CN114107218B - Hybridoma cell secreting goat endemic intranasal tumor virus monoclonal antibody and application thereof - Google Patents
Hybridoma cell secreting goat endemic intranasal tumor virus monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of cell engineering. The invention provides a hybridoma cell secreting goat local intranasal tumor virus monoclonal antibody and application thereof, wherein the preservation name of the hybridoma cell is hybridoma cell line 6E4, and the preservation date is as follows: 2021, 10, 27, deposit unit: china center for type culture Collection, accession number: CCTCC NO: C202181. The monoclonal antibody has good specificity, high antibody titer of 1:819200, simple purification method and high purity, can be used for preparing a rapid detection kit for goat local intranasal tumor virus ELISA and the like, is suitable for clinical rapid detection of goat local intranasal tumor virus diseases, and has important significance for purification of goat local intranasal tumor virus diseases.
Description
Technical Field
The invention relates to the technical field of cell engineering, in particular to a hybridoma cell secreting goat local intranasal tumor virus monoclonal antibody and application thereof.
Background
Goat local intranasal tumor virus (ENTV-2) belongs to the retrovirus of the genus beta-retrovirus of the subfamily of the retrovirus, the virions are spherical, the genome of the virus is single strand positive strand linear RNA, and the length is about 7.5kb. ENTV-2 is the etiology of a goat (Capra hircus) local intranasal tumor (enzootic nasal tumor, ENT). The goat local intranasal tumor is a chronic contagious disease of a sick goat, which is characterized by nasal fluid flow, facial swelling and weight loss, and is characterized by kidney flower-like tumors on one side or two sides in the nasal cavity, the morbidity of the disease is 1-40%, the mortality of the disease is 100%, the incubation period of the disease is as long as months or even years, the disease occurs in Asia and Europe, and serious economic loss is caused for the world sheep industry. The first report of the occurrence of the disease in the inner Mongolia goats in 1995 in China, and then the report of the occurrence of the disease in places such as Hunan, sichuan and Shaanxi.
At present, an in vitro culture system does not exist in ENTV-2, so that the research on the pathogen is limited, no vaccine capable of preventing the disease is available at home and abroad at present, and great difficulty is brought to the prevention and treatment of the disease. The only method at present is to realize the purification of the disease by detecting the pathogen and eliminating the flock with the poison. However, the current detection method is limited to PCR method, and there is no immunological detection method. The hybridoma cell strain capable of stably secreting the monoclonal antibody of goat local intranasal tumor virus (ENTV-2) is very important to establish an immunological detection method and other rapid detection methods.
Disclosure of Invention
The invention aims to provide a hybridoma cell secreting goat local intranasal tumor virus monoclonal antibody and application thereof, which lay a foundation for establishing an immunological method for rapidly detecting goat local intranasal tumor virus.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a hybridoma cell secreting goat local intranasal tumor virus monoclonal antibody, the preservation name of the hybridoma cell is hybridoma cell line 6E4, and the preservation date is as follows: 2021, 10, 27, deposit unit: china center for type culture Collection, accession number: CCTCC NO: C202181.
The invention also provides a goat local intranasal tumor virus monoclonal antibody secreted by the hybridoma cells.
The invention also provides application of the goat local intranasal tumor virus monoclonal antibody in preparation of a goat local intranasal tumor virus detection kit.
The invention also provides a goat local intranasal tumor virus infection detection kit, which comprises a goat local intranasal tumor virus monoclonal antibody.
Preferably, the kit is further packagedComprises coating buffer solution, sealing solution, dilution solution, PBST washing solution, peroxidase marked goat anti-mouse IgG (H+L), TMB chromogenic solution and 2% H 2 SO 4 。
The invention provides a hybridoma cell secreting goat local intranasal tumor virus monoclonal antibody and application thereof, wherein the preservation name of the hybridoma cell is hybridoma cell line 6E4, and the preservation date is as follows: 2021, 10, 27, deposit unit: china center for type culture Collection, accession number: CCTCC NO: C202181. The monoclonal antibody has good specificity, the antibody titer is as high as 1:819200, the purification method is simple, the purity is high, the monoclonal antibody can be used for preparing a rapid detection kit for goat local intranasal tumor virus ELISA and the like, and the monoclonal antibody is suitable for clinical rapid detection of goat local intranasal tumor virus diseases.
Drawings
FIG. 1 shows the results of monoclonal antibody subclass identification;
FIG. 2 shows the result of SDS-PAGE analysis of purified monoclonal antibodies.
Preservation description
Hybridoma cell line 6E4, deposited with the chinese collection at the address: university of martial arts, china, 10 months and 27 days of 2021, deposit number: CCTCC NO: C202181.
