CN113249334A - Hybridoma cell strain SFTSN5G12 secreting monoclonal antibody against fever-associated thrombocytopenia syndrome virus - Google Patents
Hybridoma cell strain SFTSN5G12 secreting monoclonal antibody against fever-associated thrombocytopenia syndrome 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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Abstract
The invention discloses a hybridoma cell strain SFTSN5G12 secreting monoclonal antibodies against fever-associated thrombocytopenia syndrome virus, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2020133, wherein the monoclonal antibodies of the nucleocapsid proteins of the fever-associated thrombocytopenia syndrome virus (SFTS) secreted by the hybridoma cell strain SFTSN5G12 are used for preparing a kit for detecting or diagnosing SFTS 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 SFTSN5G12 secreting monoclonal antibodies against fever with thrombocytopenia syndrome virus.
Background
SFTS Virus (SFTSV), which is a pathogen of feber with thrombocytopenia syndrome (SFTS), is a novel virus of phlebovirus genus of bunyaviridae, a novel virus first found in china in 2009, and then found in japan, korea, and the like. SFTS is an important new infectious disease threatening the health and public health safety of people, the clinical manifestations of the SFTS are mainly characterized by fever with platelets, leucopenia and digestive tract symptoms, the severe mortality rate reaches 15-30%, and the health and life safety of people are seriously affected. So far, 26 cases are found in 26 provinces such as Henan, Jiangsu, Hubei, Shandong, Anhui and Liaoning in China, thousands of cases exist each year, and hundreds of people die. The virus is successfully separated in units such as Chinese national disease control center, Henan province disease control center, Jiangsu province disease control center and the like, and the pathogeny, epidemiology, clinical medicine and the like of the virus are researched. The virus sequence is already elucidated at present, but no specific drug treatment and vaccine prevention are available for SFTS.
SFTS is a natural epidemic disease commonly suffered by human and animals, and mostly occurs in areas with relatively poor medical and health conditions such as mountainous areas and hills, and the SFTS is easily confused with multi-system diseases such as blood, digestion, respiration and the like, and brings difficulty to diagnosis and differential diagnosis of the disease due to the lack of cognition of clinical doctors.
Disclosure of Invention
Aiming at the technical problems, the invention provides a hybridoma cell strain SFTSN5G12 secreting monoclonal antibody against fever-associated thrombocytopenia syndrome virus.
The technical scheme of the invention is as follows:
a hybridoma cell strain SFTSN5G12 secreting monoclonal antibody against fever with thrombocytopenia syndrome virus is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2020133.
Further, the preparation method of the hybridoma cell strain SFTSN5G12 comprises the following steps:
s1, inoculating a Vero-E6 cell to an SFTS virus Japanese isolate Yamakuchi strain, culturing for 7 days, taking a culture supernatant, carrying out reverse transcription on total RNA extracted according to the Trizol reagent specification to synthesize a cDNA first chain, and designing a specific primer by taking the cDNA first chain as a template, wherein the sequence is as follows:
P1：5’-GGAGCATGCATGTCGGAGTGGTCCAGG-3' (SphI cleavage site underlined)
P2：5′5’-AATAAGCTTTTACAGGTTTCTGTAAGCA-3 '-3' (HindIII cleavage site is underlined)
S2, PCR amplifying the SFTS virus full-length N gene segment by taking P1 and P2 as primers, 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, performing double enzyme digestion on the PCR product and a pQE30 expression vector by using SphI and Hind III respectively, and recovering a target gene fragment and a vector fragment;
s3, connecting with T4 DNA ligase, transforming a connecting product into competent escherichia coli XL1-blue, coating an LB ampicillin plate, statically culturing at 37 ℃ overnight, respectively selecting single colonies the next day, inoculating the single colonies into 3ml of LB culture solution, performing shaking culture at 37 ℃ for 16h, extracting plasmids, performing double digestion identification, performing agarose gel electrophoresis analysis, and performing gene sequencing on positive plasmids;
