CN110903385B

CN110903385B - H1N1 influenza virus antibody and preparation method and application thereof

Info

Publication number: CN110903385B
Application number: CN201911113930.7A
Authority: CN
Inventors: 董金华; 董航; 单喜军; 陈丽梅; 李海梅
Original assignee: Shandong Kuanhezheng Bio Medicine Co ltd; Weifang Medical University
Current assignee: Shandong Kuanhezheng Bio Medicine Co ltd; Weifang Medical University
Priority date: 2019-11-14
Filing date: 2019-11-14
Publication date: 2021-03-23
Anticipated expiration: 2039-11-14
Also published as: CN110903385A

Abstract

The invention discloses an H1N1 influenza virus antibody and application thereof, the structure comprises a heavy chain variable region, a first heavy chain hypervariable region, a second heavy chain hypervariable region, a third heavy chain hypervariable region, a light chain variable region, a first light chain hypervariable region, a second light chain hypervariable region and a third light chain hypervariable region; the H1N1 influenza virus antibody can be specifically combined with H1N1 influenza virus, is used for detecting, preventing and treating H1N1 influenza, provides a new method for preventing and treating H1N1 influenza, and has very important economic and social meanings.

Description

H1N1 influenza virus antibody and preparation method and application thereof

Technical Field

The invention relates to the technical field of influenza virus antibodies in biotechnology, in particular to an H1N1 influenza virus antibody and a preparation method and application thereof.

Background

Influenza virus (inflenzavirus) is an infectious disease pathogen that often causes influenza, and highly pathogenic influenza viruses also cause death in humans and animals. The virus belongs to the family Orthomyxoviridae (Orthomyxoviridae), the genus influenza. Influenza viruses are classified into A, B, C types according to nucleoprotein and matrix protein antigenicity. The surfaces of virus particles have two important proteins, namely Hemagglutinin (HA) and Neuraminidase (NA), and influenza viruses can be divided into a plurality of subtypes according to the difference of the two proteins. For the type A protein example, there are 18 subtypes of HA (H1-H18) and 11 NA (NA 1-NA 11) that have been found. The surface protein of the influenza A virus is easy to have antigenic mutation and difficult to control, and a plurality of large influenza viruses are caused by the influenza A virus in history, and the large influenza viruses cause about 30 to 5 million deaths of people, thereby causing great threat to the life safety and the economic property of people.

The current methods for treating H1N1 influenza mainly comprise drug therapy and antibody therapy, wherein the drug therapy mainly adopts an ion channel inhibitor and a neuraminidase inhibitor. The former includes amantadine, rimantadine and derivatives thereof, which not only can generate side effects of gastrointestinal tract and central nervous system, but also can easily result in drug-resistant strains with unreduced toxicity and transmission performance after long-term use; the latter mainly comprises zanamivir and oseltamivir, and the medicines have good treatment effect on patients with H1N1 influenza at early stage, but the treated patients are easy to generate drug resistance to the medicines. Compared with drug therapy, antibody therapy has great advantages by blocking the combination of viruses and target cells and inducing immune cells to kill cells infected by the viruses, thereby achieving the aim of quickly treating H1N1 influenza.

Therefore, the development of antibodies against H1N1 influenza is a challenge to solve the problem of meeting the need for H1N1 virus detection, and prevention and treatment of H1N1 viral influenza.

Disclosure of Invention

In order to solve the problems, the invention provides an H1N1 influenza virus antibody and a preparation method and application thereof.

In order to achieve the purpose, the invention is realized by the following technical method:

preparing an influenza H1N1 antibody comprising a heavy chain variable region, a first heavy chain hypervariable region, a second heavy chain hypervariable region, a third heavy chain hypervariable region, a light chain variable region, a first light chain hypervariable region, a second light chain hypervariable region and a third light chain hypervariable region;

wherein the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 2;

the first heavy chain hypervariable region has the amino acid sequence shown in SEQ ID No. 3;

the second heavy chain hypervariable region has the amino acid sequence shown in SEQ ID No. 4;

the third heavy chain hypervariable region has the amino acid sequence shown in SEQ ID NO. 5;

the light chain variable region has an amino acid sequence shown in SEQ ID NO. 7;

the first light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 8;

the second light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 9;

the third light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 10.

