CN116178532A - Monoclonal antibody of Kelvin virus nucleoprotein and application thereof - Google Patents
Monoclonal antibody of Kelvin virus nucleoprotein and application thereof Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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Abstract
The invention provides a monoclonal antibody of a Kelvin virus nucleoprotein and application thereof, relates to the fields of biotechnology and virology, and the monoclonal antibody obtained by screening has broad spectrum, can identify the nucleoprotein of the Kelvin virus, especially can identify a 6 peptide antigen epitope in the Kelvin virus nucleoprotein, can also cross-identify the nucleoprotein of Dabiedada virus, and further defines the amino acid sequence of the antigen epitope which can be identified by the monoclonal antibody. The monoclonal antibody provided by the invention can be used for preparing detection reagents related to the Banda virus and the Dabanda virus, is favorable for the auxiliary diagnosis of 2 virus infection, and has important significance for basic research, clinical research, flow regulation and the like of the Golgi virus and the Dabanda virus in the Banda virus.
Description
Technical Field
The invention relates to the technical field of biotechnology and virology, in particular to a monoclonal antibody of a Golgi virus and application of the monoclonal antibody, in particular to common detection of the Golgi virus and Dabiedada virus, and particularly to related detection of the Golgi virus and related anti-Golgi virus medicines.
Background
Gu Ertu virus (Guertu bandavirus, GTV) is a new tick-borne virus, gu Ertu virus is separated from a sample of a tsugae tick (Dermacentor nuttalli ticks) collected in Xinjiang in 2018, a main host is the tsugae tick in the Xinjiang Golgi region, the tsugae tick is a dominant tick species in a certain region of the west of China, and is also a main transmission medium of the Golgi virus, the average positive rate is 7.89% which is higher than the positive rate (2.1-5.4%) of SFTSV carried by the Chinese long-angle tick, in vivo and vitro experiments prove that the Golgi virus can infect various mammal and human cell lines, and can cause pathological changes of brain, lung, kidney and spleen of mice, and the detection of human serum in the Xinjiang Golgi region also proves that the Golgi virus can be transmitted among ticks, animals and humans, and is a new virus with potential danger. The genome of the Golgi apparatus virus consists of single-strand and negative-strand RNA, which is divided into three sections of large (L), medium (M) and small (S), and respectively encodes RNA-dependent polymerase, precursor envelope glycoproteins (Gn and Gc, GP proteins for short) and Nucleoprotein (NP), wherein the GP proteins have important receptor binding sites and antigen epitopes for the virus to invade host cells and can stimulate the organism to generate specific immune responses; nucleoprotein stimulates early humoral and cellular immunity and only NP protein can be detected in the body at an early stage of infection.
Dabiebanda virus (original fever with thrombocytopenia syndrome virus Severe fever with thrombocytopenia syndrome Virus, SFTSV) is a new tick-borne virus closest to the Gu Ertu virus in evolutionary relationship, all belonging to the order Bunyavirales, the family of the white fiber viruses (Phenuiviridae), and the genus Bandavirus (bantavir). In 2010, SFTSV was found and named in cases of fever with thrombocytopenia syndrome (Severe fever with thrombocytopenia syndrome, SFTS) from unknown causes, and has been currently popular in the regions of Hubei, henan, zhejiang, shandong and the like in the eastern part of China and in Japan and Korea, and clinical symptoms such as hyperpyrexia, anorexia, muscle pain, chills, lymphadenopathy, leukopenia, thrombocytopenia and the like are caused after infection of human bodies, and the death rate is about 5% -30%. The fly is mainly transmitted by the biting way of ticks, and the occurrence of people's event is reported in literature. According to the latest catalogue of pathogenic microorganisms (solicited opinion manuscript) for infection among people, the national health committee adjusts the hazard degree of 5 viruses such as 'Dabiedada virus' from the original 'second class' to the 'third class'.
At present, regarding monoclonal antibodies of the large allopad virus (SFTSV), related researches utilize phage antibody libraries to separate more than 400 monoclonal antibodies aiming at the nuclear protein of the large allopad virus from patients in the recovery period of the large allopad virus, highlight the importance of the nuclear protein in inducing humoral immunity, and show the importance of developing monoclonal antibodies taking the nuclear protein as a target point as a novel virus detection means. In related researches, spleen cells of a mouse infected by the Davida virus are fused with myeloma cells, monoclonal antibodies aiming at the Nuclear proteins of the Davida virus are screened and obtained, and a sandwich ELISA detection method is established for detecting serum of a patient with the Davida virus. However, no research report on the Kelvin monoclonal antibody of the Golgi virus exists at present, and no specific detection means for the Golgi virus exists. Since both the gulf-map virus and the large allopan virus belong to RNA viruses, genome replication lacks a proofreading mechanism, virus self-mutation easily causes changes in virulence, adaptation to host capacity and epidemiology, and different strains may recombine to produce new viruses adapted to other vectors, thereby changing the distribution range of epidemic and leading to the generation of new diseases. Furthermore, effective drugs and vaccines for preventing these 2 viral infections are still in the research stage, nor are there effective diagnostic methods or reagents. Therefore, the method provides a simple, convenient, rapid and high-sensitivity detection reagent or therapeutic drug for the infection of new viruses such as Gu Ertu virus and the like, and becomes the primary problem to be solved in the current clinical diagnosis/treatment of the diseases. In order to prevent the possible epidemic and transmission of the Golgi virus and the cross infection of the same genus virus, the research of the monoclonal antibody of the virus is of great significance to the prevention and treatment of the Golgi virus and other members of the genus Banda.
