CN116410308A - Monoclonal antibody of Hatland virus and application thereof - Google Patents
Monoclonal antibody of Hatland virus and application thereof Download PDFInfo
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- CN116410308A CN116410308A CN202310044495.7A CN202310044495A CN116410308A CN 116410308 A CN116410308 A CN 116410308A CN 202310044495 A CN202310044495 A CN 202310044495A CN 116410308 A CN116410308 A CN 116410308A
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Abstract
The invention provides a monoclonal antibody of a Hatland virus and application thereof, which relate to the fields of biotechnology and virology, and the monoclonal antibody screened by the invention can specifically recognize GP protein of Ha Telan virus, can specifically recognize spatial epitope of Ha Telan virus only, and has specific neutralization activity on Ha Telan virus. The monoclonal antibody provided by the invention has strong specificity, can not cross-identify GP proteins of the Dabieda virus and the Golgi virus, can be used for preparing a kit for detecting the Ha Telan virus, and has a half effective concentration of 15.80 mug/mL for identifying the virus; the monoclonal antibody provided by the invention has strong specific neutralization activity, the half effective inhibition concentration of the neutralization Ha Telan virus is 8.386 mug/mL, and the monoclonal antibody can be used for preparing specific antibody medicaments of Cheng Kangha Telan virus, is beneficial to eliminating Ha Telan virus before the virus is epidemic or becomes a greater difficult problem, and has important significance for the prevention and treatment of the virus.
Description
Technical Field
The invention relates to the technical fields of biotechnology and virology, in particular to a monoclonal antibody of a Hatland virus and application of the monoclonal antibody, in particular to detection of Ha Telan virus and related medicaments for resisting Ha Telan virus.
Background
Ha Telan virus (HRTV) is a new tick-borne virus, 2 men in north west of missouri in 2009 were diagnosed in Ha Telan medical center, and the isolated virus was observed by electron microscopy to belong to bunyavirus particles, and was identified as a new sand fly virus by sequencing and phylogenetic analysis, and named as heart disease, and the main host is the soliton tick in north america. Ha Telan symptoms such as hyperpyrexia, anorexia, muscle pain, chills, lymphadenectasis, leukopenia, thrombocytopenia and the like can appear after the infection of the virus Ha Telan. In 2022, 1 case of death after Ha Telan virus infection appears in U.S. georgia, and currently, in every eastern state in the united states, more than 50 cases of infection have been caused, the pathogenicity and transmission mode of Ha Telan virus are similar to those of SFTSVs which cause fever with thrombocytopenia syndrome, severe cases of multiple organ failure and even death appear, and the death rate is about 5%. The U.S. disease control officials express: we are constantly aware of everything about the virus and eliminate it before it becomes a greater challenge. However, there is no relevant detection reagent or therapeutic product approved for the Ha Telan virus on the market at home and abroad.
The Dabiebanda virus (original fever with thrombocytopenia syndrome virus Severe fever with thrombocytopenia syndrome Virus, SFTSV) and Gu Ertu virus (Guertu bandavirus, GTV) are two other new tick-borne viruses closest to the Ha Telan virus in evolutionary relationship, both belonging to the Bunyavirales order, the family of white-fiber viruses (Phenuiviridae), and the genus Bandavirus. 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 prevalent in the eastern China in Hubei, henan, zhejiang, shandong and other areas, japan, korea, and causes clinical symptoms similar to Ha Telan virus after infection of human bodies, and the death rate is about 5% -30%. 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' and 'Ha Telan virus' from the original 'second class' to the 'third class'. Regarding Gu Ertu virus, gu Ertu virus is separated from a sample of a grassland tick (Dermacentor nuttalli ticks) collected in Xinjiang in 2018, the main host is the grassland tick in the Golgi region of Xinjiang, the grassland tick is a dominant tick species in a certain region of the west part of China, the average positive rate of the grassland tick is 7.89%, the average positive rate is higher than the positive rate (2.1-5.4%) of SFTSV carried by the Chinese middle long-angle blood tick, and in-vitro and in-vivo experiments prove that GTV can be infected and replicated in tissue cells of various mammals and human, and can infect mice to cause diseases; serological investigation of healthy humans found that neutralizing antibody responses further suggested that GTV was a potentially dangerous new virus.
