CN106146657B

CN106146657B - Recombinant antibody fragment capable of combining influenza virus A in broad spectrum and preparation method and application thereof

Info

Publication number: CN106146657B
Application number: CN201610543191.5A
Authority: CN
Inventors: 史晋海; 余馨; 何明跃
Original assignee: Jinming Tianjin Biomedical Technology Development Co ltd
Current assignee: Jinming Tianjin Biomedical Technology Development Co ltd
Priority date: 2016-07-12
Filing date: 2016-07-12
Publication date: 2020-07-17
Anticipated expiration: 2036-07-12
Also published as: CN106146657A

Abstract

The invention discloses a recombinant antibody capable of combining influenza virus A in a broad spectrum manner, and a preparation method and application thereof. One of them is a single chain antibody (scFv format), the variable region of which has the amino acid sequence shown by SEQ ID NO:1 and the nucleotide sequence shown by SEQ ID NO: 2. The other is a single domain heavy chain (VH form) without a light chain, the variable region of which has the amino acid sequence shown in SEQ ID NO 3 and the nucleotide sequence shown in SEQ ID NO 4. Disclosed are amino acid sequences SEQ ID NO:5 and nucleotide sequences SEQ ID NO:6 encoding single domain antibody VH trimer (VH-trimer) antibodies. The antibody fragment can be specifically combined with a conserved region of an influenza A (influenza A virus) protein, and the affinity of the antibody fragment is not less than 10^‑7And M. The antibody fragment has potential application potential in treating human epidemic infectious diseases caused by influenza A virus.

Description

Recombinant antibody fragment capable of combining influenza virus A in broad spectrum and preparation method and application thereof

Technical Field

The invention belongs to the technical field of antibody application. Relates to the potential application of the design and preparation of the human recombinant antibody of the broad-spectrum influenza virus A. In particular to a recombinant antibody segment which is combined with influenza virus A in a broad spectrum and a preparation method and application thereof.

Background

Influenza virus (influenza virus) is divided into 3 types A (A), B (B) and C (C), wherein the influenza virus A can not only infect human, but also infect mammals such as horses and pigs, birds and poultry which are closely related to human life, so that the influenza virus A can cause wide-range influenza which crosses and bursts, and seriously threatens the life health of human beings. The outer membrane of the influenza virus HAs two proteins, one is Hemagglutinin (HA) and the other is Neuraminidase (NA), both of which are easy to be mutated, so that the influenza virus HAs a plurality of serotypes and a high mutation speed, and the influenza virus becomes a confusion for preventing and treating influenza. Therefore, there is an important clinical need to develop broad-spectrum anti-influenza antibodies.

HA is a glycoprotein that plays an important role when the virus infects cells. The head of the HA protein is combined with sialic acid of a cell mask, viruses are endocytosed into cells by the cells, and then the conformation change of the HA is generated, so that the virus envelope is fused with a cell membrane, and virus genes are released into the cells. Anti-influenza virus HA antibodies in current clinical use are primarily directed to head binding of HA, since the HA head is a highly variable region. Thus, many antibodies do not have the effect of a broad spectrum against influenza.

CR6261 is a neutralizing antibody obtained by screening using phage display technology in recent years, and is capable of resisting various subtypes of influenza viruses, including H1, H2, H5, H6, H8 and H9 subtypes of viruses [1 ]. It HAs been reported that the CR6261 antibody inhibits the membrane fusion process of virus by binding to the well-conserved A helix in HA2, exerts antiviral efficacy, and the antibody light chain is not associated with the neutralizing effect of virus [2-5 ]. Therefore, development of a wide spectrum of influenza therapeutic antibodies against the conserved region of the influenza virus protein HA is expected.

Based on the interaction structure-activity relationship [3-5] between the HA conserved region of influenza virus protein and CR6261, the invention designs the antibody fragment sequence of the HA conserved region of anti-influenza virus protein, namely VH and scFv sequences, by adopting computer design and combining with the current antibody bioinformatics, and designs and expresses the molecular forms of trimer, etc. of VH antibody on the basis of the VH sequence, thus obtaining the novel micromolecular antibody of the broad-spectrum HA conserved region of anti-influenza virus protein.

Disclosure of Invention

In order to achieve the aim, the invention discloses amino acid sequences and nucleotide sequences of two types of antibodies of VH and scFv aiming at a conserved region of influenza virus A, and different molecular structures of VH-trimers of the antibodies, and relates to a preparation method and application of the influenza antibodies.