Disclosure of Invention
The invention aims to at least partially overcome the defects of the prior art and provides a broad-spectrum monoclonal antibody for recognizing the Golgi virus.
The invention also aims to fill the blank of the detection reagent for the Golgi viruses in China, and provides 3 Golgi virus detection methods and the reagent for the Golgi virus detection.
The invention also aims to provide a monoclonal antibody of the Golgi apparatus virus, which provides a research basis of an alternative antibody for auxiliary diagnosis of the Golgi apparatus virus infection.
In order to achieve the above object or one of the purposes, the present invention provides the following technical solutions:
in a first aspect, the present invention provides a monoclonal antibody of a nucleoprotein of the gulfig virus, which is capable of recognizing a linear epitope having the amino acid sequence shown in SEQ ID No.1, and is also capable of recognizing a spatial epitope having the amino acid sequence shown in SEQ ID No. 1.
In a second aspect, the 3 complementarity determining regions CDRs (HCDR 1, HC DR2, HCDR 3) of the heavy chain variable region of the monoclonal antibody have the amino acid sequences respectively: HCDR1 shown in SEQ ID No. 6; HCDR2 shown in SEQ id No. 7; HCDR3 shown in SEQ ID No. 8; the 3 complementarity determining regions CDRs (LCDR 1, LCDR2, LCDR 3) of the light chain variable region of the monoclonal antibody have the following amino acid sequences: LCDR1 as shown in SEQ ID No. 9; LCDR2 as shown in amino acid sequence KAS; LCDR3 as shown in SEQ ID NO. 10.
Preferably, the heavy chain variable region of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO. 4.
Preferably, the heavy chain variable region of the monoclonal antibody is encoded by the nucleic acid sequence shown in SEQ ID NO. 2.
Preferably, the light chain variable region of the monoclonal antibody has the amino acid sequence shown in SEQ ID NO. 5.
Preferably, the light chain variable region of the monoclonal antibody is encoded by the nucleic acid sequence shown in SEQ ID NO. 3.
In a third aspect, the present invention provides a polynucleotide comprising a monoclonal antibody to the above-described gulf-image virus nucleoprotein.
In a fourth aspect, the present invention provides a vector comprising a monoclonal antibody to the above-described Kelvin virus nucleoprotein.
In a fifth aspect, the present invention provides a host cell comprising a monoclonal antibody to the above-described gulf-image virus nucleoprotein.
In a sixth aspect, the invention also provides the use of a monoclonal antibody to a nucleoprotein of the Golgi virus in the preparation of a polynucleotide, a recombinant vector, an expression vector, a recombinant protein, a genetically engineered host cell, and a detection reagent associated with the Golgi virus.
The invention has the beneficial effects that:
the invention obtains a broad-spectrum monoclonal antibody 22G1 (abbreviated as monoclonal antibody 22G 1) of the Golgi virus, wherein the monoclonal antibody 22G1 is a monoclonal antibody derived from the nucleoprotein of the Golgi virus and can identify a 6 peptide epitope (LM) in the nucleoprotein of the Golgi virus; in addition, the nucleoprotein of the large allophana virus can be cross-identified. The monoclonal antibody 22G1 is verified by western immunoblotting detection, indirect immunofluorescence detection and enzyme-linked immunosorbent assay, and the monoclonal antibody 22G1 can recognize both the Golgi virus and the Dabiedada virus, however, the enzyme-linked immunosorbent assay (ELISA) result of the monoclonal antibody 22G1 shows that the detection efficiency of the monoclonal antibody 22G1 on the Golgi virus is slightly higher than that on the Dabiedada virus.
The active epitope of the monoclonal antibody 22G1 for broad-spectrum identification of the Golgi virus and the Dabiebanda virus of the Banda virus genus is mainly aimed at nucleoprotein, can be prepared into a detection reagent of the Banda virus genus or used for detecting a pathogen related to the Banda virus genus, and can distinguish whether the pathogen in a sample contains the Golgi virus and the Dabiebanda virus. The protein immunoblotting detection (WB), indirect immunofluorescence detection (IFA) and enzyme-linked immunosorbent assay (ELISA) 3 detection platforms provided by the invention can be used for the heavy fields of basic research, clinical research, flow regulation and the like of the Golgi virus and the Dabieda virus in the Banda virus genus.