s4, transforming XL1-blue bacteria by the recombinant expression plasmid with correct sequencing, inoculating the XL1-blue bacteria into an LB culture medium containing 100 mu g/ml Amp, and carrying out shake culture at 37 ℃ for overnight; respectively inoculating the strain in a 5ml LB medium pipe containing 100 mu g/ml Amp and a 200ml LB 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 performing shaking culture and induction for 4 hours at 37 ℃; the collected thalli are subjected to ultrasonic disruption and centrifugation, and the supernatant is taken and purified by using an Ni2+ affinity chromatography column. Carrying out 12% SDS-PAGE and Western blot identification analysis on the purified expression product to obtain purified recombinant SFTS nucleoprotein;
s5, immunizing according to the dose of 100 micrograms per mouse, emulsifying by using Freund complete adjuvant for the first immunization, emulsifying by using incomplete adjuvant for the second immunization and the third immunization, wherein the immunization interval time is two weeks, and measuring the titer of serum antibodies by collecting blood from the tail of the mouse after 7 days of the third immunization; 3d, strengthening immunity once before fusion, taking immune mouse spleen cells and SP2/0 to perform cell fusion according to a conventional method, and screening and culturing fused hybridoma cells by using HAT selective medium;
s6, detecting the supernatant of the growing hybridoma cells by indirect ELISA and indirect immunofluorescence to screen the hybridoma cells secreting the anti-SFTS virus nucleocapsid protein monoclonal antibody;
s7, inoculating the hybridoma cells secreting the anti-SFTS virus nucleocapsid protein monoclonal antibody to the abdominal cavity of a mouse to prepare ascites, and identifying the ELISA, immunofluorescence titer, specificity of the monoclonal antibody and secreted monoclonal antibody subclass of the ascites.
The invention also provides application of the hybridoma cell strain SFTSN5G12 in preparation of a reagent for detecting or diagnosing SFTS virus infection.
Furthermore, the anti-SFTS virus nucleocapsid protein monoclonal antibody secreted by the hybridoma cell strain SFTSN5G12 has antigen-antibody reaction with the N protein of the SFTS virus, and belongs to IgG type antibodies.
The invention also provides a kit for detecting or diagnosing SFTS virus infection, which comprises the anti-SFTS virus nucleocapsid protein monoclonal antibody secreted by the hybridoma cell strain SFTSN5G 12.
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 adsorbs an anti-SFTS N protein 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 the anti-SFTS 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 is obtained by coating an anti-SFTS N protein monoclonal antibody 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 SFTS virus detection 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 is an ELISA kit.
The invention has the beneficial effects that: the invention clones the full length of the SFTSV N protein, expresses the recombinant N protein in escherichia coli, purifies the protein, immunizes a Balb/c mouse by using the purified recombinant nucleoprotein, fuses immune mouse spleen lymphocytes and mouse myeloma SP2/0 cells, and screens out 1 hybridoma cell strain secreting the anti-SFTS virus nucleocapsid protein monoclonal antibody. The hybridoma cell strain can stably secrete monoclonal antibodies (MAb) for resisting SFTS viruses, and the reactivity and specificity of the monoclonal antibodies (MAb) are identified, so that a foundation is laid for the research of diagnosis of virus infection, an infection mechanism and vaccine development in the future.
Drawings
FIG. 1 shows the results of 12% SDS-PAGE analysis of the-N protein product expressed by recombinant plasmids in E.coli. Column 1: a standard molecular weight protein; column 2: crushing the Escherichia coli, and then, obtaining an Escherichia coli supernatant; column 3: the crushed escherichia coli sediments; column 4: and (4) purifying to obtain the recombinant SFTSV N protein.
FIG. 2 shows the reaction results of the monoclonal antibody secreted by the hybridoma cell strain SFTSN5G12 with uninfected Vero-E6 cells and SFTS virus-infected Vero-E6 cells.
