A monoclonal antibody against Gurtu virus nucleoprotein and its application

Abstract

The present invention provides a monoclonal antibody of Gurtu virus nucleoprotein and its application, which relates to the fields of biotechnology and virology. The monoclonal antibody screened by the present invention has a broad spectrum, can recognize the nucleoprotein of Gurtu virus, especially can recognize the 6-peptide antigen epitope in the nucleoprotein of Gurtu virus, and can also cross-recognize the nucleoprotein of Dabie Banda virus. The present invention further clarifies the amino acid sequence of the antigen epitope that the monoclonal antibody can recognize. The monoclonal antibody provided by the present invention can be used to prepare detection reagents related to Banda virus and Dabie Banda virus, which is conducive to the auxiliary diagnosis of these two virus infections, and has important significance for the basic research, clinical research, and epidemiological investigation of Gurtu virus and Dabie Banda virus in the genus Banda virus.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology and virology, and specifically to a monoclonal antibody for Gurtu virus and application of the monoclonal antibody, in particular to the joint detection of Gurtu virus and Dabiebandha virus, in particular to the related detection of Gurtu virus and related drugs against Gurtu virus. Background Art

[0002] Guertu bandavirus (GTV) is an emerging tick-borne virus. In 2018, Guertu virus was isolated from specimens of Dermacentor nuttalli ticks collected in Xinjiang. The main host is the Dermacentor nuttalli tick in the Guertu area of ​​Xinjiang. The Dermacentor nuttalli tick is a dominant tick species in a certain area in western China and is also the main vector of Guertu virus. Its average positive rate is 7.89%, which is higher than the positive rate of SFTSV carried by long-horned ticks in central China (2.1-5.4%). In vitro and in vivo experiments have shown that Guertu virus can infect a variety of mammals and human cell lines, and can cause lesions in the brain, lungs, kidneys, and spleen of mice. Detection of human serum in the Guertu area of ​​Xinjiang has also proved that Guertu virus can be transmitted between ticks, animals, and humans. It is a potentially dangerous emerging virus. The genome of Gurtu virus is composed of single-stranded, negative-strand RNA, which is divided into three segments: large (L), medium (M), and small (S), which encode RNA-dependent polymerase, precursor envelope glycoprotein (Glycoprotein, including Gn and Gc, referred to as GP protein) and nucleoprotein (Nuclear protein, NP), respectively. GP protein has important receptor binding sites and antigenic epitopes for viruses to invade host cells, and can stimulate the body to produce specific immune responses; nucleoprotein can stimulate the body to produce early humoral immunity and cellular immunity, and only NP protein can be detected in the body in the early stage of infection.

[0003] Dabie Banda virus (formerly known as Severe fever with thrombocytopenia syndrome virus, SFTSV) is a new tick-borne virus that is most closely related to Gurtu virus in evolution. Both belong to the Bunyavirales order, Phenuiviridae family, and bandavirus genus. In 2010, SFTSV was discovered and named from samples of Severe fever with thrombocytopenia syndrome (SFTS) cases. It is currently prevalent in Hubei, Henan, Zhejiang, Shandong and other regions in central and eastern China, as well as Japan and South Korea. After infection, it can cause clinical symptoms such as high fever, anorexia, muscle pain, chills, swollen lymph nodes, leukopenia, and thrombocytopenia, with a mortality rate of about 5% to 30%. It is mainly transmitted through tick bites, and there are also reports of human-to-human transmission. According to the latest "Catalogue of Pathogenic Microorganisms Transmitted Among Humans (Draft for Comments)", the National Health Commission adjusted the hazard level of five viruses including the "Dabiebanda virus" from the original "Second Category" to the "Third Category".

[0004] At present, regarding monoclonal antibodies to the Dabiebanda virus (SFTSV), related studies have used phage antibody libraries to isolate more than 400 monoclonal antibodies against the Dabiebanda virus nucleoprotein from patients in the recovery period of the Dabiebanda virus, highlighting the important role of nucleoprotein in inducing humoral immunity and demonstrating the importance of developing monoclonal antibodies targeting nucleoprotein as a new virus detection method. There are also related studies that used the spleen cells of Dabiebanda virus-infected mice to fuse with myeloma cells, screened and obtained monoclonal antibodies against the Dabiebanda virus nucleoprotein, and established a sandwich ELISA detection method for the detection of Dabiebanda virus patient serum. However, there are currently no research reports on monoclonal antibodies to the nucleoprotein of the Gurtu virus, nor are there specific detection methods for the Gurtu virus. Since both the Gurtu virus and the Dabiebanda virus are RNA viruses, the genome replication lacks a proofreading mechanism, and the virus's own mutations are prone to changes in virulence, host adaptation, and epidemiology. Different strains may recombine to produce new viruses that adapt to other media, thereby changing the distribution range of the epidemic and leading to the emergence of new diseases. In addition, effective drugs and vaccines to prevent infection with these two viruses are still in the research stage, and there are no effective diagnostic methods or diagnostic reagents. Therefore, providing a simple, rapid, and highly sensitive detection reagent or therapeutic drug for infection with emerging viruses such as Gurtu virus has become the primary problem to be solved in the current clinical diagnosis / treatment of such diseases. In order to prevent the possible spread and cross-infection of Gurtu virus with viruses of the same genus in the future, the study of monoclonal antibodies against this virus is of great significance for the prevention and control of Gurtu virus and other members of the genus Bandavirus. Summary of the invention