The invention also comprises a DNA segment for coding the H1N1 influenza virus antibody, wherein the heavy chain variable region has a gene sequence shown in SEQ ID NO.1, and the light chain variable region has a gene sequence shown in SEQ ID NO. 6.

The invention also comprises an expression vector which comprises at least one copy of DNA fragments of a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a gene sequence shown in SEQ ID NO.1, and the light chain variable region has a gene sequence shown in SEQ ID NO. 6;

the invention also includes a host cell comprising the above-described expression vector.

The invention also comprises the application of the H1N1 influenza virus antibody in the aspect of H1N1 virus detection.

The invention also includes the application of the H1N1 influenza virus antibody in the prevention and treatment of H1N1 viral influenza. Compared with the prior art, the invention has the following advantages:

the H1N1 influenza virus antibody can be specifically combined with the H1N1 influenza virus, and can be used for detecting, preventing and treating H1N1 influenza;

the acquisition of the H1N1 influenza virus antibody provides a new method for preventing and treating H1N1 influenza, and has very important economic and social significance.

Drawings

FIG. 1 is a graph showing the comparison of the H1N1 influenza virus antibody content in serum before and after immunization of mice;

FIG. 2 is an agarose gel electrophoresis of PCR amplified mouse antibody variable region genes;

FIG. 3 is a diagram showing the results of ELISA tests on the binding performance of the antibody library antibodies with H1N1 virus and streptavidin obtained from various steps in the panning process;

FIG. 4 is a diagram showing the result of ELISA analysis of anti-H1N 1 virus monoclonal antibody;

FIG. 5 is a schematic diagram showing the result of polyacrylamide gel electrophoresis of a purified Fab fragment of antibody B2;

FIG. 6 is a schematic diagram showing the results of detection of virus-binding properties of anti-H1N 1 virus monoclonal antibody B2;

FIG. 7 is a diagram showing the results of detecting a trace amount of H1N1 virus in a cell lysate using the B2 antibody.

Detailed description of the preferred embodiments

The invention aims to provide an H1N1 influenza virus antibody and a preparation method and application thereof, and the preparation method is realized by the following technical method:

The invention also includes the application of the H1N1 influenza virus antibody in the prevention and treatment of H1N1 viral influenza. The invention is further described with reference to specific examples.

Examples

The method comprises the steps of immunizing a mouse by using inactivated H1N1 virus particles, taking the spleen of the mouse after influenza virus antibodies appear in the blood of the mouse, extracting total ribonucleic acid (RNA), synthesizing DNA (cDNA) with complementary base sequences by using the RNA as a template, and amplifying antibody genes by performing Polymerase Chain Reaction (PCR) by using antibody specific primers. The treated antibody gene was cloned into a phage display vector pDeng 1, and E.coli TG-1 was further transformed with the constructed vector to prepare a phage display antibody library. Panning against the H1N1 virus yielded an antibody B2 that binds to the H1N1 virus but not to bovine serum albumin, and thus has H1N1 virus specificity.

The specific process is as follows:

first, immunization of mice and confirmation of anti-H1N 1 antibody in mouse serum after immunization

Mice were first immunized with inactivated H1N1A/Beijing/262/95 virus. The mice are female BALB/C mice with the age of 3 weeks, and are immunized by a subcutaneous injection method, wherein the immunization dose is 10 mu g/time of virus, and the immunization is carried out at intervals of weeks for 3 times. After one week of the last immunization, the mice are cut off the tail and blood is taken, enzyme linked immunosorbent assay is carried out, and whether the blood of the mice contains H1N1 influenza virus antibody is detected.

