CN113249333A - Hybridoma cell strain RSVN4C3 secreting monoclonal antibody against respiratory syncytial virus - Google Patents
Hybridoma cell strain RSVN4C3 secreting monoclonal antibody against respiratory syncytial virus Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
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- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
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Abstract
The invention discloses a hybridoma cell strain RSVN4C3 secreting anti-respiratory syncytial virus monoclonal antibody, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2020254, wherein the monoclonal antibody secreting anti-respiratory syncytial virus nucleocapsid protein from the hybridoma cell strain RSVN4C3 is used for preparing a kit for detecting or diagnosing respiratory syncytial virus infection, and the kit can be an immune colloidal gold test strip or an ELISA kit.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a hybridoma cell strain RSVN4C3 for secreting monoclonal antibodies against respiratory syncytial virus.
Background
Human Respiratory Syncytial Virus (RSV) is a major cause of acute lower respiratory tract infections (ALRI) worldwide, resulting in hospitalization of children under 5 years of age and immunocompromised adults. RSV infection is associated with high morbidity and mortality from acute lower respiratory tract infections (ali) in children in developed and developing countries. In developed countries, it is the primary cause of hospitalization of infants in one month of birth; while in developing countries, RSV is the leading cause of infant death, approximately 3400 million children under 5 years of age develop severe RSV disease each year, with 350 million children requiring hospitalization and 6.6 to 19.9 million deaths, with 99% occurring in developing countries. Although RSV contributes to a significant disease burden, no licensed vaccines are currently on the market and the world health organization has listed the development of RSV vaccines as one of the first development programs for the global vaccine program.
RSV is a non-segmented, single-stranded, negative-strand RNA virus belonging to the genus Pneumovirus, the family Paramyxoviridae. The RSV genome is approximately 15.2kb in length and encodes 11 proteins, NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2 and L10. Of all proteins encoded by the RSV genome, the nucleoprotein (N protein), whether between the A, B subtypes or within the same subtype, is the most conserved protein. Many studies at home and abroad report that the N protein gene is used as a target gene for molecular biological detection. The N protein is an important component of the RNA-dependent RNA polymerase complex in RSV and at the same time is one of the important antigens recognized by cytotoxic T cells. Therefore, the research of the N gene and the coding protein thereof has important significance for further researching the pathogenic mechanism and the immune mechanism of the RSV and developing an RSV rapid diagnostic reagent. But at present, the research on the N protein is very little in China.
Disclosure of Invention
Aiming at the technical problems, the invention provides a hybridoma cell strain RSVN4C3 for secreting monoclonal antibodies against respiratory syncytial virus.
The technical scheme of the invention is as follows:
a hybridoma cell strain RSVN4C3 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2020254.
Further, the preparation method of the hybridoma cell strain RSVN4C3 comprises the following steps:
s1, separating a strain B133 strain Hep-2 cell culture supernatant in 2017 of RSV Guizhou, and extracting total RNA of the RSV; synthesizing a first strand cDNA by reverse transcription, and designing a specific primer by taking the first strand cDNA as a template, wherein the sequence is as follows:
P1：5′-AATGGATCCATGGCTCTTAGCAAAGTC-3' (the underlined part is the BamHI cleavage site),
P2：5′-ATTGTCGACAAGCTCTACATCATTATC-3' (Sall cleavage site is underlined),
s2, using P1 and P2 as primers, amplifying RSV N gene segment by PCR, wherein the reaction conditions are as follows: 30s at 94 ℃; 30s at 52 ℃ and 1min at 72 ℃ for 35 cycles; obtaining a 908bp PCR amplification product, carrying out double enzyme digestion on the PCR product and a pQE30 expression vector respectively by using BamHI and Sall, and recovering a target gene fragment and a vector fragment;
