CN113817687B

CN113817687B - Hybridoma cell strain, influenza A virus nucleoprotein monoclonal antibody and application thereof

Info

Publication number: CN113817687B
Application number: CN202111104857.4A
Authority: CN
Inventors: 乔传玲; 陈化兰; 杨焕良; 陈艳
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2021-09-22
Filing date: 2021-09-22
Publication date: 2023-07-14
Anticipated expiration: 2041-09-22
Also published as: CN113817687A

Abstract

The invention provides a hybridoma cell strain, an influenza A virus nucleoprotein monoclonal antibody and application thereof, belonging to the technical field of swine influenza virus detection. The invention provides a hybridoma cell strain capable of secreting an influenza A virus nucleoprotein monoclonal antibody, which is preserved in China Center for Type Culture Collection (CCTCC) NO: C2021165. The monoclonal antibody of the influenza A virus secreted by the hybridoma cell strain has the advantages of good specificity, high titer and the like, and can be used for identifying whether the virus to be detected is the influenza A virus, identifying whether a sample to be detected is infected with the influenza A virus and identifying whether the serum to be detected contains the antibody of the influenza A virus. IFA detection results show that the hybridoma cell strain capable of stably secreting the anti-nucleoprotein monoclonal antibody can specifically react with H1N1, H1N2, H3N2, H5N1, H7N9 and H9N2 subtype influenza viruses, and has broad-spectrum biological activity.

Description

Hybridoma cell strain, influenza A virus nucleoprotein monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of swine influenza virus detection, and particularly relates to a hybridoma cell strain 5E2 and influenza A virus nucleoprotein monoclonal antibody and application thereof.

Background

Swine Influenza (SI) refers to an infectious respiratory disease in pigs caused by influenza a virus, which has a clinically high incidence. According to the research result of SI epidemiology, viruses of subtype H1N1, H1N2, H3N2 and the like are mainly popular in pig groups in China. Pig is the only common susceptible host of fowl, pig and human influenza virus, and pig influenza virus (Swine influenza virus, SIV) has the capability of infecting fowl and human at the same time, so that another big hazard of pig influenza is that the pig influenza is closely related to human influenza, the big outbreaks of human influenza in the last century are closely and indiscriminately related to pig influenza, 2009/H1N1 influenza which causes global outbreaks in 4 months in 2009, and the important role of pig as an intermediate host and in the cross-species transmission of influenza viruses is highlighted again. SI is thus not only an important veterinary infectious disease, but also has important public health implications.

The genome of SIV is 8-segment, single-strand, negative-strand RNA, encoding at least 10 proteins; the Nucleoprotein (NP) encoded by the fragment 5 is a main component forming a virus nucleocapsid, is very conserved among different subtype influenza viruses, induces cellular immunity generated by an organism to resist infection of the different subtype influenza viruses, has good cross-reactivity among seeds, and is the basis of influenza virus typing and diagnosis.

Laboratory diagnosis of influenza a virus (Influenza A virus, IAV) can be divided into direct (antigen) and indirect (antibody) detection, the antigen detection is mainly performed by collecting corresponding samples before or after death, nasal secretions or oral water can be collected before life by cotton swabs, and necropsy samples are mainly tissues (nasal turbinates, tonsils, trachea, lungs) and respiratory secretions are collected through tracheal or bronchial lavages. The autopsy samples can be used directly for IAV detection as well as for histopathological detection. Furthermore, immunohistochemistry (IHC) can also be used to detect viruses present in airway epithelial cells and has proven to be a reliable influenza virus diagnostic technique. Serological detection means of influenza include HI, IFA, SN and ELISA, most commonly HI and ELISA, which have the technical advantage of high throughput compared to the two.

Nowadays, the co-epidemic situation of influenza virus strains with various subtypes and different genotypes in pig groups makes the diagnosis of SI (SI) become particularly complex, so that it is urgently required to establish a universal diagnosis method capable of ensuring rapid diagnosis at the first time of occurrence of diseases, thereby better preventing epidemic spread.

Disclosure of Invention

Accordingly, the present invention has an object to provide a hybridoma cell line capable of secreting a monoclonal antibody against influenza A virus nucleoprotein with good specificity, high titer and broad-spectrum biological activity, and capable of being used for detecting swine influenza virus and swine influenza virus TCID ₅₀ Is measured.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a hybridoma cell strain which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C2021165.