Drawings
FIG. 1 is a Western blot detection of Gurley virus mab 22G1, wherein 22G1 is mab 22G1, PC is a positive control, i.e., polyclonal antibody to the Kelvy virus nucleoprotein, and NC is a negative control, i.e., healthy mouse serum;
FIG. 2 is an indirect immunofluorescence assay of the Golgi apparatus virus mab 22G1, 22G1 mab 22G1, PC as a positive control, i.e., polyclonal antibody to the Golgi apparatus virus nucleoprotein, and NC as a negative control, i.e., healthy mouse serum;
FIG. 3 is a Western blot analysis of the nuclear protein 16 peptide of the Golgi virus from the section of the Golgi 22G1, wherein P26, 27, 28, 29, 30, 31, 32 represent the number of the 16 peptide formed by repeated arrangement of 8 amino acids of the nuclear protein of the Golgi virus, respectively, and NC is the negative control, i.e., serum of healthy mice;
FIG. 4 is a Western blot analysis of the nucleoprotein 8 peptide of the Golgi virus from the section of the Golgi virus, wherein P33, 34, 35, 36, 37, 38, 39, 40, 41 represent the number of the 8 peptide formed by repeated arrangement of 7 amino acids of the nucleoprotein of the Golgi virus, respectively, and NC is the negative control, i.e., serum of healthy mice;
FIG. 5 is a Western blot analysis of the Kelvin virus monoclonal antibody 22G1 and prokaryotic expressed nucleoprotein, wherein PC is a positive control, i.e. polyclonal antibody of Kelvin virus nucleoprotein, NC is a negative control, i.e. healthy mouse serum, A is viral protein expressed by recombinant vector pET28a-GTV-NP, B is viral protein expressed by recombinant vector pET28 a-SFTSV-NP;
FIG. 6 is an indirect immunofluorescence assay of a Gurley image virus mab 22G1 with eukaryotic expressed nucleoprotein, wherein PC is a positive control, i.e., polyclonal antibody to the Gurley image virus nucleoprotein, NC is a negative control, i.e., healthy mouse serum, the first line of pictures corresponds to viral protein expressed by recombinant vector pCAGGS-GTV-NP, and the second line of pictures corresponds to viral protein expressed by recombinant vector pCAGGS-SFTSV-NP;
FIG. 7 is a graph showing the results of ELISA detection of the Gurley-directed virus monoclonal antibody 22G1 and eukaryotic-expressed nucleoprotein, with the abscissa representing the log (base 2 log) of the dilution of the Gurley-directed virus monoclonal antibody 22G1 and the ordinate representing the OD of the ELISA 450-630 The measured absorbance values, wherein 22G1-GTV represent ELISA detection results after incubation of the GTV-infected Vero cells with the concentration of different mab 22G1, and 22G1-SFTSV represent ELISA detection results after incubation of the SFTSV-infected Vero cells with the concentration of different mab 22G1, cut off line (Cut off line) =0.024.
Detailed Description
Exemplary embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein the same or similar means the same concept, such as Gu Ertu virus=gtv, dobbad virus=fever with thrombocytopenia syndrome virus=sftsv, monoclonal antibody=mab, and the like.
In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the present disclosure. The following examples are only illustrative of the present invention and should not be construed as limiting the scope of the invention.
The experimental methods used in the examples described below are conventional methods unless otherwise indicated, and materials, reagents, etc. used, unless otherwise indicated, are commercially available. Wherein, freund's complete adjuvant, freund's incomplete adjuvant, HAT medium and the like are purchased from SIGMA-Aldrich in the United states; serum-free DMEM medium, QIXzol nucleic acid extraction reagent, etc. are purchased from new longitudinal virus disease engineering company, north of lake; PEG was purchased from Glpbio company, U.S.A.; vero et al cells were purchased from ATCC; HRP-labeled goat anti-mouse or rabbit IgG secondary antibody, HRP-labeled goat anti-rabbit IgG H&L-fluorescent secondary antibodies were purchased from Ai Bokang (Shanghai) trade Co., ltd (abcam); chemiluminescence imaging analyzer Image Quant TM LAS4000 purchaseGE Healthcare from the united states; TOPO-Blunt Blunt end cloning kit (TOPO-Blunt Cloning Kit) was purchased from Nanjinovirzan Biotech Co., ltd; high content cell imaging analysis systems were purchased from PerkinElmer corporation of the united states; the primers related in the invention are all consigned to be synthesized by the division of biological engineering (Shanghai); the sequence determination of the invention is completed by Beijing qingke biotechnology limited company (Wuhan division company); gu Ertu Virus (GTV) and Dabiebanda Virus (SFTSV) were maintained by the national virus resource library of the institute of Chinese sciences, wuhan Virus. It may be evident, however, that one or more embodiments may be practiced without these specific details, with the specific details not being set forth in the embodiments, as is conventional or suggested by the manufacturer.
The molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j. The application object of the invention is derived from biological products to be detected, and belongs to inanimate samples; the direct purpose of the detection is to ensure the safety of the biological product to be detected, thereby being beneficial to perfecting the quality control standard of the biological product, and no process of obtaining the diagnosis result or health condition of the disease exists. Therefore, the invention does not belong to a disease diagnosis method and meets the basic requirements of patent laws on patent protection objects.
The invention provides a monoclonal antibody of a Golgi apparatus virus, a preparation method and application thereof, wherein the cell strain capable of secreting monoclonal antibody 22G1 is screened by using inactivated virus of the Golgi apparatus virus as a target through the fusion technology of spleen cells and myeloma cells of mice immunized by the Golgi apparatus virus; through molecular biology technology, the amino acid sequence of the monoclonal antibody 22G1 for recognizing the linear epitope, the amino acid sequence of the monoclonal antibody 22G1 and the coded nucleic acid sequence are identified, eukaryotic and prokaryotic expression vectors containing the Golgi virus and the Dabieda virus are respectively constructed, and the obtained recombinant vectors respectively construct 3 detection platforms of protein immunoblotting detection (WB), indirect immunofluorescence detection (IFA) and enzyme-linked immunosorbent assay (ELISA), so that the monoclonal antibody 22G1 can recognize the nucleoprotein of the Golgi virus and can cross-recognize the nucleoprotein of the Dabieda virus; and the monoclonal antibody 22G1 can recognize the linear epitope of the nucleoprotein and also can recognize the spatial epitope of the nucleoprotein, and has broad-spectrum recognition capability. The following describes specific embodiments of the present invention, and the specific contents are as follows:
example 1 preparation of monoclonal antibody 22G1
After a concentrated and inactivated Golgi virus is immunized on a mouse, the separated spleen cells and myeloma cells are subjected to cell fusion on feeder cells, the Golgi virus infected cells are taken as positive hole targets, hybridoma cell strains are screened out, and finally monoclonal cell strains 22G1 secreting anti-Golgi virus nucleoprotein are screened out from the positive holes. The preparation process of the hybridoma cell strain comprises animal immunization, spleen cell separation, cell fusion and monoclonalization, and the specific preparation process is as follows:
1) Immunization of animals
According to the established animal immunization scheme, SPF grade C57/BL6 mice with the age of 6-8 weeks are selected as immunized animals, and the immunogen is concentrated and inactivated Gu Ertu virus; after emulsifying the virus with equal volume of Freund's complete adjuvant, immunization was performed twice by intraperitoneal and subcutaneous multipoint injection, with two weeks of immunization interval, with an immunization volume of 200. Mu.L/v, wherein the virus amount was 8×10 7 TCID 50 /only.
2) Isolation of spleen cells
Taking immunized C57/BL6 mice, carrying out orbital exsanguination and sacrifice after anesthesia, soaking the sacrificed mice in 75% alcohol for 5 minutes, dissecting the mice in a biosafety cabinet by using a sterile instrument, taking the spleens of the mice, removing surrounding connective tissues, placing in a sterile homogenizer, firstly adding 5mL of serum-free DMEM culture medium for grinding, then adding 10mL of serum-free DMEM culture medium for fully mixing, standing for 10 minutes, taking the grinding supernatant, centrifuging at 1000rpm for 10 minutes, discarding the supernatant, adding 10mL of serum-free DMEM culture medium for resuspension cells, counting the washed splenocytes by trypan blue, taking immune splenocyte suspension (1X 10) 8 ) And (5) standby.
3) Cell fusion and monoclonalization
Suspension of immune spleen cells (1X 10) 8 ) With myeloma cells (2X 10) 7 ) (the ratio of the number of spleen cells to myeloma cells=5:1) was thoroughly 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℃water bath, 1mL of preheated 50% PEG was slowly added with mixing, then 10mL of preheated DMEM medium was slowly added over 5 minutes, and after 10 minutes of standing at 37℃the tube was centrifuged at 1000rpm for 8 minutes, and the supernatant was discarded. Resuspension of mixed cells in 50mLHAT culture medium, spreading the mixed cell-containing suspension in 96-well cell plate, adding 100 μl of the cell suspension into each well, limiting dilution to make each well in 96-well cell plate single-cell state, and standing at 37deg.C and 5% CO 2 Culturing in a cell incubator for 7-10 days, collecting supernatant of the fused cells, unexpectedly screening monoclonal antibody 22G1 from positive holes, and performing the next monoclonal antibody verification.
