CN112553168A - Hybridoma cell 3A6 strain secreting monoclonal antibody against feline parvovirus VP2 protein and application thereof - Google Patents
Hybridoma cell 3A6 strain secreting monoclonal antibody against feline parvovirus VP2 protein and application thereof Download PDFInfo
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Abstract
A hybridoma cell 3A6 strain secreting monoclonal antibody against feline parvovirus VP2 protein and application thereof belong to the field of biology, the hybridoma cell 3A6 strain is preserved in China center for type culture collection (CCTCC NO): C2020248. the hybridoma cell 3A6 strain can react with VP2 protein expressed by eukaryotic cells and feline parvovirus, has very excellent biological performance, and the prepared mouse ascites monoclonal antibody has the virus neutralization titer as high as 5 multiplied by 108The reaction titer to the VP2 protein is as high as 108And for various kinds of cat parvo diseasesThe virus strain has equivalent neutralizing capacity, is used for clinical treatment of cats with feline parvovirus infection, and has the effective rate of 100 percent.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly discloses a hybridoma cell 3A6 strain secreting monoclonal antibody against feline parvovirus VP2 protein and application thereof.
Background
Feline parvovirus disease, also known as Feline distemper and Feline panleukopenia syndrome, is a virulent infectious disease mainly characterized by severe vomiting, diarrhea and stinky feces caused by Feline Parvovirus (FPV). The disease is characterized by high morbidity, strong infectivity, fast disease course and high mortality of young cats, and is one of the most main infectious virus diseases of the felines. FPV is a single negative strand DNA virus without envelope of parvovirus family, the genome has a total length of 5.2kb, and encodes two non-structural proteins of NS1 and NS2 and three structural proteins of VP1, VP2 and VP 3. The antigen epitope of the virus is usually positioned on the capsid protein of the virus, and the capsid of the FPV virus consists of 60 structural proteins, wherein 53-54 VP2 proteins account for 90 percent of the total amount of the capsid protein and play a decisive role in the antigenicity of the FPV.
Most of the vaccines used for preventing the disease are attenuated vaccines at present, but many young cats still have the disease after the attenuated vaccines are applied. The reason for the analysis is related to maternal antibodies, vaccine titer, method of use and virus variation. The animals immunized by the attenuated vaccine can not be completely protected, and a plurality of immune animal groups outbreak the feline parvovirus disease, so the loss is very tragic. Thus, in addition to positive prophylaxis against feline parvovirus disease, there is a need for effective treatment of diseased animals to minimize losses. The hyperimmune serum of feline parvovirus is commonly used for treating feline parvovirus diseases, but has higher preparation cost, easy transmission of other viruses, uneven antibody neutralization titer and difficult standardization.
Disclosure of Invention
The invention aims to provide a hybridoma cell 3A6 strain secreting monoclonal antibody against feline parvovirus VP2 protein and application thereof. The hybridoma cell 3A6 strain secreting the monoclonal antibody against the feline parvovirus VP2 protein lays a foundation for developing monoclonal antibody-mediated treatment application of specific feline parvovirus diseases.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the hybridoma cell 3A6 strain secreting the monoclonal antibody against the feline parvovirus VP2 protein is preserved in the China Center for Type Culture Collection (CCTCC) within 12 months and 10 days of 2020, with the preservation numbers as follows: CCTCC NO: C2020248.
the hybridoma cell 3A6 strain of the invention is applied to the preparation of a therapeutic agent of the feline parvovirus monoclonal antibody 3A 6.
A therapeutic agent of feline parvovirus monoclonal antibody 3A6, which is prepared using hybridoma 3A6 strain of the present invention.
The feline parvovirus VP2 protein monoclonal antibody prepared by the hybridoma cell 3A6 strain of the invention.
The invention discloses an application of a feline parvovirus VP2 protein monoclonal antibody in preparation of a therapeutic agent of a feline parvovirus disease monoclonal antibody 3A 6.