Detailed Description
The invention provides a hybridoma cell secreting goat local intranasal tumor virus monoclonal antibody, the preservation name of the hybridoma cell is hybridoma cell line 6E4, and the preservation date is as follows: 2021, 10, 27, deposit unit: china center for type culture Collection, accession number: CCTCC NO: C202181.
The invention also provides a goat local intranasal tumor virus monoclonal antibody secreted by the hybridoma cells.
The invention also provides application of the goat local intranasal tumor virus monoclonal antibody in preparation of a goat local intranasal tumor virus detection kit.
The invention also provides a goat local intranasal tumor virus infection detection kit, which comprises a goat local intranasal tumor virus monoclonal antibody.
In the invention, the kit also comprises a coating buffer solution, a sealing solution, a diluent, a PBST washing solution, a peroxidase-marked goat anti-mouse IgG (H+L), a TMB chromogenic solution and 2% H 2 SO 4 。
In the present invention, the coating buffer is preferably a carbonate buffer of 0.04 to 0.06mol/L, more preferably a carbonate buffer of 0.05 mol/L.
In the present invention, the blocking solution is preferably 1 to 5% bovine serum albumin, more preferably 3% bovine serum albumin.
In the present invention, the diluent is preferably PBS.
In the present invention, the pH of the PBS is preferably 7.0 to 7.5, more preferably 7.2.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 preparation of goat local intranasal tumor Virus (ENTV-2) monoclonal antibody
1. Preparation of immunogens
The purified virus was used as an immunizing antigen. Collecting goat nasal fluid of natural infection cases of goat endemic intranasal tumor (Enzootic nasal adenocarcinoma, ENA), diluting nasal fluid by PBS (phosphate buffered saline) by 1:1, and fully and uniformly stirring; centrifuging nasal solution at 4deg.C for 20min at 6000r/min, and collecting supernatant; supernatant 12000r/min, centrifuging at 4deg.C for 30min, and removing precipitate to obtain supernatant; centrifuging the supernatant at 4 ℃ for 3 hours at the rotating speed of 36000r/min, and discarding the supernatant. Dissolving the precipitate with PBS buffer solution, and placing in a refrigerator at 4deg.C for use; 60%, 50%, 40%, 30% and 20% sucrose was sequentially injected into the bottom of the tube, and virus was added. Then, sucrose density gradient centrifugation was performed at 36000r/min at 4℃for 4h. Carefully sucking 40% -50% sucrose band (containing virus), adding appropriate amount of PBS, centrifuging at 4deg.C at 38000r/min for 2h, and discarding supernatant. The purified virus was obtained by resuspension with PBS.
2. Immunization of animals
4 female Balb/c mice with the age of 6-8 weeks are immunized, the proteins are mixed with an adjuvant in an equal volume during the first three times of immunization, blood is collected after 7d of the third immunization, serum is separated, and the titer is measured, and when the ELISA titer is more than 1:5000, the cell fusion is realized. 3d before fusion, and enhancing immunity. Immunization procedures are shown in tables 1,3d later for cell fusion.
TABLE 1
| Immunization time | Adjuvant | Immunity amount | Immunization mode |
| 6 weeks of age | Freund's |
100 mug/g only | Subcutaneous multipoint injection |
| 8 weeks of age | Freund's |
120 mug/g only | Subcutaneous multipoint injection |
| 10 weeks of age | Freund's |
120 mug/g only | Subcutaneous multipoint injection |
| 3d before fusion | No |
100 mug/g only | Intraperitoneal injection |
3. Establishment of hybridoma cell lines
3.1 preparation of myeloma cells
Taking 6 bottles of SP2/0 cells in the logarithmic growth phase, washing the cells from the walls of the bottles by using DMEM incomplete culture solution during fusion, collecting all SP2/0 cells into a 50mL sterilizing centrifuge tube, centrifuging at 1200rpm for 10min, collecting cell sediment, and counting.
3.2 spleen cell preparation
Taking 2 BALB/c mice with antibody titer of more than 1:102400 and carrying out booster immunization, removing eyeballs for blood collection, and separating serum to be used as positive control serum during monoclonal antibody detection. Mice were sacrificed by pulling the neck, and sterilized by soaking in ethanol for 5min. On a sterile console, mice were fixed on plastic plates, and the abdominal skin and peritoneum were lifted to expose the spleen. Taking out spleen with ophthalmic scissors and ophthalmic forceps, transferring to a sterilization plate, adding a small amount of incomplete culture solution into the plate, extruding spleen cells into the incomplete culture solution with a 200-mesh copper mesh, carefully sucking into a 50-mL sterilization centrifuge tube, centrifuging at 1300rpm for 8min, collecting precipitated cells, and counting.
