CN110777121A - Monoclonal antibody hybridoma cell 3B5 strain secreting anti-canine distemper virus H protein - Google Patents
Monoclonal antibody hybridoma cell 3B5 strain secreting anti-canine distemper virus H protein Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- 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
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
- A61K2039/507—Comprising a combination of two or more separate antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Abstract
The invention discloses a monoclonal antibody hybridoma cell 3B5 strain capable of secreting high-middle-activity anti-canine distemper virus H protein, and belongs to the technical field of biology. Screening out a hybridoma cell strain 3B5 with high neutralization activity from the established 127-strain hybridoma cell bank, wherein the epitope is amino acid 228-237 of H protein of canine distemper virus. Neutralizing titer of 2 for antibody combined therapeutic agent consisting of monoclonal antibody 3B5 and monoclonal antibodies 1D7 and 2G3
13Compared with the neutralizing titer of the single use of 3B5, 1D7 and 2G3, the neutralizing potency of the monoclonal antibody combination therapeutic agent is improved by 8-32 times, the clinical application test result of the monoclonal antibody combination therapeutic agent is improved from 98 percent to 100 percent compared with the effect of the combined use of the monoclonal antibodies 1D7 and 2G3,the treatment period is shortened from 4 days to 2 days. Can be used for clinical treatment of Canine Distemper (CD) pathogenic animals, and has the advantages of reduced production cost and improved therapeutic effect.
Description
Technical Field
The invention discloses a monoclonal antibody hybridoma cell 3B5 strain secreting anti-canine distemper virus H protein, and belongs to the technical field of biology.
Background
Canine distemper (canine distemper) is an acute, thermal and highly contagious disease caused by Canine Distemper Virus (CDV), often causes symptoms such as abnormalities of digestive tract, respiratory tract and nervous system, and can cause immune suppression of the body. The natural infection host of canine distemper is expanded from traditional canines, ferrets and raccoons to various animals such as carnivora animals, artiodactyla pigs, primates, named as monkey and fin-podales seal, the morbidity can almost reach 100 percent, the mortality rate is more than 80 percent, and the canine distemper is one of important infectious diseases which endanger the canine industry and the economic animal breeding industry.
CDV belongs to Paramyxoviridae, measles virus, and has circular or elliptical virus particle, sometimes long filament, and diameter of 150-300 nm. The particle center contains a spiral nucleocapsid with a width of about 15-17 nm, the outer surface is coated with a film with approximate double-layer outline, and 1.3nm rod-shaped fiber protrusions are arranged on the film. The genome of the virus is nonsegmented negative strand RNA, the total length is 15,616nt, and a 3 'end leader sequence, a nucleocapsid protein gene (N), a phosphoprotein gene (P), a matrix protein gene (M), a fusion protein gene (F), a hemagglutinin protein gene (H), a large protein gene (L) and a 5' end leader sequence are sequentially arranged from the 3 'end to the 5' end of the virus. P, N, L is medulla protein, which is involved in RNA transcription and replication, and N protein has the function of wrapping and protecting internal genes, and P protein may have the function of polymerase. M, F, H is a coating protein, which is located on the outside of the N protein, and is embedded in the M protein by the hydrophobic region near the N-terminus of the H protein. Wherein, the total length of the H gene is 1947nt, only contains an open reading frame, and the encoded H protein is one of glycoprotein on the surface of CDV envelope, can interact with receptor (such as SLAM and nectin-4) on the surface of target cell, and mediates the virus to invade host; meanwhile, the H protein has a plurality of neutralizing epitopes and is a main antigen for inducing the body to generate neutralizing antibodies.
At present, most of vaccines used for preventing the disease are attenuated vaccines, but the disease still occurs in a plurality of farms for dogs, minks, foxes and the like after the attenuated vaccines are applied. The reason for the analysis is related to maternal antibodies, vaccine titer, method of use and virus variation. Animals immunized by the attenuated vaccine cannot be completely protected, and a plurality of immune animal groups suffer from canine distemper and are very disastrous. Therefore, in addition to positive prevention of canine distemper, effective treatment of diseased animals is necessary to minimize losses. The hyperimmune serum of canine distemper virus is commonly used for treating canine distemper, but has high preparation cost, easy transmission of other viruses, uneven antibody neutralization titer and difficult standardization. Therefore, the monoclonal antibody has good preparation specificity, high neutralizing potency of the antibody, easy standardization and low preparation cost, and is inevitably selected.
Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone that are directed against only a particular epitope (open clouds, et al. monoclonal antibody technology related patent analysis [ J ] biotechnology, 2012(6): 78-86.). Monoclonal antibodies recognize specific epitopes (i.e., the amino acid sequences of the antibody that bind to the antigen). Therefore, analysis of the epitope recognized by the monoclonal antibody is of great significance to subsequent canine distemper virus protein function research, treatment, diagnosis and detection and other aspects.