The invention discloses a broad-spectrum recombinant antibody combined with influenza virus A, which is characterized by comprising a group of humanized recombinant influenza virus A antibody fragments; the antibody fragments include recombinant forms of VH, (VH) 2, (VH) 3, scFv, Fab ', F (ab')_、Form of IgG.

The antibody fragment is in the form of fusion with other proteins or polypeptides or small molecule markers.

Wherein the recombinant single-chain antibody fragment comprises a light chain, a heavy chain and a structural flexible connecting peptide, and the structural flexible connecting peptide has an amino acid sequence shown in SEQ ID NO. 1 and a nucleotide sequence shown in SEQ ID NO. 2.

The single-domain recombinant antibody of the recombinant antibody fragment only contains a heavy chain variable region (VH), does not contain a light chain variable region, has an amino acid sequence shown by an ID NO. 3, and has a nucleotide sequence shown by an SEQ ID NO. 4.

The heavy chain variable region VH of the heavy group antibody single domain fragment can be fused with other proteins, such as isoleucine zipper (isoleucin-zipper), to form an amino acid sequence shown in SEQ ID NO.5 and a nucleotide sequence shown in SEQ ID NO. 6.

The antibody fragment is fused to form a homologous VH trimer, wherein the VH trimer refers to three homologous VH, a non-covalent bond homotrimer is formed through an isoleucine zipper, and the weight part ratio of the three homotrimer is 1: 1: 1.

the recombinant antibody capable of combining with influenza virus A in broad spectrum is characterized in that the antibody is specifically combined with hemagglutinin of influenza virus A, and the affinity of the antibody is not less than 10^-7M。

The invention further discloses a pharmaceutical composition comprising a recombinant antibody that binds influenza A virus in a broad spectrum and a pharmaceutically acceptable carrier compatible with the antibody. Including intramuscular injection, intravenous injection or topical administration, transdermal absorption administration, and spray administration.

The invention further discloses application of the recombinant antibody capable of combining influenza A in broad spectrum in preparing two antibody medicines of VH and scFv for treating influenza A conserved regions.

The test results show that:

(1) the VH and scFv antibodies have specific binding force against influenza A antigen, such as common influenza A antigen N5H1, N5H2, etc., and have affinity of not less than 10^-7M。

(2) After the VH antibody is prepared into trimer, the binding force to the antigen is improved by 10 times.

(3) After the VH antibody is assembled into the scFv form, the antibody is more stable, and the binding force to the antigen reaches 10^-10M。

The recombinant antibody which is disclosed by the invention and has broad spectrum combination with influenza A virus can also be used as a kit for detecting influenza A virus.

The invention also provides VH antibody trimeric (VH-trimer) forms. In addition, other forms of recombinant antibodies may be developed for VH antibodies and scFv antibody sequences, which may also be in fusion with other proteins or polypeptides or small molecule markers, such as Fc fragments or PEG-modified antibodies

The nucleotide sequence of the antibody of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. A simple and feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. The present invention prepares target gene sequence with VH and scFv antibody as the synthesizing process.

The relevant sequences are as follows:

SEQ ID NO:1 single chain scFv antibody fragment amino acid sequence

EIVMTQSPSTLSASVGDRVIITCSGSSSNIGNDYVSWYQQKPGKAPKLLIYDNNKRPSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCATWDRRPTAYVVFGQGTKLTVLKRGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGGPFRSYAISWVRQAPGKGLEWVGGIIPIFGTTKYAPKFQGRFTISKDDFAGTVYLQMNSLRAEDTAVYYCARHMGYQVRETMDVWGQGTLVTVSSHHHHHHLGGCDE