[0005] The object of the present invention is to at least partially overcome the deficiencies of the prior art and provide a broad-spectrum monoclonal antibody that recognizes Gurtu virus.

[0006] The purpose of the present invention is also to fill the gap in domestic detection reagents for Gurtu virus, and provide three Gurtu virus detection methods and reagents for Gurtu virus detection.

[0007] The present invention also aims to provide a monoclonal antibody of Gurtu virus, providing a research basis for an alternative antibody for auxiliary diagnosis of Gurtu virus infection.

[0008] In order to achieve the above purpose or one of the purposes, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a monoclonal antibody to the nucleoprotein of Gurtu virus, which can recognize a linear epitope having an amino acid sequence as shown in SEQ ID NO.1, and can also recognize a spatial epitope having an amino acid sequence as shown in SEQ ID NO.1.

[0010] In the second aspect, the three complementary determining regions CDR (HCDR1, HC DR2, HCDR3) of the heavy chain variable region of the monoclonal antibody respectively have the following amino acid sequences: HCDR1 shown in SEQ ID NO.6; HCDR2 shown in SEQ ID NO.7; HCDR3 shown in SEQID NO.8; the three complementary determining regions CDR (LCDR1, LCDR2, LCDR3) of the light chain variable region of the monoclonal antibody have the following amino acid sequences: LCDR1 shown in SEQ ID NO.9; LCDR2 shown in the amino acid sequence KAS; LCDR3 shown in SEQID NO.10.

[0011] Preferably, the heavy chain variable region of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO.4.

[0012] Preferably, the heavy chain variable region of the monoclonal antibody is encoded by the nucleic acid sequence shown in SEQ ID NO.2.

[0013] Preferably, the light chain variable region of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO.5.

[0014] Preferably, the light chain variable region of the monoclonal antibody is encoded by the nucleic acid sequence shown in SEQ ID NO.3.

[0015] In a third aspect, the present invention provides a polynucleotide containing a monoclonal antibody against the above-mentioned Gurtu virus nucleoprotein.

[0016] In a fourth aspect, the present invention provides a vector containing a monoclonal antibody against the above-mentioned Gurtu virus nucleoprotein.

[0017] In a fifth aspect, the present invention provides a host cell containing the monoclonal antibody against the above-mentioned Gurtu virus nucleoprotein.

[0018] In a sixth aspect, the present invention also provides the use of monoclonal antibodies against the nucleoprotein of the Gurtu virus in the preparation of polynucleotides, recombinant vectors, expression vectors, recombinant proteins, genetically engineered host cells, and Gurtu virus-related detection reagents.

[0019] Beneficial effects of the present invention:

[0020] The present invention obtains a broad-spectrum monoclonal antibody 22G1 (abbreviated as monoclonal antibody 22G1) for Gurtu virus, which is a monoclonal antibody derived from the nucleoprotein of Gurtu virus and can recognize the 6-peptide antigen epitope (Linear epitope, LM) in the nucleoprotein of Gurtu virus; in addition, it can also cross-recognize the nucleoprotein of Dabiebandha virus. Monoclonal antibody 22G1 is verified by protein immunoblotting detection, indirect immunofluorescence detection and enzyme-linked immunosorbent assay, and monoclonal antibody 22G1 can recognize both Gurtu virus and Dabiebandha virus, but the enzyme-linked immunosorbent assay (ELISA) result of monoclonal antibody 22G1 shows that the detection efficiency of monoclonal antibody 22G1 for Gurtu virus is slightly higher than that for Dabiebandha virus.

