CN110845607A

CN110845607A - H1N1 influenza virus antibody and application thereof in H1N1 virus ultramicro-detection

Info

Publication number: CN110845607A
Application number: CN201911113018.1A
Authority: CN
Inventors: 董金华; 董航; 单喜军; 李海梅; 陈丽梅
Original assignee: Shandong Kuanhe Zheng Biomedical Co Ltd; Weifang Medical University
Current assignee: Shandong Kuanhe Zheng Biomedical Co Ltd; Weifang Medical University
Priority date: 2019-11-14
Filing date: 2019-11-14
Publication date: 2020-02-28
Anticipated expiration: 2039-11-14
Also published as: CN110845607B

Abstract

The invention discloses an H1N1 influenza virus antibody and application thereof in H1N1 virus ultramicro detection, wherein the H1N1 influenza virus antibody 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 ultramicro detection of H1N1 influenza, realizes early prevention and treatment of H1N1 influenza, and has very important economic and social meanings.

Description

H1N1 influenza virus antibody and application thereof in H1N1 virus ultramicro-detection

Technical Field

The invention relates to the technical field of influenza virus antibodies in biotechnology, in particular to an H1N1 influenza virus antibody and application thereof in ultra-micro detection of H1N1 viruses.

Background

Influenza virus (inflenzavirus) is an infectious disease pathogen that causes influenza and highly pathogenic influenza viruses can cause death of 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 are provided with two important proteins of Hemagglutinin (HA) and Neuraminidase (NA), influenza viruses can be divided into a plurality of subtypes according to the difference of the two proteins, wherein the type A protein is taken as an example, currently found HA HAs 18 subtypes (H1-H18), and NA HAs 11 subtypes (NA 1-11). 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 two types of ion channel inhibitors and neuraminidase inhibitors. Ion channel inhibitors including amantadine and rimantadine and derivatives thereof, which produce gastrointestinal and central nervous system side effects over prolonged periods of time and result in the production of resistant strains with undiminished virulence and transmission performance; the neuraminidase inhibitor mainly comprises zanamivir and oseltamivir, the medicine has good treatment effect on early-stage patients with H1N1 influenza, but the treated patients are easy to generate drug resistance to the neuraminidase inhibitor. In contrast, antibody therapy of H1N1 influenza has great advantages, because the active antibody can block the combination of virus and target cells, induce immune cells to kill virus-infected cells, and thus, the aim of rapidly treating H1N1 influenza is achieved.

Therefore, the development of antibodies against the H1N1 influenza virus is urgent to solve the problem that people can detect the H1N1 virus in an ultramicro manner and realize early prevention and treatment of the H1N1 viral influenza.

Disclosure of Invention

In order to solve the problems, the invention provides an H1N1 influenza virus antibody and application thereof in ultra-micro detection of H1N1 virus.

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

an influenza H1N1 virus antibody comprising a unique and novel sequence of 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 includes a DNA fragment encoding 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 includes an expression vector comprising at least one copy of a DNA segment as described above.

The invention also includes a host cell comprising an expression vector as described above.

The invention also comprises the application of the H1N1 influenza virus antibody in the ultra-micro detection of the H1N1 virus.

Compared with the prior art, the invention has the following advantages:

the H1N1 influenza virus antibody can be specifically combined with H1N1 influenza virus, is used for ultramicro detection of H1N1 influenza virus, provides a new method for early prevention and treatment of H1N1 influenza, and has very important economic and social meanings.

Drawings

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

FIG. 2 is an agarose electrophoresis of PCR products of the amplified mouse antibody variable region gene;

FIG. 3 is a graph showing the binding performance of the antibodies in the antibody library with H1N1 virus and streptavidin;

FIG. 4 is a diagram showing the results of screening for monoclonal antibodies against H1N1 virus;

FIG. 5 is a diagram showing the result of polyacrylamide gel electrophoresis performed on the purified antibody fragment;

FIG. 6 is a schematic diagram showing the results of testing the binding properties of the H1N1 virus-resistant monoclonal antibody F1 against H1N 1;

FIG. 7 is a schematic diagram of the principle of detecting H1N1 influenza virus based on enzyme-linked immunosorbent assay of F1 antibody;

FIG. 8 is a standard curve for detecting H1N1 influenza virus in an enzyme-linked immunosorbent assay based on F1 antibody;

reference numerals

M：DNA marker；V_L: an antibody light chain variable region gene; v_H: antibody heavy chain variable region gene.

Detailed Description

The invention aims to provide an H1N1 influenza virus antibody and application thereof in H1N1 virus ultramicro detection, which are realized by the following technical scheme:

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;

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

The invention is further described with reference to specific examples.