Example 2 verification of monoclonal antibody 22G1
1) Western blotting detection (WB)
The Vero cells are infected by the Golgi virus and amplified in the Vero cells, the Vero cells and cell supernatants after the infection of the Golgi virus are collected, beta-propiolactone is added for inactivation respectively, viral proteins are separated by 12% SDS-PAGE gel electrophoresis, transferred to a 0.2 mu m polyvinylidene fluoride membrane, and blocked by 5% (w/v) skimmed milk powder prepared by PBS for 1 hour at 37 ℃, and PCR detection shows that the Golgi virus has positive amplification in both the Vero cells and cell supernatants.
Positive control was a polyclonal antibody to rabbit anti-gulfig virus nucleoprotein, diluted with primary antibody 1:2000 dilution; negative control was healthy mouse serum, diluted with primary antibody 1:2000 dilution; taking the supernatant of the fused cells as primary antibodies, respectively adding the primary antibodies to the blocked polyvinylidene fluoride membranes, incubating for 2 hours at 37 ℃, washing for 3 times by using TBS-T, and respectively adding the primary antibodies into the membranes according to the following formula 1:2000 dilution of HRP labeled goat anti-mouse or rabbit IgG antibody, again at 37 degrees C after 1 hours incubation, immunoblotting display, in the chemiluminescent imaging analyzer Image Quant LAS4000 imaging.
The result of western blotting detection of the monoclonal antibody 22G1 obtained by screening and the Golgi virus separated after inactivation is shown in figure 1, and the result proves that the monoclonal antibody 22G1 can recognize Gu Ertu virus separated from gel after inactivation, namely the monoclonal antibody 22G1 can recognize linear epitope of the Kelvin virus nucleoprotein.
2) Indirect immunofluorescence assay (IFA)
Virus culture: vero cells were grown at 1X 10 4 Uniformly spreading the cells/holes into 96-hole cell plates, adding a DMEM culture medium containing 2% fetal calf serum according to 100 mu/hole, and discarding the supernatant when the cell density is over 80%; mu.L of cell supernatant containing Golgi virus was added at a multiplicity of infection MOI=1, and at 37℃and 5% CO 2 Is cultured in a cell culture incubator for 3 days.
In-situ fixation: taking out the cell plate, washing the cell plate with sterile PBS for 1 time, adding 4% paraformaldehyde according to 300 mu L/hole, fixing for 15 minutes, and washing with PBS for 3 times again; the fixed cell plates were permeabilized for 10 minutes by adding 0.4% Triton X-100 at 100. Mu.L/well and washed again with PBS for 4 times; the permeabilized cell plates were blocked at 37℃for 1 hour by adding 5% BSA to 100. Mu.L/well and the blocking solution was discarded.
IFA detection: the positive control was a polyclonal antibody to the rabbit anti-gulfig virus nucleoprotein, with 1% bsa at 1:2000 dilution; the negative control was healthy mouse serum with 1% bsa at 1:2000 dilution; taking the supernatant of the fusion cell secreting the monoclonal antibody 22G1 as a primary antibody, adding the treated 96-well cell plate according to 100 mu/well, and incubating at 37 ℃ for 2 hours; the secondary antibody was an HRP-labeled goat anti-rabbit IgG (h+l) fluorescent antibody, with 1% bsa at 1:2000 dilution, adding diluted secondary antibody according to 50 mu L/hole, and incubating for 1 hour at 37 ℃; the fluorescent dye Hoechest 33258 was then added at 50. Mu.L/well, left at room temperature for 5 minutes, and the results were observed using a high content cell imaging analysis system.
The indirect immunofluorescence detection results of the monoclonal antibody 22G1 obtained by screening and the inactivated in-situ immobilized Golgi virus are shown in FIG. 2, and the monoclonal antibody 22G1 can be proved to be capable of effectively recognizing the spatial epitope of the Golgi virus.
Example 3 recognized epitope of monoclonal antibody 22G1
1) Short peptides of truncated gul-figure viral nucleoprotein
Based on the amino acid sequence of the nucleoprotein of the gul-graph virus, 32 truncated 16 peptides were prepared, numbered P1-P32. The front and back 16 peptides comprise 8 identical amino acid arrangements, and the first amino acid of the latter 16 peptide is identical to the ninth amino acid of the former 16 peptide, so that 32 16 peptides are prepared by sequential arrangement and combination.
Referring to the method of western blot detection (WB) of example 2, the WB detection screens out 16 peptides that reacted positively with mab 22G1. The results are shown in FIG. 3, which representatively shows the result of the positive reaction, namely, the number P31 of the 16 peptide of the GTV nucleoprotein recognized by monoclonal antibody 22G1.