[0021] The active epitopes of the broad-spectrum recognition of the monoclonal antibody 22G1 obtained by the present invention on Gurtu virus and Dabie Banda virus of the genus Banda virus are mainly directed to nucleoprotein, and can be prepared into a detection reagent for the genus Banda virus, or used for pathogen detection related to the genus Banda virus, and can distinguish whether the pathogen in the sample contains Gurtu virus and Dabie Banda virus. The three detection platforms provided by the present invention, namely, protein immunoblotting (WB), indirect immunofluorescence detection (IFA) and enzyme-linked immunosorbent assay (ELISA), can be used in major fields such as basic research, clinical research, and epidemiological investigation of Gurtu virus and Dabie Banda virus in the genus Banda virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a protein immunoblot detection image of Gurtu virus monoclonal antibody 22G1, wherein 22G1 is monoclonal antibody 22G1, PC is a positive control, i.e., a polyclonal antibody against Gurtu virus nucleoprotein, and NC is a negative control, i.e., healthy mouse serum;

[0023] Figure 2 This is an indirect immunofluorescence detection image of Gurtu virus monoclonal antibody 22G1, 22G1 is monoclonal antibody 22G1, PC is the positive control, i.e., the polyclonal antibody of Gurtu virus nucleoprotein, and NC is the negative control, i.e., the serum of healthy mice;

[0024] Figure 3 It is a protein immunoblot detection diagram of Gurtu virus monoclonal antibody 22G1 and truncated Gurtu virus nucleoprotein 16 peptides, wherein P26, 27, 28, 29, 30, 31, 32 represent the numbers of 16 peptides formed by the repeated arrangement of 8 amino acids in the nucleoprotein of Gurtu virus, and NC is the negative control, i.e., healthy mouse serum;

[0025] Figure 4It is a protein immunoblot detection image of Gurtu virus monoclonal antibody 22G1 and truncated Gurtu virus nucleoprotein 8 peptides, wherein P33, 34, 35, 36, 37, 38, 39, 40, 41 represent the numbers of 8 peptides formed by the repeated arrangement of 7 amino acids of Gurtu virus nucleoprotein, and NC is the negative control, i.e., healthy mouse serum;

[0026] Figure 5 It is a protein immunoblot detection diagram of Gurtu virus monoclonal antibody 22G1 and prokaryotic expressed nucleoprotein, wherein PC is a positive control, i.e., a polyclonal antibody against Gurtu virus nucleoprotein, NC is a negative control, i.e., healthy mouse serum, A is a viral protein expressed by the recombinant vector pET28a-GTV-NP, and B is a viral protein expressed by the recombinant vector pET28a-SFTSV-NP;

[0027] Figure 6 It is an indirect immunofluorescence detection diagram of Gurtu virus monoclonal antibody 22G1 and eukaryotic expressed nucleoprotein, wherein PC is a positive control, i.e., a polyclonal antibody against Gurtu virus nucleoprotein, NC is a negative control, i.e., healthy mouse serum, the first row of pictures corresponds to the viral protein expressed by the recombinant vector pCAGGS-GTV-NP, and the second row of pictures corresponds to the viral protein expressed by the recombinant vector pCAGGS-SFTSV-NP;

[0028] Figure 7 This is the result of the enzyme-linked immunosorbent assay of Gurtu virus monoclonal antibody 22G1 and eukaryotic expressed nuclear protein. The horizontal axis is the logarithmic value of the dilution multiple of Gurtu virus monoclonal antibody 22G1 (logarithmic value with base 2), and the vertical axis is the OD value of the microplate reader. 450-630 The absorbance values ​​measured, wherein 22G1-GTV represents the ELISA test results after incubation of Vero cells infected with GTV with different concentrations of monoclonal antibody 22G1, 22G1-SFTSV represents the ELISA test results after incubation of Vero cells infected with SFTSV with different concentrations of monoclonal antibody 22G1, and Cut off line (Cut offline) = 0.024. DETAILED DESCRIPTION

[0029] Exemplary embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein the same or similar represents the same concept, such as Gurtu virus = GTV, Dabiebanda virus = fever with thrombocytopenia syndrome virus = SFTSV, monoclonal antibody = monoclonal antibody, etc.

[0030] In the following detailed description, for ease of explanation, numerous specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. The following embodiments are only used to illustrate the present invention and should not be considered to limit the scope of the present invention.

[0031] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used are commercially available unless otherwise specified. Among them, Freund's complete adjuvant, Freund's incomplete adjuvant, HAT medium, etc. were purchased from SIGMA-Aldrich in the United States; serum-free DMEM medium, QIXzol nucleic acid extraction reagent, etc. were purchased from Hubei Xinzongke Viral Disease Engineering Technology Co., Ltd.; PEG was purchased from Glpbio in the United States; Vero cells were purchased from ATCC; HRP-labeled goat anti-mouse or rabbit IgG secondary antibodies, HRP-labeled goat anti-rabbit IgG H&L fluorescent secondary antibodies and other antibodies were purchased from Abcam (Shanghai) Trading Co., Ltd. (abcam); chemiluminescent imaging analyzer Image Quant TM LAS4000 was purchased from GE Healthcare in the United States; TOPO-Blunt flat-end cloning kit (TOPO-Blunt Cloning Kit) was purchased from Nanjing Novozyme Biotechnology Co., Ltd.; high-content cell imaging analysis system was purchased from PerkinElmer in the United States; the primers involved in the present invention were commissioned to be synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.; the sequence determination of the present invention was commissioned to Beijing Qingke Biotechnology Co., Ltd. (Wuhan Branch); Gurtu virus (GTV) and Dabiebanda virus (SFTSV) are preserved by the National Virus Resource Bank of Wuhan Institute of Virology, Chinese Academy of Sciences. However, it is obvious that one or more embodiments can also be implemented without these specific details. If the specific conditions are not indicated in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0032] The molecular biology experimental methods not specifically described in the following examples are all carried out in accordance with the specific methods listed in the book "Molecular Cloning Experiment Guide" (3rd edition) by J. Sambrook, or in accordance with the kit and product instructions. The application objects of the present invention are derived from the biological products to be tested, which are inanimate samples; the direct purpose of the detection is to ensure the safety of the biological products to be tested, thereby facilitating the improvement of the quality control standards of biological products, and there is no process for obtaining the diagnosis results of the disease or the health status. Therefore, the present invention does not belong to the diagnosis method of the disease, and meets the basic requirements of the Patent Law for the subject of patent protection.