The enzyme-linked immunosorbent assay was performed as follows: adding 100 mu L of PBS (phosphate buffer solution) containing H1N1A/Beijing/262/95 virus (0.5 mu g/mL) into a 96-hole enzyme label plate hole, staying overnight at 4 ℃, discarding the antigen solution the next day, adding 200 mu L of 2% skimmed milk powder solution, incubating for 2 hours at 25 ℃, and sealing the enzyme label plate;

washing the ELISA plate with 0.1% Tween 20PBS solution for 3 times, adding 1000, 2000, 4000, 8000, 16000, 32000, and 64000 times diluted mouse serum before and after immunization, incubating at 25 deg.C for 1 hr, washing the ELISA plate with PBST solution, adding rabbit anti-mouse IgG antibody labeled with horseradish peroxidase (HRP), incubating for 1 hr, washing the plate with PBST, adding HRP substrate 3,3',5,5' -tetramethylbenzidine (TMBZ; prepared with pH6.0 sodium acetate solution, containing 1/10000 diluted 30% H₂O₂) After developing, measuring the absorbance at 450nm by using an enzyme-labeling instrument, drawing a curve, and comparing the antibody content in the mouse serum before and after immunization.

The result of ELISA is shown in FIG. 1, the absorbance of the serum of mice diluted 1000 times after immunization at 450nm is 1.2, the absorbance gradually decreases with the increase of the dilution times, and the absorbance of the sample diluted 32000 times and later has no obvious change. The absorbance of the serum of the mice before and after the immunization is always maintained at a lower level and has little change. The results of this experiment indicate that antibodies against H1N1 virus are present in the sera of mice after immunization.

Amplification of mouse antibody variable region gene and construction of phage display antibody

The experimental process comprises the following steps: after confirming that the serum of the mouse contains the anti-H1N 1 influenza virus antibody, the mouse is sacrificed, the spleen of the mouse is picked up, total RNA is extracted by using TRIzol reagent, cDNA is synthesized by using the total RNA as a template, and the variable region gene of the antibody is amplified by further utilizing the specific primer of the antibody gene to execute polymerase chain reaction.

The double-digested (SalI/NotI) mouse light chain variable region gene was cloned into a phage display vector pDONG 1. Successfully screening, cloning, amplifying, culturing and extracting plasmids, then carrying out double enzyme digestion (SfiI/XhoI), connecting with the heavy chain variable region gene of the antibody which is also cut by the SfiI/XhoI to construct a vector, and transforming the escherichia coli competence TG-1.

Culturing at 37 deg.C to transform Escherichia coli to OD₆₀₀At 0.5, add 10⁹pfu helper phage KM13 was infected at 37 ℃ for 1 hour, centrifuged at 3000g for 30 minutes, the supernatant was discarded, 40mL of a 2YT medium (1.6% Tryptone, 1% Yeast Extract, 0.5% NaCl) containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1% glucose was used to suspend the cells, shaken at 30 ℃ and 250rpm for 16 hours the following day, the culture broth was centrifuged at 5000g for 30 minutes the next day, the supernatant was recovered, 1/5 volumes of PEG/NaCl solution were added to the supernatant, mixed well, and then placed on ice for 30 minutes, 5000g and centrifuged for 30 minutes, the supernatant was discarded, and 2mL of sterile PBS solution was added to dissolve and precipitate, thereby preparing a phage display antibody library solution.

The agarose gel electrophoresis of the PCR amplified mouse antibody variable region gene is shown in FIG. 2, the mouse antibody variable region gene amplification is successful, wherein the length of the heavy chain variable region gene is about 400bp, the length of the light chain variable region gene is about 350bp, the gene fragments are cloned to a phage display vector pDONG1, a phage is manufactured by using Escherichia coli competence TG-1, and finally, a gene containing 10 bp is obtained⁸Phage display antibody libraries of seed antibodies.