Two hybridoma cell strains 1D7 and 2G3 secreting high neutralizing activity canine distemper virus monoclonal antibody (a hybridoma cell strain 1D7 secreting high neutralizing activity canine distemper virus monoclonal antibody, national invention patent application No. 201210081170.8, publication No. CN102618503B, publication No. 2013-06-12, a monoclonal antibody and antibody composition for neutralizing canine distemper virus, national invention patent application No. 201310036576.9, publication No. CN103059133B, publication No. 2015-12-09) are screened, and the monoclonal antibody 1D7 or/and 2G3 prepared by the method can neutralize CDV strains of different animal sources, and the range of anti-canine distemper virus strains is wide.
However, it is not clear to which viral structural protein the monoclonal antibodies (1D7 and 2G3) are directed, and the epitopes of the monoclonal antibodies have not been resolved. The clinical treatment capability of Canine Distemper (CD) pathogenic animals needs to be enhanced, the production cost needs to be reduced, the treatment effect needs to be improved, and the clinical application range needs to be wider.
Disclosure of Invention
Technical problem
The monoclonal antibody hybridoma cell strain secreting the anti-canine distemper virus H protein provided by the invention has better biological characteristics, and a monoclonal antibody 3B5 prepared from the monoclonal antibody has stronger virus neutralizing capacity with an antibody combination therapeutic agent consisting of other canine distemper virus monoclonal antibodies 1D7 and 2G3, can be used for clinical treatment of Canine Distemper (CD) pathogenic animals, wherein the capacity of neutralizing the canine distemper virus is enhanced, the production cost is reduced, the treatment effect is improved, and the clinical application range is wider.
Technical scheme
In order to achieve the purpose, the method is realized by the following technical scheme:
a monoclonal antibody hybridoma cell 3B5 strain secreting anti-canine distemper virus H protein, wherein the hybridoma cell 3B5 strain is preserved in the China center for type culture Collection in 7 and 9 months in 2019, and the addresses are as follows: the preservation number of the preservation center of Wuhan university in Wuhan, China is CCTCC NO: c2019143, classification naming: the monoclonal antibody hybridoma cell line 3B5 for resisting the canine distemper virus H protein is characterized in that the epitope recognized by the canine distemper virus H protein monoclonal antibody prepared from the hybridoma cell line 3B5 is at the 228 th-237 th amino acid of the canine distemper virus H protein, and the polypeptide sequence of the epitope is YGKTYLLVPD.
The hybridoma cell 3B5 secreting the canine distemper virus H protein monoclonal antibody is applied to preparation of a canine distemper virus monoclonal antibody combined therapeutic agent.
And the monoclonal antibody 3B5 of the H protein of the canine distemper virus, which is prepared by using the hybridoma cell 3B5 strain. The monoclonal antibody 3B5, the monoclonal antibody 1D7 secreted by the hybridoma cell 1D7 strain and the monoclonal antibody 2G3 secreted by the hybridoma cell 2G3 strain are mixed uniformly according to the ratio of 1:1:1 to prepare the monoclonal antibody combination therapeutic agent.
The monoclonal antibody combined therapeutic agent can be used for clinical treatment of canine distemper.
Advantageous effects
It is not clear in the prior art to which viral structural protein the monoclonal antibodies (1D7 and 2G3) are directed, nor is the epitope of the monoclonal antibody resolved. The invention screens another monoclonal antibody hybridoma cell 3B5 strain (Sunwei, etc.. the preparation and identification of a canine distemper virus H protein monoclonal antibody [ J/OL ] Chinese veterinary science, first network 2019-08-16, https:// doi.org/10.16656/j.issn.1673-4696.2019.0182.), and the prepared monoclonal antibody 3B5 is directed against the canine distemper virus H protein, and the screening of the recognition epitope of the monoclonal antibody shows that the epitope of 3B5, 2G3 and 1D7 combined with CDV is different. And the epitope recognized by the monoclonal antibody is determined by a peptide scanning method to be amino acids 228-237 of the H protein of CDV, the polypeptide sequence of the epitope is YGKTYLLVPD, and the epitope is not reported and belongs to a newly discovered canine distemper virus neutralizing epitope.