[0033] The present invention provides a monoclonal antibody of Gurtu virus, a preparation method and its application. By using the cell fusion technology of spleen cells of mice immunized with Gurtu virus and myeloma cells, the inactivated virus of Gurtu virus is used as the target, and the cell strain capable of secreting monoclonal antibody 22G1 of the present invention is screened; by using molecular biology technology, the amino acid sequence of linear epitopes recognized by monoclonal antibody 22G1, as well as the amino acid sequence and the encoded nucleic acid sequence of monoclonal antibody 22G1 itself are identified, and eukaryotic and prokaryotic expression vectors containing Gurtu virus and Dabiebandha virus are respectively constructed, and the obtained recombinant vectors are respectively constructed into three detection platforms of protein immunoblotting detection (WB), indirect immunofluorescence detection (IFA) and enzyme-linked immunosorbent assay (ELISA), further verifying that monoclonal antibody 22G1 can recognize the nucleoprotein of Gurtu virus and can also cross-recognize the nucleoprotein of Dabiebandha virus; and monoclonal antibody 22G1 can recognize both the linear epitope of nucleoprotein and the spatial epitope of nucleoprotein, and has a broad-spectrum recognition ability. The specific embodiments of the present invention are described below, and the specific contents are as follows:

[0034] Example 1. Preparation of monoclonal antibody 22G1

[0035] The present invention immunizes mice with concentrated and inactivated Gurtu virus, and then separates spleen cells and myeloma cells for cell fusion on feeder cells, takes Gurtu virus infected cells as positive well targets, screens hybridoma cell lines, and finally screens out the monoclonal cell line 22G1 that secretes anti-Gurtu virus nucleoprotein from the positive wells. The process of preparing hybridoma cell lines includes animal immunization, separation of spleen cells, cell fusion and monoclonalization, and the specific preparation process is as follows:

[0036] 1) Animal immunization

[0037] According to the established animal immunization plan, 6-8 week old SPF grade C57 / BL6 mice were selected as immunized animals, and the immunogen was concentrated and inactivated Gurtu virus. After the virus was emulsified with an equal volume of Freund's (in)complete adjuvant, two immunizations were performed by intraperitoneal and subcutaneous multi-point injections, with an interval of two weeks between each immunization. The immunization volume was 200 μL / mouse, of which the virus amount was 8×10 7 TCID 50 / Only.

[0038] 2) Isolation of splenocytes

[0039] Immunized C57 / BL6 mice were taken, and the orbital bleeding was performed after anesthesia. The mice were soaked in 75% alcohol for 5 minutes, and then the mice were dissected using sterile instruments in a biosafety cabinet. The spleen of the mice was taken, the surrounding connective tissue was stripped, and the spleen was placed in a sterile homogenizer. 5 mL of serum-free DMEM medium was added to grind, and then 10 mL of serum-free DMEM medium was added to mix thoroughly. After standing for 10 minutes, the grinding supernatant was taken, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was added with 10 mL of serum-free DMEM medium to resuspend the cells. The washed spleen cells were counted with trypan blue, and the immune spleen cell suspension (1×10 8 )spare.

[0040] 3) Cell fusion and monoclonalization

[0041] The immune spleen cell suspension (1×10 8 ) and myeloma cells (2×10 7 ) (the ratio of spleen cells: myeloma cells = 5:1) was fully mixed, centrifuged at 1600rpm for 10 minutes, and the supernatant was discarded to obtain mixed cells. The centrifuge tube containing the mixed cells was placed in a 37°C water bath environment, and 1mL of preheated 50% PEG was slowly added, mixed while adding, and then 10mL of preheated DMEM medium was slowly added within 5 minutes. After standing at 37°C for 10 minutes, centrifuged at 1000rpm for 8 minutes, and the supernatant was discarded. The centrifuge tube containing the mixed cells was resuspended with 50mL of HAT medium, and the suspension containing the mixed cells was evenly spread in a 96-well cell plate, 100μL of cell suspension was added to each well, and each well in the 96-well cell plate was made into a single cell state after limiting dilution, and placed in a 37°C, 5% CO2 cell culture incubator for 7-10 days. The supernatant of the fused cells was taken, and the monoclonal antibody 22G1 was unexpectedly screened out from the positive wells for the next step of monoclonal antibody verification.