The invention has the beneficial effects that:
the invention uses purified feline parvovirus antigen to immunize BALB/c mice, prepares immune splenocytes, fuses with SP2/0 myeloma cell line, and uses eukaryotic expression VP2 protein and purified feline parvovirus to carry out double screening to obtain a monoclonal antibody hybridoma cell strain which can react with eukaryotic expression VP2 protein and feline parvovirus, and the strain is named as 3A 6.
Compared with the prior art, the invention has the following advantages:
1. the BALB/C mouse immunogen used in the present invention is prepared from the currently circulating strain of FPV. The screened VP2 protein monoclonal antibody immunogen is reported to be VP2 protein expressed by a prokaryotic expression system, while the VP2 protein adopted by the invention is derived from an insect baculovirus expression system (a eukaryotic expression system), although the VP2 protein amino acid sequence is highly conserved, the protein produced by different expression systems has different protein post-translational modification levels, so that the immune antigen has certain difference.
2. The invention screens out a hybridoma cell 3A6 strain from a established hybridoma cell bank in which 55 strains secrete anti-FPV monoclonal antibodies, the biological performance is very good, and the mouse ascites monoclonal antibodies prepared by the hybridoma cell have the virus neutralization titer as high as 5 multiplied by 108The reaction titer to the VP2 protein is as high as 108The titer is obviously higher than the parameter values reported by the existing data, the neutralization capacity of various feline parvovirus strains is equivalent, the feline parvovirus strain neutralization solution is used for clinical treatment of cats infected with feline parvovirus, and the effective rate reaches 100%.
3. The therapeutic agent of the feline parvovirus monoclonal antibody 3A6 prepared by the hybridoma cell 3A6 strain has obvious treatment effect, the cure rate reaches 100 percent, and the treatment period is about 5 days.
Detailed Description
The hybridoma cell 3A6 strain secreting the monoclonal antibody against the feline parvovirus VP2 protein is preserved in the China Center for Type Culture Collection (CCTCC) in 12 months and 10 days of 2020 at the address of: eight-path Wuhan university school (Wuhan university collection center) 299 in Wuchang area, Wuhan city, Hubei province has the collection number: CCTCC NO: C2020248.
the hybridoma cell 3A6 strain of the invention is applied to the preparation of a therapeutic agent of the feline parvovirus monoclonal antibody 3A 6.
A therapeutic agent of feline parvovirus monoclonal antibody 3A6, which is prepared using hybridoma 3A6 strain of the present invention.
The feline parvovirus VP2 protein monoclonal antibody prepared by the hybridoma cell 3A6 strain of the invention.
The invention discloses an application of a feline parvovirus VP2 protein monoclonal antibody in preparation of a therapeutic agent of a feline parvovirus disease monoclonal antibody 3A 6.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 establishment of monoclonal antibody hybridoma cell 3A6 Strain
1. Preparation of FPV antigen
Inoculating a currently popular FPV virulent LYG-18 strain (stored in a biological veterinary drug research laboratory of veterinary research institute of agricultural academy of sciences of Jiangsu province, virus genome sequence information is uploaded to a GenBank database and numbered as MW017574) to FK81 cells (purchased from an ATCC cell bank), collecting infection supernatant after the cells are completely diseased, repeatedly freezing and thawing for three times, and centrifuging at 8000r/min for 3 minutes to remove cell fragments; the supernatant was collected and PEG was added6000(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 LTNE solution (pH 7.8). Adding the heavy suspension into a sucrose gradient solution with the concentration of 50% -30% -10%, ultracentrifuging at 60000r/min for 3 hours, collecting layered protein bands (between 30% -10%), absorbing the virus bands, observing the virus morphology through electron microscope negative staining, determining the virus concentration by using a BCA protein quantitative detection kit (purchased from Saimer Feishell science and technology company), 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 the age of 8 weeks, 50. mu.g/mouse, were immunized with the purified FPV immunizing antigen by intraperitoneal injection. 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. Collecting blood after 3 rd immunization at 6 th day, measuring serum antibody titer by indirect ELISA method, selecting serum antibody titer > 1063 days before fusion, re-boosting with purified FPV antigenAnd (5) epidemic disease.