Screening of antibody library

Adding 100 μ L PBS containing 1 μ g/ml H1N1 virus into 10 wells of 96-well microplate, incubating overnight at 4 deg.C, discarding antigen solution the next time, adding 200 μ L2% skimmed milk powder PBS solution into each well, incubating and sealing at 25 deg.C for 2 hr, washing with PBST for 3 times, adding 200 μ L phage solution into each well (R0; each well contains 10 μ L phage solution)⁹cfu phage) were incubated at room temperature for 2 hours, and after washing with PBST, phage bound to H1N1 virus were eluted by adding 100 μ L trypsin per well;

culturing TG-1 E.coli to OD₆₀₀Taking 4mL, adding 500 mu L of dissolved phage solution, infecting at 37 ℃ for 30 minutes, centrifuging for 20 minutes at 5000g, discarding the supernatant, suspending the thalli by using 4mL of 2YT culture medium containing 100 mu g/mL ampicillin, 50 mu g/mL kanamycin and 0.1% glucose, shaking the thalli at 30 ℃, 250rpm for 16 hours, next day 5000g, centrifuging for 30 minutes, recovering the supernatant, adding 1/5 volumes of PEG/NaCl solution into the supernatant, uniformly mixing, standing on ice for 30 minutes, 5000g, centrifuging for 30 minutes, discarding the supernatant, adding 200 mu L of sterilized PBS solution, and obtaining phage solution (R1) after the first enrichment;

repeating the operation twice to respectively obtain phage solutions R2 and R3; and performing enzyme-linked immunosorbent assay, and verifying the binding specificity and binding performance of the phage display antibody library obtained in the panning process and the H1N1 virus.

The enzyme-linked immunosorbent assay was performed as follows: mu.L of PBS containing H1N1A/Beijing/262/95 virus (0.5. mu.g/mL) or Streptavidin (Streptavidin) was added to the 96-well plate, overnight at 4 ℃, the antigen solution was discarded the next day, 200. mu.L of 2% skim milk powder solution was added, and the plate was incubated at 25 ℃ for 2 hours. The microplate was washed 3 times with PBS containing 0.1% Tween 20, and diluted solutions of R0, R1, R2 and R3 phage (10)⁹cfu/well), incubation at 25 ℃ for 1 hour, washing the microplate with PBST solution, addition of HRP-labeled mouse anti-M13 antibody, incubation for 1 hour, washing the plate with PBST, addition of HRP substrate TMBZ (prepared with sodium acetate solution pH6.0, containing 1/10000 diluted 30% H₂O₂) And after color development, measuring the absorbance at 450nm by using an enzyme labeling instrument, drawing a histogram, and comparing the binding performance of the phage antibody, the H1N1 virus and streptavidin obtained in each step.

The results of the enzyme-linked immunosorbent assay are shown in fig. 3, and the results of comparing the binding capacities of the phage libraries R0, R1, R2 and R3 obtained in the phage panning process with the H1N1 show that the binding capacity of the phage solution R3 obtained in the third panning process with the H1N1 virus is significantly increased compared with that of R0, R1 and R2, and the binding performance with streptavidin is very weak and unchanged, which indicates that the anti-H1N 1 virus antibody in the constructed phage display antibody library is enriched.

Fourth, monoclonal antibody screening

Culturing TG-1 E.coli to OD₆₀₀0.4, 100 μ L panning sieve R2 phage antibody library dissolved out phage solution, infected 200 μ L Escherichia coli bacterial liquid, 37 degrees C after incubation for 30 minutes, the bacterial liquid is coated to containing 100 μ g/mL ampicillin, 50 μ g/mL kanamycin and 1% glucose 2YT medium plate, 37 degrees C overnight culture, the next day choose 48 colonies to inoculate 96 hole culture plate, 37 degrees C culture to OD₆₀₀Is 0.4, 10 are added per well⁸pfuKM13 phage, infecting for 30 minutes, centrifuging for 20 minutes at 5000g, discarding supernatant, adding 200 μ L2 YT culture medium containing 100 μ g/mL ampicillin, 50 μ g/mL kanamycin and 0.1% glucose into each well, suspending thallus, culturing at 30 deg.C and 250rpm for 16 hours; 5000g the next day, centrifuging for 30 minutes, recovering the supernatant, performing enzyme-linked immunosorbent assay, and verifying the binding specificity and binding performance of each monoclonal antibody and H1N1 virus.