Preferably, the preparation method of the hybridoma cell strain comprises the following steps: connecting a swine influenza virus nucleoprotein gene with a nucleotide sequence shown as SEQ ID NO.1 with a vector pCAGGS to obtain a recombinant plasmid pCAGGS-NP; the recombinant plasmid pCAGGS-NP is used as an immunogen, and a hybridoma cell fusion technology is adopted to obtain the hybridoma cell strain.

Preferably, the swine influenza virus nucleoprotein gene is derived from swine influenza virus A/swing/Guangxi/18/2011 (H1N 1).

The invention also provides an influenza A virus nucleoprotein monoclonal antibody with broad-spectrum biological activity, which is secreted and produced by the hybridoma cell strain.

The invention also provides application of the hybridoma cell strain in preparation of swine influenza virus products.

The invention also provides application of the monoclonal antibody in preparation of swine influenza virus products.

The invention also provides a method for preparing the swine influenza virus TCID by using the monoclonal antibody ₅₀ The application in the product was determined.

Preferably, the swine influenza virus comprises H1N1, H1N2, H3N2, H5N1, H7N9, and H9N2 subtype influenza viruses.

Preferably, the product comprises an immunological diagnostic reagent, a test strip and a kit.

Preferably, the kit comprises an enzyme-linked immunosorbent assay diagnosis kit and a colloidal gold kit.

The invention has the beneficial effects that:

the hybridoma cell strain provided by the invention can stably secrete the influenza A virus nucleoprotein monoclonal antibody with good specificity, high titer and broad-spectrum biological activity, and can generate influenza virus subtype H1N1, H1N2, H3N2, H5N1, H7N9 and H9N2Specific reaction. The influenza A virus nucleoprotein monoclonal antibody provided by the invention can be used for identifying whether the virus to be detected is the influenza A virus, identifying whether a sample to be detected is infected with the influenza A virus and identifying whether the serum to be detected contains the antibody of the influenza A virus. The monoclonal antibody of the invention can also be used for preparing immunological diagnostic reagents of influenza A virus or antibodies thereof, such as enzyme-linked immunosorbent assay (ELISA) diagnostic kits, colloidal gold kits and the like, and is used for detecting whether animal (including pigs, birds and the like) excreta, oral and nasal secretions and feces contain influenza A virus or not and whether animal serum contains antibodies of influenza A virus or not. The influenza A virus nucleoprotein monoclonal antibody provided by the invention can be used for influenza virus TCID ₅₀ The sensitivity is improved by a factor of about 47 compared to the detection result of the Hemagglutination (HA) assay.

Preservation description

The hybridoma cell strain 5E2 provided by the invention is preserved in China center for type culture collection, the preservation time is 2021, 6 and 23 days, the preservation address is in the eight-path 299-number university of Wuhan in Wuchang district of Wuhan, hubei province, and the preservation number is CCTCC NO: C2021165.

Drawings

FIG. 1 shows the results of the pCAGGS-NP protein expression test, wherein A is the result of the recombinant plasmid pCAGGS-NP IFA test, B is the result of the pCAGGS empty vector IFA test, C is the result of the blank IFA test, D is the result of the Western-blot test, M in the D chart represents the relative molecular weight standard of the protein, 1 represents the pCAGGS-NP transfected 293T cell, 2 represents the pCAGGS transfected 293T cell;

FIG. 2 shows the Western blot detection results of 5E2 MAbs, where M is the protein relative molecular mass standard, 1 is 5E2 MAb incubation, 2 is SP2/0 supernatant incubation;

FIG. 3 is a graph showing the detection of 5E2 MAb antigen binding activity, wherein A is H1N1 subtype influenza virus; b is H1N2 subtype influenza virus; c is H3N2 subtype influenza virus; d is H5N1 subtype influenza virus; e is H7N9 subtype influenza virus; f is H9N2 subtype influenza virus.

Detailed Description

The invention provides a hybridoma cell strain which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C2021165. The hybridoma cell strain has a preservation time of 2021, 6 and 23 days and a preservation address of eight paths 299 of university of Wuhan in Wuhan, the university of Wuhan, the Hubei province.

In the present invention, the preparation method of the hybridoma cell line preferably comprises the steps of: connecting a swine influenza virus nucleoprotein gene with a nucleotide sequence shown as SEQ ID NO.1 with a vector pCAGGS to obtain a recombinant plasmid pCAGGS-NP; the recombinant plasmid pCAGGS-NP is used as an immunogen, and a hybridoma cell fusion technology is adopted to obtain the hybridoma cell strain.