3. Cell fusion
Adopting PEG cell fusion method, mixing SP2/0 myeloma cell (purchased from ATCC cell bank) with good growth state and spleen cell of immunized BALB/C mouse at a ratio of 1:5, centrifuging at 1000r/min for 10 min, discarding supernatant, tapping the tube bottom with palm to make the cells loose and uniform, preheating in 40 deg.C water bath, adding 50% PEG preheated to 40 deg.C with 1mL suction tube within 45 s4000(Sigma Co., Ltd.) 1mL, gently shaking while adding, adding 15mL of fetal calf serum-free RPMI-1640 medium preheated to 37 ℃ over 90 seconds, standing at room temperature for 10 minutes, centrifuging at 1000r/min for 10 minutes, discarding the supernatant, adding 10% Fetal Calf Serum (FCS) (Seimer Feishell technology Co., Ltd.) and HAT (Sigma Co., Ltd.) in RPMI-1640 medium, resuspending in 96-well plates containing feeder cells (feeder cells are obtained from peritoneal macrophages of non-immunized BALB/C mice), and placing in a 5% CO-free culture medium2Culturing in an incubator. After 3 days, the cells were supplemented with RPMI-1640 medium containing HAT (Sigma) and 10% FCS (Seimer Feishal technologies), and 5 days, the cells were replaced with RPMI-1640 medium containing HT (Sigma) and 10% FCS (Seimer Feishal technologies), and 10 days, the cells were replaced with 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 for subsequent antibody detection.
4. Screening of hybridoma cell lines
Determining the coating concentration of the purified FPV antigen according to a square matrix method, carrying out multiple dilution on the purified FPV antigen by using a carbonate buffer solution with a coating solution of 0.05mol/L, pH 9.6.6, coating an ELISA plate by using the amount of the FPV antigen diluted to 800 times, coating 100 mu L/hole, coating overnight at 4 ℃, washing 3 times by PBST, each time for 5 minutes, 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 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 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 2mol/L sulfuric acid in an amount of 50. mu.L per well. OD determination by enzyme-linked immunosorbent assay450nmValues were zeroed for blank control, P is the value of each well, N is the OD of the negative reference serum450nmValue when OD of negative reference serum450nmValue less than or equal to 0.1, OD of positive reference serum450nmValue and OD of negative reference serum450nmThe 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, detection is carried out once after 2-3 days, and hybridoma cells with positive detection results in two times are cloned.
5. Cloning of hybridoma cells
The viable cells in the positive wells were first stained and counted with trypan blue, diluted to 100 cells/15 mL with RPMI-1640 medium containing 10% FCS (product of Saimer Feishell technology Co., Ltd.), and the diluted cell suspension was added to a 96-well cell culture plate at 0.15 mL/well, 37 ℃ and 5% CO2Culturing in an incubator, observing the formation of clone cells under a microscope after 4-5 days, recording only a single clone growth hole, taking cell supernatant at 8-9 days, and performing ELISA detection in time. Selecting positive monoclonal cells, cloning for more than 3 times until all cell wells are positive and each well detects OD450nmThe values are closer. And (3) performing expanded culture on the cloned FPV specific monoclonal antibody hybridoma cell strain, and freezing and storing. After 20 times of cell fusion, screening, cloning and identification, a 55-strain hybridoma cell bank which stably secretes the FPV specific monoclonal antibody is established.