Wherein, panel A shows the reaction between the monoclonal antibody secreted by the SFTSN5G12 hybridoma cell and the SFTS virus infected Vero-E6 cell, and the specific immunofluorescence is presented in the cytoplasm; panel B shows that SFTSN5G12 hybridoma cells secrete monoclonal antibodies that are non-reactive with uninfected Vero-E6 cells.
FIG. 3 shows the result of Western blot analysis of the positive reaction between SFTSN-5G12 monoclonal antibody and purified and expressed recombinant SFTSV-N protein. Column 1: a standard molecular weight protein; column 2: purifying the recombinant SFTSV N protein.
FIG. 4 shows the results of the specificity test of the antigen detection diagnostic reagent prepared from the colloidal gold-labeled SFTSN-5G12 monoclonal antibody. The test strip only reacts with SFTS virus and presents a positive strip, but does not react with four serotype dengue fever viruses (D1, D2, D3 and D4), Japanese Encephalitis Virus (JEV), Yellow Fever Virus (YFV) and Rift Valley Fever Virus (RVFV).
FIG. 5 shows the result of the test on the sensitivity of the colloidal gold antigen-detecting reagent for the SFTSN-5G12 monoclonal antibody. The test strip can detect 105More than one PFU of viral particles.
FIG. 6 is a graph showing the results of one-time detection of the IgM antibody detection kit based on the SFTSN-5G12 monoclonal antibody capture method. The IgM antibody detection kit by the capture method has the advantages of deep color reaction of positive serum, low background of negative serum, obvious positive and negative contrast and capability of judging a result by naked eyes.
Detailed Description
Example 1
The hybridoma cell strain SFTSN5G12 secreting monoclonal antibody against fever with thrombocytopenia syndrome 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 days, and the preservation number is CCTCC NO: C2020133.
The hybridoma cell strain SFTSN5G12 is prepared by the following method:
1. materials and methods
S1, inoculating a Vero-E6 cell to an SFTS virus Japanese isolate Yamakuchi strain, culturing for 7 days, taking a culture supernatant, carrying out reverse transcription on total RNA extracted according to the Trizol reagent specification to synthesize a cDNA first chain, and designing a specific primer by taking the cDNA first chain as a template, wherein the sequence is as follows:
P1：5’-GGAGCATGCATGTCGGAGTGGTCCAGG-3' (SphI cleavage site underlined)
P2：5′5’-AATAAGCTTTTACAGGTTTCTGTAAGCA-3 '-3' (HindIII cleavage site is underlined)
S2, PCR amplifying the SFTS virus full-length N gene segment by taking P1 and P2 as primers, 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, performing double enzyme digestion on the PCR product and a pQE30 expression vector by using SphI and Hind III respectively, and recovering a target gene fragment and a vector fragment;
s3, connecting with T4 DNA ligase, transforming a connecting product into competent escherichia coli XL1-blue, coating an LB ampicillin plate, statically culturing at 37 ℃ overnight, respectively selecting single colonies the next day, inoculating the single colonies into 3ml of LB culture solution, performing shaking culture at 37 ℃ for 16h, extracting plasmids, performing double digestion identification, performing agarose gel electrophoresis analysis, and performing gene sequencing on positive plasmids;
s4, transforming XL1-blue bacteria by the recombinant expression plasmid with correct sequencing, inoculating the XL1-blue bacteria into an LB culture medium containing 100 mu g/ml Amp, and carrying out shake culture at 37 ℃ for overnight; respectively inoculating the strain in a 5ml LB medium pipe containing 100 mu g/ml Amp and a 200ml LB 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 performing shaking culture and induction for 4 hours at 37 ℃; collecting thallus, ultrasonic crushing, centrifuging, collecting supernatant, and adding Ni2+Purifying with affinity chromatography column. The purified expression product is identified by 12 percent SDS-PAGE and Western blotAnd (6) analyzing.