At present, regarding a neutralization monoclonal antibody of large allopad virus (SFTSV), a serum antibody library platform of a patient infected by the SFTSV is utilized for screening and preparing a humanized neutralization monoclonal antibody of the SFTSV, and a Gn protein immunization two-peak camel is utilized for screening and obtaining a nano neutralization monoclonal antibody of the SFTSV GP by utilizing a phage library, a nano monoclonal antibody and other technical platforms for the related research, and the humanized antibody is constructed. However, no studies have been reported on Ha Telan virus (HRTV) neutralizing mab. Since Ha Telan virus, dabiedada virus and Golgi virus all belong to RNA virus, genome replication lacks a proofreading mechanism, virus self-mutation easily causes toxicity, adaptation to host capacity and epidemiology changes, and different strains can recombine to generate new viruses adapting to other media, so that the epidemic distribution range is changed, and new diseases are generated. Furthermore, effective drugs and vaccines for preventing these 3 viral infections are still in the research stage, nor are there effective diagnostic methods or reagents. Therefore, the method provides a simple, convenient, rapid, sensitive and high-specificity detection reagent or therapeutic drug for the infection of the novel viruses such as Ha Telan 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 Ha Telan virus and the cross infection of the virus with the same genus, the research of neutralizing monoclonal antibody of the virus is of great significance to the control work of Ha Telan 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 monoclonal antibody specifically recognizing Ha Telan virus.
The invention also aims to fill the blank of the Ha Telan virus detection reagent in China, and provides a Hatland virus detection method and a kit for Ha Telan virus detection.
The invention also aims to provide a monoclonal antibody which can be used for specifically neutralizing Ha Telan virus, and provides a research basis for providing an alternative specific antiviral antibody or treatment method for Ha Telan virus infection treatment.
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 the hart virus, wherein the 3 complementarity determining regions CDRs (HCDR 1, HCDR2, HCDR 3) of the heavy chain variable region of the monoclonal antibody have the following amino acid sequences, respectively: HCDR1 shown in SEQ ID No. 5; HCDR2 as shown in SEQ ID No. 6; HCDR3 shown in SEQ ID No. 7; the 3 complementarity determining regions CDRs (LCDR 1, LCDR2, LC DR 3) of the light chain variable region of the monoclonal antibody have the following amino acid sequences: LCDR1 as shown in SEQ ID NO. 8; LCDR2 as shown in amino acid sequence LVS; LCDR3 as shown in SEQ ID NO. 9.
Preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 1.
Preferably, the heavy chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO. 3.
Preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 2.
Preferably, the light chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO. 4.
In a second aspect, the present invention provides a polynucleotide comprising a monoclonal antibody to the Ha Telan virus described above.
In a third aspect, the present invention provides a vector comprising a monoclonal antibody to the Ha Telan virus described above.
In a fourth aspect, the invention provides a host cell comprising a monoclonal antibody to the Ha Telan virus described above.
In a fifth aspect, the invention provides an immunoconjugate comprising a monoclonal antibody to the Ha Telan virus described above.
In a sixth aspect, the invention also provides the use of a monoclonal antibody against Ha Telan virus in the preparation of polynucleotides, recombinant vectors, expression vectors, recombinant proteins, genetically engineered host cells, immunoconjugates, and detection reagents for Ha Telan virus or anti-Ha Telan virus drugs.
The invention has the beneficial effects that:
the invention obtains a monoclonal antibody 10D6 (abbreviated as monoclonal antibody 10D 6) of the Hatland virus, wherein the monoclonal antibody 10D6 is a monoclonal antibody of envelope Glycoprotein (GP, abbreviated as GP protein) of the Ha Telan virus, can specifically identify spatial epitopes of Ha Telan virus, but cannot identify linear epitopes of Ha Telan virus, and cannot cross-identify Dabiedada virus and Golgi virus. The specificity of the monoclonal antibody 10D6 is strong, and half Effective Concentration (EC) of the specific recognition of the monoclonal antibody 10D6 on the spatial epitope of Ha Telan virus is obtained through indirect immunofluorescence detection 50 ) 15.80 mug/mL, can be used for preparing a Ha Telan virus detection reagent.