SEQ ID NO:2 single chain scFv antibody fragment nucleotide sequence

GAAATCGTTATGACCCAGTCTCCGTCTACCCTGTCTGCGTCTGTTGGTGACCGTGTTATC ATCACCTGCTCTGGTTCTTCTTCTAACATCGGTAACGACTACGTTTCTTGGTACCAGCAG AAACCGGGTAAAGCGCCGAAACTGCTGATCTACGACAACAACAAACGTCCGTCTGGTGTT CCGTCTCGTTTCTCTGGTTCTGGTTCTGGTACCGAATTCACCCTGACCATCTCTTCTCTG CAGCCGGACGACTTCGCGACCTACTACTGCGCGACCTGGGACCGTCGTCCGACCGCGTAC GTTGTTTTCGGTCAGGGTACCAAACTGACCGTTCTGAAACGTGGTGGTGGTGGTGGTTCT GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGAAGTTCAGCTGGTT GAATCTGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCACCGTTTCT GGTGGTCCGTTCCGTTCTTACGCGATCTCTTGGGTTCGTCAGGCGCCGGGTAAAGGTCTG GAATGGGTTGGTGGTATCATCCCGATCTTCGGTACCACCAAATACGCGCCGAAATTCCAG GGTCGTTTCACCATCTCTAAAGACGACTTCGCGGGTACCGTTTACCTGCAGATGAACTCT CTGCGTGCGGAAGACACCGCGGTTTACTACTGCGCGCGTCACATGGGTTACCAGGTTCGT GAAACCATGGACGTTTGGGGTCAGGGTACCCTGGTTACCGTTTCTTCTCACCACCACCAC CACCACCTGGGTGGTTGCGACGAA

SEQ ID NO:3 single domain VH heavy chain antibody fragment amino acid sequence

QVQLVESGGGLVQPGGSLRLSCKASGGPFRSYAISWVRQAPGKGLEWMGGIIPIFGTTKYAPKFQGRFTISRDDFAGTVYLQMNSLRAEDTAMYYCAKHMGYQVRETMDVWGQGTLVTVSSLGGCDPHHHHHH*

SEQ ID NO:4 single domain VH heavy chain antibody fragment nucleotide sequence

CAGGTTCAGCTGGTTGAATCTGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGT CTGTCTTGCAAAGCGTCTGGTGGTCCGTTCCGTTCTTACGCGATCTCTTGGGTTCGTCAG GCGCCGGGTAAAGGTCTGGAATGGATGGGTGGTATCATCCCGATCTTCGGTACCACCAAA TACGCGCCGAAATTCCAGGGTCGTTTCACCATCTCTCGTGACGACTTCGCGGGTACCGTT TACCTGCAGATGAACTCTCTGCGTGCGGAAGACACCGCGATGTACTACTGCGCGAAACAC ATGGGTTACCAGGTTCGTGAAACCATGGACGTTTGGGGTCAGGGTACCCTGGTTACCGTT TCTTCTCTGGGTGGTTGCGACCCGCACCACCACCACCACCACTAA

SEQ ID NO：5

Single domain VH heavy chain antibody fragment trimer amino acid sequence

EQKLISEEDLM QVQLVESGGGLVQPGGSLRLSCKASGGPFRSYAISWVRQAPGKGLEWMGGIIPIFGTTKYAPKFQGRFTISRDDFAGTVYLQMNSLRAEDTAMYYCAKHMGYQVRETMDVWGQGTLVTVSSGGGSQRMKQIEDKIEEILSKIYHIENEIARIKKLVGEHHHHHH**

SEQ ID NO：6

Single domain VH heavy chain antibody fragment trimer nucleotide sequence

GAACAGAAACTGATCTCTGAAGAAGACCTG CAGGTTCAGCTGGTTGAATCTGGTGGTGGTCTGGTTCAGCCGGGTGGTTCTCTGCGTCTGTCTTGCAAAGCGTCTGGTGGTCCGTTCCGT TCTTACGCGATCTCTTGGGTTCGTCAGGCGCCGGGTAAAGGTCTGGAATGGATGGGTGGT ATCATCCCGATCTTCGGTACCACCAAATACGCGCCGAAATTCCAGGGTCGTTTCACCATC TCTCGTGACGACTTCGCGGGTACCGTTTACCTGCAGATGAACTCTCTGCGTGCGGAAGAC ACCGCGATGTACTACTGCGCGAAACACATGGGTTACCAGGTTCGTGAAACCATGGACGTT TGGGGTCAGGGTACCCTGGTTACCGTTTCTTCTGGTGGTGGTTCTCAGCGTATGAAACAG ATCGAAGACAAAATCGAAGAAATCCTGTCTAAAATCTACCACATCGAAAACGAAATCGCG CGTATCAAAAAACTGGTTGGTGAACACCACCACCACCACCACTAATAA

Description of the drawings: the diagonal sequence is a universal Myc detection tag introduced at the N-terminal when the trimer is constructed.