Examples

The inactivated H1N1 virus particles are used for immunizing mice, after influenza virus antibodies appear in the blood of the mice, the spleen of the mice is picked, total ribonucleic acid (RNA) is extracted, DNA (cDNA) with a complementary base sequence with an RNA chain is synthesized, and Polymerase Chain Reaction (PCR) is carried out by using antibody specific primers to amplify antibody genes. After the antibody gene was treated, it was cloned into a phage display vector pDONG1, and Escherichia coli TG-1 was further transformed with the constructed vector to prepare a phage display antibody library. And (3) panning against the H1N1 virus to obtain an antibody F1, wherein the antibody can be combined with the H1N1 virus but not with bovine serum albumin, and has the specificity of the H1N1 virus. 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 3 weeks of age, and the immunization adopts a subcutaneous injection method, the dosage is 10 mu g of virus each time, and the immunization is carried out 3 times every other week. After one week of the last immunization, the mice are cut off the tail and blood is taken, and enzyme-linked immunosorbent assay is carried out to confirm that the blood of the mice contains H1N1 influenza virus antibodies.

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 ℃, abandoning 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 3 times with PBS solution containing 0.1% Tween 20, 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 sodium acetate solution of pH6.0, containing 1/10000 diluted 30% H₂O₂) After the development, the absorbance at 450nm is measured by an enzyme-labeling instrument, a curve is drawn, and the content of the antibody in the serum of the mouse before and after the immunization is compared.

The results of ELISA are shown in FIG. 1, and the absorbance at 450nm of the serum of the mouse diluted 1000 times after immunization is 2.4, the absorbance gradually decreases with the increase of the dilution times, and the absorbance at 64000 times dilution is close to the absorbance generated by the serum before immunization. The serum of the mice before immunization does not change greatly before and after dilution and is always maintained at a lower level. The results of this experiment indicate that antibodies against H1N1 virus are present in the sera of immunized mice.

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 mouse light chain variable region gene was treated with restriction enzyme SalI/NotI and cloned into phage display vector pDONG 1. After successful cloning, the plasmid containing the light chain variable region gene was amplified, and further the antibody heavy chain variable region gene treated with SfiI/XhoI was cloned into pDeng 1 already containing the light chain, and E.coli TG-1 was transformed.

Culturing at 37 deg.C to transform Escherichia coli to OD₆₀₀To 0.5, a helper phage M13 was added, the mixture was infected at 37 ℃ for 1 hour, centrifuged at 3000g for 30 minutes, the supernatant was discarded, the cells were suspended in 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, shaken at 30 ℃ and 250rpm for 16 hours, centrifuged at 5000g and 30 minutes the next day to collect the supernatant, 1/5 volumes of PEG/NaCl solution were added to the supernatant, the mixture was mixed well, and then placed on ice for 30 minutes, centrifuged at 5000g and 30 minutes to discard the supernatant, and the precipitate was dissolved by adding 2mL of sterile PBS solution to prepare a phage display antibody library solution.

The agarose electrophoresis pattern of the PCR product of the amplified mouse antibody variable region gene is shown in FIG. 2, the mouse antibody variable region gene is successfully amplified, wherein the length of the heavy chain variable region gene is about 400bp, the length of the light chain variable region gene is about 370bp, the gene fragments are cloned to a phage display vector pDeng 1, a phage is prepared by using Escherichia coli TG-1, and finally, a PCR product containing about 10 genes is obtained⁷Phage display antibody libraries of seed antibodies.

Screening of antibody library

Add 100. mu.L of PBS containing 1. mu.g/ml H1N1 virus to 10 wells of a 96-well microplate, incubate overnight at 4 ℃ and discard the antigen solution the next time, add each wellAdding 200 μ L PBS containing 2% skimmed milk powder, incubating at 25 deg.C for 2 hr for blocking, washing with PBST for 3 times, adding 200 μ L phage solution (R0; each well contains 10)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 of trypsin per well;

culturing TG-1 E.coli to OD₆₀₀To 0.4, 4mL of bacterial solution was taken, 500. mu.L of the dissolved phage solution was added to the bacterial solution, infected at 37 ℃ for 30 minutes, centrifuged at 5000g for 20 minutes, the supernatant was discarded, the cells were suspended in 2YT medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1% glucose, and shaken at 30 ℃ and 250rpm for 16 hours; centrifuging the culture solution for 30 minutes at 5000g the next day, separating and recovering the supernatant, adding 1/5 volumes of PEG/NaCl solution into the supernatant solution, uniformly mixing, placing on ice for 30 minutes, centrifuging for 30 minutes at 5000g, discarding the supernatant, and adding 200 mu L of sterilized PBS solution to serve as phage solution (R1) after the first enrichment;

repeating the steps 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 solution containing H1N1A/Beijing/262/95 virus (0.5. mu.g/mL) or Streptavidin (Streptavidin) is added to a 96-well plate, the solution is overnight at 4 ℃, the antigen solution is discarded the next day, 200. mu.L of solution containing 2% skimmed milk powder is added, the plate is incubated at 25 ℃ for 2 hours, and the plate is blocked. 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) After color development, absorbance at 450nm is measured by a microplate reader, a histogram is drawn, and the binding performance of the phage antibody, the H1N1 virus and streptavidin is obtained by comparing each step.

