Single-domain antibody combined with immunoglobulin, anti-avian influenza single-domain antibody, bifunctional antibody and application thereof

An immunoglobulin and single-domain antibody technology, which is applied to immunoglobulin-binding single-domain antibodies, anti-avian influenza single-domain antibodies, bifunctional antibodies and their application fields, and can solve the problems of short elimination half-life and small molecules.

Active Publication Date: 2021-09-03
北京纽安博生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Single-domain antibodies have the advantages mentioned above, but because of their small size, they are quickly cleared from the blood circulation in vivo, resulting in a short elimination half-life in vivo

Method used

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  • Single-domain antibody combined with immunoglobulin, anti-avian influenza single-domain antibody, bifunctional antibody and application thereof
  • Single-domain antibody combined with immunoglobulin, anti-avian influenza single-domain antibody, bifunctional antibody and application thereof
  • Single-domain antibody combined with immunoglobulin, anti-avian influenza single-domain antibody, bifunctional antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Construction of Human Immunoglobulin (Ig) Specific Single Domain Antibody Gene Library

[0070] 1. Alpaca immunization procedure, monitoring of specific single domain antibody production

[0071] (1) Alpaca immunization procedure: multi-point subcutaneous injection of alpaca neck and back, human IgG and IgA antigens and Freund's complete adjuvant equal volumes were prepared into an emulsion, and the first immunization interval was 4 weeks; For 4 immunizations, an equal volume of human IgG and IgA antigens and Freund's incomplete adjuvant was prepared into an emulsion, and the interval between each immunization was 3 weeks.

[0072] (2) From the second immunization, peripheral blood was collected one week after each immunization, and ELISA was used to monitor the antibody production level of Ig antigen. The results are shown in figure 1 , the serum titer was the highest after the fourth immunization. Use protein G column chromatography to separate the heavy ...

Embodiment 2

[0105] Example 2 Expression and purification of avian influenza virus nucleoprotein and construction of avian influenza virus nucleoprotein-specific single domain antibody gene library

[0106] 1. Cloning, expression and purification of avian influenza virus nucleoprotein: the gene is synthesized from Translate DNA SequencePet30arc+T7#3.seq (5076,5561), and the gene sequence is (sequence table SEQ ID NO.46):

[0107] 2. Construction of the expression vector: PCR amplified products were separated and recovered by agarose gel electrophoresis, digested with BamHI (BioLab Company), XhoI (BioLab Company), and treated with the same enzyme carrier pET-28a (Sigma) by 3 :1 end ratio for cohesive end ligation. Ligation product by CaCl 2 The transformation method was used to transform competent Escherichia coli JM109 (Sigma), and kanamycin resistance was used to screen recombinant clones. Plasmid DNA of positive clones was extracted for colony PCR. The correct positive recombinants wi...

Embodiment 3

[0111] Example 3 Screening of anti-Ig single domain antibodies

[0112] 1. Screening of anti-IgA specific single domain antibody

[0113] Use 100ug / ml of purified human IgG and IgA to coat the polystyrene microwells with high adsorption capacity of Thermo Electric Company, 150ul / well, overnight at 4°C. Aspirate the coated antigen, add 350ul 2% MPBST (2% skimmed milk, 0.05M, pH7.2-7.4PBS, 0.05% T20), block at room temperature for 2 hours, remove the blocking solution, add the VHH in the above example 1 Phage 5X10 for GenBank 11 , combined at room temperature for 2 hours, rinsed with PBST and PBS 10 times each, added TEA and allowed to stand at room temperature for 10 minutes, eluted specifically bound phage, neutralized TEA with 1M pH8.0 Tris-HCl, and kept on ice. Infect the bacteriophage with semi-logarithmic phase growth TG1, take appropriate amount of bacterial solution and dilute it, spread it on AMP / LB plate, incubate at 32°C, measure the titer of the eluent, and superin...

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Abstract

The invention discloses a single-domain antibody combined with immunoglobulin, an anti-avian influenza single-domain antibody, a bifunctional antibody and applications thereof. The present invention screens and obtains single-domain antibodies capable of mediating binding to immunoglobulins from the constructed immune nanobody gene library, the amino acid sequences of which are shown in SEQ ID NO.1‑4, and this group of single-domain antibodies can specifically bind Human IgA, with very low binding affinity to human IgG and mouse IgG. The present invention further provides the anti-avian influenza single domain antibody shown in SEQ ID NO.5. In the present invention, the single-domain antibody is also fused with other antibodies against tumors, viral pathogens or single-domain antibodies to construct a multifunctional specific fusion protein. Animal experiments have proved that the constructed fusion protein has the function of prolonging the function of the antibody or single-domain antibody. The half-life time in animals can effectively improve the bioavailability of therapeutic antibodies, and significantly enhance the effects of therapeutic antibody biological targeting therapy.

Description

technical field [0001] The present invention relates to single-domain antibodies, in particular to a group of single-domain antibodies that mediate binding to immunoglobulins, anti-avian influenza single-domain antibodies, and bifunctional antibodies obtained by fusion of single-domain antibodies and anti-avian influenza single-domain antibodies. One relates to their application in the treatment or detection of diseases such as tumors and pathogenic microorganism infections, and belongs to the field of single-domain antibodies that mediate binding to immunoglobulins and their applications. Background technique [0002] Belgian scientist Raymond Hamers found that in addition to the common four-chain antibody in camel blood, there is also a naturally occurring heavy chain antibody that lacks the light chain. When the heavy chain antibody gene is recombined, the heavy chain stable region is cut off The gene of the CH1 region, thus the expressed heavy chain lacks the CH1 region,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/42C07K16/10C12N15/13G01N33/574G01N33/569A61K39/395A61K39/42A61P35/00A61P31/04A61P31/16
CPCA61K2039/505A61P31/04A61P31/16A61P35/00C07K16/1018C07K16/42C07K2317/31C07K2317/35C07K2317/569G01N33/56911G01N33/56983G01N33/57484
Inventor 叶青
Owner 北京纽安博生物技术有限公司
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