The invention utilizes an indirect ELISA detection method established by taking recombinant H protein as a coating antigen to screen out a hybridoma cell strain 3B5 with high neutralization activity from an established 127-strain hybridoma cell bank, the biological performance of the hybridoma cell strain is excellent, the epitope recognized by the monoclonal antibody is determined to be amino acids 228-237 of the H protein of CDV by a peptide scanning method, the polypeptide sequence of the epitope is YGKTYLLVPD, and the epitope is not reported yet. The antibody combination therapeutic agent composed of the prepared monoclonal antibody 3B5 and other canine distemper virus monoclonal antibodies 1D7 and 2G3 has stronger virus neutralizing capacity, can be used for clinical treatment of Canine Distemper (CD) pathogenic animals, has enhanced capability of neutralizing canine distemper viruses, reduces the production cost, improves the treatment effect, and has wider clinical application range.
The invention has the following characteristics and advantages:
1. the hybridoma cell 3B5 strain of the canine distemper virus H protein monoclonal antibody is screened from a hybridoma cell bank in which 127 strains secrete anti-CDV monoclonal antibodies by an indirect ELISA detection method established by taking recombinant H protein as a coating antigen, has very excellent biological performance, and is directed against the virus protein H protein.
2. The canine distemper virus H protein monoclonal antibody 3B5 prepared from the hybridoma cell 3B5 strain recognizes an epitope at amino acids 228-237 of a canine distemper virus H protein, the polypeptide sequence of the epitope is YGKTYLLVPD, and the epitope is not reported.
3. The monoclonal antibody 3B5 of the canine distemper virus H protein is different from the epitope recognized by the monoclonal antibody 1D7 secreted by the hybridoma cell 1D7 strain and the epitope recognized by the monoclonal antibody 2G3 secreted by the hybridoma cell 2G3 strain, and the three are uniformly mixed according to the ratio of 1:1:1 to prepare the monoclonal antibody combination therapeutic agent which can overcome the singleness of the monoclonal antibody recognizing the epitope and improve the neutralizing activity of the monoclonal antibody against CDV.
4. The neutralizing potency of the monoclonal antibody combination therapeutic agent of the present invention is 2
13The neutralizing potency of the antibody therapeutic agent is obviously higher than that of the antibody therapeutic agent used alone or in combination of two monoclonal antibodies (the neutralizing potency of the monoclonal antibodies 3B5, 1D7 and 2G3 is 2 respectively
8、2
10、2
10The neutralizing potency of the two combined antibody combination therapeutic agents 3B5+1D7, 3B5+2G3 and 1D7+2G3 is 2
11、2
11、2
12) The combined monoclonal antibody combination therapeutic agent has stronger virus neutralizing capacity, and the virus neutralizing capacity is improved by 8-32 times compared with that of the single use of 3B5, 1D7 and 2G 3.
5. The monoclonal antibody combined therapeutic agent is used for clinical treatment, the clinical application test result of the monoclonal antibody combined therapeutic agent is improved from 98% to 100% compared with the effect of the combined monoclonal antibodies 1D7 and 2G3, and the treatment period is shortened from 4 days to 2 days. Can be used for clinical treatment of Canine Distemper (CD) pathogenic animals, reduces production cost, improves treatment effect, and has wider clinical application range.
Biological preservation
The hybridoma 3B5 strain was deposited in the chinese type culture collection on 7/9/2019 at address: the preservation number of the preservation center of Wuhan university in Wuhan, China is CCTCC NO: c2019143, classification naming: monoclonal antibody hybridoma cell strain 3B5 for resisting canine distemper virus H protein.
Detailed Description
Establishment of monoclonal antibody hybridoma cell strain
Preparation of CDV antigen
Inoculating CDV Onderstepoort strain (product of International Inc. of England, Inc.) in Vero cell (purchased from ATCC cell bank), collecting infection supernatant after the cell has complete lesion, repeatedly freezing and thawing for three times, centrifuging at 8000r/min for 3 min, and removing cell debris; the supernatant was collected and PEG was added
6000(product of Sigma Co.) was added to a final concentration of 9%, sodium chloride was further added to a final concentration of 3%, and the mixture was stirred at room temperature to dissolve and precipitated at 4 ℃ overnight. The next day, centrifugation was carried out at 8000r/min for 1 hour, and the supernatant was discarded and resuspended in 5mL of TNE solution (pH 7.8). Adding the heavy suspension into 60% -40% -20% sucrose gradient solution, ultracentrifuging at 60000r/min for 3 hours, collecting layered protein bands (between 60% -40%), absorbing virus bands, observing virus morphology by electron microscope negative staining, and measuring virus concentration by a spectrophotometer and storing at-80 ℃ for later use.
2. Animal immunization
Female BALB/C mice (purchased from the university of Yangzhou, comparative medicine laboratory center) at 8 weeks of age were immunized with purified CDV immunizing antigen by intraperitoneal injection at 200. mu.g/mouse. The antigen was emulsified with an equal volume of Freund's complete adjuvant (product of Sigma) and then immunized 2 times every 14 days with Freund's incomplete adjuvant (product of Sigma) such as floral antigen. Blood is collected by cutting off the tail on the 6 th day after 3 rd immunization, the serum antibody titer is measured by an indirect ELISA method, a mouse with the antibody titer reaching more than 1:10000 is selected, and the purified CDV antigen is used for boosting immunization again 3 days before fusion.