s3, connecting a target gene fragment and a vector fragment by using T4 DNA ligase, converting a connection product into competent escherichia coli XL1-blue, coating an LB ampicillin plate, performing static culture at 37 ℃ overnight, respectively selecting single colonies the next day, inoculating the single colonies into 3ml of LB culture solution, performing oscillation culture at 37 ℃ for 16h, extracting plasmids, performing double enzyme digestion identification, performing agarose gel electrophoresis analysis, and performing gene sequencing on positive plasmids;
s4, transforming escherichia coli XL1-blue bacteria by the recombinant expression plasmid with correct sequencing, inoculating the escherichia coli XL1-blue bacteria into an LB culture medium containing 100 mu g/ml ampicillin (Amp), and performing shaking culture at 37 ℃ for overnight; according to 1 percent of inoculation amountRespectively inoculating the strain in a 5ml LB bacterial culture medium tube containing 100 mu g/ml Amp and a 200ml LB culture medium triangular flask for amplification culture, carrying out shake culture at 37 ℃ until the value of the bacterial liquid A600 is about 0.6, adding IPTG with the final concentration of 100 mu g/ml, and carrying out shake culture induction at 37 ℃ for 4 h; collecting thallus, ultrasonic crushing, centrifuging, collecting supernatant, and adding Ni2+Purifying with affinity chromatography column; carrying out 12% SDS-PAGE and Western blot identification analysis on the purified expression product to obtain purified recombinant RSV nucleoprotein;
s5, immunizing a BALA/C mouse with purified recombinant RSV nucleoprotein, immunizing a mouse according to the dose of 100 micrograms per time, emulsifying the first immunization with a Freund' S complete adjuvant, emulsifying the second immunization and the third immunization with an incomplete adjuvant, wherein the immunization interval time is two weeks, collecting blood from the tail of the mouse after 7 days of the third immunization to measure the titer of serum antibodies, performing boosting immunization once 3 days before fusion, performing cell fusion on splenocytes of the immunized mouse and SP2/0 according to a conventional method, and screening and culturing fused hybridoma cells by using an HAT selection culture medium;
s6, detecting the supernatant of the hybridoma cells by the grown hybridoma cells through an indirect ELISA method, an indirect immunofluorescence method and a western-blot method, and screening hybridoma cell strains secreting the anti-respiratory syncytial virus nucleocapsid protein monoclonal antibodies.
The invention also provides application of the hybridoma cell strain RSVN4C3 in preparation of a reagent for detecting or diagnosing respiratory syncytial virus infection.
Furthermore, the monoclonal antibody of the anti-respiratory syncytial virus nucleocapsid protein secreted by the hybridoma cell strain RSVN4C3 has antigen-antibody reaction with the RSV N protein, and belongs to an IgG type antibody. The monoclonal antibody of the anti-respiratory syncytial virus nucleocapsid protein secreted by the hybridoma cell strain RSVN4C3 has antigen-antibody reaction with the RSV N protein, and belongs to an IgG type antibody.
The invention also provides a kit for detecting the respiratory syncytial virus antigen by an indirect immunofluorescence method and detecting the respiratory syncytial virus antigen by a colloidal gold method so as to diagnose the respiratory syncytial virus infection, a kit for detecting the respiratory syncytial virus antigen by an ELISA method so as to diagnose the respiratory syncytial virus infection and a method for detecting the respiratory syncytial virus specific IgM antibody of the patient serum by an IgM capture method so as to diagnose the respiratory syncytial virus infection. The 4 methods and the kit comprise the monoclonal antibody of the anti-respiratory syncytial virus nucleocapsid protein secreted by the hybridoma cell strain RSVN4C 3.
Optionally, the kit is an immune colloidal gold test strip.
Furthermore, the test strip sequentially comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, absorbent paper and a PVC bottom plate which is positioned below and used as an assembly platform according to a connection sequence, wherein the nitrocellulose membrane is overlapped with the colloidal gold pad, the nitrocellulose membrane is overlapped with the absorbent paper, the colloidal gold pad is composed of a glass cellulose membrane which is adsorbed with a colloidal gold-labeled anti-RSVN 4C3 monoclonal antibody, and the nitrocellulose membrane is provided with a quality control line coated by goat anti-mouse IgG polyclonal antibody and a detection line coated by anti-RSV N protein monoclonal antibody.