Once the relevant sequences have been obtained, other combinations of molecules of interest, such as sequence dimers, IgG, etc., can also be obtained in large quantities by recombinant methods, followed by cloning their DNA sequences into vectors (e.g., pET22(B), pET 32), transformation into cells (e.g., B L21 (DE3), Rosseta cells), and isolation from the propagated host cells by conventional methods.

The invention also relates to the design of vectors comprising appropriate DNA sequences as described above together with appropriate control sequences for promoters, dimers and trimers which may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces, fungal cells such as yeast, mammalian cells such as CHO, COS or 293 cells (commercially available).

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Process or electrotransfer techniques, the steps used being well known in the art. When the host is a eukaryotic cell, the following DNA transfection methods may be used: calcium phosphate coprecipitation methods, conventional mechanical methods such as microinjection, electroporation, liposomes, and the like.

The obtained transformant can be cultured by a conventional method to obtain the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from conventional media or modified media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant proteins can be isolated and purified by various separation methods using physical, chemical and other properties, if desired, these methods are well known to those skilled in the art, including but not limited to, conventional renaturation treatment, treatment with protein precipitant (salting-out method), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HP L C) and other various liquid chromatography techniques and combinations thereof.

The invention further discloses a pharmaceutical composition which contains the broad-spectrum recombinant antibody capable of combining with the influenza virus A and a pharmaceutically acceptable carrier compatible with the antibody. A pharmaceutically acceptable carrier should be compatible with, i.e., capable of being blended with, the antibody of the invention without substantially diminishing the effectiveness of the pharmaceutical composition under normal circumstances, the pharmaceutical formulation being compatible with the mode of administration. Pharmacologically acceptable carriers include one or more pharmaceutically acceptable excipients such as binders, diluents, disintegrants, preservatives, dispersants, glidants and lubricants. Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof are saline, buffers, sugars such as lactose, glucose and sucrose; malt, gelatin; polyols, such as propylene glycol, glycerol, sorbitol, glycerol sugar alcohol and polyethylene glycol, alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a stabilizer; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like.

The injection of the invention mainly comprises intramuscular injection, such as split needle injection and water injection, and can be intravenous injection or local administration, transdermal absorption administration, spray administration, medicinal membrane and the like. Intravenous injections include large infusion, small infusion, skin test, powder injection, etc.

The concentration of active ingredient in the injection is 50mg/ml-0.5mg/ml, preferably 5-30 mg/ml. Ok

2. The excipient is one or more of mannitol, low molecular dextran, sorbitol, polyethylene glycol, tween, glucose, lactose, galactose and glycine, preferably mannitol, lactose and glucose. Ok

3. The pH regulator is: tris, phosphoric acid, and the like non-volatile acids. And physiologically acceptable organic or inorganic acids and bases and salts such as sodium hydroxide, sodium carbonate or potassium or ammonium salt, sodium phosphate or potassium or ammonium salt, sodium acetate or ammonium salt, etc., preferably citric acid, phosphoric acid, potassium hydroxide, sodium hydroxide.

4. The solubilizer is: PEG6000, PEG4000, Tween 20-80, sodium dodecyl sulfate, tris (hydroxymethyl) aminomethane, etc., preferably PEG6000, PEG4000, and arginine.

5. The antioxidant is inorganic sulfur or organic sulfur containing antioxidant; the inorganic sulfur antioxidant is sulfite such as sodium sulfite, potassium sulfite, sodium pyrosulfite, sodium thiosulfate, sodium bisulfite, and potassium bisulfite; the organic sulfur antioxidant is thioglycerol, thiourea, 2-mercaptoethanol, 2-mercaptopropanol, and 1-thiosorbitol; the antioxidant of the present invention must be pharmaceutically acceptable and not biologically or pharmacologically active per se, and particularly preferably a sulfite such as sodium sulfite, potassium sulfite, sodium metabisulfite, sodium thiosulfate, sodium bisulfite, thioglycerol, thiourea.

6. The osmotic pressure regulator is one or a combination of sodium chloride and potassium chloride.

The broad-spectrum recombinant antibody combined with influenza virus A, the preparation method and the application thereof disclosed by the invention have the positive effects that:

(1) the antibody is humanized, has small molecule, low immunogenicity and high safety.