3. Cell fusion
Adopting PEG cell fusion method to obtain S with good growth stateMixing P2/0 myeloma cells (purchased from ATCC cell bank) and immunized BALB/C mouse spleen cells at a ratio of 1:5, centrifuging at 1000r/min for 10 min, discarding supernatant, flicking the bottom of the tube with palm to make the cells loose and uniform, preheating in 40 deg.C water bath, adding 50% PEG preheated to 40 deg.C within 45 s with 1mL pipette
4000(Sigma Co., Ltd.) 1mL of the medium was gently shaken while adding the medium, 15mL of fetal calf serum-free RPMI-1640 medium preheated to 37 ℃ was added over 90 seconds, the mixture was left to stand at room temperature for 10 minutes, centrifuged at 1000r/min for 10 minutes, the supernatant was discarded, resuspended in 10% Fetal Calf Serum (FCS) (Seimer Feishi technology Co., Ltd.) and HAT (Sigma Co., Ltd.) in RPMI-1640 medium, and the resulting suspension was dispensed onto a 96-well plate containing feeder cells and placed in a 5% CO-free plate
2Culturing in an incubator. After 3 days, the cells were supplemented with RPMI-1640 medium containing HAT (Sigma) and 10% FCS (Seimer Feishal technologies), and after 5 days, the cells were cultured in RPMI-1640 medium containing HT (Sigma) and 10% FCS (Seimer Feishal technologies), and after 10 days, the cells were cultured in RPMI-1640 medium containing 10% FCS (Seimer Feishal technologies), and when the fused cells had grown to 1/5 of the bottom area of the well of 96-well plate, the supernatant was collected and subjected to antibody detection.
4. Screening of hybridoma cell lines
Determining the coating concentration of the purified CDV antigen according to a matrix method, performing double dilution on the purified CDV antigen by using a coating solution which is 0.05mol/L of pH9.6 carbonate buffer solution, coating an ELISA plate by using the amount of the CDV antigen diluted to 400 times, coating 100 mu L/hole, placing at 4 ℃ overnight, washing 3 times by using PBST (phosphate buffer solution containing 0.05% Tween-20 and having pH 7.4) for 5 minutes each time, and finally drying by beating; blocking each well with PBST containing 10% calf serum, 200 μ L/well, standing at 37 deg.C for 2 hr, washing with PBST for 3 times, each time for 5 min, and drying at the last time; adding cell supernatant 12 days after fusion, immune mouse positive serum diluted 1:1000 and mouse negative serum diluted 1:1000 into corresponding holes, acting at 37 ℃ for 1 hour, washing for 3 times by PBST (PBST), each time for 5 minutes, and finally patting dry; adding Horse Radish Peroxidase (HRP) -labeled goat anti-mouse IgG (Shanghai Biyuntian biotechnology Co., Ltd.) diluted at 1:5000 into the mixture at a concentration of 100. mu.L/well, standing at 37 deg.C for 1 hr, washing with PBST for 5 min for 3 times,patting dry for the last time; adding TMB substrate, 100 mu L/hole, and developing for 10 minutes at room temperature in a dark place; the reaction was stopped by adding 50. mu.L of 2mol/L sulfuric acid per well. OD determination by enzyme-linked immunosorbent assay
450nmValues were zeroed for blank control, P is the value of each well, N is the OD of the negative reference serum
450nmValue when OD of negative reference serum
450nmValue less than or equal to 0.1, OD of positive reference serum
450nmValue and OD of negative reference serum
450nmThe ratio of the values is more than or equal to 2.1, namely the detection holes with the P/N more than or equal to 2.1 are judged to be positive under the premise that negative and positive controls are established, the detection is carried out once after every 2 days, and hybridoma cells with positive detection results of the two times are cloned.
5. Cloning of hybridoma cells
Viable cells in positive wells were stained and counted with trypan blue, diluted to 100 cells/15 mL in RPMI-1640 medium containing 10% FCS (Seimer Feishell technology Co., Ltd.), the diluted cell suspension was added to a 96-well cell culture plate, 0.15mL in each well, 37 ℃ and 5% CO2, and after 4 days, formation of clonal cells was observed under a microscope, only single clonal growth wells were recorded, and cell supernatants were collected at 8 days for timely ELISA detection. Selecting positive monoclonal cells, cloning for more than 3 times until all cell wells are positive and each well detects OD
450nmThe values are closer. And (3) performing expanded culture on the cloned CDV specific monoclonal antibody hybridoma cell strain, and freezing and storing. After 20 times of cell fusion, screening, cloning and identification, a 127-strain hybridoma cell bank capable of stably secreting the NDV specific monoclonal antibody is established.