Further, the control line was obtained by coating goat anti-mouse IgG polyclonal antibody on a nitrocellulose membrane at a concentration of 0.8 mg/ml; the test line was obtained by coating a monoclonal antibody against RSV N protein on a nitrocellulose membrane at a concentration of 0.5 mg/ml.
Further, the detection line and the quality control line are separated by 0.5 cm.
Furthermore, the detection method of the immune colloidal gold test strip for detecting the human respiratory syncytial virus comprises the following steps:
s1, dropping 200 mu L of test sample into the sample dropping hole, reacting for 10-15min and observing the result;
s2, judging the result:
the quality control line and the detection line are positive if red strips appear;
a red strip appears only on the quality control line, and the strip is negative;
if no strip appears in the quality control line, the result is judged to be invalid no matter whether the detection line has a red strip or not.
Optionally, the kit for detecting or diagnosing respiratory syncytial virus is an ELISA kit.
The invention has the beneficial effects that: the invention clones the full-length gene of RSV N protein, expresses recombinant N protein in colon bacillus, purifies, immunizes BALB/C mice with the purified recombinant nucleoprotein, fuses immune mouse spleen lymphocytes and mouse myeloma SP2/0 cells, and screens out hybridoma cell strains secreting monoclonal antibodies against respiratory syncytial virus nucleocapsid protein. The hybridoma cell strain can stably secrete the monoclonal antibody (MAb) for resisting the respiratory syncytial virus, identifies the reactivity and the specificity of the monoclonal antibody (MAb), and lays a foundation for the research of the diagnosis of RSV virus infection, an infection mechanism and vaccine development in the future.
Drawings
FIG. 1 is an SDS-PAGE analysis of recombinant RSV nucleocapsid proteins expressed and purified in E.coli.
FIG. 2 is a diagram showing the result of Western blot reaction between the monoclonal antibody secreted by hybridoma cell strain RSVN4C3 and recombinant RSV nucleocapsid protein expressed and purified in Escherichia coli.
FIG. 3 is a graph showing the immunofluorescence results of RSV-infected Hep-2 cells specifically recognized by the monoclonal antibody secreted by hybridoma cell line RSVN4C 3. Wherein, A: normal uninfected Hep-2 cells; RSV infects Hep-2 cells.
FIG. 4 shows the results of the specificity test of the antigen detection diagnostic reagent prepared from the colloidal gold-labeled RSVN4C3 monoclonal antibody.
FIG. 5 shows the results of the sensitivity test of the reagent for detecting colloidal gold antigen of the RSVN4C3 monoclonal antibody.
Detailed Description
Example 1
A hybridoma cell strain RSVN4C3 secreting monoclonal antibody against respiratory syncytial virus is preserved in China Center for Type Culture Collection (CCTCC) with the address of Wuhan, China, the preservation date of 2020, 12 months and 1 day, and the preservation number is CCTCC NO: C2020254.