The enzyme-linked immunosorbent assay was performed as follows: 100. mu.L of PBS containing H1N1A/Beijing/262/95 virus (0.5. mu.g/mL) was added to a 96-well plate, overnight at 4 ℃, the antigen solution was discarded the next day, 200. mu.L of 2% skim milk powder solution was added, and the plate was incubated at 25 ℃ for 2 hours and blocked. Washing an ELISA plate 3 times by using PBS (phosphate buffer solution) containing 0.1% Tween 20, adding a phage solution, incubating for 1 hour at 25 ℃, washing the ELISA plate by using PBST solution, adding a mouse anti-M13 antibody marked by HRP (horse radish peroxidase), incubating for 1 hour, washing the plate by using PBST, adding an HRP substrate TMBZ, measuring the absorbance at 450nm by using an ELISA reader after color development, drawing a histogram, and comparing the binding performance of the phage antibody prepared by each clone with H1N1 virus and bovine serum albumin.

The experimental results are as follows: as a result, as shown in FIG. 4, the phage antibody prepared from 31 out of 48 clones had stronger binding ability to H1N1 virus than to bovine serum albumin, indicating the antigen specificity of the antibody. Selecting clone B2 with strong binding ability with virus, culturing, extracting plasmid, analyzing the cloned sequence, wherein the heavy chain variable region gene and amino acid sequence are shown as SEQ ID NO.1 and SEQ ID NO.2, the antibody light chain variable region gene and amino acid sequence are shown as SEQ ID NO.6 and SEQ ID NO.7,

the hypervariable region in the heavy chain variable region is divided into a first heavy chain hypervariable region, a second heavy chain hypervariable region and a third heavy chain hypervariable region, and the amino acid sequences of the hypervariable regions are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the hypervariable regions in the light chain variable region are divided into a first light chain hypervariable region, a second light chain hypervariable region and a third light chain hypervariable region, and the amino acid sequences thereof are respectively shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.

The first heavy chain hypervariable region has the amino acid sequence shown in SEQ ID NO. 3: GYTFTDYW;

the second heavy chain hypervariable region has the amino acid sequence shown in SEQ ID No. 4: IFPSDGYT;

the third heavy chain hypervariable region has the amino acid sequence shown in SEQ ID No. 5: TRSTTVVAEDWYFDV, respectively;

the first light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 8: SSISSNY;

the second light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 9: RTS;

the third light chain hypervariable region has the amino acid sequence shown in SEQ ID No. 10: QQGSSIPRT are provided.

The B2 antibody is a novel antibody because no sequence identical to the present antibody gene is found by aligning the sequences with the antibody sequences registered in the antibody gene library.

Expression and purification of monoclonal antibody B2

Selecting the B2 escherichia coli bacterial colony obtained in the fourth step, extracting pDOng1 carrier after amplification culture, converting non-inhibition escherichia coli HB2151, selecting a single bacterial colony, inoculating the single bacterial colony into 4mL LB liquid culture medium, overnight culture at 37 ℃, inoculating the culture bacterial liquid into 500mL LB liquid culture medium containing 100 mu g/mL ampicillin, and culturing at 37 ℃ and 200rpm until OD is reached₆₀₀At 0.4, IPTG was added to a final concentration of 1mM, and the mixture was cultured at 30 ℃ for 16 hours. The culture supernatant was collected by centrifugation, and after adding a 75% ammonium sulfate-adjusted solution and allowing to stand at 4 ℃ for 2 hours, the mixture was centrifuged at 6000rpm for 20 minutes, the supernatant was discarded, the precipitate was dissolved in 10mL of TALON buffer, and the Fab fragment of the B2 antibody was purified by TALON resin. Performing polyacrylamideThe purified antibody fragment was confirmed by gel electrophoresis and stained with Coomassie brilliant blue.

After destaining the gel, two protein bands were observed as shown in FIG. 5. The upper band has a molecular weight of 27kDa, and is the variable region and the first constant region protein of the antibody heavy chain, and the lower band is the antibody light chain. This result showed that the B2 Fab fragment was expressed in e.coli and purified using talen resin.