6. Expression and purification of canine distemper virus H protein
A nucleic acid sequence (synthesized by general biosystems (Anhui) Co., Ltd.) optimized by E.coli codon was artificially synthesized based on the H gene sequence of Onderstepoort strain (accession No.: AF378705) published in GenBank, and NheI and HindIII cleavage sites were introduced into both ends of the sequence. Subsequently, the artificially synthesized sequence and prokaryotic expression plasmid pET28a (+) (product of Beijing Solebao science and technology Co., Ltd.) were subjected to NheI and HindIII (product of Baori physician's technology (Beijing) Co., Ltd.) enzymes, respectivelyCutting and digesting, identifying by agarose gel electrophoresis, respectively recovering fragments by using a gel recovery kit (a product of Tiangen Biochemical technology (Beijing) Co., Ltd.), incubating for 1 hour at 4 ℃ under the connection effect of T4 ligase (a product of Baori doctor Tech Co., Ltd.), and transforming to E.coli Rosetta competence (a product of Baori doctor Tech Co., Ltd.), thereby obtaining the strain capable of expressing the H protein of the canine distemper virus. The strain was cultured to OD
600nmWhen the value is about 1.0, IPTG (product of Baori doctor's technology (Beijing) Co., Ltd.) with a final concentration of 1mmol/L is added, induced expression is carried out at 30 ℃ for 6 hours, the thalli are collected and purified by a Ni-NTA affinity chromatography column (product of GE Co., Ltd.), purified recombinant protein is collected, the concentration of the purified recombinant protein is measured by a BCA protein quantitative determination kit (product of Saimer Feishell science Co., Ltd.), and the recombinant protein is stored at-70 ℃ for later use.
7. Screening of hybridoma cell strain of canine distemper virus H protein monoclonal antibody
Determining the coating concentration of the purified H protein according to a matrix method, diluting the purified H protein by using a coating solution which is 0.05mol/L of carbonate buffer solution with pH9.6 in a multiple ratio, diluting to 0.1 mu g/hole, coating overnight at 4 ℃, washing for 3 times by PBST (PBST), washing for 5 minutes each time, and finally beating to dry; blocking each well with PBST containing 10% calf serum, 200 μ L/well, standing at 37 deg.C for 2 hr, washing with PBST for 3 times, each time for 5 min, and drying at the last time; adding 127 cell supernatants (1:1000 dilution) stably secreting CDV specific monoclonal antibody into corresponding wells, reacting at 37 deg.C for 1 hr, washing with PBST for 3 times, each time for 5 min, and drying; adding 1:5000 diluted Horse Radish Peroxidase (HRP) -labeled goat anti-mouse IgG (Shanghai Biyuntian biotechnology Co., Ltd.), 100 μ L/well, standing at 37 deg.C for 1 hr, washing with PBST for 3 times (5 min each time), and drying by patting for the last time; adding TMB substrate, 100 mu L/hole, and developing for 10 minutes at room temperature in a dark place; the reaction was stopped by adding 50. mu.L of 2mol/L sulfuric acid per well. OD determination by enzyme-linked immunosorbent assay
450nmValue, selecting OD
450nmThe CDV specific monoclonal antibody hybridoma cell strain corresponding to the highest value. The results of 3 repetitions of the above experiment revealed that the OD of the monoclonal antibody of hybridoma 3B5 strain
450nmThe value is highest.
8. Preparation of ascites
Injecting sterilized liquid paraffin into BALB/C mice (purchased from the university of Yangzhou, comparative medicine experiment center) with the age of 8-10 weeks, 0.5 mL/mouse, injecting hybridoma cell strain into the abdominal cavity of the mice after 7 days, wherein each hybridoma cell strain is 0.2mL (containing 2 x 10 of hybridoma cell strain)
6～5×10
6Individual hybridoma cells), after 7-10 days, taking ascites of a mouse with obviously swollen abdomen, centrifuging for 10 minutes at 3000r/min, collecting supernatant, subpackaging and storing at-20 ℃ for later use.