In the invention, the process for obtaining the swine influenza virus nucleoprotein gene with the nucleotide sequence shown in SEQ ID NO.1 preferably comprises the following steps: inoculating 10-day-old specific pathogen-free (SPF) chick embryo (purchased from experimental animal center of Harbin veterinary research institute) with virus A/wire/Guangxi/18/2011 (H1N 1) (isolated, identified and stored by Harbin veterinary research institute), proliferating for 72 hours, harvesting allantoic fluid, extracting RNA, performing reverse transcription to obtain virus Nucleoprotein (NP) gene cDNA, and performing PCR amplification by taking the cDNA as a template. In the present invention, the primer sequences used for the PCR amplification are preferably: an upstream primer NPF 5'-CATCGATATGGCGTCTCAAGGCACCAA-3'; the downstream primer NPR is 5'-GCTCGAGTTAGTTGTCATACTCCTCTG-3'; the PCR reaction conditions are preferably as follows: pre-denaturation at 95℃for 5min, 1min at 94℃for 30s at 55℃for annealing and extension at 72℃for 1min for 35 cycles.

The specific mode of connecting the swine influenza virus nucleoprotein gene and the vector pCAGGS is not particularly limited, and in the specific embodiment, claI and XhoI are used for respectively carrying out double digestion treatment on the NP gene and the plasmid pCAGGS, the concentration is respectively measured after purification, and the DNA is incubated for 16 hours at 16 ℃ by using T4 DNA ligase. The invention connects NP gene into carrier pCAGGS, after transformation, picking single colony and enlarging culture, after sequencing verification, confirming to obtain recombinant expression plasmid pCAGGS-NP.

In the invention, recombinant plasmid pCAGGS-NP is taken as an immunogen, and in the process of obtaining the hybridoma cell strain by adopting a hybridoma cell fusion technology, the immunized animal is preferably a BALB/c mouse. The specific method of the hybridoma cell fusion technique is not particularly limited, and any conventional hybridoma cell fusion technique in the art can be adopted.

The influenza A virus nucleoprotein monoclonal antibody is characterized by IgM by Ig subclass, kappa chain for light chain, and is preferably obtained by inoculating hybridoma cell strain cells into abdominal cavity of experimental animal, preferably BALB/c mouse, to generate ascites. The titer of the culture supernatant of the hybridoma cell is 1:10 ² Ascites titer is 1:10 ⁵ . The influenza A virus nucleoprotein monoclonal antibody (MAb 5E 2) can specifically react with NP protein of influenza virus, and IFA detection results show that the MAb 5E2 can specifically react with H1N1, H1N2, H3N2, H5N1, H7N9 and H9N2 subtype influenza virus.

The invention also provides an application of the hybridoma cell strain or the monoclonal antibody in preparation of swine influenza virus products. The invention also provides a method for preparing the swine influenza virus TCID by using the monoclonal antibody ₅₀ The application in the product was determined.

In the invention, the swine influenza virus preferably comprises H1N1, H1N2, H3N2, H5N1, H7N9 and H9N2 subtype influenza viruses, the product preferably comprises an immunological diagnosis reagent, a test paper strip and a kit, and the kit preferably comprises an enzyme-linked immunosorbent assay diagnosis kit and a colloidal gold kit. The invention purifies MAb 5E2 and then uses the purified MAb 5E2 in influenza virus TCID ₅₀ The sensitivity was improved by a factor of about 47 compared to the detection results of the HA assay.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Construction and identification of recombinant expression plasmid pCAGGS-NP

Virus a/wire/guanxi/18/2011 (H1N 1) (isolated, identified and stored by the haelsholtzia veterinary institute) was inoculated with 10-day-old specific pathogen-free (SPF) chick embryos (purchased from the experimental animal center of the haelsholtzia veterinary institute), allantoic fluid was harvested after 72H of proliferation, RNA was extracted using a TIANamp Virus RNA Kit viral RNA extraction kit, and viral NP gene cDNA was obtained by reverse transcription using universal primer uni12 (sequence: 5'-AGCAAAAGCAGG-3') (SEQ ID No. 2). Polymerase Chain Reaction (PCR) amplification of NP gene using cDNA as template, primers used: an upstream primer NPF 5'-CATCGATATGGCGTCTCAAGGCACCAA-3' (SEQ ID NO. 3); the downstream primer NPR 5'-GCTCGAGTTAGTTGTCATACTCCTCTG-3' (SEQ ID NO. 4); the PCR reaction conditions were: pre-denaturation at 95℃for 5min, followed by annealing at 94℃for 1min,55℃for 30s, extension at 72℃for 1min,35 cycles. And (3) recovering PCR products by using the gel, respectively carrying out double enzyme digestion treatment on the NP gene and the plasmid pCAGGS by using ClaI and XhoI, respectively measuring the concentration after purification, connecting the NP gene into the vector pCAGGS by using T4 DNA ligase at 16 ℃ for 16h, picking single bacterial colony after transformation, amplifying and culturing, and confirming to obtain the recombinant expression plasmid pCAGGS-NP after sequencing verification.

