CN110627901B

CN110627901B - Monoclonal antibody of influenza virus matrix protein M1 and application thereof

Info

Publication number: CN110627901B
Application number: CN201910912806.0A
Authority: CN
Inventors: 蒲娟; 佟琪; 宗亚楠; 王明暘; 曲润康; 刘金华
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-09-25
Filing date: 2019-09-25
Publication date: 2021-02-23
Anticipated expiration: 2039-09-25
Also published as: CN110627901A

Abstract

The invention relates to the technical field of biology, in particular to a monoclonal antibody of influenza virus matrix protein M1 and application thereof. The invention provides a monoclonal antibody of influenza virus matrix protein M1, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown in SEQ ID NO. 1-3; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NO. 4-6. The monoclonal antibody can specifically recognize and combine influenza virus matrix protein M1 of various subtypes, has broad-spectrum applicability among influenza virus subtypes, has high specificity and sensitivity, can be well applied to technologies such as immunoblotting experiments, indirect immunofluorescence experiments, immunoprecipitation and the like, and has high application value.

Description

Monoclonal antibody of influenza virus matrix protein M1 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a monoclonal antibody of an influenza virus matrix protein M1, a hybridoma cell secreting the monoclonal antibody of the influenza virus matrix protein M1 and application thereof.

Background

Antibody technology is largely divided into monoclonal antibody technology and polyclonal antibody technology. An antigen is determined by a plurality of antigenic determinants, one antigenic determinant stimulates the body, and a B lymphocyte receives an antibody produced by the antigen, which is called a monoclonal antibody. Stimulation of the body by multiple epitopes correspondingly produces a wide variety of monoclonal antibodies, which when mixed together are polyclonal. Polyclonal antibodies may exist in multiple subtypes, resulting in poor specificity compared to monoclonal antibodies. When the polyclonal antibody is used for an immunity experiment, a background value is generated, and the experiment result is influenced to different degrees. The characteristics of high specificity and sensitivity of the monoclonal antibody enable the monoclonal antibody to be widely applied to molecular experiments and clinical diagnosis.

The M1 protein of the influenza virus is the most abundant protein in the virus particles, and the M1 protein can maintain the integrity of the morphological structure of the influenza virus particles and plays an important role in replication and transcription of virus genomes and assembly and budding of viruses. Furthermore, the M1 protein is highly conserved between influenza virus subtypes. Therefore, the development of a broad-spectrum monoclonal antibody capable of specifically recognizing M1 protein of various subtypes of influenza viruses is beneficial to the diagnosis of influenza and the deep research of molecular mechanisms, and has important significance.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide a broad-spectrum monoclonal antibody which has higher sensitivity and specificity and can specifically bind to the matrix protein M1 of a plurality of subtype influenza viruses and a hybridoma cell for producing the monoclonal antibody.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides a monoclonal antibody against influenza virus matrix protein M1, said monoclonal antibody comprising a heavy chain variable region and a light chain variable region, capable of specifically binding to influenza virus matrix protein M1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID nos. 1-3, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NO. 4-6.

Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is any one of the following:

(1) as shown in SEQ ID NO. 7;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 7;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.7 and has the same function; the homology is preferably at least 85%; more preferably at least 95%.

The amino acid sequence of the variable region of the light chain of the monoclonal antibody is any one of the following:

(1) as shown in SEQ ID NO. 8;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 8;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.8 and has the same function. The homology is preferably at least 85%; more preferably at least 95%.

The monoclonal antibody of the invention can specifically bind to a matrix protein M1 of a plurality of subtype influenza viruses, and the antigen recognition region of the monoclonal antibody is positioned in the 169-252 amino acid region of the M1 protein.

More preferably, the subtype of the monoclonal antibody is IgG1 with kappa chain light chain.

Specifically, the amino acid sequence of the heavy chain of the monoclonal antibody is any one of the following:

(1) as shown in SEQ ID NO. 9;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 9;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.9 and has the same function. The homology is preferably at least 85%; more preferably at least 95%.

The amino acid sequence of the light chain of the monoclonal antibody is any one of the following:

(1) as shown in SEQ ID NO. 10;

(2) the amino acid sequence of the protein with the same function is obtained by deletion, substitution or insertion of one or more amino acids of the sequence shown as SEQ ID NO. 10;

(3) an amino acid sequence of a protein which has at least 70 percent of homology with the sequence shown as SEQ ID NO.10 and has the same function. The homology is preferably at least 85%; more preferably at least 95%.