The inventionThe obtained monoclonal antibody 10D6 is further verified by a micro neutralization test: the monoclonal antibody 10D6 can specifically neutralize Ha Telan virus, and has no cross neutralization activity on virus members of two banda viruses, namely big banda virus and Golgi virus. Half-effective Inhibitory Concentration (IC) of monoclonal antibody 10D6 for neutralizing Ha Telan virus 50 ) Is 8.386 mug/mL, and can be used for preparing medicines for resisting Ha Telan virus.
The specific recognition and neutralization active epitope of the monoclonal antibody 10D6 is mainly aimed at GP protein of Ha Telan virus, especially space epitope, has strong specificity, can be prepared into a Ha Telan virus detection kit, is used for detecting a relevant pathogen of Banda virus, and can distinguish the pathogen in a sample: is a mere Ha Telan virus? Or is mixed with Ha Telan virus? The monoclonal antibody 10D6 can also be used for neutralizing the active site of Ha Telan virus so as to block Ha Telan virus infection, and is hopeful to prepare an immunoconjugate from the monoclonal antibody 10D6 of the invention, and the immunoconjugate is used for preparing an antibody drug for preventing or treating Ha Telan virus, so that the monoclonal antibody is applied to clinic for preventing and treating Ha Telan virus-caused diseases, and the monoclonal antibody is fully recognized and eliminated before the virus becomes a greater problem, so that the harm of the virus to life health of people is reduced as much as possible; can also be used in the serious fields of basic research, clinical research, flow regulation and the like of Ha Telan virus.
Drawings
FIG. 1 is a Western blot analysis of Ha Telan virus mab 10D6, wherein C is Ha Telan virus-infected Vero cells and V is the culture supernatant of Vero cells not infected with Ha Telan virus, i.e., a Vero cell reference control;
FIG. 2 is an indirect immunofluorescence assay of Ha Telan virus mab 10D6, PC as positive control and NC as negative control;
FIG. 3 is an indirect immunofluorescence assay of Ha Telan virus mab 10D6 at different concentrations with 3 recombinant baculoviruses, respectively, PC as positive control, NC as negative control, the first row of pictures corresponding to recombinant baculovirus vAc-HRTV-GP, the second row of pictures corresponding to recombinant baculovirus vAc-SFTSV-GP, and the third row of pictures corresponding to recombinant baculovirus vAc-GTV-GP;
FIG. 4 is a graph of nonlinear regression fit plotted according to fluorescence intensity of FIG. 3 with intermediate immunofluorescence detection plotted on the abscissa as a logarithmic value of Ha Telan virus mab 10D6 concentration and on the ordinate as a percentage of recognition efficiency;
FIG. 5 is a graph of a non-linear regression fit plotted according to the results of indirect immunofluorescence measurements of Vero cells infected with different concentrations of Ha Telan virus mab 10D6 after neutralization incubation with 3 Banda viruses (HRTV, GTV, SFTSV), respectively, with the concentration of Ha Telan virus mab 10D6 on the abscissa and the percentage of inhibition on the ordinate.
Detailed Description
Exemplary embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein the same or similar represent the same concepts, such as Ha Telan virus=hrtv, dobbad virus=fever with thrombocytopenia syndrome virus=sftsv, gu Ertu virus=gtv, monoclonal antibody=mab, etc.