Respectively transfecting 293T cells with pCAGGS-NP and pCAGGS according to the instructions of a Lipofectamine LTX transfection kit; 293T cells transfected for 60h were fixed with 4% paraformaldehyde for 1h, and were observed under a fluorescent microscope with chicken anti-SIV polyclonal serum as primary antibody and fluorescent-labeled anti-chicken antibody as secondary antibody. As a result, as shown in FIG. 1A, it was revealed from FIG. 1A that 293T cells transfected with pCAGGS-NP exhibited green fluorescence, whereas no green fluorescence was observed in the empty vector pCAGGS and 293T cells not transfected with plasmid (FIG. 1B, FIG. 1C), indicating that the NP gene of the constructed eukaryotic expression vector pCAGGS-NP was expressed in eukaryotic cells.

Samples of 293T cells transfected with pCAGGS-NP and pCAGGS for 60h were subjected to SDS-PAGE, transferred to NC membrane, and Western blot identification was performed using chicken anti-SIV polyclonal serum, and the results are shown in FIG. 1D. As can be seen from FIG. 1D, 293T cell samples transfected with pCAGGS-NP exhibited a distinct band at 56ku, consistent with the expected size; the empty vector does not have the reaction band, and the NP protein is further proved to be expressed in eukaryotic cells.

Example 2

Preparation of immunogenic DNA and mouse immunization

The pCAGGS-NP was purified using polyethylene glycol and its DNA content was measured to be about 10.8. Mu.g/. Mu.L by a spectrophotometer. pCAGGS-NP was immunized by leg intramuscular injection at a dose of 100. Mu.g/mouse for 4-5 weeks old BALB/c mice (purchased from Peking Vitre Lihua laboratory animal technologies Co., ltd.) 3 times at 2-week intervals, and 3d before cell fusion was again injected with 150. Mu.g/mouse for leg muscle.

Screening of positive hybridoma cells

Preparation of feeder cells

The day before cell fusion, the non-immunized BALB/c mice were sacrificed by cervical removal and immersed in 75% alcohol for 5min. Fixing the sterilized mouse abdomen on an dissecting plate upwards, cutting the skin of the mouse abdomen, then blunt stripping to expose the peritoneum, replacing scissors, cutting a small opening at the peritoneum, sucking 2-3ml RPMI Medium1640 culture medium into the mouse abdomen, sucking for several times, obtaining abdominal macrophages, centrifuging at 800rpm for 10min, suspending the supernatant with HAT-containing culture medium, adding the supernatant into a 96-well cell culture plate, and placing the mixture in a cell culture box for culture at 100 mu.l/well.

Culture of S2/0 cells

The SP2/0 cells were removed from the liquid nitrogen, rapidly thawed in a 37℃water bath, centrifuged at 800rpm for 10min at room temperature to pellet the cells, resuspended in DMEM medium with 20% serum concentration, supplemented with the medium and cultured in a cell incubator.

Preparation of immune spleen cells

The immunized BALB/c mice were collected, and then were subjected to cervical removal and sacrifice, and immersed in 75% alcohol for 5min. The abdomen of the soaked mice is fixed upwards, the spleen is taken out after the abdominal cavity is opened, the mice are placed in a culture dish filled with DMEM basic culture solution, spleen cells are released by grinding the spleen, and the spleen cells are collected into a 50ml sterile centrifuge tube.

Cell fusion

(1) The collected spleen cells were centrifuged at 800rpm for 10min, the culture solution was discarded, the cells were resuspended in DMEM medium, and the cells were counted after air-aspiration and mixing.

(2) SP2/0 cells were collected, centrifuged at 800rpm for 10min, the culture broth was discarded, the cell pellet was resuspended in DMEM broth, and the mixture was blow-sucked and counted.