In a second aspect, the present invention provides a hybridoma cell producing the monoclonal antibody to influenza virus matrix protein M1.

In a third aspect, the present invention provides a nucleic acid encoding the monoclonal antibody to influenza virus matrix protein M1.

Preferably, the nucleic acid sequence encoding the heavy chain of the monoclonal antibody is as shown in SEQ ID NO.11 or is the complementary sequence thereof; the nucleic acid sequence of the light chain of the encoding monoclonal antibody is shown as SEQ ID NO.12 or is a complementary sequence thereof.

In a fourth aspect, the invention provides a labeling complex obtained by biochemical labeling of the monoclonal antibody of influenza virus matrix protein M1.

Preferably, the biochemical label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a radioactive label.

In a fifth aspect, the invention provides an application of the monoclonal antibody of the influenza virus matrix protein M1 or the hybridoma cell or the labeled complex of the monoclonal antibody of the influenza virus matrix protein M1 in preparation of a kit for detecting influenza virus or influenza virus matrix protein M1.

In a sixth aspect, the invention provides an application of the monoclonal antibody of the influenza virus matrix protein M1 or the labeled complex of the hybridoma cell or the monoclonal antibody of the influenza virus matrix protein M1 in preparing a kit for detecting influenza virus antibodies.

In a seventh aspect, the present invention provides a use of the monoclonal antibody against influenza virus matrix protein M1 or the hybridoma cell or the labeled complex against the monoclonal antibody against influenza virus matrix protein M1 for the preparation of a medicament for the prevention or treatment of influenza virus.

In an eighth aspect, the invention provides an application of the monoclonal antibody of the influenza virus matrix protein M1 or the hybridoma cell or the labeled complex of the monoclonal antibody of the influenza virus matrix protein M1 in quality control of influenza virus vaccines.

In a ninth aspect, the present invention provides a medicament comprising the monoclonal antibody to influenza virus matrix protein M1.

The medicine can also comprise other active ingredients or auxiliary materials allowed in the pharmaceutical field besides the monoclonal antibody of the influenza virus matrix protein M1.

In a tenth aspect, the present invention provides an influenza virus detection kit comprising the monoclonal antibody to the influenza matrix protein M1 or a labeled complex of the monoclonal antibody to the influenza matrix protein M1.

The detection kit for the influenza virus can also comprise other reagents or materials required for detection, including but not limited to buffer solution, secondary antibody and the like, besides the labeled complex of the monoclonal antibody of the influenza virus matrix protein M1 or the monoclonal antibody of the influenza virus matrix protein M1.

The invention has the beneficial effects that: the monoclonal antibody of the influenza virus matrix protein M1 provided by the invention can specifically recognize and combine influenza virus M1 proteins of various subtypes, has broad spectrum among influenza virus subtypes, has high specificity among influenza viruses and other viruses, and has higher sensitivity, so that the monoclonal antibody can be well applied to immunoblotting experiments (Western Blot, the maximum dilution multiple can reach 8 multiplied by 10⁴Multiple), indirect immunofluorescence experiment (IFA, maximum dilution factor can reach 5X 10⁵Multiple) and immunoprecipitation (IP, the minimum usage amount is only 1 mu g), and the like, and has higher application value; meanwhile, the method lays a technical foundation for the deep research of the pathogenic mechanism of the influenza virus.

Drawings

FIG. 1 shows the result of the specificity analysis of the monoclonal antibody 2C5 for Western blot detection in example 2 of the present invention, wherein the ratio of the sequences in lane 1: a sample of cell lysate not infected with virus; lanes 2-5: cell lysate samples infected with H9N2, H7N9, H5N1, H1N1 subtype influenza virus were in order.

FIG. 2 shows the result of the sensitivity analysis of the monoclonal antibody 2C5 for Western blot detection in example 2 of the present invention, wherein lane 1 is the lysate of infected virus cells and lane 2 is the lysate of uninfected virus cells.

FIG. 3 shows the result of analysis of the specificity of monoclonal antibody 2C5 for IFA detection in example 3 of the present invention, wherein A, B, C, D represents the detection results of infection with influenza viruses of H9N2, H7N9, H5N1 and H1N1 subtypes, monoclonal antibody 2C5 as a primary antibody, and E represents a negative control well of uninfected virus.

FIG. 4 is a graph showing the results of analysis of the sensitivity of the monoclonal antibody 2C5 for IFA detection in example 3 of the present invention, in which A, B, C, D, E, F indicates the dilution levels of the monoclonal antibody 2C5 in the order of 1: 1X 10³、1:5×10³、1:1×10⁴、1:5×10⁴、1:1×10⁵、1:5×10⁵The detection result of (1).