According to the 16 amino acid sequence of 16 peptide number P31, 9 truncated 8 peptides were further prepared, numbered P33-P41. Each 8 peptide is formed by sequentially selecting 8 amino acids from 16 amino acid sequences, wherein the first amino acid of the 8 peptide with the last number is identical to the second amino acid of the 8 peptide with the previous number, and 98 peptides are prepared by sequentially arranging and combining the two amino acids. According to the coding nucleic acid sequence of 8 peptide, bamH I and Sal I enzyme cutting sites are respectively added before and after the truncated 8 peptide nucleotide sequence, the synthesis of DNA fragment by the family of the engine is entrusted, the synthesized DNA fragment is cloned into a pXXGST prokaryotic expression plasmid vector, and then the plasmid is electrically transformed into the competence of escherichia coli BL21, so as to obtain the monoclonal colony for expressing the recombinant plasmid. Culturing in 3mL of liquid LB medium at 37 ℃ and 220rpm for 12 hours; transferring 30 mu L of culture bacterial liquid into 3mL of fresh liquid LB culture medium, continuing amplification culture for 4 hours at 30 ℃, taking out the amplified culture bacterial liquid, inducing protein expression at 42 ℃ and 220rpm, centrifuging at 5000rpm for 5 minutes, removing supernatant, re-suspending bacterial cells by using 200 mu L of 1×loading buffer, separating protein by 12% SDS-PAGE gel electrophoresis, and identifying the expression of truncated 8 peptide after staining by coomassie brilliant blue staining liquid.
2) WB identification of truncated 8 peptide
Referring to the method of western blot detection (WB) of example 2, WB detection screened 8 peptide positive for mab 22G1 reaction. The results are shown in FIG. 4, wherein the numbers of the 8 peptides with positive reaction are P34-36, namely the numbers of the 8 peptides of the GTV nucleoprotein recognized by the monoclonal antibody 22G1 are P34, P35 and P36, namely the P34-36 are specific linear epitopes recognized by the monoclonal antibody 22G1. Upon analysis the truncated 8 peptides each comprise the amino acid sequence set forth in SEQ ID NO. 1: GPDGLP.
Example 4 sequence identification of monoclonal antibody 22G1
The cultured monoclonal antibody 22G1 cell strain of the Golgi apparatus virus is prepared by extracting total RNA, performing reverse transcription to obtain cDNA, amplifying and recovering fragments of a variable region (comprising a heavy chain variable region and a light chain variable region), constructing the amplified and cultured clone vector pTOPO-Blunt, and performing censoring and sequencing by using universal primers M13F and M13R, wherein the specific procedures are as follows:
1) Nucleic acid extraction:
the monoclonal antibody 22G1 cell strain obtained by the screening is taken, 1mL of QIXzol nucleic acid extraction reagent is added, and after repeated blowing and beating of the lysed cells, total RNA is extracted according to the specification of the QIXzol nucleic acid extraction reagent. The concentration and purity of the extracted RNA were measured using an ultraviolet spectrophotometer.
2) Reverse transcription:
extracting 3 μg of the above RNA, adding Oligo (dT) (500 μg/mL), 10mM dNTP Mix, 5 XFirst-StrandBuffer, 0.1M DTT, RNaseOUT TM Recombinant Ribonuclease Inhibitor (recombinant ribonuclease inhibitor) and M-MLVRT to obtain synthesized cDNA.
3) Sequencing after amplification:
the variable region was amplified by PCR: 2. Mu.L of cDNA was used as a template, primers designed by Orlandi et al (1989) for amplifying the mouse variable region gene were added, and PCR amplification was performed using high fidelity enzyme (2 XMax Mastermix) to obtain the target gene fragment, the primer sequences are shown in Table 1 below.
Table 1: variable region PCR amplification primer sequence table
PCR amplification procedure: pre-denaturation at 95℃for 3min; denaturation at 94℃for 15s, annealing at 55℃for 15s, elongation at 72℃for 30s, 30 cycles; finally, the extension is carried out for 5min at 72 ℃.
And (3) PCR product recovery: 30-50 mu L of PCR product is taken for 1% agarose electrophoresis. The PCR product was purified to a size of about 300bp according to the instructions of the DNA gel recovery kit (Gel Extraction Kit).
Cloning vector ligation transformation: the target genes of the variable regions of the heavy chain and the light chain were ligated to pTOPO-Blunt using pTOPO-Blunt as cloning vector according to the instructions of the Blunt-ended cloning kit (TOPO-Blunt Cloning Kit), respectively. 2 mu L of recombinant is transformed into competent DH10B receptor bacteria, and then the strain is coated on an LB plate containing 100 mu g/mL Ampicillin (Ampicillin) at 37 ℃ overnight to obtain positive bacteria containing light and heavy chains.
Positive colony culture and then sequencing by inspection: 5-10 positive bacteria are picked from the flat plate by a fresh sterilization gun head and placed in 1mL LB culture medium (50 mug/mL containing ampicillin), and after culturing for 8-12 h at 37 ℃ and 300rpm, bacterial liquid is sent to the liquagenase for sequencing, and the sequencing universal primers are designated as M13F and M13R.