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 was purchased from GE Healthcare in 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 system is purchased from americaPerkinElmer corporation of the country; 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); ha Telan virus (HRTV), dabiebanda virus (SFTSV), gu Ertu virus (GTV) are stored by national virus resource library of the institute of Siraitia martensii 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 Hartley blue virus, a preparation method and application thereof, wherein the cell strain capable of secreting monoclonal antibody 10D6 is screened by using an inactivated virus of Ha Telan virus as a target through a fusion technology of spleen cells and myeloma cells of mice immunized by Ha Telan virus; through molecular biology technology, the amino acid sequence and the coding nucleic acid sequence of the monoclonal antibody 10D6 are identified, a baculovirus eukaryotic expression vector containing Hartmann virus, dabiedada virus and Golgi virus is constructed, and recombinant baculovirus vAc-HRTV-GP, vAc-SFTSV-GP and vAc-GTV-GP are obtained after transfection of Sf9 cells; further, 2 detection platforms of western blotting detection (WB) and indirect immunofluorescence detection (IFA) were established, so that it was verified that the mab 10D6 specifically recognizes GP proteins of Ha Telan virus and that GP proteins of large banda virus and gulf-image virus could not be cross-recognized; confirmation by neutralization test: monoclonal antibody 10D6 specifically neutralizes Ha Telan virus, while there is no neutralizing activity against both banda virus and gulf-image virus members. The following describes specific embodiments of the present invention, and the specific contents are as follows:
example 1 preparation of monoclonal antibody 10D6
According to the invention, after a Ha Telan virus is immunized on a mouse, the separated spleen cells and myeloma cells are subjected to cell fusion on feeder cells, ha Telan virus infected cells are used as positive hole targets, hybridoma cell strains are screened out, and finally monoclonal cell strain 10D6 secreting GP protein resisting Ha Telan virus is screened out from 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 established animal immunization schemes, SPF-class female BALB/c mice with the age of 6-8 weeks are selected as immunized animals, and the immunogen is concentrated inactivated Ha Telan virus; after emulsifying the virus with an equal volume of Freund's (non) complete adjuvant (Sigma-Aldrich, USA), two immunizations were performed by intraperitoneal and subcutaneous multipoint injection, each immunization being separated by two weeks, with an immunization volume of 200. Mu.L/mouse.
2) Isolation of spleen cells
Taking immunized BALB/c 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, stripping off 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 quantity ratio isSpleen cells myeloma cells = 5:1), centrifuged at 1600rpm for 10 min, and the supernatant 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, taking the supernatant of the fused cells, screening out the monoclonal antibody 10D6 from the positive hole, and carrying out the next monoclonal antibody verification.
Example 2 validation of mab 10D6
1) Western blotting detection (WB)
And (3) taking Ha Telan virus infected Vero cells and amplifying in the Vero cells, collecting Ha Telan virus infected Vero cells and cell supernatants, respectively adding beta-propiolactone for inactivation, separating viral proteins by 12% SDS-PAGE gel electrophoresis, transferring to a 0.2um polyvinylidene fluoride membrane, sealing with 5% (w/v) skimmed milk powder prepared by PBS at 37 ℃ for 1 hour, and performing PCR detection to show that Ha Telan viruses positively amplify in the Vero cells and cell supernatants.
Positive control was polyclonal antibody against the rabbit anti Ha Telan viral nucleoprotein, diluted with primary antibody 1:2000 dilution; the reference control of Vero cells was beta-Actin murine monoclonal antibody, diluted with primary antibody diluent 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 obtained mab 10D6 and Ha Telan virus separated after inactivation is shown in fig. 1, which proves that mab 10D6 cannot recognize Ha Telan virus separated from gel after inactivation, i.e., mab 10D6 cannot recognize linear epitope of Ha Telan virus.
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 Ha Telan virus-containing cell supernatant was added at a multiplicity of infection MOI=1, and at 37℃with 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 rabbit anti Ha Telan viral nucleoprotein, 1 with 1% bsa: 2000 dilution; the negative control was healthy mouse serum with 1% bsa at 1:2000 dilution; taking the supernatant of the fusion cells as a primary antibody, adding the treated 96-well cell plate according to 100 mu/well, and incubating for 2 hours at 37 ℃; 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 Hoechest33258 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 obtained monoclonal antibody 10D6 and the in-situ immobilized, inactivated and Ha Telan viruses are shown in figure 2, and the monoclonal antibody 10D6 is proved to be capable of specifically recognizing the spatial epitope of Ha Telan viruses.