[0042] Example 2: Validation of mAb 22G1

[0043] 1) Western blotting (WB)

[0044] Vero cells were infected with Gurtu virus and amplified in Vero cells. Vero cells and cell supernatants infected with Gurtu virus were collected and inactivated by adding β-propiolactone. The viral proteins were separated by 12% SDS-PAGE gel electrophoresis and transferred to a 0.2 μm polyvinylidene fluoride membrane. The membrane was blocked with 5% (w / v) skimmed milk powder prepared in PBS at 37°C for 1 hour. PCR detection showed that Gurtu virus was positively amplified in both Vero cells and cell supernatants.

[0045] The positive control was a rabbit polyclonal antibody against Gurtu virus nucleoprotein, diluted 1:2000 with primary antibody diluent; the negative control was healthy mouse serum, diluted 1:2000 with primary antibody diluent; the supernatant of the above-mentioned fusion cells was taken as the primary antibody and added to the above-mentioned blocked polyvinylidene fluoride membrane, incubated at 37°C for 2 hours, washed 3 times with TBS-T, and then added with HRP-labeled goat anti-mouse or rabbit IgG antibody diluted 1:2000, incubated again at 37°C for 1 hour, and then immunoblotted and imaged on the chemiluminescence imaging analyzer Image Quant LAS4000.

[0046] The results of protein immunoblotting of the screened monoclonal antibody 22G1 and the isolated Gurtu virus after inactivation are as follows: Figure 1 As shown, it is proved that monoclonal antibody 22G1 can recognize the Gurtu virus separated from the gel after inactivation, that is, monoclonal antibody 22G1 can recognize the linear epitope of the Gurtu virus nucleoprotein.

[0047] 2) Indirect immunofluorescence assay (IFA)

[0048] Virus culture: Vero cells were cultured at 1×10 4 / well were evenly spread into a 96-well cell plate, and DMEM culture medium containing 2% fetal bovine serum was added at 100μ / well. When the cell density grew to more than 80%, the supernatant was discarded; 100μL of cell supernatant containing Gurtu virus was added at a multiplicity of infection MOI=1, and the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 3 days.

[0049] In situ fixation: remove the cell plate, wash it once with sterile PBS, add 4% paraformaldehyde at 300 μL / well, fix for 15 minutes, and then wash it three times with PBS; add 0.4% TritonX-100 at 100 μL / well to the fixed cell plate, permeabilize for 10 minutes, and wash it four times with PBS; add 5% BSA at 100 μL / well to the permeabilized cell plate, block at 37°C for 1 hour, and discard the blocking solution.

[0050] IFA detection: The positive control was a rabbit polyclonal antibody against Gurtu virus nucleoprotein, diluted 1:2000 with 1% BSA; the negative control was healthy mouse serum, diluted 1:2000 with 1% BSA; the supernatant of the fusion cells secreting the above-mentioned monoclonal antibody 22G1 was taken as the primary antibody, added to the above-treated 96-well cell plate at 100μ / well, and incubated at 37°C for 2 hours; the secondary antibody was HRP-labeled goat anti-rabbit IgG (H+L) fluorescent antibody, diluted 1:2000 with 1% BSA, added to 50μL / well of the diluted secondary antibody, and incubated at 37°C for 1 hour; then, the fluorescent dye Hoechest 33258 was added at 50μL / well, left at room temperature for 5 minutes, and the results were observed using a high-content cell imaging analysis system.

[0051] The results of indirect immunofluorescence detection of the screened monoclonal antibody 22G1 with inactivated and in situ fixed Gurtu virus are as follows Figure 2 As shown, it is proved that monoclonal antibody 22G1 can effectively recognize the spatial epitope of Gurtu virus.

[0052] Example 3: Antigenic epitopes recognized by monoclonal antibody 22G1

[0053] 1) Truncated short peptides of Gurtu virus nucleoprotein

[0054] According to the amino acid sequence of the nucleoprotein of Gurtu virus, 32 truncated 16-peptides were prepared and numbered as P1 to P32. The two 16-peptides before and after contain 8 identical amino acid arrangements, and the first amino acid of the latter numbered 16-peptide is the same as the ninth amino acid of the previous numbered 16-peptide, and 32 16-peptides are prepared in this order.