(3) Spleen cells were mixed with SP2/0 cells at a ratio of 4:1, centrifuged at 800rpm for 10min, and the upper culture broth was carefully aspirated. The centrifuge tube was gently pulled over the EP tube rack to loosen and thoroughly mix the cell pellet.

(4) While gently agitating the cells, 0.8ml of PEG preheated at 37℃was added, and the PEG was added over 1 min.

(5) After the centrifuge tube was held by hand and left to stand for 1min, the reaction was terminated by slowly adding the preheated DMEM medium at 37℃and 1ml of DMEM medium was added dropwise within 1min, 2ml of DMEM medium was added at 2 min and 3ml of DMEM medium was added at 3 min, to terminate the PEG action.

(6) The cells were allowed to stand in a 37℃cell incubator for 10min at 800rpm, the upper layer of culture broth was discarded, the cells were resuspended in 40ml of HAT culture broth, added to a 96-well cell culture plate in which feeder cells were previously cultured, and cultured in an incubator at 100. Mu.l/well.

Screening of positive hybridoma cells

And (3) operating according to a conventional indirect ELISA method, determining the optimal antigen coating concentration, the optimal serum dilution and the optimal acting time of the primary antibody and the secondary antibody by a matrix method, and establishing an ELISA screening method of monoclonal antibodies (MAbs). The specific operation is as follows:

(1) Purified GX18 virus protein was used as coating antigen, diluted to 1. Mu.g/ml, 2. Mu.g/ml, 4. Mu.g/ml, 6. Mu.g/ml, 8. Mu.g/ml, 10. Mu.g/ml, ELISA plates were coated, 100. Mu.l/well, and left to coat overnight at 4 ℃. The next day, the coating solution was thrown out, and the plates were washed 3 times with PBST for 5 min/time.

(2) ELISA plates were blocked by adding 5% skim milk, 200. Mu.l/well, blocked for 2h at 37℃and washed 3 times with PBST for 5 min/time.

(3) Negative and positive sera were diluted 1:100, 1:200, 1:400, 1:800, 1:1 by 600-fold, 100 μl/well, and incubated at 37deg.C for 1h, and plates were washed 3 times with PBST for 5 min/time.

(4) Sheep anti-mouse HRP-IgG was added at 1:4000 dilution, incubated at 37℃for 1h, and PBST was repeatedly washed 3 times for 5 min/time.

(5) Added in an amount of 100. Mu.l/wellAdding TMB substrate color development solution, placing in a 37 deg.C incubator for 4min, adding 2mol/L H ₂ S0 ₄ Stop reaction, 50. Mu.l/well, read OD _450nm 。

(6) ELISA results are judged, and the antigen coating concentration and serum dilution with positive hole OD value closest to 1, negative hole OD value smaller than 0.2 and P/N value larger than or equal to 2.1 are used as the optimal working concentration.

After 10d of fusion, hybridoma cells were screened by established ELISA, and positive culture well cells were subjected to cloning purification by limiting dilution until 100% of all cloned cells were positive.

Example 3

Identification of MAb subclasses

MAb subtype was identified using the SBAClon-typingTM Systemm/HRP antibody subtype kit. The specific method comprises the following steps:

(1) Coating: the capture antibody was diluted to a final coating concentration of 7. Mu.g/ml, and 100. Mu.l per well was added to ELISA plates overnight (about 16 h) at 4 ℃.

(2) Closing: the coating was discarded and the plates were washed 3 times with 5min each with PBST. 5% skim milk was added to wells, 200 μl each, and the plates were washed 3 times with PBST at 37deg.C for 2 h.

(3) An antibody: positive hybridoma cell supernatants were primary antibodies, 100 μl per well, incubated 1h at 37 ℃, and pbst plates washed 3 times.

(4) And (2) secondary antibody: sheep anti-mouse secondary antibodies IgGl, igG2b, igG2a, igG3, igA, igM, lambda, kappa were added at 1:500 dilution, 100 μl per well incubated for 1h at 37deg.C, and PBST plates were washed 3 times.

(5) Color development: adding 100 μl of TMB color developing solution into each well, keeping away from light at 37deg.C for 10min, adding 50 μl of stop solution into each well after color development is completed, and OD _450nm The values are read.

The antibody subclass of the MAb secreted by the hybridoma cell line (5E 2) of the invention was determined to be IgM type, and the light chain was determined to be kappa chain.