Biological Properties of monoclonal antibody 3B5
1. Chromosome analysis of hybridoma cell lines
The hybridoma cells were chromosome-counted by giemsa staining. SP2/0 myeloma cells (purchased from ATCC cell bank) and positive hybridoma cells were cultured, grown to logarithmic phase, and colchicine was added to the cell flask to a final concentration of 0.1. mu.g/ml, and then placed in a cell culture chamber for further culture for 4 hours. The cells were blown up and mixed with 5mL of 0.075mol/L KCL hypotonic solution pre-warmed at 37 ℃, placed in a 37 ℃ incubator for 30 minutes, added with the newly prepared fixative (methanol: glacial acetic acid 3:1) lmL, mixed while dropping, and centrifuged at 1000r/min for 10 minutes. The cell pellet was left by discarding the supernatant, the cells were blown up with 5mL of the fixative, acted at 37 ℃ for 30 minutes, centrifuged at 1000r/min for 10 minutes, and the above operation was repeated once. The cell sediment is suspended and mixed evenly by lmL fixing solution, 1 drop of the suspension is absorbed by a dropper, dropped on a pre-frozen glass slide, laid on the glass slide and dried naturally. Staining for 10 min with newly prepared giemsa staining solution, washing with tap water, air drying, and observing under microscope. The number of chromosomes of the hybridoma cell strain is 94, the number of chromosomes of myeloma cells is 54-64, and the number of chromosomes of mouse spleen cells is 40, so that the obtained hybridoma cell strain is proved to be the result of fusion of the two cells.
2. Characterization of the specificity of monoclonal antibodies
Experiments were performed in 24-well cell culture plates. Respectively inoculating a CDV Onderstepoort strain (product of International Limited International Inc. in the Netherlands), a canine influenza virus (purchased from China institute of veterinary drugs) and a canine parvovirus (purchased from China institute of veterinary drugs) to Vero cells (purchased from ATCC cell bank), culturing for 72 hours, removing a cell culture solution by suction, washing for 2 times by using a serum-free culture solution, adding 1 mL/hole of absolute ethyl alcohol precooled at-20 ℃ into a cell culture hole, fixing for 30 minutes at 4 ℃, washing for 3 times by using PBS, and patting to be dry; adding culture supernatant of hybridoma cell 4F6, incubating at 200 μ L/well for 1 hr at 37 deg.C, washing with PBS for 3 times, and drying; a200-fold dilution of FITC-labeled goat anti-mouse IgG antibody (product of Wuhan Dr. Debioengineering, Ltd.) was added thereto, and the mixture was incubated at 200. mu.L/well for 1 hour at 37 ℃ and washed 5 times with PBS, and then observed under a fluorescence microscope. Under a fluorescence microscope, the monoclonal antibody can only react with Vero cells infected by CDV Ondesteopoort strain to generate fluorescence, but does not have fluorescence with Vero cells infected by other pathogens, and the specificity of the monoclonal antibody to CDV is proved.
3. Determination of monoclonal antibody type
The subclass of the monoclonal antibody secreted by hybridoma cell 3B5 strain was determined using a monoclonal antibody subclass identification kit (seimer feishell scientific), and the result showed that the subclass of the monoclonal antibody was IgG1 κ.
4. Stability assay for monoclonal antibodies
Continuously culturing and subculturing the obtained hybridoma cell 3B5 strain for 50 times, freezing and storing by liquid nitrogen and recovering, and continuously detecting antibody titer in hybridoma cell culture supernatant by indirect ELISA method to be 2
12It was confirmed that the hybridoma 3B5 strain can continuously and stably secrete anti-CDV monoclonal antibodies.
5. Determination of virus neutralization potency of monoclonal antibodies
Vero cells are digested by a method of fixing virus to dilute antibodies and then inoculated into a 96-well cell plate. Respectively mixing the cell culture supernatant and mouse ascites of 10-fold serial diluted monoclonal antibody with the same volume of 200TCID
50Mixing the CDV suspension, acting at 37 deg.C for 1 hr, inoculating 0.1ml of the disease-antibody suspension per well into the 96-well cell plate, setting CDV and normal Vero cell control, standing at 37 deg.C and 5% CO
2The result of the incubator culture was observed that the neutralization titer of the supernatant of the monoclonal antibody 3B5 cell culture was 2
8Neutralizing effect on ascites of miceA price of 10
6。
6. Recognition epitope screening of monoclonal antibodies
The difference in binding of monoclonal antibody 3B5 to the epitope previously prepared 2G3 and 1D7 was analyzed using an indirect ELISA addition assay. According to the ELISA antibody titer of the three monoclonal antibodies, the three monoclonal antibodies are respectively diluted to the saturation concentration, 50 percent of the three monoclonal antibodies are respectively and uniformly mixed under the concentration, and the OD of the mixed monoclonal antibodies and the OD of the three monoclonal antibodies under the saturation concentration are respectively measured
450nmThe value is obtained. AI values of three monoclonal antibodies superimposed on each other were calculated according to the following formula (AI (%)) [ (2A1+2/A1+ A2) -1]X 100, a1 and a2 are the absorbance values of each of the two monoclonal antibodies, and a1+2 is the absorbance value measured for 2 monoclonal antibodies plus. If AI<50 are 2 monoclonal antibodies that bind to the same antigenic site, if AI>50 are two monoclonal antibodies that bind to different antigenic sites. The experimental results showed that the mabs 2G3 and 1D7 had an AI of 75 (greater than 50), 3B5 and 1D7 had an AI of 85 (greater than 50), and 2G3 and 3B5 had an AI of 80 (greater than 50), indicating that the epitopes of 3B5, 2G3, and 1D7 that bind CDV are different.