[0055] Referring to the method of protein immunoblotting (WB) detection in Example 2, 16 peptides that reacted positively with monoclonal antibody 22G1 were screened by WB detection. Figure 3 As shown, the figure representatively shows the results including a positive reaction, that is, the 16-peptide of the GTV nucleoprotein recognized by the monoclonal antibody 22G1 is numbered as P31.

[0056] According to the 16 amino acid sequence of the 16-peptide numbered P31, 9 truncated 8-peptides were further prepared, numbered P33 to P41. Each 8-peptide was formed by selecting 8 amino acids from the 16 amino acid sequence in sequence, and the first amino acid of the latter numbered 8-peptide was the same as the second amino acid of the previous numbered 8-peptide, and 9 8-peptides were prepared by sequential arrangement and combination. According to the encoding nucleic acid sequence of the 8-peptide, BamH I and Sal I restriction sites were added before and after the nucleotide sequence of the truncated 8-peptide, respectively, and the DNA fragment was commissioned to be synthesized by Qingke Biotechnology, and the synthesized DNA fragment was cloned into the pXXGST prokaryotic expression plasmid vector, and then transformed into the competent Escherichia coli BL21 by electroporation to obtain a monoclonal colony expressing the recombinant plasmid. In 3 mL of liquid LB medium, culture at 37°C, 220 rpm for 12 hours; 30 μL of the culture solution was transferred to 3 mL of fresh liquid LB medium, and the culture was continued at 30°C, 220 rpm for 4 hours, the cultured solution was taken out and protein expression was induced at 42°C, 220 rpm, centrifuged at 5000 rpm for 5 minutes, the supernatant was removed, and the bacteria were resuspended in 200 μL of 1× Loading buffer, and the proteins were separated by 12% SDS-PAGE gel electrophoresis. After staining with Coomassie Brilliant Blue staining solution, the expression of the truncated 8-peptide was identified.

[0057] 2) WB identification of truncated octapeptides

[0058] Referring to the method of protein immunoblotting (WB) detection in Example 2, eight peptides that reacted positively with monoclonal antibody 22G1 were screened by WB detection. Figure 4 As shown in the figure, the 8 peptides with positive reactions are numbered P34-36, that is, the 8 peptides of GTV nucleoprotein recognized by monoclonal antibody 22G1 are numbered P34, P35 and P36, that is, P34-36 are specific linear epitopes recognized by monoclonal antibody 22G1. According to the analysis, these truncated 8 peptides all contain the amino acid sequence described in SEQ ID NO.1: GPDGLP.

[0059] Example 4: Sequence Identification of Monoclonal Antibody 22G1

[0060] Total RNA was extracted from the cultured Gurtu virus monoclonal antibody 22G1 cell line, and reverse transcribed into cDNA. The variable region (including the heavy chain variable region and the light chain variable region) fragments were amplified and recovered, and then constructed into the cloning vector pTOPO-Blunt. The cloned vector after amplification and culture was sent to the detection sequence with universal primers M13F and M13R. The specific process is as follows:

[0061] 1) Nucleic acid extraction:

[0062] Take the monoclonal antibody 22G1 cell line obtained by the above screening, add 1 mL of QIXzol nucleic acid extraction reagent, repeatedly blow and lyse the cells, and extract total RNA according to the instructions of QIXzol nucleic acid extraction reagent. Use a UV spectrophotometer to detect the concentration and purity of the extracted RNA.

[0063] 2) Reverse transcription:

[0064] Take 3 μg of the extracted RNA, add Oligo(dT) (500 μg / mL), 10 mM dNTP Mix, 5× First-Strand Buffer, 0.1 M DTT, RNaseOUT TM Recombinant Ribonuclease Inhibitor and M-MLVRT were used for reverse transcription to obtain synthetic cDNA.

[0065] 3) Sequencing after amplification:

[0066] PCR amplification of the variable region: Take 2 μL cDNA as a template, add primers designed by Orlandi et al. (1989) to amplify the mouse variable region gene, and use a high-fidelity enzyme (2×Max MasterMix) for PCR amplification to obtain the target gene fragment. The primer sequences are shown in Table 1 below.

[0067] Table 1: Variable region PCR amplification primer sequence list

[0068]

[0069]

[0070] PCR amplification program: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, and 30 cycles; finally extension at 72°C for 5 min.

[0071] PCR product recovery: Take 30-50 μL of the PCR product for 1% agarose electrophoresis, and purify the PCR product of about 300 bp in size according to the instructions of the DNA gel extraction kit.

[0072] Cloning vector connection transformation: pTOPO-Blunt was used as the cloning vector, and the variable region target genes of the heavy chain and light chain were connected to pTOPO-Blunt according to the instructions of the TOPO-Blunt Cloning Kit. 2 μL of the recombinant was transformed into the competent DH10B recipient bacteria, and then spread on the LB plate containing 100 μg / mL ampicillin, and incubated at 37°C overnight to obtain positive bacteria containing light and heavy chains.