FIG. 5 shows the analysis results of the specificity of monoclonal antibody 2C5 for IP assay detection in example 4 of the present invention; wherein, H1, H5, H7 and H9 respectively represent M1 protein enriched from different virus infected samples by the monoclonal antibody 2C5, Input is a cell lysis sample infected with virus, M1 is influenza virus M1 protein, and Actin is internal reference protein Actin.

FIG. 6 is the result of analysis of the sensitivity of monoclonal antibody 2C5 for IP experimental detection in example 4 of the present invention; wherein, IP is M1 protein enriched from different virus infected samples by monoclonal antibody 2C5, Input is cell lysis sample infected with virus, M1 is M1 protein of influenza virus, and Actin is reference protein Actin.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 hybridoma cell line 2C5 and acquisition of influenza M1 protein monoclonal antibody produced therefrom

1. Preparation of immunogens

The immunogen for immunizing mice is recombinant influenza virus M1 protein which is expressed by adopting a prokaryotic expression system and carries a GST tag, and the specific preparation method is as follows:

(1) construction of M1 protein prokaryotic expression vector

A M1 gene sequence of an H9 subtype influenza virus is used as a template, and a M1 protein coding gene is amplified by adopting the following primers through PCR and is connected to a pGEX-1ZT vector.

An upstream primer: 5'-CTGGTTCCGCGTGGATCCGGATCCATGAGTCTTCTAACCGAGGT-3' (containing BamH I cleavage site)

A downstream primer: 5'-GGCCGCGATATCAAGCTTAAGCTTTCACTTGAACCGCTGCAGTT-3' (containing HindIII cleavage sites)

(2) Prokaryotic expression of M1 protein

And (3) transforming the constructed prokaryotic expression vector of the M1 protein into a Trans T1 competent cell, screening to obtain a positive strain, sequencing, and extracting a plasmid after the sequencing is correct.

(3) Preparation of M1 protein immunogen

The positive plasmid successfully constructed is transferred into competent cells of escherichia coli BL21, expression protein of the positive plasmid is induced, and soluble protein expressed in the supernatant is purified to immunize a mouse.

2. Animal immunization

The immunogen prepared above is used for immunizing BALB/c mice of 6 weeks old, and the specific immunization method is as follows: the first immunization, mixing the antigen and the water-soluble adjuvant according to the volume ratio of 9:1, and performing subcutaneous multi-point injection on the neck and the back of a mouse at 100 mu g/mouse after uniform mixing; three weeks after the first immunization, the mice are immunized twice, and the method is the same as the first immunization; two weeks after the second immunization, the mice are immunized for the third time by the same method; one week after the three-immunization, the mice are subjected to blood sampling of the eye veins, serum is separated, and the serum titer of the mice is detected by an indirect ELISA method by coating an ELISA plate with HIS label M1 protein. After the titer reached the fusion requirement, the mice were boosted three days before fusion (50. mu.g/mouse were intraperitoneally injected directly with the M1 protein immunogen prepared above). After three times of immunization, mice with serum antibody titer reaching the standard are selected for boosting immunization.

3. Obtaining hybridoma cells

And taking the splenocytes of the mice after the boosting immunization and fusing the splenocytes with sp2/0 cells. An indirect ELISA method for screening hybridoma cells was established using HIS-tag-carrying recombinant M1 protein expressed in a prokaryotic system as an antigen, and the screening was performed 10-14 days after cell fusion. After 3 times of screening and subcloning of the positive hybridoma cells, a hybridoma cell strain capable of stably secreting M1 protein monoclonal antibody is obtained and named as 2C 5.

4. Potency detection of hybridoma cell 2C5 secretion monoclonal antibody

In order to determine the titer of the monoclonal antibody secreted by the hybridoma cell 2C5, the detection of the antibody titer was performed using the indirect ELISA method established in step 3 above; ascites from immunized mice were also prepared as controls. Diluting ascites from 1:400 to 1:3.277 × 10 at 4-fold ratio⁷Doubling, the hybridoma cell supernatant from 1:100 to a doubling ratio of 1: 5.12X 10⁴In addition, the antibody titer was determined by performing an indirect ELISA assay using a monoclonal antibody secreted from hybridoma cell 2C5 as a primary antibody. The result shows that the antibody titer of the hybridoma cell supernatant can reach 1: 5.12X 10⁴(ii) a The antibody titer of the ascites of the mouse can reach 1:3.277 multiplied by 10⁷。