Example 4

5E2 ascites preparation and antibody purification

Ascites preparation

Intraperitoneal injection of 0.5ml FITC-sensitized, 7 to 8 week old female BALB/c miceAfter the day, each mouse was vaccinated with about 10 ⁶ The survival condition of the mice is observed 7-10 days after inoculation of hybridoma cells (about 0.5ml of hybridoma cell suspension), and ascites is collected after the abdomen of the mice is obviously enlarged; centrifuging collected ascites at 5000rpm for 10min, removing other impurities such as oil, and freeze preserving.

Ascites purification of IgM subtype mab

MAbs were purified according to Pierce company IgM purification kit instructions as follows:

(1) MBP pre-packed column preparation: the cap on the upper part of MBP was opened, the cap on the lower part was removed, the original stock solution in the column was allowed to naturally flow down by gravity, and the column was rinsed with 5ml of MBP column Preparation Buffer.

(2) Ascites treatment: centrifuging ascites at 5000rpm for 10min, removing oil layer, collecting clarified liquid at lower part, placing into dialysis bag, dialyzing in deionized water with pH of 5.5, dialyzing at 4deg.C for 24 hr (with dialysate to be replaced 2-3 times in the middle), and mixing pretreated ascites with Binding Buffer at ratio of 1:3.

(3) Purifying ascites:

(1) the MBP pre-packed column is placed in a refrigerator at 4 ℃, 2ml of Binding Buffer is added for natural sedimentation, and the waste liquid is removed.

(2) Pretreated ascites diluted with Binding Buffer pre-chilled at 4℃was added, allowed to pass through the gel naturally, the filtrate was collected, and again added to the column, and the filtrate was collected 3-4 times in this manner (the whole procedure was performed at 4 ℃).

(3) 42ml of Binding Buffer was added, allowed to flow down naturally, unbound protein was washed away, and the elution effect was detected by collecting 3ml of filtrate from the last flow (this procedure was performed at 4 ℃ C.).

(4) MBP pre-packed column and the Elution Buffer were left at room temperature for a period of time, after which 4ml of Elution Buffer was added and allowed to flow down naturally, the bottom lid was closed and incubated upright at room temperature for at least 1h. The bottom lid was opened and the eluate collected. (elution effect was measured by adding more eluent 0.5-3 ml).

(5) The column was washed with 10ml of deionized water, then 10. 10ml Binding Buffer was added, the bottom lid was closed, then 1.5. 1.5ml Binding Buffer was added, the top lid was closed, and the column was stored upright in a 4 ℃ refrigerator.

ELISA titers for MAbs

The ELISA plate was coated with the purified whole virus protein and its ELISA titer was measured.

The specific method comprises the following steps:

(1) Coating: the whole virus protein was coated with purified, 100. Mu.l per well was added to ELISA plates at 4℃overnight (about 16 h).

(3) An antibody: positive hybridoma cell supernatant and preparing ascites as primary antibody, wherein the positive hybridoma cell supernatant is 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Diluted by times, ascites is 10 ^-1 、 10 ^-2 ……10 ^-12 Double dilution, setting SP2/0 cell supernatant as negative control, incubating for 1h at 37 ℃ per well 100 μl, and PBST washing plate 3 times.

(4) And (2) secondary antibody: HRP-labeled goat anti-mouse secondary antibody was added at 1:5000 dilution, 100 μl per well incubated at 37deg.C for 1h, and the plates were washed 3 times with PBST.

The titer of the culture supernatant of the hybridoma cells is 1:10 ² Ascites titer is 1:10 ⁵ 。

Western blot detection of MAb

The monoclonal antibody (5E 2 MAb) obtained by secretory purification of the hybridoma cell line (5E 2) was subjected to Western blot test, and the results are shown in FIG. 2. As can be seen from fig. 2, the 5e2 MAb can produce a specific band with the NP protein of influenza virus at about 56ku, while the SP2/0 supernatant did not react with it, thus demonstrating that the 5e2 MAb is a monoclonal antibody against the linear epitope of the SIV NP protein.