Sixthly, H1N1 virus binding of monoclonal antibody B2

Enzyme-linked immunosorbent assay was performed to confirm that the purified Fab antibody fragment has H1N1 virus binding activity. Adding 100 mu L PBS solution containing H1N1A/Beijing/262/95 virus (0.5 mu g/mL) or BSA (1 mu g/mL) into a 96-hole enzyme label plate, standing overnight at 4 ℃, discarding the protein solution the next time, adding 200 mu L2% skimmed milk powder solution, incubating for 2 hours at 25 ℃, and blocking the enzyme label plate; washing the ELISA plate 3 times by PBS solution containing 0.1% Tween 20, adding 100 μ L diluted Fab antibody fragment solution into each well, incubating for 1 hour at 25 ℃, washing the ELISA plate by PBST solution, adding HRP-labeled mouse anti-histidine tag antibody, incubating for 1 hour, washing the plate by PBST, adding HRP substrate TMBZ, developing, measuring absorbance at 450nm by an ELISA reader, drawing a bar chart, and comparing the binding performance of the purified Fab fragment with H1N1 virus and bovine serum albumin.

The results of the elisa are shown in fig. 6, the absorbance of the purified B2 Fab fragment bound to H1N1 virus is 0.16, and the absorbance of the purified B2 Fab fragment bound to bovine serum albumin is 0.01, so it can be concluded that the antibody B2 Fab can specifically bind to H1N1 virus, and can be used for detecting H1N1 virus.

Seventh, detection of H1N1 virus in cell lysate

A minute amount of influenza virus was added to the cell lysate, an enzyme-linked immunosorbent assay was performed, and H1N1 virus was detected using B2 Fab antibody. Culture and Collection of 5X10⁶MDCK cells were suspended in 2mL of a cell lysate (50mM Tris-HCl, 1% Triton X-100,150mM NaCl,10mM MgCl2, 0.1% SDS), incubated at 37 ℃ for 30 minutes, centrifuged at 10000g for 10 minutes, and the supernatant was collected. 500 μ L of cells were lysedTo this solution, H1N1A/Beijing/262/95 virus was added to a final concentration of 100 ng/mL. Respectively adding 100 mu L of cell lysate and lysate containing virus into a 96-hole enzyme label plate, staying overnight at 4 ℃, discarding the protein solution the next time, adding 200 mu L of 2% skimmed milk powder solution, incubating for 2 hours at 25 ℃, and sealing the enzyme label plate; washing the ELISA plate 3 times with PBS containing 0.1% Tween 20, adding 100 μ L diluted Fab antibody fragment solution (2 μ g/mL) into each well, incubating at 25 deg.C for 1 hr, washing the ELISA plate with PBST solution, adding HRP-labeled mouse anti-histidine tag antibody, incubating for 1 hr, washing the plate with PBST, adding HRP substrate TMBZ, developing, measuring absorbance at 450nm with ELISA reader, drawing column diagram, and comparing the absorbance of the coated cell lysate and the micropores containing influenza virus cell lysate.

As shown in fig. 7, the absorbance of the purified B2 Fab fragment bound to H1N1 virus in cell lysate is 0.14, while the absorbance of the lysate without virus is 0.02, which is significantly different from that of the lysate without virus, in which p is less than 0.01. Therefore, the method can be used for detecting the H1N1 influenza virus in the cells.

Sequence listing

<110> Weifang medical college

Shandong-Guanghe-Biomedicine Co Ltd

<120> H1N1 influenza virus antibody and preparation method and application thereof

<141> 2019-11-14

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 363

<212> DNA

<213> mice (Mus musculus)

<400> 1

cagatccagt tggtgcagtc tggggctgag ctggtgaggc ctggggcttc ggtgaggctg 60

tcctgcaagg cttctggcta caccttcacc gactactgga taaactgggt gaagcagagg 120

cctggacaag gccttgagtg gatcggaaat atttttcctt ctgatggtta tactaactac 180

aatcaaaagt tcaaggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca gcagcccgac atctgaggac tctgcggtct tttactgtac aagatctact 300

acggtagtag ctgaggactg gtacttcgat gtctggggcg caggaacctc agtcaccgtc 360

tcg 363

<210> 2

<211> 121

<212> PRT

<213> mice (Mus musculus)