5. Specific detection of hybridoma cell 2C5 secretion monoclonal antibody

In order to verify whether the hybridoma cell 2C5 prepared in the step 4 secretes the monoclonal antibody and the mouse ascites monoclonal antibody is a specific antibody against the M1 protein of influenza virus, H9N2, H7N9, H5N1, H1N1 subtype influenza virus, newcastle disease virus (LaSota), adenovirus and normal chick embryo allantoic fluid are added to the loading buffer solution to boil the sample, the M1 protein monoclonal antibody 2C5 is diluted 2000-fold with a primary antibody diluent to be used as a primary antibody for incubation, and the Western blot method is used to detect the specificity of the monoclonal antibody against the M1 protein of influenza virus. The result shows that the monoclonal antibody 2C5 can specifically recognize four subtypes of influenza viruses, namely H9N2, H7N9, H5N1 and H1N1, and has better specificity.

6. Identification of the antigen recognition region of monoclonal antibody 2C5

In order to identify whether the monoclonal antibody secreted by hybridoma cell 2C5 and the antigen recognition region of mouse ascites monoclonal antibody are in the same position, the 85 th and 168 th amino acids of matrix protein M1 are used as truncation points, M1 gene is divided into two sections, truncated expression vectors pGEX-1ZT-M1- > 1(1-168aa) and pGEX-1ZT-M1- > 2(85-252aa) are respectively constructed, the two truncated expression vectors are induced to express truncated M1 protein, the truncated M1 protein is used as antigen, and a murine monoclonal antibody (commercialized) aiming at GST tag and a monoclonal antibody 2C5 are respectively used as primary antibodies to carry out Western blot detection. The results show that the antigen recognition regions of the murine GST monoclonal antibody and the 2C5 monoclonal antibody are located in the same region, and are located in the amino acid region 169-252 of the M1 protein.

7. Subtype identification of 2C5 monoclonal antibody

The primary subtype of the antibody was identified using the SBA Clonotyping System-HRP kit, and the subtype of the 2C5 monoclonal antibody was IgG1 with kappa chain as the light chain.

Example 2 application of monoclonal antibody 2C5 in immunoblotting (Western blot)

1. Specificity identification of monoclonal antibody 2C5 for Western blot detection

(1) A549 cell plating: a549 cells are transferred to a 12-hole plate from a large cell bottle about 24 hours before Western blot experiment, the number of the cells needed by the experiment is calculated, and simultaneously, one more hole of cells is paved for counting, and observation is paid attention. When the cells grow to 80% -90% of the cell holes, gently removing the culture solution in the cell holes, gently washing the cells once by using PBS, and discarding the washing solution;

(2) and (3) diluting the virus: taking allantoic fluid of H9N2, H7N9, H5N1 and H1N1 subtype viruses out of a refrigerator at-80 ℃, dissolving at 4 ℃, counting cells by using a cell counter according to a conventional cell counting method, calculating the infection amount of the required virus according to MOI (molar equivalent index) of 0.2, and diluting the virus according to the calculation result;

(3) infection with virus: the diluted virus solution was added to a washed 12-well cell plate, and the plate was left at 37 ℃ with 5% CO₂Culturing in an incubator, and after the virus is adsorbed for 1h, replacing the liquid in the cell hole with a DMEM medium containing 0.2 mug/mL TPCK pancreatin and 1% double-antibody serum-free medium;

(4) collecting a sample: lysing the cell Sample by using a Sample Buffer, performing ultrasonic treatment on the cell Sample by using a cell ultrasonic instrument after instantaneous centrifugation, centrifuging to collect supernatant, and meanwhile, resuspending the precipitate by using PBS;

(5) western blot identification:

preparing glue: the glass plate, the comb, the adhesive tape and the like are cleaned in advance. Preparing 10% separation glue according to a formula, sealing the glue surface with water, pouring off the water and adding 5% concentrated glue after the separation glue is solidified, inserting a proper comb, placing the gel into an electrophoresis tank after the gel is solidified, assembling the gel into the electrophoresis tank, filling newly-configured electrophoresis liquid into an inner-layer electrophoresis tank, and filling the outer-layer electrophoresis tank with the electrophoresis liquid after the comb is pulled out;

boiling samples: respectively taking 20 mu L of the supernatant obtained after the ultrasonic lysis and prepared in the step (4) and PBS precipitation resuspension, adding 4 mu L of 6 multiplied by protein loading buffer solution, and boiling the sample in a metal bath at 100 ℃ for 10 min;

loading: adding 10 mu L of protein sample into the sample hole, and adding 10 mu L of protein Marker on two sides of the sample hole respectively;