The preparation method of the hybridoma cell strain RSVN4C3 comprises the following steps:
materials and methods
The method comprises the following steps of taking culture supernatant of a strain B133 Hep-2 cell isolated in 2017 Guizhou of RSV, extracting total RNA of the RSV according to Trizol reagent instructions, carrying out reverse transcription to synthesize a cDNA first chain, and designing specific primers by taking the cDNA first chain as a template, wherein the sequences are as follows:
P1：5′-AATGGATCCATGGCTCTTAGCAAAGTC-3' (the underlined part is the BamHI cleavage site),
P2：5′-ATTGTCGACAAGCTCTACATCATTATC-3' (Sall cleavage site is underlined);
PCR amplification of RSV N gene segment with P1 and P2 as primers and under the reaction conditions: 30s at 94 ℃; 30s at 52 ℃ and 1min at 72 ℃ for 35 cycles; obtaining a 908bp PCR amplification product, carrying out double enzyme digestion on the PCR product and a pQE30 expression vector by using BamHI and SalI respectively, and recovering a target gene fragment and a vector fragment;
connecting by using T4 DNA ligase, transforming competent escherichia coli XL1-blue by a connecting product, coating an LB ampicillin plate, statically culturing overnight at 37 ℃, respectively picking single colonies the next day, inoculating the single colonies into 3ml of LB culture solution, performing oscillation culture for 16h at 37 ℃, extracting plasmids, performing double digestion identification, performing agarose gel electrophoresis analysis, and performing gene sequencing on positive plasmids;
e.coli XL1-blue bacteria are transformed by the recombinant expression plasmid with correct sequencing, inoculated in LB culture medium containing 100 mug/ml ampicillin (Amp), and cultured overnight under shaking at 37 ℃; respectively inoculating the strain into a 5ml LB bacterial culture medium pipe containing 100 mu g/ml Amp and a 200ml LB culture medium triangular flask according to the inoculation amount of 1 percent for amplification culture, adding IPTG with the final concentration of 100 mu g/ml when the value of the bacterial liquid A600 is about 0.6 through shaking culture at 37 ℃, and carrying out shaking culture induction for 4 hours at 37 ℃; collecting the thalli, carrying out ultrasonic crushing and centrifugation, taking supernatant, purifying by using a Ni2+ affinity chromatography column, and carrying out 12% SDS-PAGE and Western blot identification and analysis on a purified expression product.
Expression and identification of recombinant RSV-N proteins
The RSV-N gene is amplified and cloned into an island expression vector pQE-30, and the RSV-N protein product expressed by the recombinant plasmid in escherichia coli is analyzed by 12 percent SDS-PAGE, so that a specific protein band with the relative molecular weight of about 30kD can be seen, and the size is consistent with the expected size. Western blot analysis shows that the purified and expressed recombinant protein has specific reaction with mouse anti-RSV-N monoclonal antibody, and the expression protein is proved to be RSV-N protein. See fig. 1 and 2.
Establishment and identification of hybridoma cell strain
The BAL A/C mouse is immunized by purified recombinant RSV nucleocapsid protein, immune spleen cells are taken to fuse with SP2/0 myeloma cells, and hybridoma cells are selected and cultured by HAT selection.
Screening positive hybridoma cells by using indirect immunofluorescence and indirect ELISA, subcloning by using a limiting dilution method for 3 times, and obtaining 1 hybridoma cell strain which stably secretes the anti-RSV-N protein monoclonal antibody and is named as RSVN-4C 3.
The monoclonal antibody secreted by the hybridoma cells of this strain showed specific immunofluorescence in the cytoplasm in the absence of reactivity with uninfected Hep-2 cells and in the presence of RSV-infected Hep-2 cells (FIG. 3, wherein A: normal uninfected Hep-2 cells; B: RSV-infected Hep-2 cells).
The indirect immunofluorescence method specifically identifies that the monoclonal antibody of the strain only reacts with RSV infected cells, and does not react with influenza virus (H1N1, PR8 strain), EV71 virus and Coxsackie virus (CA16) infected cells.
Injecting the RSVN4C3 hybridoma into mouse abdominal cavity to prepare monoclonal antibody titer of ascites, and measuring by indirect ELISA method of coating recombinant nucleoprotein, wherein the titer of RSVN4C3 ascites antibody is 1:1, 000, 000; the titer of RSVNC3 is 1:100000 as determined by indirect immunofluorescence; are obviously higher than the titer of immune serum.
Through the identification of the monoclonal antibody subclasses, the heavy chains of the RSVN4C3 monoclonal antibodies are IgG1, and the light chains are kappa chains.