The results of the enzyme-linked immunosorbent assay are shown in fig. 3, when the binding capacities of the phage libraries R0, R1, R2 and R3 obtained in the phage panning process and the H1N1 virus are compared, it is found that the binding capacity of the phage solution obtained in the second panning process and the H1N1 virus begins to increase, the binding capacity of the phage solution R3 obtained in the third panning process and the H1N1 virus is significantly increased compared with that of R0, R1 and R2, and the binding performance to streptavidin is very weak and unchanged, which indicates that the constructed phage display antibody library against the H1N1 virus is enriched.

Fourth, monoclonal antibody screening

Culturing TG-1 E.coli to OD₆₀₀Taking 100 mu L of elutriation sieve R2 phage antibody library dissolved out phage solution for infecting 200 mu L of escherichia coli bacterial solution, incubating for 30 minutes at 37 ℃, coating the bacterial solution on a 2YT culture medium plate containing 100 mu g/mL ampicillin, 50 mu g/mL kanamycin and 1% glucose, and culturing overnight at 37 ℃; the next day, 48 colonies were picked and inoculated onto 96-well plates, and cultured at 37 ℃ to OD₆₀₀To 0.4, M13 phage was added to each well, centrifuged at 5000g for 20 minutes after infection, the supernatant was removed, 200. mu.L of 2YT medium containing 100. mu.g/mL ampicillin, 50. mu.g/mL kanamycin and 0.1% glucose was added to each well, the cells were suspended, and cultured at 30 ℃ and 250rpm for 16 hours; the culture solution is centrifuged for 30 minutes at 5000g the next day, the supernatant is separated and recovered, enzyme-linked immunosorbent assay is carried out, and the binding specificity and the binding performance of each monoclonal antibody and H1N1 virus are verified.

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 a solution containing 2% skim milk powder was added, and the plate was incubated at 25 ℃ for 2 hours and then blocked. Washing the ELISA plate with PBS solution containing 0.1% Tween 20 for 3 times, adding phage solution, incubating at 25 deg.C for 1 hr, washing the ELISA plate with PBST solution, adding HRP-labeled mouse anti-M13 antibody, incubating for 1 hr, washing the plate with PBST, adding HRP substrate TMBZ (prepared with sodium acetate solution of pH6.0, containing 1/10000 diluted 30% H)₂O₂) After developing color, using enzyme-linked immunosorbent assay to measureAbsorbance at 450nm was plotted in a bar graph to compare the binding performance of phage antibodies prepared from each clone to H1N1 virus and bovine serum albumin.

As a result, as shown in FIG. 4, the phage antibody prepared from 30 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 F1 with strong virus binding ability, culturing, extracting plasmid, analyzing the clone sequence, wherein the heavy chain variable region gene and amino acid sequence are shown as SEQ ID NO.1 and SEQ ID NO.2, and the antibody light chain variable region gene and amino acid sequence are shown as SEQ ID NO.6 and SEQ ID NO. 7.

The amino acid sequence of the super-variable region in the heavy chain variable region is shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the amino acid sequence of the super-variable region in the light chain variable region is 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: GYTFTNYW;

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

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

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

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

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

The F1 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 F1

Culturing the F1 Escherichia coli colony obtained in the fourth step, extracting pDONG1 vector, transforming non-inhibitory Escherichia coli HB2151, selecting single colony, inoculating into 4mL LB liquid culture medium, culturing overnight at 37 deg.C, inoculating overnight culture solution into culture medium containing 100 μ g/mLAmpicillin was cultured in 500mL of LB liquid medium at 37 ℃ and 200rpm to OD₆₀₀At 0.4, IPTG was added to a final concentration of 1mM, the temperature was adjusted to 30 ℃ and the culture was continued for 16 hours; the culture supernatant was collected by centrifugation, and ammonium sulfate was added to adjust the concentration of the solution to 75%, and after standing at 4 ℃ for 2 hours, the solution was centrifuged at 6000rpm for 20 minutes, the supernatant was discarded, and the precipitate was dissolved in 10mL of TALON buffer solution, and the Fab fragment of F1 antibody was purified by TALON resin. Polyacrylamide gel electrophoresis was performed, and the purified antibody fragment was confirmed by staining with coomassie brilliant blue.