(4) electrophoresis: setting the program of the electrophoresis apparatus as constant voltage 65V electrophoresis for 30min, constant voltage 120V electrophoresis for 1h and constant voltage 55V; observing electrophoresis until bromophenol blue runs out of the bottom of the gel, finishing electrophoresis, gently taking out the protein gel, and washing the protein gel with distilled water to remove residual electrophoresis liquid;

(5) transfer printing: soaking filter paper and sponge in the pre-cooled transfer printing liquid in advance, after electrophoresis is finished, carefully unloading a glue block, referring to a Marker on the protein glue, cutting the glue according to the size of target protein, measuring the length and width of the protein glue to cut a PVDF film with proper size, and soaking the PVDF film in anhydrous methanol for activation; sequentially laying sponge, 4 layers of filter paper, albumin glue, a PVDF (polyvinylidene fluoride) membrane, 4 layers of filter paper and sponge in a transfer printing clamp, paying attention to the fact that each layer needs to be completely filled with bubbles by a glass rod, finally placing the transfer printing clamp in a transfer printing groove, pouring sufficient transfer printing liquid, and performing 250mA constant-current transfer printing for 1 hour;

(6) and (3) sealing: and after the transfer printing is finished, taking out the PVDF film, and observing whether a clear Marker strip exists on the PVDF film to judge whether the film transfer is successful. Placing the membrane in 5% skim milk, sealing on a horizontal shaking table at room temperature for 1h or overnight at 4 ℃;

(7) incubation of primary antibody: after the completion of the blocking, the PVDF membrane was washed clean with PBST, and the clean membrane was placed in mouse GST monoclonal antibody diluted with a primary anti-diluent and monoclonal antibody 2C5 prepared in example 1, and incubated overnight on a horizontal shaker at 4 ℃;

(8) and (3) incubation of the secondary antibody: recovering primary antibody after the primary antibody incubation is finished, washing the membrane three times by PBST (Poly Butylene succinate) for 5min each time, then adding 5% skim milk to dilute HRP-labeled goat anti-mouse IgG by 10000 times, and incubating for 1h at room temperature by using a horizontal shaker;

(9) color development: after incubation of the secondary antibody, the membrane was washed three times with PBST, 10min each, and the Tanon 5200 automated chemiluminescence image exposure apparatus was started in advance. After the membrane washing is finished, adding a proper amount of color developing liquid on the PVDF membrane until the surface of the membrane is completely covered, and developing for 2min in a dark place;

(10) exposure: the PVDF membrane was removed from the luminescent solution with tweezers, the membrane was exposed in an exposure apparatus for a period of time depending on the amount of protein expressed, and the exposure image was stored and the results were analyzed.

2. Sensitivity determination of monoclonal antibody 2C5 for Western blot detection

Virus infection and Western blot identification were carried out according to the method described in 1 above, and monoclonal antibody 2C5 was expressed at a ratio of 1: 1X 10³、1:2×10³、1:4×10³、1:8×10³、1:1×10⁴、1:4×10⁴、1:8×10⁴、1:1×10⁵After dilution, as a primary antibodyThe sensitivity of monoclonal antibody 2C5 was tested by incubation.

3. Results of the experiment

The results of the Western blot specificity tests show that the monoclonal antibody 2C5 can be specifically combined with M1 proteins of four subtypes of influenza viruses, namely H9N2, H7N9, H5N1 and H1N1 (shown in figure 1), and does not react with other cell components, and the monoclonal antibody 2C5 can be used for identifying M1 proteins of various subtypes of influenza viruses in a broad spectrum manner in the Western blot tests and has good specificity.

The result of the Western blot sensitivity test shows that when the monoclonal antibody 2C5 is diluted to 1 × 10⁵The monoclonal antibody 2C5 cannot be combined with M1 protein when being multiplied, and the maximum dilution of the monoclonal antibody 2C5 applied to Western blot detection is 1:8 × 10⁴(FIG. 2).