The monoclonal antibody can identify RSV nucleocapsid protein, and the identification amino acid sequence is as follows:
MALSKVKLNDTLNKDQLLSSSKYTIQRSTGDSIDTPNYDVQKHINKLCGMLLI TEDANHKFTGLIGMLYAMSRLGREDTIKILKDAGYHVKANGVDVTTHRQDIN GKEMKFEVLTLASLTTEIQINIEIESRKSYKKMLKEMGEVAPEYRHDSPDCGM IILCIAALVITKLAAGDRSGLTAVIRRANNVLKNEMKRYKGLLPKDIANSFYE VFEKYPHFIDVFVHFGIAQSSTRGGSRVEGIFAGLFMNAYGAGQVMLRWGVL AKSVKNIMLGHASVQAEMEQVVEVYEYAQKLGGEAGFYHILNNPKASLLSL TQFPHFSSVVLGNAAGLGIMGEYRGTPRNQDLYDAAKAYAEQLKENGVINY SVLDLTAEELEAIKHQLNPKDNDVEL
example 2
The monoclonal antibody of anti-respiratory syncytial virus nucleocapsid protein secreted by a hybridoma cell strain RSVN4C3 is used for detecting clinical respiratory syncytial virus infection by an indirect immunofluorescence method.
Taking a nasopharyngeal secretion or nasopharyngeal wash sample of a patient with respiratory tract infection, diluting the nasopharyngeal secretion or nasopharyngeal wash sample with normal saline, centrifuging the nasopharynx and nasopharynx for 5 minutes at 2000 r/min, collecting nasopharyngeal cast-off cells, suspending the cast-off cells with 100 microliters of normal saline, dripping the cells onto a glass slide, and drying the glass slide to fix the cells with acetone. RSVN4C3 monoclonal antibody diluted 1:10000 was added, the dilution was reacted with Phosphate Buffer Solution (PBS) containing 3% Bovine Serum Albumin (BSA) at 37 ℃ for 30 minutes, the slide was washed with PBS for 10 minutes, and 1: the method comprises the following steps of reacting for 30 minutes at 37 ℃ after a FITC fluorescent-labeled goat anti-mouse IgG antibody diluted by 50, washing the slide glass for 10 minutes by PBS, drying the slide glass, adding a fluorescent protective agent, adding a cover glass, sealing and fixing the periphery of the cover glass by nail polish, observing cells by a fluorescence microscope, and detecting whether green fluorescent particles are positive in the cells, namely respiratory syncytial virus infection, or detecting whether the green fluorescent particles are negative or not, wherein the green fluorescent particles are not in the cells.
Example 3
The embodiment provides a kit for detecting or diagnosing respiratory syncytial virus infection, which is an immune colloidal gold test strip and comprises a monoclonal antibody which is secreted by the hybridoma cell strain RSVN4C3 and is used for resisting the respiratory syncytial virus nucleocapsid protein.
The test strip sequentially comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, absorbent paper and a PVC bottom plate which is positioned below and used as an assembly platform according to a connection sequence, wherein an overlap joint is arranged between the nitrocellulose membrane and the colloidal gold pad, an overlap joint is arranged between the nitrocellulose membrane and the absorbent paper, the colloidal gold pad is composed of a glass cellulose membrane which adsorbs an anti-RSVN 4C3 monoclonal antibody marked by colloidal gold, and the nitrocellulose membrane is provided with a quality control line coated by a goat anti-mouse IgG polyclonal antibody and a detection line coated by an anti-RSV N protein monoclonal antibody.
The quality control line was obtained by coating goat anti-mouse IgG polyclonal antibody on nitrocellulose membrane at a concentration of 0.8 mg/ml; the test line was obtained by coating a monoclonal antibody against RSV N protein on a nitrocellulose membrane at a concentration of 0.5 mg/ml. The interval between the detection line and the quality control line is 0.5 cm.
Example 4
The embodiment provides a detection method of the immune colloidal gold test strip in the embodiment 3 for detecting the human respiratory syncytial virus, which comprises the following steps:
s1, dropping 200 mu L of test sample into the sample dropping hole, reacting for 10-15min and observing the result;
s2, judging the result:
the quality control line and the detection line are positive if red strips appear;
a red strip appears only on the quality control line, and the strip is negative;
if no strip appears in the quality control line, the result is judged to be invalid no matter whether the detection line has a red strip or not.