(2) The small molecule antibody is prepared by using E.coli cells with relatively low cost, and the manufacturing cost is low.

(3) The antibody is directed against a conserved region of the influenza virus A, and has wide application range and stable effectiveness.

Drawings

FIG. 1 binding curves of the E L ISA of VH single domain antibodies;

FIG. 2 binding curves of the E L ISA of VH single domain trimeric antibody;

FIG. 3 binding curves of the E L ISA of the single chain antibody.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

The term "antibody" fragment as used herein is a protein having the structural characteristics of an antibody in which a VH single domain antibody has only the antibody heavy chain variable region with a molecular weight of about 15000 daltons, and a scFv single chain antibody has the antibody heavy and light chain variable regions formed by a linker peptide (GGGGS) 4 with a molecular weight of about 28000. VH trimer is the product of VH single domain antibody realization by Ziplinker peptide.

Example 1.

Design and Synthesis of antibody sequences

The amino acid sequences of the antibodies SEQ ID NO. 1 and SEQ ID NO. 3 are designed respectively by a computer design means. Then, according to the codon bias of E.coli cells, a proper codon is designedE.coliThe nucleotide sequences of SEQ ID NO 2 and SEQ ID NO 4 expressed by cell heterology are sent to a gene synthesis company, the synthesized gene is connected with a pET22(B) vector, a clone plasmid is transformed from B L21 (DE3) to competent cells by electrotransformation (voltage is 1.9KV, Resistance 200 omega cut 1mm, TC 5 ms) by coating the competent cells on an ampicillin L B solid plate, 10 single colonies are picked, and the positive clone is identified by colony PCR.

Example 2.

Cloning bacteria expression identification

And (3) thallus culture, namely respectively selecting monoclonal bacteria, respectively inoculating the monoclonal bacteria into 2 ml of L B culture medium test tubes containing 50 ng/L Amp, carrying out shaking culture at 37 ℃ overnight, inoculating the monoclonal bacteria into 2YT (containing 50 ng/L Amp) culture medium in a 25 ml/100 ml triangular flask according to the inoculation amount of 5%, carrying out shaking culture at 37 ℃ at 240 rpm until logarithmic growth phase, adding isopropyl thiogalactoside (IPTG) with the final concentration of 0.5 mmol/L mmol to induce, carrying out shaking culture at 37 ℃ at 240 rpm for further 5h, centrifuging 1300 × g for 10 min, collecting thallus, extracting soluble scFv antibodies in the thallus by using Bugbuster liquid, and detecting positive expression cloning by western blot.

Example 3

Preparation of antibody samples

Antibody amplification preparation by culturing cells in a 400 ml/2000ml Erlenmeyer flask volume under the same conditions as in example 3 of example 1, cells were collected by centrifugation, and 5 ml/g of cells were resuspended in disruption buffer (0.3 mol/L NaCl, 50 mmol/L NaH)₂PO₄·2H ₂0, 10 mmol/L imidazole), sonicated 99 times (4 s work, 5 s dwell), centrifuged at 13000 g for 20 min at 4 ℃, and the supernatant was collected.

Single-domain antibody purification for VH and VH-trimers by first purifying the antibody with a hitrap his pre-column, equilibrating 5 Column Volumes (CV) with loading buffer (with disruption buffer), loading 50 ml, washing 5CV with loading buffer, and then washing with washing buffer (50 mmol/L NaH)₂PO₄0.3 mol/L NaC L, 50 mmol/L imidazole, pH 8.0) 10 CV, finally eluted with elution buffer (500 mmol/L imidazole, the rest being the same as the washing buffer), fractions were collected and pooled with bufferA (Tris 50mM, NaCl200 mM, pH 8.0). The antibody was purified using a hisscreen capto Adheree pre-packed column in the second step, bufferA samples were loaded and washed 5CV (column volume), buffer B (Tris 50mM, NaCl 1M, pH 8.0) washed 5CV, buffer C (NaAC 50mM, NaCl200 mM, pH 5.0) washed 5CV, and finally eluted with Na100% buffer D (NaAC 50mM, pH 5.0). The VH monodomain antibody was obtained at 4-6 mg/L, respectively, giving a VH trimer yield of 4-8 mg/L.