Example 3 use of monoclonal antibody 2C5 in Indirect Immunofluorescence (IFA)

1. Specific identification of monoclonal antibody 2C5 for IFA detection

(1) The four subtypes of influenza viruses, H9N2, H7N9, H5N1 and H1N1, were infected into MDCK cells according to the method for virus infection in example 2;

(2) washing: gently throwing off the culture solution in the holes, washing once by using PBS (phosphate buffer solution), gently moving to avoid cell shedding, and discarding the PBS in the holes;

(3) fixing: adding a proper amount of absolute ethyl alcohol into each hole: acetone ═ 3: 2, fixing the prepared fixing solution at room temperature for 20 min;

(4) washing: spin-drying the stationary liquid in the holes, washing for 3 times by using PBS (phosphate buffer solution), and placing the holes on a shaking table to shake and wash for 3-5min after each washing;

(5) and (3) sealing: adding appropriate amount of 5% skimmed milk to each well, sealing in a constant temperature incubator at 37 deg.C for 1h, washing with PBS for 3 times, and shaking and washing on a shaking table for 3-5 min;

(6) primary antibody incubation: diluting monoclonal antibody 2C5 1000 times with PBS only, adding appropriate amount of diluted primary antibody into each well, and standing overnight at 4 deg.C;

(7) washing: the same step (4);

(8) and (3) secondary antibody incubation: diluting FITC-labeled goat anti-mouse IgG by 400 times with PBS, adding a proper amount of diluted secondary antibody into each hole, keeping out of the sun with tinfoil paper, and incubating for 1h at 37 ℃;

(9) washing: discarding liquid in the holes, washing for 3 times by using PBST, and placing the PBST on a shaking table to shake and wash for 3-5min after each washing;

(10) and (4) observing results: when the cells were observed under a fluorescence microscope, the cells were judged to be positive if they had brighter green fluorescence as compared with the positive control.

2. Determination of IFA sensitivity of monoclonal antibody 2C5

The indirect immunofluorescence assay was performed as described in 1 above, with monoclonal antibody 2C5 at a ratio of 1: 1X 10, respectively³、1:5×10³、1:1×10⁴、1:5×10⁴、1:1×10⁵、1:5×10⁵After dilution, the cells were incubated as primary antibodies, and sensitivity of M1 monoclonal antibody was examined.

3. Results of the experiment

The IFA specificity detection result shows that clear fluorescence can be generated in the cell pores infected with the virus due to the specific binding of the virus M1 protein and the monoclonal antibody 2C5, and obvious fluorescence does not appear in the cells not infected with the virus due to the fact that the monoclonal antibody 2C5 does not react with other cell components (figure 3), so that the monoclonal antibody 2C5 can be applied to an indirect immunofluorescence experiment and has high specificity.

The IFA sensitivity test results show that the quantity of fluorescence generated in the cell hole is gradually reduced and the brightness is gradually reduced along with the increasing dilution of the primary antibody, and when the monoclonal antibody 2C5 is diluted to 1: 5X 10⁵Only a small amount of fluorescence was generated in the cell well, indicating that the maximum dilution of monoclonal antibody 2C5 for indirect immunofluorescence was 1: 5X 10⁵(FIG. 4).

Example 4 use of monoclonal antibody 2C5 in Immunoprecipitation (IP)

1. Specific identification of monoclonal antibody 2C5 for IP detection

(1) Preparation of cell lysate: opening a 4 ℃ centrifuge in advance for precooling, and taking the DTT, the Protease Inhibitor (PIC) and the PMSF out from the temperature of minus 20 ℃ for dissolution. To 1mL of lysine Buffer was added 10. mu.L of DTT, 10. mu.L of PIC, and 10. mu.L of PMSF to obtain a cell lysate.

(2) Preparation of cell lysates: infection with subtype H9N2, H7N9, H5N1, H1N1 virus was performed as described for the virus in example 2; 24h after infection, cell lysates were prepared. The DMEM medium in the cell plate was discarded, after washing once with PBS, the PBS wash was discarded, an appropriate amount of 1 × cell lysate was added to the cell wells, and the cell plates were incubated on ice for 5 minutes. The cells were scraped from the cell wells, the extract was transferred to a 1.5mL sterile centrifuge tube using a pipette gun, the cell lysate was sonicated on ice, after sonication, centrifuged at 14,000 Xg for 10 minutes at 4 ℃ and the centrifuged supernatant was transferred to a new EP tube. The supernatant is a cell lysis sample, and if no experiment is carried out on the same day, the sample can be temporarily stored at-80 ℃.

(4) Incubation of Beads: mu.L of the cell lysate sample prepared in step (3) was taken as input, and the cell lysate sample was boiled and stored at-80 ℃. Approximately 500. mu.L of lysine Buffer was filled in the system, 10. mu.L of Beads and monoclonal antibody 2C5 (murine IgG added to the control) were added to each tube, and the mixture was incubated at 4 ℃ for 3 to 4 hours.