2. Expression and identification of recombinant RSV-N proteins
the-N gene is successfully amplified and cloned into an island expression vector pQE-30, and a-N protein product expressed by the recombinant plasmid in escherichia coli is analyzed by 12% SDS-PAGE, so that a specific protein band with the relative molecular weight of about 25kD can be seen, and the size of the specific protein band is consistent with the expected size. See fig. 1, where column 1: a standard molecular weight protein; column 2: crushing the supernatant of the large intestine bacillus; column 3: the crushed escherichia coli sediments; column 4: and (4) purifying to obtain the recombinant SFTSV N protein.
3. Establishment and identification of hybridoma cell strain
The purified recombinant nucleocapsid protein is used for immunizing BALB/C mice, immune spleen cells are taken to be fused with SP2/0 myeloma cells, and hybridoma cells are selected and cultured through HAT selection. Positive hybridoma cells are screened by using indirect immunofluorescence and indirect ELISA, and after 3 times of subcloning by a limiting dilution method, 1 hybridoma cell strain which stably secretes an anti-N protein monoclonal antibody is obtained and is respectively named as SFTSN-4G 12. The monoclonal antibody secreted by 5 strains of hybridoma cells has no reaction with uninfected Vero-E6 cells and reacts with SFTS virus infected Vero-E6 cells to present specific immunofluorescence in cytoplasm, as shown in FIG. 2, wherein, the graph A shows the reaction between the monoclonal antibody secreted by SFTSN5G12 hybridoma cells and SFTS virus infected Vero-E6 cells and presents specific immunofluorescence in cytoplasm; panel B shows that SFTSN5G12 hybridoma cells secrete monoclonal antibodies that are non-reactive with uninfected Vero-E6 cells. The specific identification of the indirect immunofluorescence method shows that the monoclonal antibody of the strain only reacts with infected cells, and does not react with dengue fever virus (D1, D2, D3 and D4), epidemic encephalitis B virus, yellow fever virus and rift valley fever virus infected cells. Injecting the hybridoma cell into abdominal cavity of mouse to prepare single titer of ascites, and measuring by indirect ELISA method of coating recombinant nucleoprotein, wherein titer of 5G12 is 1: 1000000; the titer is 1:100000 determined by indirect immunofluorescence; are obviously higher than the titer of immune serum. Through the identification of the monoclonal antibody subclasses, the heavy chain of the monoclonal antibody of the strain is IgG1, and the light chain is a kappa chain.
Western blot analysis shows that the SFTSN5G12 monoclonal antibody and the purified and expressed recombinant SFTSV-N protein show positive reaction, and the SFTSV-N protein is identified as shown in a figure 3, wherein the 1 st column: a standard molecular weight protein; column 2: purifying the recombinant SFTSV N protein.
The SFTSN-5G12 monoclonal antibody recognizes the SFTSV-N protein, and the recognized amino acid sequence is as follows: MSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALIIKKLKETGGDDWVK DTKFIIVFALTRGNKIVKASGKMSNSGSKRLMALQEKYGLVERAETRLSITPV RVAQSLPTWTCAAAAALKEYLPVGPAVMNLKVENYPPEMMCMAFGSLIPTA GVSEATTKTLMEAYSLWQDAFTKTINVKMRGASKTEVYNSFRDPLHAAVNS VFFPNDVRVKWLKAKGILGPDGVPSRAAEVAAAAYRNL are provided.
Example 2
The embodiment provides a kit for detecting or diagnosing SFTS virus infection, which is an immune colloidal gold test strip and comprises an anti-SFTS nucleocapsid protein monoclonal antibody secreted by the hybridoma cell strain SFTSN5G 12.
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 a lap joint is formed between the nitrocellulose membrane and the colloidal gold pad, a lap joint is formed between the nitrocellulose membrane and the absorbent paper, the colloidal gold pad is composed of a glass cellulose membrane which adsorbs an anti-SFTS N protein 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 the anti-SFTS N protein monoclonal antibody.