Purification of single-chain antibody against scFv in a first step the antibody was purified using a protein L pre-column, the loading buffer (with disruption buffer) was equilibrated to 5 Column Volumes (CV), 50 ml was loaded, 5CV was washed with buffer A (PBS +0.1M glycine, pH 8.0), then 5CV was washed with buffer B (citric acid 50mM, pH 3.5), and finally eluted with buffer C (citric acid 50mM, pH 3.0) into the buffer A solution used in the second purification step, the antibody was purified using a hisscreen capto Adhere pre-column in the second step, the buffer A sample was loaded and 5CV was washed (column volume), buffer B (Tris 50mM, NaCl 1M, pH 8.0) 5CV was washed, buffer C (NaAC 50mM, NaCl200 mM, pH 5.0) 5CV was washed, and finally eluted with buffer D (NaAC 50mM, pH 5.0) to give a fermentation broth of 10-18 mg/L mg.

Example 4.

Enzyme-linked immunosorbent assay for detecting antigen-antibody binding force

Detecting the binding activity of a target antibody by an enzyme-linked immunosorbent assay (E L ISA), wherein influenza virus protein (H5N 2) of 10 mug/ml coats E L ISA plate holes, the temperature is 4 ℃ overnight, PBS containing 2% BSA is sealed at 37 ℃ for 1H, 100 mug purified soluble antibody is added into each hole, 1 h.100 mug is incubated at 37 ℃ and is diluted by PBST (1: 4000) to form anti-His antibody-HRP, 1 h.100 mug TMB is incubated at 37 ℃ for color development, the color development reaction is stopped by 100 mug 1 mol/L HCl, and the enzyme-linked immunosorbent assay (ELISA) reads at the wavelength of 450 nm.

(1) E L ISA binding curves of VH Single domain antibodies detailed in the E L ISA binding curves of the VH Single domain antibody of FIG. 1;

the undiluted sample had a protein concentration of 48.4ug/ml and was diluted in 10 gradients by 2-fold dilution.

Table 1 results of detection of E L ISA uptake values of VH single domain antibodies:

(2) VH single domain trimer antibody E L ISA binding curve, see figure 2. VH single domain trimer antibody E L ISA binding curve affinity KD =6.85x10^-9M。

TABLE 2 results of E L ISA uptake values of VH Single Domain trimeric antibodies

The concentration of the undiluted sample protein is 1.93ug/ml, and 8 gradients are diluted by a 2-fold dilution method;

(3) the binding curve of E L ISA of single-chain antibody is shown in detail in figure 3. the binding curve of E L ISA of single-chain antibody. affinity KD =2.0x10^-10And M. The undiluted sample protein concentration is 0.28ug/ml, and 10 gradients are diluted by 2-fold dilution method

Table 3 results of E L ISA uptake values of single chain antibodies:

the results show that:

the single-domain VH antibody and the trimer thereof and the single-chain antibody have good specificity and binding force to influenza virus protein (H5N 2).

Example 5

Taking 5ml of 2mg/ml purified antibody under aseptic condition, and adding auxiliary materials according to the following formula to obtain the final components of the stock solution: 10mg of antibody. 50mM tris, 100mM Glycine,10mM NaCl,25mM argine, pH 7.5, magnetic stirring at 4 ℃ for 10 minutes, accurately dispensing a pipette into 20ml of sterilized and pyrogen-removed penicillin bottles, capping, and freeze-drying in a freeze dryer. Freeze-drying conditions: a pre-freezing stage: 4 hours at-40 ℃. A main drying stage: 10 minutes at-40 ℃; 25 hours at-30 ℃; 9 hours at-10 ℃. And (3) final drying stage: 20 ℃ for 3 hours. And (5) after the freeze drying is finished, covering a cover under vacuum to obtain the product.

Reference documents:

1. Throsby M,Van Den Brink E,et al.heterosubtypic neutralizingmonoclonal antibodies cross-protective against H5N1 and H1N1recovered fromhuman IgM+ memory B cells.PLos One. 2008,3(12):e3942

2.Ekiert DC ,et al. Antibody recognition of a highly conservedinfluenza virus epitope.Science.2009.324(59240246-251.

3. Anvir,Y at al., (2014) Molecular Signatures of Hemagglutinin Stem-Directed HeterosubtypicHuman Neutralizing Antibodies against Influenza AViruses. PLOS Pathogens. e1004103.

4. Cui, W et al., (2012) The Molecular Mechanism of Action of theCR6261-Azichromycin Combination Found through Computational Analysis PLOS onee37790.