Example 5
This example provides the specificity test of the immune colloidal gold test strip of example 3.
The antigen detection diagnostic reagent prepared by the colloidal gold labeled RSVN4C3 monoclonal antibody specifically recognizes respiratory syncytial virus, shows positive reaction with two genotypes A and B of RSV, and has no cross reaction with influenza virus H1N1, enterovirus 71 (EV71) and coxsackie virus A16(CA16) (figure 4).
Example 6
This example provides the sensitivity test of the immune colloidal gold test strip of example 3.
The sensitivity test analysis of the antigen detection and diagnosis kit prepared from the colloidal gold-labeled RSVN4C3 monoclonal antibody shows that the antigen detection reagent can detect 105More than one PFU of viral particles (figure 5).
Example 7
The embodiment provides an ELISA kit for detecting respiratory syncytial virus antigen by a double-antibody sandwich method so as to diagnose respiratory syncytial virus infection.
Adding 100 mu L of RSVN4℃ 3 monoclonal antibody diluted by 10000 phosphate buffer solution (PBS pH7.2) into each well, adding 96-well ELISA reaction plates into each well, coating overnight at 4 ℃, adding 100 mu L of 3% BSA into each well, sealing for 1 hour at room temperature, washing the ELISA plates by PBS-Tween20 for 3 times, adding 100 mu L of patient nasopharyngeal washing liquid, reacting for 1 hour at 37 ℃, washing the ELISA plates by PBS-Tween20 for 3 times, adding 100 mu L of diluted horseradish peroxidase (HRP) -labeled RSVN4℃ 3 monoclonal antibody, reacting for 1 hour at 37 ℃, washing the ELISA plates by PBS-Tween20 for 3 times, adding HRP substrate (TMB or OPD), stopping the reaction after developing reaction for 30 minutes at 37 ℃, measuring the absorbance OD value of each sample well by using an enzyme labeling instrument, and judging that the OD value is more than 2 times higher than that of the negative control to be positive.
Example 8
The embodiment provides an ELISA kit for detecting a respiratory syncytial virus specific IgM antibody of patient serum by an IgM capture method so as to diagnose respiratory syncytial virus infection.
Adding 100 μ L of goat anti-human IgM antibody diluted with 1:500 phosphate buffer solution (PBS pH7.2) to a 96-well ELISA reaction plate per well, coating overnight at 4 deg.C, adding 100 μ L of 3% BSA per well, blocking at room temperature for 1 hr, PBS-Tween20 washes the ELISA plate 3 times, adds 100. mu.L 1:100 diluted patient serum, reacts at 37 ℃ for 1 hour, PBS-Tween20 was washed 3 times on the ELISA plate, 100. mu.L (50ng) of recombinant respiratory syncytial virus N protein was added thereto, and the reaction was carried out at 37 ℃ for 1 hour, PBS-Tween20 washing ELISA plate 3 times, diluted horseradish peroxidase (HRP) labeled RSVN4C3 monoclonal antibody, 37 degrees C reaction for 1 hours, washing the ELISA plate 3 times with PBS-Tween20, adding HRP substrate (TMB or OPD), developing at 37 deg.C for 30 min, terminating the reaction, and determining the absorbance OD value of each sample well with microplate reader, wherein the OD value is more than 2 times higher than that of the negative control, and determining the plate is positive. Respiratory syncytial virus specific IgM antibody positive allows the diagnosis of recent respiratory syncytial virus infections.
Sequence listing
<110> Guizhou province people hospital
<120> hybridoma cell strain RSVN4C3 secreting monoclonal antibody against respiratory syncytial virus
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<170> PatentIn version 3.5
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<213> Artificial sequence
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Claims (10)
1. A hybridoma cell strain RSVN4C3 secreting monoclonal antibody against respiratory syncytial virus is characterized in that the hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2020254.