[0073] After the positive colonies are cultured, they are sent for sequencing: 5 to 10 positive bacteria are picked from the above plate with a fresh sterilized pipette tip and dropped into 1 mL LB medium (containing 50 μg / mL ampicillin). After culturing at 37°C and 300 rpm for 8 to 12 hours, the bacterial solution is sent to Qingke Biotechnology for sequencing. The designated universal sequencing primers are M13F and M13R.

[0074] 4) Sequencing results

[0075] According to the sequencing feedback results, the nucleotide sequence SEQ ID NO.2 encoding the heavy chain variable region (VH) and the nucleotide sequence SEQ ID NO.3 encoding the light chain variable region (VL) of monoclonal antibody 22G1 were obtained; after converting the sequencing results into amino acid sequences, the amino acid sequence SEQ ID NO.4 of the heavy chain variable region (VH) and the amino acid sequence SEQ ID NO.5 of the light chain variable region (VL) of monoclonal antibody 22G1 were obtained; through analysis, the sequences of the three complementary determining regions CDR (HCDR1, HCDR2, HCDR3) of the heavy chain variable region of monoclonal antibody 22G1 are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively; the sequences of the three complementary determining regions CDR (LCDR1, LCDR2, LCDR3) of the light chain variable region are shown in SEQ ID NO.9, amino acid sequence KAS, and SEQ ID NO.10, respectively. The sequence of monoclonal antibody 22G1 is summarized in Table 2 below:

[0076] Table 2: Sequence list of mAb 22G1

[0077]

[0078] Blastn sequence alignment analysis showed that the light chain variable region of monoclonal antibody 22G1 belonged to the κ type immunoglobulin (Ig).

[0079] Example 5. Application of monoclonal antibody 22G1 (detection reagent)

[0080] 1) Construction and expression of recombinant plasmid containing nucleoprotein

[0081] The full-length nucleic acid sequences of the nucleoprotein of Gurtu virus (GenBank sequence number: KT328591) and Dabiebanda virus (GenBank sequence number: AFJ15061) were amplified from the cDNA of Gurtu virus and Dabiebanda virus by PCR amplification technology, and cloned into the downstream of the T7 promoter of the eukaryotic expression vector pCAGGS (with His tag) and the prokaryotic expression vector pET28a, respectively. After expression in competent cells DH5α, four recombinant plasmids pCAGGS-GTV-NP, pCAGGS-SFTSV-NP, pET28a-GTV-NP and pET28a-SFTSV-NP were obtained, respectively.

[0082] pET28a-GTV-NP and pET28a-SFTSV-NP were transformed into Escherichia coli competent cells BL21 by electroporation, and spread on LB plates containing 100 μg / mL kanamycin (Kanamycin) at 37°C overnight. Pick a single positive colony in 3mL LB liquid culture medium, culture at 37°C, 220rpm for 12 hours, take 30μL of bacterial solution and transfer to 3mL fresh LB liquid culture medium at 37°C, 220rpm for further amplification and culture for 4 hours, add 30μLIPTG to induce expression, culture at 37°C, 220rpm for 4 hours, centrifuge to remove the supernatant of the bacterial solution, use 200μL 1× Loading buffer to resuspend the bacteria, and obtain a bacterial suspension, which is subsequently used for protein immunoblotting (WB) as described in Example 2.

[0083] pCAGGS-GTV-NP and pCAGGS-SFTSV-NP were transfected into a 96-well cell plate pre-plated with Vero cells using the transfection reagent Lipo3000. After culturing in a cell culture incubator at 37°C and 5% CO2 for 3 days, the Vero cells transfected with the above recombinant plasmids were fixed in situ by referring to the indirect immunofluorescence assay (IFA) method in Example 2. The cell plates after fixation, permeabilization and blocking were subsequently prepared for indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) analysis.

[0084] 2) Western blot detection of monoclonal antibody 22G1 (WB)

[0085] The bacterial suspension expressed by the above-mentioned pET28a-GTV-NP and pET28a-SFTSV-NP was separated by 12% SDS PAGE gel electrophoresis and transferred to a 0.2um polyvinylidene fluoride membrane. Referring to the protein immunoblotting (WB) method in Example 2, monoclonal antibody 22G1 was reacted with pET28a-GTV-NP and pET28a-SFTSV-NP by WB reaction, and the results were as follows: Figure 5As shown in the figure, it can be clearly seen that monoclonal antibody 22G1 can not only recognize the linear epitopes of the protein expressed by pET28a-GTV-NP, but also cross-recognize the linear epitopes of the protein expressed by pET28a-SFTSV-NP, that is, monoclonal antibody 22G1 has the ability to recognize the linear epitopes of the nucleoprotein of Gurtu virus and Dabiebanda virus.