6. Expression and purification of FPV virulent LYG-18 strain VP2 protein
According to the sequence information of the gene of the currently popular FPV virulent LYG-18 strain VP2, a nucleic acid sequence (synthesized by general biological systems (Anhui) limited, known existing sequences can be found in a network database) optimized by insect cell codons is artificially synthesized, and Sal I and Kpn I enzyme cutting sites are respectively introduced at two ends of the artificially synthesized sequence. Subsequently, the artificially synthesized sequence and the shuttle plasmid pFastBac1TM are subjected to Sal I and Kpn I (product of Baori doctor's technology (Beijing) Co., Ltd.) digestion respectively, and are identified by agarose gel electrophoresis, then fragments are respectively recovered by using a gel recovery kit (product of Tiangen Biochemical technology (Beijing) Co., Ltd.), and the fragments are incubated for 16 hours at 4 ℃ under the connection effect of T4 ligase (product of Baori doctor's technology (Beijing) Co., Ltd.), and are transformed into E.coli DH5 alpha competence (product of Baori doctor's technology (Beijing) Co., Ltd.), so as to obtain the recombinant shuttle plasmid pFastBac-FPV-VP 2. The recombinant shuttle plasmid is transformed into E.coli DH10Bac competent cells (product of Seimer Feichell science and technology company), homologous recombination is carried out, and the recombinant bacmid-FPV-VP2 is obtained by culturing and screening for 48 hours through IPTG condition screening culture medium (40 mg/ml final concentration of IPTG and 40mg/ml final concentration of X-Gal, wherein the IPTG condition screening culture medium contains three antibodies (100. mu.g/ml final concentration of kanamycin, 50. mu.g/ml final concentration of gentamicin and 70. mu.g/ml final concentration of tetracycline), and the screening culture medium can judge whether homologous recombination occurs through colony formation color, white colonies represent successful recombination, and blue colonies do not succeed). Transfecting the recombinant bacmid to an Sf9 insect cell (product of Roche) by a liposome-mediated transfection method (X-tremeGENE HP DNAstrafection Reagent), transfecting for 72 hours, collecting cell supernatant which is the first generation recombinant baculovirus after cell lesion, then continuously inoculating the first generation recombinant baculovirus to the Sf9 insect cell, collecting the second generation recombinant baculovirus under the same culture condition, and so on, collecting the fourth generation recombinant baculovirus, and naming the fourth generation recombinant baculovirus as rBV-FPV-VP2, and storing at-20 ℃ for later use.
Culturing High-Five insect cells (Saimeishiel technologies) in 500mL suspension culture flask (Saimeishiel technologies) at 28 deg.C and 120r/min by shaking culture until cell density reaches 5 × 106At each mL, the recombinant baculovirus rBV-FPV-VP2 was used to infect cells with an MOI of 0.5 (multiplicity of infection), the cells were cultured for another 48-72 hours, the cell culture supernatant was collected and centrifuged at 8000r/min for 30 minutes to remove large cell debrisAnd (3) slicing. Adding an inactivating agent BEI (BEI is totally called as binary ethyleneimine, the principle is that virus nucleic acid is destroyed without destroying protein, and the virus antigenicity is kept, compared with the traditional formaldehyde inactivation mode, the method is more advantageous, purchased from Sigma company), the final concentration is 0.5%, after 24 hours of action, taking the liquid to carry out inactivation inspection (after inoculating the liquid into cells, observing whether the cells have pathological changes after 96 hours, repeating for three times, and determining that the inactivation is successful after no cell pathological changes appear). According to the volume of the collected liquid (500 mL as an example), the collected liquid is purified by an affinity chromatography resin material (purchased from GE company in America), diluted and stored by PBS, the obtained recombinant protein is named as recombinant feline parvovirus VP2 protein, and the protein concentration is measured by a BCA protein quantitative determination kit (product of Seimer Feishell science and technology company), and the collected liquid is stored at-70 ℃ for later use.
7. Screening of monoclonal antibody hybridoma cell 3A6 strain of FPV virulent LYG-18 strain VP2 protein
Determining the coating concentration of the purified recombinant feline parvovirus VP2 protein according to a matrix method, diluting the purified recombinant feline parvovirus VP2 protein by a carbonate buffer solution with a coating solution of 0.05mol/L, pH 9.6.6 in a multiple ratio, diluting to 0.1 mu g/hole, coating overnight at 4 ℃, washing for 3 times by PBST (PBST), each time for 5 minutes, and finally drying by beating for the last time; 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 (1:1000 dilution) of 55 strains stably secreting FPV specific monoclonal antibody into corresponding holes, acting for 1 hour at 37 ℃, washing for 3 times by PBST, each time for 5 minutes, and finally drying by beating; 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 assay450nmValue, selecting OD450nmThe FPV specific monoclonal antibody hybridoma cell strain corresponding to the highest numerical value is named as: 3a6, deposited in the chinese culture collection on 12 months and 10 days of 2020 at the address: 29 eight-way in Wuchang district of Wuhan city, Hubei provinceThe Wuhan university school 9 (Wuhan university Collection) has the preservation number: CCTCC NO: C2020248.
the results of 3 repetitions of the above experiment revealed that the OD of the monoclonal antibody of hybridoma 3A6 strain450nmThe value is highest.