5. Lingwood, D et al., (2012) Structural and genetic basis fordevelopment of broadly neutralizing influenza antibodies. Nature 489, 566-571。

<110> Gen (Tianjin) biomedical technology Co., Ltd

<120> recombinant antibody fragment capable of combining influenza virus A in broad spectrum, and preparation method and application thereof

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130

<210>4

<211>402

<212>DNA

<213> Artificial sequence

<400>4

caggttcagc tggttgaatc tggtggtggt ctggttcagc cgggtggttc tctgcgtctg 60

tcttgcaaag cgtctggtgg tccgttccgt tcttacgcga tctcttgggt tcgtcaggcg 120

ccgggtaaag gtctggaatg gatgggtggt atcatcccga tcttcggtac caccaaatac 180

gcgccgaaat tccagggtcg tttcaccatc tctcgtgacg acttcgcggg taccgtttac 240

ctgcagatga actctctgcg tgcggaagac accgcgatgt actactgcgc gaaacacatg 300

ggttaccagg ttcgtgaaac catggacgtt tggggtcagg gtaccctggt taccgtttct 360

tctctgggtg gttgcgaccc gcaccaccac caccaccact aa 402

<210>5

<211>175

<212>PRT

<213> Single Domain VH heavy chain antibody fragments

<400>5

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Met Gln Val Gln Leu Val

1 5 10 15

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

20 25 30

Cys Lys Ala Ser Gly Gly Pro Phe Arg Ser Tyr Ala Ile Ser Trp Val

35 40 45

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly Gly Ile Ile Pro

50 55 60

Ile Phe Gly Thr Thr Lys Tyr Ala Pro Lys Phe Gln Gly Arg Phe Thr

65 70 75 80

Ile Ser Arg Asp Asp Phe Ala Gly Thr Val Tyr Leu Gln Met Asn Ser

85 90 95

Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala Lys His Met Gly

100 105 110

Tyr Gln Val Arg Glu Thr Met Asp Val Trp Gly Gln Gly Thr Leu Val

115 120 125

Thr Val Ser Ser Gly Gly Gly Ser Gln Arg Met Lys Gln Ile Glu Asp

130 135 140

Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile

145 150 155 160

Ala Arg Ile Lys Lys Leu Val Gly Glu His His His His His His

165170 175

<210>6

<211>528

<212>DNA

<213> Artificial sequence

<400>6

gaacagaaac tgatctctga agaagacctg caggttcagc tggttgaatc tggtggtggt 60

ctggttcagc cgggtggttc tctgcgtctg tcttgcaaag cgtctggtgg tccgttccgt 120

tcttacgcga tctcttgggt tcgtcaggcg ccgggtaaag gtctggaatg gatgggtggt 180

atcatcccga tcttcggtac caccaaatac gcgccgaaat tccagggtcg tttcaccatc 240

tctcgtgacg acttcgcggg taccgtttac ctgcagatga actctctgcg tgcggaagac 300

accgcgatgt actactgcgc gaaacacatg ggttaccagg ttcgtgaaac catggacgtt 360

tggggtcagg gtaccctggt taccgtttct tctggtggtg gttctcagcg tatgaaacag 420

atcgaagaca aaatcgaaga aatcctgtct aaaatctacc acatcgaaaa cgaaatcgcg 480

cgtatcaaaa aactggttgg tgaacaccac caccaccacc actaataa 528

Claims

1. A recombinant antibody for binding influenza A virus, wherein the recombinant antibody is a single-domain recombinant antibody, only contains a heavy chain variable region and NO light chain variable region, and the amino acid sequence of the recombinant antibody is SEQ ID NO:3, respectively.

2. A recombinant antibody combined with influenza virus A is characterized in that the recombinant antibody is formed by fusing a heavy chain variable region VH of a single-domain antibody with an isoleucine zipper, and the amino acid sequence of the recombinant antibody is shown in SEQ ID NO. 5.

3. A recombinant antibody that binds to influenza a virus, characterized in that said recombinant antibody is a homologous VH trimer formed by fusion of the recombinant antibody of claim 2, wherein the ratio between each monomer in the trimer is 1: 1: 1.

4. a pharmaceutical composition comprising the recombinant antibody that binds to influenza a virus of any one of claims 1 to 3 and an antibody-compatible pharmaceutically acceptable carrier.