振卿焦永军张晓曾晓燕金秋汪华
Owner NANJING MEDICAL UNIV
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