8. Preparation of ascites
The sterilized liquid paraffin was intraperitoneally injected into BALB/C mice (purchased from the university of Yangzhou, comparative medicine laboratory center) of 8-10 weeks old, 0.5 mL/mouse, and after 7 days, the hybridoma cell 3A6 strain obtained above was injected into the abdominal cavity of each mouse, 0.2mL (containing 2X 10 cells)6～5×106Individual 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.
EXAMPLE 2 biological Properties of monoclonal antibodies
1. Chromosome analysis of hybridoma cell lines
The hybridoma cells were chromosome-counted by giemsa staining. Respectively culturing SP2/0 myeloma cells and positive hybridoma cells, growing to logarithmic phase, adding colchicine into a cell bottle to make the final concentration of the colchicine to be 0.1 mu g/ml, and then placing the cell bottle into a cell culture box for continuous culture for 4-5 hours. The cells were blown up and mixed with 5mL of 0.075mol/LKCI hypotonic solution pre-warmed at 37 ℃, placed in a 37 ℃ incubator for 30 minutes, added with a newly prepared fixative (containing methanol and glacial acetic acid, the volume ratio of methanol to glacial acetic acid is 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 l mL of 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 3A6 strain is 96, 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 3A6 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 the currently popular FPV virulent LYG-18 strain, feline herpesvirus, feline calicivirus and feline adenovirus (the strains are all stored in a biological veterinary drug research laboratory of veterinary research institute of agricultural academy of sciences of Jiangsu province), culturing for 72 hours, sucking and discarding cell culture solution, washing for 2 times by serum-free culture solution, adding 1 mL/hole of-20 ℃ precooled absolute ethanol into the cell culture hole, fixing for 30 minutes at 4 ℃, washing for 3 times by PBS, and patting to dry; adding culture supernatant of hybridoma cell 3A6, incubating at 200 μ L/well for 1 hr at 37 deg.C, washing with PBS 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 fluorescent microscope, the monoclonal antibody secreted by the hybridoma cell 3A6 strain can only react with FK81 cells infected by FPV to generate fluorescence, but does not react with FK81 cells infected by other pathogens to generate fluorescence, and the monoclonal antibody secreted by the hybridoma cell 3A6 strain is proved to have specificity on FPV.
3. Determination of monoclonal antibody type
The subclass of the monoclonal antibody secreted by hybridoma cell 3a6 strain was determined using a monoclonal antibody subclass identification kit (seimer feishell scientific), and the result showed that the subclass of the monoclonal antibody secreted by hybridoma cell 3a6 strain was IgG2b κ.
4. Stability assay for monoclonal antibodies
Continuously culturing the obtained hybridoma cell 3A6 strain for 50 times, freezing with liquid nitrogen, and recovering, and continuously detecting antibody titer in hybridoma cell culture supernatant by indirect ELISA method, wherein the antibody titer is 5 × 108It was confirmed that the hybridoma cell 3A6 strain can continuously and stably secrete a monoclonal antibody against the protein of feline parvovirus VP 2.