(5) Washing the Beads: opening the 4 ℃ centrifuge in advance; the sample was left to stand on ice for 3min and centrifuged using a 4 ℃ centrifuge at 3000rpm for 3 min. The supernatant was aspirated using a 1mL gun taking care not to aspirate the Beads. Using a previously prepared washing reagent (5. mu.L of DTT added to 1mL lysine Buffer), the sample was inverted 3min, and the washing, standing and centrifugation were repeated 3 times in this order.

(6) After 3min on ice, centrifugation was carried out at 3000rpm for 3min at 4 ℃, the supernatant was carefully aspirated off using a gun, a final concentration of 1 × loading buffer was added to the sample, the sample was denatured by metal bath boiling for 10min, and the protein sample enriched by monoclonal antibody 2C5 and input were removed after flash centrifugation for Western Blot identification.

(7) Western Blot identification: western Blot was performed using the method in example 2.

2. Monoclonal antibody 2C5 for sensitivity determination for IP detection

The immunoprecipitation experiment was carried out as described in 1 above, and 10. mu.g, 5. mu.g, 1. mu.g, and 0.5. mu.g of monoclonal antibody 2C5 were added to four groups of cell samples, H9N2, H7N9, H5N1, and H1N1, respectively, to incubate them to examine the minimum dose of monoclonal antibody 2C5 for IP experiments.

3. Results of the experiment

(1) Specificity: the monoclonal antibody 2C5 can be combined with M1 protein in a cell sample, and M1 protein is detected by means of immunoblotting, which indicates that the monoclonal antibody 2C5 prepared in example 1 can be applied to IP experiment for detecting M1 protein of influenza virus (FIG. 5).

(2) Sensitivity: the enriched M1 protein band could not be detected when the monoclonal antibody 2C5 was added at a dose of 0.5. mu.g, indicating that the minimum dose of monoclonal antibody 2C5 for IP experiments was 1. mu.g (FIG. 6).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Ile Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

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

35 40 45

Gly Met Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Gly Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Glu Tyr Asp Gly Asp Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 8

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Asp Leu Lys

100 105 110

<210> 9

<211> 462

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Ala Val Leu Gly Leu Leu Phe Cys Leu Gly Thr Phe Pro Ser Cys

1 5 10 15

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

20 25 30

Pro Ser Gln Ser Leu Ser Ile Thr Cys Ile Val Ser Gly Phe Ser Leu

35 40 45

Thr Gly Tyr Gly Val Asn Trp Leu Arg Gln Pro Pro Glu Lys Gly Leu

50 55 60

Glu Trp Leu Gly Met Ile Trp Gly Asp Gly Ile Thr Asp Tyr Asn Ser

65 70 75 80

Ala Leu Lys Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln

85 90 95

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

100 105 110

Tyr Cys Ala Arg Glu Tyr Asp Gly Asp Ser Ala Trp Phe Ala Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro

130 135 140

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

145 150 155 160

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

195 200 205

Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His

210 215 220

Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys

225 230 235 240

Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe

245 250 255

Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro

260 265 270

Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val

275 280 285

Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr

290 295 300

Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu

305 310 315 320

Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys

325 330 335

Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser

340 345 350

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

355 360 365

Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile

370 375 380

Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly

385 390 395 400

Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp

405 410 415

Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp

420 425 430

Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His

435 440 445

Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 10

<211> 233

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Val Leu Met Leu Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Asp

1 5 10 15

Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln

20 25 30

Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser Gly

35 40 45

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

50 55 60

Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro

85 90 95

Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg Glu

100 105 110

Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Asp Leu Lys Arg Ala

115 120 125

Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu

130 135 140

Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro

145 150 155 160

Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn

165 170 175

Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His

195 200 205

Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile

210 215 220

Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230

<210> 11

<211> 1389

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atggctgtct tggggctgct cttctgcctg ggaacattcc caagctgtat cctttcccag 60