Further, the epitope recognized by the prepared monoclonal antibody was screened by a peptide scanning method, and a peptide fragment of 15 amino acids in length was synthesized based on the amino acid sequence of the H protein to screen the epitope, each synthetic peptide was migrated as 5 peptides, and each of the synthetic peptides had 5 amino acid repeats with the former and latter synthetic peptides (synthesized by general biosystems (Anhui) Co., Ltd.). Synthesizing 80 continuous overlapping short peptides, respectively coating an ELISA plate with 100 ng/hole, taking the prepared monoclonal antibody 3B5(1:10000) as a primary antibody and HRP-IgG as a secondary antibody, detecting by an indirect ELISA method (see the detailed implementation mode of the invention, (I) establishing a monoclonal antibody hybridoma cell strain, 7. screening section contents of the canine distemper virus H protein monoclonal antibody hybridoma cell strain), and detecting according to the detected OD
450nmThe recognition epitope of the monoclonal antibody is preliminarily identified. The result shows that the epitope recognized by the monoclonal antibody 3B5 is at amino acids 228-237 of the H protein, the polypeptide sequence of the epitope is YGKTYLLVPD, and the epitope is not reported.
(III) monoclonal antibody combination therapeutic agent and neutralization activity assay
1. Preparation of monoclonal antibodies 3B5, 1D7, 2G3
Monoclonal antibodies 3B5, 1D7, 2G3 were prepared by cell culture supernatant method. Monoclonal antibody hybridoma 3B5 strain secreting anti-canine distemper virus H protein, monoclonal antibody hybridoma 1D7 strain secreting canine distemper virus specificity and hybridoma 2G3 strain (a hybridoma 1D7 strain secreting high neutralizing activity canine distemper virus monoclonal antibody, national invention patent application No.: 201210081170.8, authorization notice number: CN102618503B, date of authorized announcement: 2013-06-12, a monoclonal antibody and an antibody composition for neutralizing canine distemper virus, and the national invention patent application number: 201310036576.9, authorization notice number: CN103059133B, date of authorized announcement: 2015-12-09, the above cell lines were purchased from Nanjing Tianbang Biotech Co., Ltd.) and cultured in cell culture flasks with RPMI-1640 medium containing 10% FCS (Seimer Feishell scientific Co., Ltd.) until the cell density reached 80% -90% (cell concentration was about 2X 10).
6one/mL), the medium was replaced with serum-free RPMI-1640 medium, the cells were cultured in a 5% CO2 incubator until all cells were dead, the culture medium was centrifuged at 1000r/min for 10 minutes, and the supernatant was stored at-20 ℃ for further use.
2. Preparation of monoclonal antibody combination therapeutic agent
Monoclonal antibody 3B5, monoclonal antibody 1D7 and monoclonal antibody 2G3 prepared by the cell culture supernatant method were mixed well in a 1:1:1 volume ratio to prepare a monoclonal antibody combination therapeutic agent (3B5+1D7+2G 3). Meanwhile, monoclonal antibody 3B5, monoclonal antibody 2G3 and monoclonal antibody 1D7 were combined two by two and mixed well in a 1:1 volume ratio to prepare antibody combination therapeutics (3B5+1D7, 3B5+2G3, 1D7+2G 3).
3. Monoclonal antibodies and determination of neutralizing Activity of compositions
The neutralizing titers of monoclonal antibodies 3B5, 1D7, 2G3 and monoclonal antibody combination therapeutics (3B5+1D7+2G3, 3B5+1D7, 3B5+2G3, 1D7+2G3) prepared by the cell culture supernatant method were determined separately using the method of fixing the virus dilution antibody (see the present invention, "embodiments, (ii) biological properties of monoclonal antibody 3B5, 5. determination of virus neutralizing titer of monoclonal antibody" section content). The results are shown in Table 1 below,
the neutralizing titer of the monoclonal antibodies 3B5, 1D7 and 2G3 is 2 respectively
8、2
10、2
10The neutralizing potency of the two combined antibody combination therapeutic agents 3B5+1D7, 3B5+2G3 and 1D7+2G3 is 2
11、2
11、2
12The neutralizing titer of the antibody combination therapeutic agent 3B5+1D7+2G3 was 2
13The combined monoclonal antibody combination therapeutic agent has stronger virus neutralizing capacity (which is obviously higher than that of the combined monoclonal antibody combination therapeutic agent alone or combined monoclonal antibody combination therapeutic agent two by two), and the virus neutralizing capacity is improved by 8-32 times compared with that of the combined monoclonal antibody combination therapeutic agent singly using 3B5, 1D7 and 2G 3.