After destaining the stained 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 F1Fab fragment was expressed in e.coli and purified using talen resin.

Sixthly, H1N1 virus binding of monoclonal antibody F1

Enzyme-linked immunosorbent assay was performed to confirm that the purified Fab antibody fragment has H1N1 virus binding activity. Adding 100 mu L of 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, 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 blocking the enzyme label plate; after washing the microplate 3 times with PBS containing 0.1% Tween 20, 100. mu.L of diluted Fab antibody fragment solution was added to each well, incubated at 25 ℃ for 1 hour, the microplate was washed with PBST solution, HRP-labeled mouse anti-histidine tag antibody was added, and after incubation for 1 hour, the microplate was washed with PBST, and HRP substrate TMBZ (prepared with sodium acetate solution pH6.0, containing 1/10000 diluted 30% H) was added₂O₂) After color development, absorbance at 450nm is measured by a microplate reader, a bar graph is drawn, and the binding performance of the purified Fab fragment with H1N1 virus and bovine serum albumin is compared.

As shown in fig. 6, the purified F1Fab fragment binds to H1N1 virus with an absorbance of 0.25, while the BSA-coated wells have an absorbance of 0.01, thus concluding that the antibody F1Fab can specifically bind to H1N1 virus.

Seventh, H1N1 influenza virus detection based on F1 antibody

H1N1 influenza virus was detected based on phage display F1 antibody using the method shown in figure 7. The method comprises the steps of coating an anti-H1N 1 antibody in the pores of a 96-pore microplate, sealing the microplate, adding an inactivated H1N1 virus, adding a phage display F1 antibody after washing the plate, adding an anti-phage antibody labeled with horseradish peroxidase, and adding a substrate for color development after washing the plate. When no virus exists in the sample, the reaction system does not develop color; the system developed in the presence of virus and the more H1N1 virus in the sample, the more F1 phage was captured by the virus to the microplate, the more anti-phage antibody was captured accordingly, and the darker the color developed with the enzyme added substrate. The virus content of the solution can be determined by plotting a standard curve of virus concentration versus absorbance.

The specific operation is as follows. Adding an anti-H1N 1 antibody (M149, Baori Biotechnology Co., Ltd.) into the wells of a 96-well ELISA plate, standing overnight at 4 ℃, discarding the liquid in the wells the next time, adding 200. mu.L of 2% skimmed milk powder solution, standing at room temperature for two hours, and sealing the ELISA plate. After washing the plate, adding 100 mu L of inactivated avian influenza H1N1 virus solution or Bovine Serum Albumin (BSA) solution containing different concentrations (0, 0.1, 1, 10, 100, 1000, 10000ng/mL) into the micropore, incubating at 25 ℃ for 1 hour, removing the solution in the pore, washing the plate with PBS solution (PBST solution) containing 0.1% Tween, adding F1 antibody solution for phage display, incubating at 25 ℃ for 1 hour, washing the plate, adding anti-phage antibody solution (1 mu g/mL) labeled with Horse Radish Peroxidase (HRP), incubating at 25 ℃ for 1 hour, removing the solution in the pore, washing the plate with PBST for 3 times, finally adding HRP substrate 3,3,5, 5-tetramethylbenzidine hydrochloride (TMBZ) solution for color development, measuring the absorbance of the solution in the pore at 450nm, and preparing a standard curve.

The results are shown in FIG. 8. The horizontal axis represents the concentration of H1N1 virus or BSA solution, the concentration of the virus solution is from 0 to 10000ng/mL, and the vertical axis represents the absorbance of the solution in each concentration corresponding to the well. The absorbance of the solution is gradually increased along with the gradual increase of the virus concentration in the solution, and the absorbance of the solution is in a linear corresponding relation, while the absorbance of the wells added with BSA as a negative control is not changed due to the increase of the BSA concentration, which indicates that the F1 antibody has H1N1 virus specificity, and the content of H1N1 virus in a sample can be determined according to the curve, namely the F1 antibody developed in the research can be used for detecting the content of H1N1 influenza virus in the sample, and can be detected when the H1N1 virus is ultramicro-dose, and the minimum detection limit is 1 ng/mL.

Sequence listing

<110> Weifang medical college

Shandong-Guanghe-Biomedicine Co Ltd

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Claims

1. An influenza H1N1 virus antibody characterized by: 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;

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: the expression vector comprising at least one copy of the DNA segment of claim 2.

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

5. The use of the H1N1 influenza virus antibody of claim 1, wherein: the application in the ultramicro detection of the H1N1 virus.