The 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 SFTS N-5G12 against SFTS N protein on a nitrocellulose membrane at a concentration of 0.5 mg/ml. And (3) adding the colloidal gold-labeled SFTSN-5G12 monoclonal antibody on the binding pad, and if the sample contains the SFTSV antigen, enabling the liquid to move to the binding pad to be combined with the colloidal gold-labeled SFTSN-5G12 monoclonal antibody, and then moving to the detection line to form an immune complex of the double-antibody sandwich antigen, so that a red strip visible to naked eyes is formed. If the sample does not contain the SFTSV antigen, the sample does not contain gold-labeled antigen-antibody complex and does not contain a red strip.
Example 3
The present embodiment provides a diagnostic reagent for antigen detection prepared by using SFTSN-5G12 monoclonal antibody labeled with colloidal gold, which is used for detecting SFTS virus, and 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 4
This example is a specific assay performed on the kit of example 2
As shown in FIG. 4, the colloidal gold labeled SFTSN-5G12 monoclonal antibody was used to prepare the antigen detection diagnostic reagent, and the colloidal gold diagnostic reagent specifically recognizes SFTS virus, and has no cross reaction with 4 serotypes of dengue fever virus (D1, D2, D3, D4), epidemic encephalitis B virus, yellow fever virus and rift valley fever virus.
Example 5
This example is a sensitivity test performed on the kit of example 2
As shown in FIG. 5, the SFTSN5G12 monoclonal antibody colloidal gold antigen detection reagent sensitivity analysis shows that the antigen detection reagent can detect 105More than one PFU of viral particles.
Example 6
The embodiment provides an ELISA kit for detecting SFTS viral antigen by a double-antibody sandwich method so as to diagnose SFTS viral infection.
Adding 100 mu L of SFTSN5G12 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 serum, reacting for 1 hour at 37 ℃, washing the ELISA plates by PBS-Tween20 for 3 times, adding 100 mu L of diluted SFTSN5G12 monoclonal antibody marked by horseradish peroxidase (HRP), reacting for 1 hour at 37 ℃, washing the ELISA plates by PBS-Tween20 for 3 times, adding HRP substrates (ABTS or TMB or OPD), performing chromogenic reaction for 30 minutes at 37 ℃, stopping the reaction, 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 negative control as positive.
Example 7
This example provides an ELISA kit for detecting serum IgM antibodies specific to SFTS virus in patients by IgM capture method for diagnosing SFTS 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 washing ELISA plate 3 times, adding 100 u L (25ng) recombinant SFTSV-N protein, 37 ℃ reaction for 1 hours, the ELISA plate was washed 3 times with PBS-Tween20, diluted horseradish peroxidase (HRP) -labeled SFTSN5G12 monoclonal antibody, reacted at 37 ℃ for 1 hour, washing the ELISA plate with PBS-Tween20 for 3 times, adding HRP substrate (ABTS or TMB or OPD), performing color reaction at 37 ℃ for 30 minutes, terminating the reaction, and determining the absorbance OD value of each sample well by using an enzyme-labeling instrument, wherein the OD value is more than 2 times higher than that of a negative control, and the positive is judged. SFTS virus specific IgM antibody positive can diagnose the recent infection of SFTS virus. 115 suspected patient sera in an SFTS virus epidemic area of Henan province are used for testing the detection effect of ELISA by using an IgM capture method, 85 positive IgM antibodies and 30 negative IgM antibodies are detected, and the result is completely consistent with the result of a kit for detecting the total antibodies of the patient sera in the market. FIG. 6 is a graph showing the results of one-time detection of the IgM antibody detection kit based on the SFTSN-5G12 monoclonal antibody capture method. As can be seen from FIG. 6, the IgM antibody detection kit by the capture method has deep color reaction of positive serum, low background of negative serum, obvious positive and negative contrast, and the result can be judged by naked eyes.