[0086] 3) Indirect immunofluorescence assay (IFA) of monoclonal antibody 22G1

[0087] The cell plates fixed in situ after the above-mentioned pCAGGS-GTV-NP and pCAGGS-SFTSV-NP transfected Vero cells were taken and incubated with monoclonal antibody 22G1 respectively. The indirect immunofluorescence detection (IFA) method in Example 2 was referred to. The positive controls were mouse anti-Gurtu virus nucleoprotein polyclonal antibody and mouse anti-Dabiebanda virus nucleoprotein polyclonal antibody, which were diluted 1:2000 with 1% BSA respectively; the negative control was healthy mouse serum, which was diluted 1:2000 with 1% BSA. The IFA test results are shown in Figure 2. Figure 6 As shown in the figure, it can be clearly seen that monoclonal antibody 22G1 can not only recognize the spatial epitope of the protein expressed by pCAGGS-GTV-NP, but also cross-recognize the spatial epitope of the protein expressed by pCAGGS-SFTSV-NP, that is, monoclonal antibody 22G1 also has the ability to recognize the spatial epitopes of the nucleoprotein of Gurtu virus and Dabiebanda virus.

[0088] 4) Enzyme-linked immunosorbent assay (ELISA) of monoclonal antibody 22G1

[0089] The cell lines that can detect monoclonal antibody 22G1 were cultured and expanded. When the cell density reached 80%, the cell supernatant was collected. The cell supernatant stock solution was diluted with the primary antibody diluent in multiple ratios, from 1:2 to 1:2. 13 Different concentrations of cell supernatant dilutions were used as primary antibodies; the negative control was healthy mouse serum, which was diluted 1:2000 with primary antibody diluent; 50 After 48 hours of in situ fixation of Vero cells, diluted primary antibody and negative control were added at 50 μL / well, incubated at 37°C for 2 hours, washed 6 times with PBS-T, and added with 1:2000 diluted HRP-labeled goat anti-mouse IgG antibody. After incubation at 37°C for 1 hour, TMB colorimetric solution was added, incubated at 37°C for 10 minutes, TMB stop solution was immediately added, and dual-wavelength absorbance at 450 / 630 nm was measured using an enzyme reader. OD 450-630 The value that is 2 times or more higher than the negative control is judged as the Cut off line (Cut offline), that is, above the Cut off line is positive.

[0090] The results of ELISA of monoclonal antibody 22G1 are as follows Figure 7 As shown in the figure, monoclonal antibody 22G1 can effectively detect Gurtu virus and Dabiebandha virus, and the detection efficiency of Gurtu virus is higher than that of Dabiebandha virus.

[0091] The monoclonal antibody 22G1 for Gurtu virus provided by the present invention can recognize the nucleoprotein of Gurtu virus and Dabiebanda virus, and can recognize not only the linear epitopes of Gurtu virus and Dabiebanda virus, but also the spatial epitopes of Gurtu virus and Dabiebanda virus. The enzyme-linked immunosorbent assay (ELISA) result of the monoclonal antibody 22G1 provided by the present invention shows that the detection efficiency of monoclonal antibody 22G1 for Gurtu virus is higher than that for Dabiebanda virus, and can be used to prepare Gurtu virus and Dabiebanda virus related detection reagents, which is beneficial to the research and prevention of Gurtu virus and Dabiebanda virus.

[0092] The specific embodiments of the present invention have been described in detail so that those skilled in the art will be easily understood. However, according to all the descriptions disclosed, different modifications or replacements can be made to those details, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A monoclonal antibody to the nucleoprotein of Gurtu virus, characterized in that: The three complementary determining regions of the heavy chain variable region of the monoclonal antibody are: HCDR1 shown in SEQ ID NO.6; HCDR2 shown in SEQ ID NO.7; HCDR3 shown in SEQ ID NO.8; the three complementary determining regions of the light chain variable region of the monoclonal antibody are: LCDR1 shown in SEQ ID NO.9; LCDR2 shown in the amino acid sequence KAS; LCDR3 shown in SEQ ID NO.

10.

2. The monoclonal antibody to the nucleoprotein of Gurtu virus according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

4.

3. The monoclonal antibody to the nucleoprotein of Gurtu virus according to claim 1, characterized in that: The heavy chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO.

2.

4. The monoclonal antibody to the nucleoprotein of Gurtu virus according to claim 1, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO.

5.

5. The monoclonal antibody to the nucleoprotein of Gurtu virus according to claim 1, characterized in that: The light chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO.

3.

6. A polynucleotide encoding a monoclonal antibody against the Gurtu virus nucleoprotein according to any one of claims 1 to 5. A vector comprising the polynucleotide according to claim 6. A host cell comprising the vector according to claim 7.

9. A detection reagent comprising a monoclonal antibody against the nucleoprotein of Gurtu virus as claimed in any one of claims 1 to 5, for detecting Gurtu virus and / or Dabiebandha virus.

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