gtgcagctga aggagtcagg tcctggcctg gtggcgccct cacagagcct gtccatcaca 120

tgcatcgtgt caggattctc attaaccggc tatggtgtaa actggcttcg ccagcctcca 180

gaaaagggtc tggagtggct gggaatgata tggggtgatg gaatcacaga ctataattca 240

gctctcaaat ccagactgag catcagcaag gacaactcca agagccaagt tttcttaaaa 300

atgaacagtc tgcaaactgg tgacacagcc aggtactact gtgccagaga gtatgatggt 360

gactccgcct ggtttgctta ctggggccaa gggactctgg tcactgtctc tgcagccaaa 420

acgacacccc catctgtcta tccactggcc cctggatctg ctgcccaaac taactccatg 480

gtgaccctgg gatgcctggt caagggctat ttccctgagc cagtgacagt gacctggaac 540

tctggatccc tgtccagcgg tgtgcacacc ttcccagctg tcctgcagtc tgacctctac 600

actctgagca gctcagtgac tgtcccctcc agcacctggc ccagcgagac cgtcacctgc 660

aacgttgccc acccggccag cagcaccaag gtggacaaga aaattgtgcc cagggattgt 720

ggttgtaagc cttgcatatg tacagtccca gaagtatcat ctgtcttcat cttcccccca 780

aagcccaagg atgtgctcac cattactctg actcctaagg tcacgtgtgt tgtggtagac 840

atcagcaagg atgatcccga ggtccagttc agctggtttg tagatgatgt ggaggtgcac 900

acagctcaga cgcaaccccg ggaggagcag ttcaacagca ctttccgctc agtcagtgaa 960

cttcccatca tgcaccagga ctggctcaat ggcaaggagt tcaaatgcag ggtcaacagt 1020

gcagctttcc ctgcccccat cgagaaaacc atctccaaaa ccaaaggcag accgaaggct 1080

ccacaggtgt acaccattcc acctcccaag gagcagatgg ccaaggataa agtcagtctg 1140

acctgcatga taacagactt cttccctgaa gacattactg tggagtggca gtggaatggg 1200

cagccagcgg agaactacaa gaacactcag cccatcatgg acacagatgg ctcttacttc 1260

gtctacagca agctcaatgt gcagaagagc aactgggagg caggaaatac tttcacctgc 1320

tctgtgttac atgagggcct gcacaaccac catactgaga agagcctctc ccactctcct 1380

ggtaaataa 1389

<210> 12

<211> 702

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atggtcctta tgttactgct gctatgggtt ccaggttcca ctggtgacat tgtgctgaca 60

cagtctcctg cttccttagc tgtatctctg gggcagaggg ccaccatctc atgcagggcc 120

agtaaaagtg tcagtacatc tggctatagt tatctgcact ggtaccaaca gaaaccagga 180

cagccaccca aactcctcat ctatcttgca tccaacctag aatctggggt ccctgccagg 240

ttcagtggca gtgggtctgg gacagacttc accctcaaca tccatcctgt ggaggaggag 300

gatgctgcaa cctattactg tcagcacagt agggagcttc ctctcacgtt cggtgctggg 360

accaagctgg acctgaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc 420

agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 480

aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 540

agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 600

accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 660

acttcaccca ttgtcaagag cttcaacagg aatgagtgct aa 702

<210> 13

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ctggttccgc gtggatccgg atccatgagt cttctaaccg aggt 44

<210> 14

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggccgcgata tcaagcttaa gctttcactt gaaccgctgc agtt 44

Claims

1. The monoclonal antibody of the influenza virus matrix protein M1, which comprises a heavy chain variable region and a light chain variable region, and is capable of specifically binding to the influenza virus matrix protein M1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown in SEQ ID NO. 1-3; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in SEQ ID NO. 4-6.

2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No.7 and the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 8.

3. The monoclonal antibody according to claim 1 or 2, wherein the heavy chain of the monoclonal antibody has the amino acid sequence shown in SEQ ID No.9 and the light chain has the amino acid sequence shown in SEQ ID No. 10.

4. A nucleic acid encoding the monoclonal antibody of any one of claims 1 to 3.

5. The nucleic acid of claim 4, wherein the nucleic acid sequence encoding the heavy chain of the monoclonal antibody is set forth in SEQ ID No.11 and the nucleic acid sequence encoding the light chain of the monoclonal antibody is set forth in SEQ ID No. 12.

6. A labeled complex obtained by biochemically labeling the monoclonal antibody according to any one of claims 1 to 3.

7. The labeling complex of claim 6, wherein the biochemical label is selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.

8. Use of the monoclonal antibody of any one of claims 1 to 3 or the labeled complex of claim 6 or 7 for the preparation of a kit for detecting influenza virus, influenza virus matrix protein M1 or influenza virus antibodies.

9. Use of the monoclonal antibody of any one of claims 1 to 3 or the labeled complex of claim 6 or 7 for the preparation of a medicament for the prevention or treatment of influenza virus or for quality control of an influenza virus vaccine.

10. A medicament comprising a monoclonal antibody according to any one of claims 1 to 3.

11. An influenza virus detection kit comprising the monoclonal antibody according to any one of claims 1 to 3 or the labeled complex according to claim 6 or 7.