Humanized antibody for resisting avian influenza H5N1 hemagglutinin antigen, and preparation method and application thereof

A humanized antibody and avian influenza technology, applied in the biological field, can solve problems such as time-consuming transformation, reduced affinity activity, and other problems, and achieve the effect of reducing immunogenicity, high neutralization activity, and wide application prospects

Active Publication Date: 2014-04-23
SHANGHAI INST OF IMMUNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is undeniable that the humanized transformation of antibodies is time-consuming and laborious. After transformation, there are often situations such as reduced affinity activity and specificity, and there are more or less immunogenic problems. Therefore, humanized antibodies are gradually growing rapidly.

Method used

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  • Humanized antibody for resisting avian influenza H5N1 hemagglutinin antigen, and preparation method and application thereof
  • Humanized antibody for resisting avian influenza H5N1 hemagglutinin antigen, and preparation method and application thereof
  • Humanized antibody for resisting avian influenza H5N1 hemagglutinin antigen, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Humanized transformation of murine antibody mH5M9

[0047] The variable region sequence of the murine antibody mH5M9 is published in the NCBI database (heavy chain GenBank No.AGX28126.1, light chain GenBank No.AGX28125.1), see figure 1 . Use the Kabat method to find out the CDR sequence, see figure 1 underlined part. The murine antibody mH5M9 was humanized by CDR grafting. The most suitable template was searched by BLASTP-search against the non-redundant protein database, and the results showed that the heavy and light chains of human anti-FabOX108 (PDB No.3DGG_B and No.3DGG_A) had the highest homology, reaching 73% and 75%. Then, the heavy chain and light chain CDR regions of the murine antibody mH5M9 were grafted onto the FR frameworks of the human templates VH and VL, respectively, to obtain the original humanized antibody A1. For the sequence comparison results, see figure 2 .

[0048] Based on the simulated spatial structure of the initial version ...

Embodiment 2

[0051] Example 2. Construction of humanized antibody expression vector

[0052] Insert the humanized antibody hH5M9 heavy and light chain variable region genes (SEQ ID NO: 3 and SEQ ID NO: 4) into the corresponding sites of the eukaryotic expression vector to obtain positive recombinant plasmids IFH-VH and IFL -VL, the expression vector is preserved by our laboratory. Expression vector plasmid map such as image 3 Shown, wherein, A is the heavy chain eukaryotic expression vector IFH; B is the light chain eukaryotic expression vector IFL.

Embodiment 3

[0053] Example 3. Expression of Humanized Antibodies

[0054] The humanized antibody expression vectors IFH-VH and IFH-VL constructed in the above Example 2 were transformed into Escherichia coli DH5α, and positive clones were picked and inoculated in 500ml LB medium for amplification. Using the plasmid extraction kit from MACHEREY-NAGEL Xtra Mid), the DNA was extracted and purified according to the manufacturer's instructions. Using Invitrogen's FreeStyle TM MAX Reagent transfection The above plasmid DNA was co-transfected into 293F cells (host cell 293F cells, purchased from Invitrogen), and the operation method was performed according to the manufacturer's instructions. Culture supernatants were collected 6-7 days after transfection.

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PUM

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Abstract

The invention discloses a humanized antibody for resisting avian influenza H5N1 hemagglutinin (HA) antigen. A heavy chain variable region of the humanized antibody has an amino acid sequence shown in SEQ ID No.1; a light chain variable region of the humanized antibody has an amino acid sequence shown in SEQ ID No.2. In addition, the invention also discloses a preparation method and application of the humanized antibody. The humanized antibody is extensive and widespread in coagulation inhibition activity for avian influenza H5N1 virus, high in affinity and neutralization activity for various subtypes of the HA antigen of the avian influenza H5N1, and capable of recognizing a special conserved region of one H5HA; the humanized antibody is high in neutralization activity and capable of reducing immunogenicity; a recognized epitope is highly conserved in the H5 type influenza virus; the humanized antibody is extensive in application prospect in prevention and treatment and vaccine design of the avian influenza H5N1 in the future.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a humanized antibody against the hemagglutinin (Hemagglutinin, HA) antigen of avian influenza H5N1. In addition, the present invention also relates to the preparation method and use of the antibody. Background technique [0002] Avian influenza is an infectious disease syndrome caused by a subtype of influenza A virus. Influenza A virus is divided into 16 subtypes from H1 to H16 according to the difference of their surface proteins. Avian influenza around the world is mainly caused by the highly pathogenic H5 and H7 subtypes, while humans are susceptible to the H1 and H3 subtypes. The highly pathogenic avian influenza H5N1 virus has killed tens of thousands of poultry in Asia, and the outbreak has spread to more than 50 countries in the Middle East, Europe and Africa. Originally, avian influenza was only transmitted among poultry animals, but with evolution, some animal influenza ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/63C12N5/10A61K39/395A61P31/16G01N33/53C07K14/11
Inventor 王颖熊斐斐夏立亮吴标王登宇赵国屏
Owner SHANGHAI INST OF IMMUNOLOGY
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