Example 3 sequence identification of mab 10D6
The cultured monoclonal antibody 10D6 cell strain of Ha Telan 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 on the amplified and cultured clone vector by using universal primers M13F and M13R, wherein the specific procedures are as follows:
1) Nucleic acid extraction:
the monoclonal antibody 10D6 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 DH10B receptor bacteria are coated on an LB plate containing 100 mu g/mL Ampicillin, and the positive bacteria containing light and heavy chains are obtained after overnight at 37 ℃.
Positive colony culture and then sequencing by inspection: 5-10 positive bacteria are picked from the 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 liquagmetaceae organism for sequencing, and the sequencing general primers are designated as M13F and M13R.
4) Sequencing results
According to sequencing feedback results, obtaining a nucleotide sequence SEQ ID NO.3 of a heavy chain variable region (VH) and a nucleotide sequence SEQ ID NO.4 of a light chain variable region (VL) of the monoclonal antibody 10D 6; converting the sequencing result into an amino acid sequence, and obtaining the amino acid sequence SEQ ID NO.1 of a heavy chain variable region (VH) and the amino acid sequence SEQ ID NO.2 of a light chain variable region (VL) of the monoclonal antibody 10D 6; by analysis, the sequences of the 3 complementarity determining regions CDRs (HCDR 1, HCDR2, HCDR 3) of the heavy chain variable region of mab 10D6 are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, respectively; the sequences of the 3 complementarity determining regions CDRs (LCDR 1, LCDR2, LCDR 3) of the light chain variable region are shown as SEQ ID NO.8, amino acid sequence LVS, SEQ ID NO.9, respectively. The sequence for mab 10D6 is summarized in table 2 below:
table 2: sequence listing of mab 10D6
The light chain variable region of mab 10D6 belongs to a kappa-type immunoglobulin (Ig) by Blastn sequence alignment.
EXAMPLE 4 application of monoclonal antibody 10D6 (detection reagent)
1) Eukaryotic expression of GP proteins containing 3 viruses
The full-length nucleic acid sequences of GP proteins of Ha Telan viruses (GenBank serial number: NC 024494), dabanda viruses (GenBank serial number: KY 440773) and Gu Ertu viruses (GenBank serial number: KT 328592) are amplified from cDNA of 3 viruses of Ha Telan viruses, dabanda viruses and Golgi viruses by PCR amplification technology, cloned to the downstream of the Polyhedrin promoter of eukaryotic expression plasmid vector pFast-Bac-Dual, and 3 recombinant plasmids pFBD-HRTV-GP, pFBD-SFTSV-GP and pFBD-GTV-GP are obtained respectively through expression of competent cells DH5 alpha. The 3 recombinant plasmids are transfected into Sf9 cells by using Cellfectin transfection reagent, cell supernatants are collected after 3-5 days of culture in a cell culture box at 27 ℃ to obtain 3 recombinant baculoviruses vAc-HRTV-GP, vAc-SFTSV-GP and vAc-GTV-GP respectively, the Sf9 cells are infected by the 3 recombinant baculoviruses, and the culture is continued for 3 days in the cell culture box at 27 ℃.
Referring to example 2, the recombinant baculovirus-infected Sf9 cells described above were treated by the method of intermediate immunofluorescence assay (IFA), and the cell plates after fixation, permeabilization and blocking were used for subsequent indirect immunofluorescence assay (IFA).
2) Detection of recombinant baculovirus by monoclonal antibody 10D6 (IFA)
The monoclonal antibody 10D6 is diluted to 100 mug/mL, and then twice gradient dilution is sequentially carried out until the final concentration is 0.195 mug/mL, and 10 concentration gradients are provided in total: 100. Mu.g/mL, 50. Mu.g/mL, 25. Mu.g/mL, 12.5. Mu.g/mL, 6.25. Mu.g/mL, 3.125. Mu.g/mL, 1.563. Mu.g/mL, 0.781. Mu.g/mL, 0.390. Mu.g/mL, and 0.195. Mu.g/mL.