TABLE 1 comparison of neutralizing titers of antibodies
Clinical application of (IV) monoclonal antibody combined therapeutic agent
1. Treatment of clinical CD sick dogs
The prepared monoclonal antibody combination therapeutic agent is used for treating outpatient canine distemper disease dogs, a subcutaneous injection method is adopted, 1mL of monoclonal antibody combination therapeutic agent is injected into each kilogram of body weight once a day, symptomatic treatment is assisted, a canine distemper virus detection test paper strip is adopted for measurement every day, and the calculated effective rate, the cure rate and the treatment period are shown in table 2. As a result, it was found that the cure rate of the antibody combination therapeutic agent 3B5+1D7+2G3 reached 100% (higher than others), and the treatment cycle was about 2 days (shorter than others).
TABLE 2 comparison of clinical applications of monoclonal antibody combination therapeutics
The hybridoma cell strain 3B5 of the H protein monoclonal antibody of the canine distemper virus has very excellent biological performance, and the epitope recognized by the monoclonal antibody prepared by the monoclonal antibody is amino acids 228-237 of the H protein of CDV, the polypeptide sequence of the epitope is YGKTYLLVPD, and the epitope is not reported. The antibody combination therapeutic agent composed of the prepared monoclonal antibody 3B5 and other canine distemper virus monoclonal antibodies 1D7 and 2G3 has stronger virus neutralizing capacity, and the virus neutralizing capacity of the monoclonal antibody combination therapeutic agent combined with the three monoclonal antibodies is stronger than that of the monoclonal antibody combination therapeutic agent singly used 3B5, 1D7 and 2G3 is improved by 8-32 times. The monoclonal antibody combined therapeutic agent can be used for clinical treatment of canine distemper animals, and compared with the combined use of the monoclonal antibodies 1D7 and 2G3, the effect of the clinical application test result of the monoclonal antibody combined therapeutic agent is improved from 98% to 100%, the treatment period is shortened from 4 days to 2 days, the treatment effect is remarkable, the canine distemper virus neutralizing capacity is enhanced, the production cost is reduced, and the clinical application range is wider.
Claims (5)
1. A monoclonal antibody hybridoma cell 3B5 strain secreting anti-canine distemper virus H protein, wherein the hybridoma cell 3B5 strain is preserved in the China center for type culture Collection in 7 and 9 months in 2019, and the addresses are as follows: the preservation number of the preservation center of Wuhan university in Wuhan, China is CCTCC NO: c2019143, classification naming: the monoclonal antibody hybridoma cell line 3B5 for resisting the canine distemper virus H protein is characterized in that the epitope recognized by the canine distemper virus H protein monoclonal antibody prepared from the hybridoma cell line 3B5 is at the 228 th-237 th amino acid of the canine distemper virus H protein, and the polypeptide sequence of the epitope is YGKTYLLVPD.
2. The use of the monoclonal antibody hybridoma cell 3B5 which secretes canine distemper virus H protein according to claim 1.
3. The use of the hybridoma cell 3B5 secreting canine distemper virus H protein monoclonal antibody of claim 1 for the preparation of a canine distemper virus monoclonal antibody combination therapeutic agent.
4. The monoclonal antibody 3B5 against the H protein of canine distemper virus, prepared from the hybridoma cell strain 3B5 of claim 1.
5. The use of claim 3, wherein the monoclonal antibody 3B5 of the H protein of canine distemper virus is mixed with monoclonal antibody 1D7 secreted by hybridoma cell 1D7 and monoclonal antibody 2G3 secreted by hybridoma cell 2G3 at a ratio of 1:1:1 to prepare the monoclonal antibody combination therapeutic agent.
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| CN111849923A (en) * | 2020-07-30 | 2020-10-30 | 江苏省农业科学院 | Hybridoma cell 2D12 strain secreting monoclonal antibody against canine distemper virus H protein |
| CN114088944A (en) * | 2022-01-19 | 2022-02-25 | 山东畜牧兽医职业学院 | Canine distemper virus antigen detection test strip based on nano antibody and preparation method thereof |
| CN114088944B (en) * | 2022-01-19 | 2022-04-01 | 山东畜牧兽医职业学院 | Canine distemper virus antigen detection test strip based on nano antibody and preparation method thereof |
| CN114805586A (en) * | 2022-04-14 | 2022-07-29 | 天津华科泰生物技术有限公司 | anti-MxA monoclonal antibody composition and application thereof |
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