5. ELISA Titers and Virus neutralization Titers of monoclonal antibodies
Indirect ELISA method using FPV virulent LYG-18 strain as coating antigen (see example 1 for a single experiment)Screening of hybridoma cell line of step 4 in establishment of cloned antibody hybridoma cell line 3A 6) the titers of culture supernatant of hybridoma and ascites of mouse were determined, and it was revealed that the ELISA titer (i.e., reaction titer) of culture supernatant of hybridoma cell line 3A6 was 5X 106Ascites reaction titer is 1X 109。
The titers of the culture supernatants of the hybridoma cells and the ascites of the mice were determined by an indirect ELISA method using VP2 protein as the envelope antigen (see screening of 7 th step of the monoclonal antibody hybridoma cell 3A6 strain for FPV virulent LYG-18 strain VP2 protein monoclonal antibody hybridoma cell 3A6 strain in the establishment of monoclonal antibody hybridoma cell 3A6 strain in example 1), and the results showed that the culture supernatant of hybridoma cell 3A6 strain had a reaction titer of 108Ascites reaction titer is 109。
FK81 cells were digested by a method of fixing virus dilution antibody, and then seeded in 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 200TCID50Mixing the FPV 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 FK81 cell control, standing at 37 deg.C and 5% CO2The neutralization titer of the supernatant of the monoclonal antibody 3A6 cell culture was 5X 10 as observed in the incubator6The mouse ascites neutralization potency is 5 multiplied by 108。
EXAMPLE 3 efficacy testing of monoclonal antibody therapeutics
1. Preparation of monoclonal antibody 3A6 therapeutic agent
Monoclonal antibody 3a6 was prepared by cell culture supernatant method. Hybridoma cell 3A6 strain secreting monoclonal antibody specific to feline parvovirus VP2 protein was cultured in cell culture flasks in RPMI-1640 medium containing 10% FCS (Seimer Feishell science) 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. Clinical application of monoclonal antibody 3A6 therapeutic agent
The prepared monoclonal antibody 3A6 therapeutic agent is used for treating outpatient cat with parvovirus disease, a subcutaneous injection method is adopted, 1mL of monoclonal antibody 3A6 therapeutic agent is injected into per kilogram of body weight once a day, symptomatic treatment is assisted, a cat parvovirus detection test paper strip (purchased from Beijing Shijiheng animal epidemic prevention technology Co., Ltd.) is adopted for measurement every day, and the effective rate and the cure rate are calculated. As a result, the cure rate of the monoclonal antibody 3A6 therapeutic agent is 100%, and the treatment period is about 5 days.
The hybridoma cell 3A6 strain secreting the anti-feline parvovirus VP2 protein specific monoclonal antibody has very excellent biological performance, and the mouse ascites monoclonal antibody prepared by the hybridoma cell has the virus neutralization titer as high as 5 multiplied by 108The reaction titer to the VP2 protein is as high as 108The titer is obviously higher than the parameter values reported by the existing data, the neutralization capacity of the cat parvovirus strains is equivalent, the cat parvovirus strain neutralization solution is used for clinical treatment of cat infected and diseased cats, and the effective rate reaches 100%.
The invention discloses a hybridoma cell 3A6 strain secreting a monoclonal antibody specific to a feline parvovirus VP2 protein and application thereof, and can be realized by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.
Claims (5)
1. The hybridoma cell 3A6 strain secreting monoclonal antibody against feline parvovirus VP2 protein is characterized in that the hybridoma cell 3A6 strain is deposited in China Center for Type Culture Collection (CCTCC) 12.10.2020, with the collection numbers: CCTCC NO: C2020248.
2. use of the hybridoma cell strain 3a6 according to claim 1 for the preparation of a therapeutic agent comprising feline parvovirus monoclonal antibody 3a 6.
3. A therapeutic agent of feline parvovirosis monoclonal antibody 3A6 prepared by using hybridoma cell 3A6 strain according to claim 1.
4. A monoclonal antibody against VP2 protein of feline parvovirus produced by using the hybridoma 3A6 strain of claim 1.
5. The use of the monoclonal antibody against feline parvovirus VP2 protein according to claim 4 in the preparation of a therapeutic agent comprising feline parvovirus monoclonal antibody 3A 6.
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| CN116640189A (en) * | 2023-04-13 | 2023-08-25 | 广东光峰生物技术有限公司 | Kit for rapidly detecting pathogenic microorganisms and application thereof |
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| CN112877297A (en) * | 2021-03-27 | 2021-06-01 | 哈尔滨元亨生物药业有限公司 | Method for preparing cat distemper virus monoclonal antibody by using bioreactor |
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| CN116732045A (en) * | 2023-07-13 | 2023-09-12 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Anti-cat IgG monoclonal antibody and application thereof |
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