Positive control: polyclonal antibodies to the Gn protein of the murine anti Ha Telan virus, to the Gn protein of the murine anti large banda virus and to the Gn protein of the murine anti gul diagram virus, respectively, were raised against 1% bsa at 1:2000 dilution; the negative control was healthy mouse serum with 1% bsa at 1:2000 dilution.
Referring to example 2, the method of intermediate immunofluorescence assay (IFA) was used to incubate with different concentrations of mab 10D6 with 3 recombinant baculoviruses, vAc-HRTV-GP, vAc-SFTSV-GP and vAc-GTV-GP, respectively, and the results were observed using a high content cell imaging analysis system to obtain the fluorescence intensity values for each well.
3) IFA detection result analysis
Fluorescence intensity value obtained by IFA detection, and substitutionBinding of mab 10D6 to 3 recombinant baculoviruses is shown. The relative fluorescence intensities of the fluorescence intensity values of each well and the fluorescence intensity values of the positive control wells are defined as recognition efficiencies, and the recognition efficiencies for 3 recombinant baculoviruses are respectively recorded as: anti-HRTV, anti-SFTSV, anti-GTV, and nonlinear regression curve of dose-efficiency was drawn using GraphPad Prism8 software to further calculate half-Effective Concentration (EC) of monoclonal antibody 10D6 for 3 recombinant baculoviruses 50 )。
The results of indirect immunofluorescence detection of mab 10D6 on 3 recombinant baculoviruses, vAc-HRTV-GP, vAc-SFTSV-GP and vAc-GTV-GP, are shown in fig. 3, fig. 3 showing that specific recognition was evident for each of 6 representative concentrations of mab 10D6 and recombinant baculovirus, vAc-HRTV-GP.
Based on the fluorescence intensity values of the mab 10D6 and 3 recombinant baculovirus IFA detection reactions in fig. 3, a non-linear fit curve was plotted as shown in fig. 4. As can be seen from fig. 4, the recognition efficiencies of the mab 10D6 on the 3 recombinant baculoviruses are different, wherein the recognition efficiency of the recombinant baculoviruses vAc-HRTV-GP is positively correlated with the concentration of the mab 10D6, i.e., it is further demonstrated that the mab 10D6 can specifically recognize the GP protein of the Ha Telan virus. Calculation of median effective concentration EC of mab 10D6 for specific recognition of recombinant baculovirus vAc-HRTV-GP according to FIG. 4 50 =15.80 μg/mL, but no specific recognition was made for recombinant baculoviruses vAc-SFTSV-GP and vAc-GTV-GP containing GP proteins of SFTSV and GTV.
EXAMPLE 5 application (neutralizing Activity) of monoclonal antibody 10D6
To evaluate the specific neutralizing activity of mab 10D6 against Ha Telan virus and cross-neutralizing activity against large banda virus and gulf-graph virus, the following virus-antibody neutralization assay was performed, with the following specific experimental steps:
1) Vero cells were grown at 1X 10 4 Uniformly spreading the cells into 96-well cell plates, adding 50 mu L of DMEM medium containing 2% fetal calf serum into each well, and keeping the cell density to 80% for later use;
2) The monoclonal antibody 10D6 is diluted to 100 mug/mL, and then twice gradient dilution is sequentially carried out until the final concentration is 0.195 mug/mL, and 10 concentration gradients are provided in total: 100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 3.125 μg/mL, 1.563 μg/mL, 0.781 μg/mL, 0.390 μg/mL, and 0.195 μg/mL;
(3) mixing the 10 diluted monoclonal antibody 10D6 with 50 μl of 3 kinds of Banda virus (HRTV, GTV, SFTSV) (virus titer TCID) with the same volume 50 =100), and incubating at 37 ℃ for 2 hours to obtain virus neutralization suspension; positive control wells were 3 virus suspensions, ha Telan virus, dabanda virus, golgi virus, virus titre TCID 50 =100; negative control was uninfected healthy Vero cell suspension;
(4) adding the processed suspension into the well plate hole containing Vero cells obtained in the step 1), and adding 5% CO at 37 DEG C 2 Culturing in a cell incubator for 3 days;