Example 5

Detection of MAb antigen binding Activity

Respectively infecting MDCK single-layer cells with H1N1, H1N2, H3N2, H5N1, H7N9 and H9N2 subtype influenza diseases, fixing the MDCK cells after 72H infection with 4% paraformaldehyde for 1H, and washing with PBST for 3 times; 5E2 MAb is used as primary antibody, the primary antibody acts for 3 hours at 37 ℃, and PBST is washed for 3 times; sheep anti-mouse IgG antibody (IgG-FITC) was used as the secondary antibody, and after washing with PBST three times at room temperature, it was observed under a fluorescence microscope. The results are shown in FIG. 3, and all virus-infected cell wells showed obvious green fluorescence, which indicates that the monoclonal antibodies of the invention can specifically react with H1N1, H1N2, H3N2, H5N1, H7N9 and H9N2 subtype influenza viruses and have broad-spectrum biological activity.

Example 6

5E2 MAb infection in viral tissue half-size (TCID ₅₀ ) Application in determination

HA test determination method

Subculturing MDCK cells, spreading 96-well cell culture plate, and placing at 37deg.C and 5% CO ₂ Culturing in incubator, and measuring TCID for virus when cell confluence reaches 80% ₅₀ The method comprises the steps of carrying out a first treatment on the surface of the The virus to be tested is diluted by 10 times according to the ratio of 1:10, 1:100, 1:1000 and the like, and the specific method is as follows: adding 135 μl of cell maintenance solution (adding TPCK-pancreatin with final concentration of 1 μg/mL into DMEM nutrient solution) into 96-well cell culture plate, adding 15 μl of virus solution into the first column, arranging 4 repeated wells, blowing, mixing, sucking 15 μl into the second column, diluting to 12 column, and standing at 4deg.C; discarding the culture solution from the 96-well cell culture plate, adding 100 mu L of maintenance solution into each well, washing for 2 times, and discarding the maintenance solution; transferring diluted virus into cell culture plate at 37deg.C with 5% CO ₂ Culturing in an incubator. Observing cytopathy during the period, measuring HA activity after 72h, summarizing HA positive results and corresponding virus dilution factors, and calculating TCID according to a Reed-Muench method ₅₀ 。

Immunocytochemistry (ICC) test measurement method

Solution preparation

Washing liquid: PBS containing 0.05% tween (1 ml tween+2 LPBS); fixing solution: 4% paraformaldehyde; color development liquid: AEC staining kit; stop solution: distilled water; an antibody: NP mab, 1: diluting by 500; and (2) secondary antibody: HRP-labeled goat anti-mouse secondary antibody 1: and (5) diluting by 1000.

Operating procedure

(1) And (3) paving: subculturing MDCK cells, spreading 96-well cell culture plate, and placing at 37deg.C and 5% CO ₂ Culturing in incubator, and measuring TCID of virus when cell confluence reaches 90% ₅₀ ；

(2) Virus dilution: the virus to be tested is diluted by 10 times according to the ratio of 1:10, 1:100, 1:1000 and the like, and the specific method is as follows: adding 135 μl of cell maintenance solution (adding TPCK-pancreatin with final concentration of 1 μg/mL into DMEM nutrient solution) into 96-well cell culture plate, adding 15 μl of virus solution into the first column, arranging 4 repeated wells, blowing, mixing, sucking 15 μl into the second column, diluting to 12 column, and standing at 4deg.C;

(3) Washing the plate: discarding the culture solution from 96-well cell culture plate, adding 100 μl of maintenance solution into each well, washing for 2 times, discarding the maintenance solution, transferring diluted virus into cell culture plate, and transferring into 5% CO at 37deg.C ₂ Culturing in an incubator during which cytopathy is observed;

(4) Fixing: after 72h, discarding the redundant supernatant of the 96-well plate, washing the plate twice, adding 100 mu L of fixing solution into each well, and standing for 1h at room temperature;

(5) Incubating primary antibodies: removing the supernatant, washing the plate 3 times with washing solution for 5 min/time, adding 100 mu LNP antibody into each hole, and allowing the mixture to act for 1h at room temperature;

(6) Incubating a secondary antibody: discarding the supernatant, washing with washing solution for 3 times and 5 min/time, adding 100 μl of secondary antibody into each well, and standing at room temperature for 10min;

(7) Color development: discarding the supernatant, washing with washing solution for 3 times and 5 min/time, adding 100 μl of color-developing agent (for use at present) into each hole, and allowing the mixture to act at room temperature for 60min; removing supernatant, adding 50 μl of distilled water into each well, observing under a mirror, counting corresponding wells containing stained cells and their corresponding viral dilution factors, and calculating TCID according to Reed-Muench method ₅₀ 。

With the two methods described above,TCID of two strains of influenza viruses GX18 and GD9 were determined separately ₅₀ The results are shown in Table 1. As can be seen from Table 1, the sensitivity of the virus detected using the monoclonal antibody of the present invention (ICC detection) was improved by about 47 times over that of the Hemagglutination (HA) detection.