4) Sequencing results
According to the sequencing feedback result, obtaining a nucleotide sequence SEQ ID NO.2 of a heavy chain variable region (VH) and a nucleotide sequence SEQ ID NO.3 of a light chain variable region (VL) of the monoclonal antibody 22G 1; converting the sequencing result into an amino acid sequence, and obtaining an amino acid sequence SEQ ID NO.4 of a heavy chain variable region (VH) and an amino acid sequence SEQ ID NO.5 of a light chain variable region (VL) of the monoclonal antibody 22G 1; by analysis, the sequences of the 3 complementarity determining regions CDRs (HCDR 1, HCDR2, HCDR 3) of the heavy chain variable region of mab 22G1 are shown as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, respectively; the sequences of the 3 CDRs (LCDR 1, LCDR2, LCDR 3) of the light chain variable region are shown as SEQ ID NO.9, amino acid sequence KAS, SEQ ID NO.10, respectively. The sequence of mab 22G1 is summarized in table 2 below:
table 2: sequence listing of mab 22G1
The light chain variable region of mab 22G1 belongs to the kappa-type immunoglobulin (Ig) as analyzed by Blastn sequence alignment.
EXAMPLE 5 application of monoclonal antibody 22G1 (detection reagent)
1) Construction and expression of recombinant plasmids containing nucleoprotein
The nucleic acid sequences of the full length of the nucleoprotein of the virus Gu Ertu (GenBank serial number: KT 328591) and the large allopad virus (GenBank serial number: AFJ 15061) are amplified from the cDNA of the Golgi virus and the large allopad virus by a PCR amplification technology, cloned to the downstream of the T7 promoter of the eukaryotic expression vector pCAGGS (with His tag) and the prokaryotic expression vector pET28a respectively, and 4 recombinant plasmids pCAGGS-GTV-NP, pCAGGS-SFTSV-NP, pET28a-GTV-NP and pET28a-SFTSV-NP are obtained respectively through the expression of competent cells DH5 a.
pET28a-GTV-NP and pET28a-SFTSV-NP were plated onto LB plates containing 100. Mu.g/mL Kanamycin (Kanamycin) by electrotransformation into E.coli competent cells BL21 overnight at 37 ℃. Selecting single positive colony, culturing in 3mL LB liquid medium at 37 ℃ and 220rpm for 12 hours, transferring 30 mu L of bacterial liquid into 3mL of fresh LB liquid medium, continuing amplification culture at 37 ℃ and 220rpm for 4 hours, adding 30 mu LIPTG for induction expression, culturing at 37 ℃ and 220rpm for 4 hours, removing bacterial liquid supernatant by a centrifuge, and re-suspending bacterial bodies by using 200 mu L of 1×loading buffer to obtain bacterial body suspension, and subsequently preparing for protein immunoblotting detection (WB) described in example 2.
The pCAGGS-GTV-NP and pCAGGS-SFTSV-NP were transfected into 96-well cell plates previously plated with Vero cells by the transfection reagent Lipo3000 at 37℃with 5% CO 2 After 3 days of culture in a cell incubator, the Vero cells transfected with the recombinant plasmids are fixed in situ by the method of intermediate immunofluorescence assay (IFA) of example 2, and the fixed, permeabilized and blocked cell plates are subsequently prepared for analysis by indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA), respectively.
2) Western immunoblotting detection (WB) of monoclonal antibody 22G1
The bacterial suspensions expressed by pET28a-GTV-NP and pET28a-SFTSV-NP are separated by 12% SDS PAGE gel electrophoresis, transferred to a 0.2um polyvinylidene fluoride membrane, and subjected to WB reaction by referring to a method of Western immunoblotting detection (WB) in example 2, and the result is shown in FIG. 5, wherein the monoclonal antibody 22G1 can be used for recognizing not only the linear epitope of the protein expressed by pET28a-GTV-NP but also the linear epitope of the protein expressed by pET28a-SFTSV-NP, namely, the monoclonal antibody 22G1 has the recognition capability on the linear epitope of the nucleoprotein of the Golgi virus and the Dabiedada virus.
3) Indirect immunofluorescence assay (IFA) for mab 22G1
After the above-mentioned pCAGGS-GTV-NP and pCAGGS-SFTSV-NP were used to transfect Vero cells, the cell plates were fixed in situ, and incubated with monoclonal antibody 22G1, respectively, the method of intermediate immunofluorescence assay (IFA) was used in reference to example 2, and positive controls were a polyclonal antibody against murine anti-Golgi virus nucleoprotein and a polyclonal antibody against murine anti-Dabiebanda virus nucleoprotein, respectively, at 1% BSA: 2000 dilution; the negative control was healthy mouse serum with 1% bsa at 1:2000 dilution. The IFA detection result is shown in FIG. 6, and the figure shows that the monoclonal antibody 22G1 can not only recognize the spatial epitope of the pCAGGS-GTV-NP expression protein, but also can cross-recognize the spatial epitope of the pCAGGS-SFTSV-NP expression protein, namely, the monoclonal antibody 22G1 has the recognition capability on the spatial epitope of the nucleoprotein of the Golgi virus and the Dabanda virus.