(5) the neutralization activity of the virus-antibody was examined by the method of intermediate immunofluorescence assay (IFA) in reference example 2. The secondary antibodies correspond to polyclonal antibodies against HRP-labeled rabbit anti-Ha Telan viral nucleoprotein, rabbit anti-dobbad viral nucleoprotein, rabbit anti-golgi viral nucleoprotein, respectively with 1% bsa at 1:2000 dilution, adding diluted secondary antibody according to 50 mu L/hole, and incubating for 1 hour at 37 ℃;
(6) the results were observed using a high content cell imaging analysis system to obtain the fluorescence intensity values for each well. The fluorescence intensity values obtained by IFA detection represent the number of Vero cells infected with viruses in each well, represent the infection inhibition rates of different concentrations of the monoclonal antibody 10D6 on different viruses, and are respectively recorded as follows: anti-HRTV, anti-SFTSV, anti-GTV. Dose-inhibited nonlinear regression curves were plotted using GraphPad Prism8 software to further calculate the median effective inhibitory concentration (IC 50 )。
As shown in FIG. 5, the inhibition rates of the monoclonal antibody 10D6 on 3 kinds of Banda viruses are different, wherein the identification efficiency of the HRTV is positively correlated with the concentration of the monoclonal antibody 10D6, which proves that the monoclonal antibody 10D6 can specifically identify and neutralize Ha Telan viruses, namely, the 10D6 only has specific neutralization activity on Ha Telan viruses, but cannot neutralize large BandaViruses and gul-graph viruses. Calculation of half-effective inhibitory concentration IC of mab 10D6 against HRTV according to FIG. 5 50 = 8.386 μg/mL, but there was no cross-neutralizing capacity for large banda virus and gulf-map virus.
The monoclonal antibody 10D6 of the Ha Telan virus provided by the invention can specifically recognize the GP protein of Ha Telan virus, can specifically recognize only the spatial epitope of Ha Telan virus, has strong specificity, can be used for preparing Ha Telan virus detection reagent, and has half effective concentration of 15.80 mug/mL; the monoclonal antibody 10D6 of Ha Telan virus provided by the invention can specifically neutralize Ha Telan virus, and half effective inhibition concentration of the neutralized Ha Telan virus is 8.386 mug/mL, so that the monoclonal antibody can be used for preparing specific antibody medicines for resisting Ha Telan virus, and is beneficial to research and control of Ha Telan 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 hartlet blue virus, 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. 5; HCDR2 as shown in SEQ ID No. 6; HCDR3 shown in SEQ ID No. 7; 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. 8; LCDR2 as shown in amino acid sequence LVS; LCDR3 as shown in SEQ ID NO. 9.
2. The monoclonal antibody of Ha Telan virus according to claim 1, wherein: the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 1.
3. The monoclonal antibody of Ha Telan virus according to claim 1, wherein: the heavy chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO. 3.
4. The monoclonal antibody of Ha Telan virus according to claim 1, wherein: the light chain variable region has an amino acid sequence shown in SEQ ID NO. 2.
5. The monoclonal antibody of Ha Telan virus according to claim 1, wherein: the light chain variable region is encoded by the nucleic acid sequence shown in SEQ ID NO. 4.
6. A polynucleotide encoding a monoclonal antibody to the Ha Telan virus of any one of claims 1-5.
7. A vector comprising the polynucleotide of claim 6.
8. A host cell comprising the vector of claim 7.
9. An immunoconjugate comprising a monoclonal antibody to the Ha Telan virus of any one of claims 1-5.
10. Use of a monoclonal antibody against a Ha Telan virus as claimed in any one of claims 1 to 5 in the manufacture of a Ha Telan virus detection reagent or an anti-Ha Telan virus medicament.
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| CN202310044495.7A CN116410308A (en) | 2023-01-30 | 2023-01-30 | Monoclonal antibody of Hatland virus and application thereof |
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| CN202310044495.7A CN116410308A (en) | 2023-01-30 | 2023-01-30 | Monoclonal antibody of Hatland virus and application thereof |
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