2. The hybridoma cell line RSVN4C3 according to claim 1, wherein the preparation method comprises:
s1, separating a strain B133 strain Hep-2 cell culture supernatant of the RSV, and extracting total RNA of the RSV;
s2, amplifying the RSV N gene segment by adopting PCR to obtain a PCR amplification product, carrying out double enzyme digestion on the PCR product and the pQE30 expression vector by using BamHI and Sall respectively, and recovering a target gene segment and a vector segment;
s3, connecting a target gene fragment and a vector fragment by using T4 DNA ligase, converting a connecting product into competent escherichia coli XL1-blue, coating an LB ampicillin flat plate, respectively selecting single colonies after culture, inoculating and culturing, extracting plasmids, carrying out double enzyme digestion identification, carrying out agarose gel electrophoresis analysis, and carrying out gene sequencing on positive plasmids;
s4, converting the recombinant expression plasmid with correct sequencing into escherichia coli XL1-blue bacteria, inoculating, culturing and expanding, collecting bacteria, ultrasonically crushing, centrifuging, taking supernatant, carrying out chromatography, and purifying; carrying out 12% SDS-PAGE and Western blot identification analysis on the purified expression product to obtain purified recombinant RSV nucleoprotein;
s5, immunizing a BALA/C mouse by using the purified recombinant RSV nucleoprotein, performing cell fusion on splenocytes of the immunized mouse and SP2/0, and screening and culturing fused hybridoma cells by using HAT selective culture medium;
s6, detecting the hybridoma cell by indirect ELISA method, indirect immunofluorescence method and western-blot method, and screening hybridoma cell strain secreting monoclonal antibody against respiratory syncytial virus nucleocapsid protein.
3. Use of a hybridoma cell line RSVN4C3 according to claim 1 or 2 in the preparation of a reagent for detecting or diagnosing respiratory syncytial virus infection.
4. The monoclonal antibody against respiratory syncytial virus nucleocapsid protein secreted by the hybridoma cell strain RSVN4C3 according to claim 1, wherein the monoclonal antibody is antigen-antibody reactive with RSV N protein and is an IgG type antibody.
5. A kit for detecting or diagnosing respiratory syncytial virus infection comprising the monoclonal antibody of claim 4.
6. The kit for detecting or diagnosing respiratory syncytial virus infection according to claim 5, wherein the kit is an immune colloidal gold test strip.
7. The kit for detecting or diagnosing respiratory syncytial virus infection according to claim 6, wherein the test strip comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, a water absorbing paper and a PVC bottom plate as an assembly platform positioned below in sequence, the nitrocellulose membrane and the colloidal gold pad are overlapped, the nitrocellulose membrane and the water absorbing paper are overlapped, the colloidal gold pad is composed of a glass cellulose membrane absorbed with a colloidal gold-labeled RSVN4C3 monoclonal antibody, the nitrocellulose membrane is provided with a quality control line coated with goat anti-mouse IgG polyclonal antibody and a detection line coated with anti-RSV N protein monoclonal antibody.
8. The kit for detecting or diagnosing respiratory syncytial virus infection, according to claim 7, wherein said quality control line is obtained by coating goat anti-mouse IgG polyclonal antibody on a nitrocellulose membrane at a concentration of 0.8 mg/ml; the test line was obtained by coating a monoclonal antibody against RSV N protein on a nitrocellulose membrane at a concentration of 0.5 mg/ml.
9. The kit for detecting or diagnosing respiratory syncytial virus infection according to claim 7, wherein the detection method of the kit for human respiratory syncytial virus detection is as follows:
s1, dropping 200 mu L of test sample into the sample dropping hole, reacting for 10-15min and observing the result;
s2, judging the result:
the quality control line and the detection line are positive if red strips appear;
a red strip appears only on the quality control line, and the strip is negative;
if no strip appears in the quality control line, the result is judged to be invalid no matter whether the detection line has a red strip or not.
10. The kit for detecting or diagnosing respiratory syncytial virus infection according to claim 5, wherein said kit is an ELISA kit.
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