Sequence listing
<110> Guizhou province people hospital
<120> hybridoma cell strain SFTSN5G12 secreting monoclonal antibody against fever-induced thrombocytopenia syndrome virus
<130> do not
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 27
<212> DNA
<213> Artificial sequence
<221> misc_feature
<222> (1)..(27)
<223> specific primers for HRSV N gene fragment amplification designed according to experimental requirements
<400> 1
ggagcatgca tgtcggagtg gtccagg 27
<210> 2
<211> 28
<212> RNA
<213> Artificial sequence
<221> misc_feature
<222> (1)..(28)
<223> specific primers for HRSV N gene fragment amplification designed according to experimental requirements
<400> 2
aataagcttt tacaggtttc tgtaagca 28
Claims (10)
1. A hybridoma cell strain SFTSN5G12 secreting monoclonal antibody against fever with thrombocytopenia syndrome 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: C2020133.
2. The hybridoma cell strain SFTSN5G12 according to claim 1, which is prepared by the following steps:
s1, inoculating the Yamakuchi strain of the SFTS virus isolate to Vero-E6 cells, culturing, taking supernate, and extracting total RNA of the SFTS;
s2, amplifying the full-length N gene fragment of the SFTS virus 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 SphI and Hind III respectively, 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 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 XL1-blue bacteria, performing inoculation culture, amplification culture and shaking culture induction, collecting the bacteria, performing ultrasonic disruption and centrifugation, taking supernatant, performing chromatography and purification; carrying out 12% SDS-PAGE and Western blot identification analysis on the purified expression product to obtain purified recombinant SFTS nucleoprotein;
s5, immunizing a mouse by using the purified recombinant SFTS nucleoprotein, taking splenocytes of the immunized mouse to fuse with SP2/0 cells, and screening and culturing fused hybridoma cells by using HAT selective culture medium;
s6, detecting the supernatant of the growing hybridoma cells by indirect ELISA and indirect immunofluorescence to screen the hybridoma cells secreting the anti-SFTS nucleocapsid protein monoclonal antibody;
s7, inoculating the hybridoma cells secreting the anti-SFTS nucleocapsid protein monoclonal antibody to the abdominal cavity of a mouse to prepare ascites, and identifying ELISA and immunofluorescence titer of the ascites, specificity of the monoclonal antibody and subclass of the secreted monoclonal antibody.
3. The use of the hybridoma cell line SFTSN5G12 of claim 1 or 2 in the preparation of a reagent for detecting or diagnosing SFTS virus infection.
4. The monoclonal antibody against SFTS virus nucleocapsid protein secreted by the hybridoma cell strain SFTSN5G12 as claimed in claim 1, wherein the monoclonal antibody reacts with the N protein of SFTS virus by antigen-antibody reaction and belongs to IgG type antibody.
5. A kit for detecting or diagnosing SFTS viral infection comprising the monoclonal antibody of claim 4.
6. The kit for detecting or diagnosing SFTS virus infection according to claim 5, wherein the kit is an immune colloidal gold test strip.
7. The kit for detecting or diagnosing SFTS virus infection according to claim 6, it is characterized in that 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 the connection sequence, the cellulose nitrate membrane is lapped with the colloidal gold pad, the cellulose nitrate membrane is lapped with the absorbent paper, the colloidal gold pad is composed of a glass cellulose membrane adsorbed with colloidal gold labeled anti-SFTS N protein monoclonal antibody, the nitrocellulose membrane is overlapped with the absorbent paper, the colloidal gold pad is composed of a glass cellulose membrane absorbed with colloidal gold labeled anti-SFTS N protein monoclonal antibody, 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-SFTS N protein monoclonal antibody.
8. The kit for detecting or diagnosing SFTS viral infection according to claim 7, wherein said 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 is obtained by coating the anti-SFTS N protein monoclonal antibody on a nitrocellulose membrane at the concentration of 0.5 mg/ml.
9. The kit for detecting or diagnosing SFTS virus infection according to claim 7, wherein the detection method of the kit for SFTS 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 SFTS virus infection according to claim 5, wherein the kit is an ELISA kit.
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