<400> 2

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

1 5 10 15

Ser Val Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Phe Pro Ser Asp Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys

85 90 95

Thr Arg Ser Thr Thr Val Val Ala Glu Asp Trp Tyr Phe Asp Val Trp

100 105 110

Gly Ala Gly Thr Ser Val Thr Val Ser

115 120

<210> 3

<211> 8

<212> PRT

<213> mice (Mus musculus)

<400> 3

Gly Tyr Thr Phe Thr Asp Tyr Trp

1 5

<210> 4

<211> 8

<212> PRT

<213> mice (Mus musculus)

<400> 4

Ile Phe Pro Ser Asp Gly Tyr Thr

1 5

<210> 5

<211> 15

<212> PRT

<213> mice (Mus musculus)

<400> 5

Thr Arg Ser Thr Thr Val Val Ala Glu Asp Trp Tyr Phe Asp Val

1 5 10 15

<210> 6

<211> 330

<212> DNA

<213> mice (Mus musculus)

<400> 6

gaaaatgtgc tcacccagtc tccaaccacc atggctgcat ctcccgggga gaagatcact 60

atcacctgca gtgccagctc aagtataagt tccaattact tgcattggta tcagcagaag 120

ccaggattct cccctaaact cttgatttat aggacatcca atctggcttc tggagtccca 180

gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaattgg caccatggag 240

gctgaagatg ttgccactta ctactgccag cagggtagta gtataccacg gacgttcggt 300

ggaggcacca agctggaaat caaacgtgcg 330

<210> 7

<211> 110

<212> PRT

<213> mice (Mus musculus)

<400> 7

Glu Asn Val Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly

1 5 10 15

Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Phe Ser Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu

65 70 75 80

Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Ser Ile Pro

85 90 95

Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala

100 105 110

<210> 8

<211> 7

<212> PRT

<213> mice (Mus musculus)

<400> 8

Ser Ser Ile Ser Ser Asn Tyr

1 5

<210> 9

<211> 3

<212> PRT

<213> mice (Mus musculus)

<400> 9

Arg Thr Ser

1

<210> 10

<211> 9

<212> PRT

<213> mice (Mus musculus)

<400> 10

Gln Gln Gly Ser Ser Ile Pro Arg Thr

1 5

Claims

1. An influenza H1N1 virus antibody characterized by: comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region further comprises a first heavy chain hypervariable region, a second heavy chain hypervariable region and a third heavy chain hypervariable region, and the light chain variable region further comprises a first light chain hypervariable region, a second light chain hypervariable region and a third light chain hypervariable region;

wherein the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 2; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 7;

the amino acid sequence of the first heavy chain hypervariable region is shown as SEQ ID NO.3, the amino acid sequence of the second heavy chain hypervariable region is shown as SEQ ID NO.4, and the amino acid sequence of the third heavy chain hypervariable region is shown as SEQ ID NO. 5;

the amino acid sequence of the first light chain hypervariable region is shown in SEQ ID NO.8, the amino acid sequence of the second light chain hypervariable region is shown in SEQ ID NO.9, and the amino acid sequence of the third light chain hypervariable region is shown in SEQ ID NO. 10.

2. A DNA fragment encoding the H1N1 influenza virus antibody of claim 1, wherein: the heavy chain variable region has a gene sequence shown in SEQ ID NO.1, and the light chain variable region has a gene sequence shown in SEQ ID NO. 6.

3. An expression vector, characterized in that: comprising at least one copy of the DNA fragment of claim 2.

4. A host cell, characterized in that: comprising the expression vector of claim 3.

5. The use of the H1N1 influenza virus antibody of claim 1, wherein: the application in preparing the medicines for detecting the H1N1 virus.

6. The use of the H1N1 influenza virus antibody of claim 1, wherein: the application in preparing the medicine for preventing and treating the H1N1 viral influenza.