TABLE 1 two strains of influenza virus TCID ₅₀ Is measured according to the measurement results of (2)

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> Harbin veterinary institute of Chinese academy of agricultural sciences (Harbin division center of Chinese animal health and epidemiology center)

<120> hybridoma cell strain, influenza A virus nucleoprotein monoclonal antibody and application thereof

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<213> Artificial sequence (Artificial Sequence)

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aatgctactg aaatcagagc atctgttggg agaatgattg gaggaattgg aagattctac 120

atacagatgt gcaccgaact ccaactcagt gactatgaag ggaggctgat ccaaaacagt 180

ataacaatag agagaatggt cctctctgca tttgatgaga ggagaaacaa atacctagag 240

gaacatccca gtgcggggaa agatccaaag aaaacgggag gaccaatcta caaaaagaga 300

gacggaaaat ggatgagaga gctgattctg tatgacaaag atgaaatcag aagaatttgg 360

cgccaagcga acaatggtga agatgctact gctggtctca ctcacctgat gatttggcat 420

tccaacctga atgatgccac atatcagaga acaagagctt tagtgcgtac tggaatggat 480

cccagaatgt gttctctgat gcaaggttca actctcccaa ggagatctgg agctgctggt 540

gcagcagtaa agggagttgg gacgatggta atggaactga ttcggatgat aaagcggggt 600

atcaatgatc ggaacttctg gagaggcgag aatggacgaa gaacaagaat tgcatatgag 660

aggatgtgca acatcctcaa agggaagttc cagacagcag cgcagcgagc aatgatggac 720

caggtgcggg aaagcaggaa tcccgggaat gctgagattg aagatctcat ctttctggca 780

cgatctgcac tcattctgag aggatcagtg gcccataaat catgcctacc tgcttgtgta 840

tatggacttg ttgtagcaag tggatatgac tttgaaagag aagggtactc tctagtcgga 900

atagatcctt tccgtctgct tcaaagcagt caggtgttca gcctcattag accaaatgag 960

aatccagtac ataagagcca gctgatatgg atggcatgtc attctgcagc atttgaagac 1020

ctgagagtgt caagttttat cagagggaca aaggtggtcc caagaggaca attgtccacc 1080

agaggggttc aaattgcgtc aaatgaaaac atggaaacaa tggattccat tactcttgaa 1140

ctgagaagca aatactgggc tataagaacc aggagcggag ggaacaccaa tcaacagaga 1200

tcatccgcag ggcaaattag tgtacaacct actttctcag tacagagaaa ccttcctttc 1260

gagagaacga ccatcatggc agcatttact ggaaacactg aaggcagaac atctgacatg 1320

aggactgaaa tcataagaat gatggaaagc gccagaccag aagatgtgtc cttccagggg 1380

cggggagtct tcgagctctc ggacgaaaag gcaacgaacc cgatcgtgcc ttcctttgac 1440

atgagtaatg aaggatctta tttcttcgga gacaatgcag aggagtatga caactaa 1497

<210> 2

<211> 12

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

agcaaaagca gg 12

<210> 3

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

catcgatatg gcgtctcaag gcaccaa 27

<210> 4

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gctcgagtta gttgtcatac tcctctg 27

Claims

1. The hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of C2021165.

2. An influenza a nucleoprotein monoclonal antibody having broad spectrum biological activity, wherein said monoclonal antibody is secreted by the hybridoma cell line of claim 1.

3. Use of the hybridoma cell line of claim 1 for the preparation of a swine influenza virus detection product.

4. Use of the monoclonal antibody of claim 2 for preparing a swine influenza virus product.

5. Use of the monoclonal antibody of claim 2 for preparing a swine influenza virus TCID50 assay product.

6. The use according to any one of claims 3 to 5, wherein the swine influenza virus comprises H1N1, H1N2, H3N2, H5N1, H7N9 and H9N2 subtype influenza virus.

7. The use according to any one of claims 3-5, wherein the product comprises an immunological diagnostic reagent, a test strip, a kit.

8. The use according to claim 7, wherein the kit comprises an enzyme-linked immunosorbent assay diagnostic kit, a colloidal gold kit.