4) ELISA detection of monoclonal antibody 22G1
And (3) amplifying and culturing cell strains capable of respectively resisting the monoclonal antibodies 22G1, and collecting cell supernatants when the cell density reaches 80%. The cell supernatant stock was diluted in a multiple ratio with an anti-dilution solution, sequentially from 1:2 dilution to 1:2 13 Cell supernatant dilutions of different concentrations were used as primary antibodies; the negative control was healthy mouse serum diluted with primary antibody at 1:2000 dilution; is infected by GTV and SFTSV respectively (100 TCID 50 Well) after 48 hours Vero cells were plated in situ by adding diluted primary and negative controls at 50. Mu.L/wellAfter incubation at 37℃for 2 hours, after 6 washes with PBS-T, 1:2000 dilution of HRP marked goat anti-mouse IgG antibody, after 1 hour incubation at 37 ℃, adding TMB color development solution, incubation at 37 ℃ for 10 minutes, immediately adding TMB stop solution, using enzyme-labeled instrument to make 450/630nm dual wavelength absorbance measurement, OD 450-630 A judgment that the value is 2 times or more higher than that of the negative control is a Cut off line (Cut off line), that is, a positive value is not less than the Cut off line.
The results of enzyme-linked immunosorbent assay (ELISA) of the monoclonal antibody 22G1 are shown in FIG. 7, wherein the monoclonal antibody 22G1 can effectively detect the Golgi virus and the Dabiedada virus, and the detection efficiency of the Golgi virus is higher than that of the Dabiedada virus.
The monoclonal antibody 22G1 of the Golgi virus provided by the invention can recognize the nucleoprotein of the Golgi virus and the Dabieda virus, can recognize the linear epitope of the Golgi virus and the Dabieda virus, and can also recognize the spatial epitope of the Golgi virus and the Dabieda virus. The ELISA result of the monoclonal antibody 22G1 provided by the invention shows that the detection efficiency of the monoclonal antibody 22G1 on the Golgi virus is higher than that of the monoclonal antibody 22G1 on the Dabiedada virus, and the monoclonal antibody can be used for preparing the related detection reagents of the Golgi virus and the Dabiedada virus, thereby being beneficial to the research and the prevention and the control of the Golgi virus and the Dabiedada virus.
Specific embodiments of the present invention have been described in detail so that those skilled in the art will readily understand. Various modifications or substitutions of details may be made in accordance with all that has been disclosed, and such modifications and alterations are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (10)
1. A monoclonal antibody to a nucleoprotein of a gulf-map virus, characterized in that: can recognize both linear epitope with the amino acid sequence shown in SEQ ID NO.1 and spatial epitope with the amino acid sequence shown in SEQ ID NO. 1.
2. The monoclonal antibody to the nucleoprotein of the gulf-figure virus according to claim 1, characterized in that: the 3 complementarity determining region CDRs of the heavy chain variable region of the monoclonal antibody have the following amino acid sequences: HCDR1 shown in SEQ ID No. 6; HCDR2 shown in SEQ ID No. 7; HCDR3 shown in SEQ ID No. 8; the 3 complementarity determining region CDRs of the light chain variable region of the monoclonal antibody have the following amino acid sequences: LCDR1 as shown in SEQ ID No. 9; LCDR2 as shown in amino acid sequence KAS; LCDR3 as shown in SEQ ID NO. 10.
3. The monoclonal antibody to the nucleoprotein of the gulf-figure virus according to claim 2, characterized in that: the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 4.
4. The monoclonal antibody to the nucleoprotein of the gulf-figure virus according to claim 2, characterized in that: the heavy chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO. 2.
5. The monoclonal antibody to the nucleoprotein of the gulf-figure virus according to claim 2, characterized in that: the light chain variable region has an amino acid sequence shown in SEQ ID NO. 5.
6. The monoclonal antibody to the nucleoprotein of the gulf-figure virus according to claim 2, characterized in that: the light chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO. 3.
7. A polynucleotide encoding a monoclonal antibody to the gulfig virus nucleoprotein of any of claims 1-6.
8. A vector comprising the polynucleotide of claim 7.
9. A host cell comprising the vector of claim 8.
10. Use of a monoclonal antibody to a nucleoprotein of the gulf-figure virus according to any of claims 1-6 for the preparation of a gulf-figure virus-related detection reagent.
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