3.3 cell fusion
Feeder cells were prepared the day prior to cell fusion and conditioned with HAT medium to 2 x 10 5 Each mL of the cell suspension was dispensed onto a cell fusion plate at 1 drop/well. 37 ℃,5% CO 2 Culturing for standby. Spleen cells and SP2/0 myeloma cells at 7:1, mixing with 50% PEG1500 as fusion agent, inoculating into 96-well cell culture plate, and culturing at 37deg.C under 5% CO 2 . After 3 days of culture after fusion, the growth of the cells was observed, and after 5 days, 1/2 of the medium was replaced with HAT medium. After 7-10 days, HAT medium was changed out with HT medium, and after two weeks, normal DMEM complete medium (containing 10% fetal bovine serum, 1% diabody) was changed. And sucking out the supernatant for antibody detection when the cell covers more than 1/10 of the bottom area of the hole.
3.4 screening of Positive hybridoma cells
The supernatant is detected by established indirect ELISA method, and cells with positive detection are subjected to expansion culture and cloning. Indirect ELISA method: coating of ENTV-2 purified virus (0.5. Mu.g/well), coating overnight at 4℃and washing 3 times with PBST, adding culture supernatant of hybridoma cells, incubating for 45min at 37℃and washing 3 times with PBST, adding 1: goat anti-mouse IgG (H+L) was labeled with peroxidase at 12000 dilution, incubated at 37℃for 45min, washed 3 times with PBST, developed for 10min with TMB as a substrate, and OD450 values were measured on a microplate reader after termination of the reaction. After 3 subclones, hybridoma cells secreting goat endemic intranasal tumor virus monoclonal antibody were selected and designated 6E4.
4. Preparation of mouse ascites and determination of antibody titer
Taking BALB/c mice, injecting 0.5 mL/mouse of sterilized Freund's incomplete adjuvant into the abdominal cavity, and injecting 6E4 monoclonal antibody hybridoma cells 10 into the mice after 1 week 6 The abdominal cavity of the mice is obviously enlarged about 1 week per mouse (without serum), at this time, the ascites of the mice is extracted, the mice are centrifuged for 10min at 3000r/min, the supernatant is taken, and the precipitate is discarded. And taking supernatant liquid of the sample to detect by using an established indirect ELISA method, wherein the determination result takes the maximum dilution of the antibody with the P/N more than or equal to 2.1 as the final value of the antibody titer, and the result shows that the detected antibody titer is 1:819200.
5. Monoclonal antibody subclass identification
Referring to Mouse MonoclonalAntibody Isotyping Kit kit instructions, 6E4 ascites 1:20000 dilutions were used to determine the subclass of ascites. The subclass of the monoclonal antibody 6E4 is identified as kappa chain, igG1 subclass (see FIG. 1).
6. Purification of ascites
Reference to
The purification was performed using the ProteinA Resin (Beijing full gold Biotechnology Co., ltd.) kit instructions. The purified 6E4 monoclonal antibody was subjected to denaturing SDS-PAGE analysis, and the results are shown in FIG. 2.
7. Monoclonal antibody specific detection
Coating: purifying virus by using ENTV-2, treating ENTV-2 sickness with sheep nose liquid,Aphtha virus, mycoplasma ovipneumoniae, mycoplasma filiformis goat subspecies, DMEM, PBS, ddH containing 10% fetal bovine serum 2 O-coated ELISA plate, 100. Mu.L/well, coated overnight at 4℃and washed 5 times with PBST wash solution and dried by pipetting. Closing: blocking solution, 200. Mu.L/well, was added, blocked at 37℃for 2h and pipetted off. An antibody: 6E4 purified monoclonal antibody (1.5. Mu.g/well) was added, incubated at 37℃for 45min, washed 5 times with PBST wash, and patted dry. And (2) secondary antibody: peroxidase-labeled goat anti-mouse IgG (H+L) (1:12000 dilution) was added, incubated at 37℃for 45min, washed 5 times with PBST washes, and patted dry. Color development: TMB color development was added and incubated for 10min at 100. Mu.L/well. And (3) terminating: adding 2% H 2 SO 4 OD was read at 450nm at 50. Mu.L/well. The results show that: the monoclonal antibody can only react with the ENTV-2 purified virus and the ENTV diseased sheep nose fluid, and does not react with the negative control and the blank control. The 6E4 monoclonal antibody was shown to be specific for the ENTV-2 virus.
Example 2 kit for detection of goat endemic intranasal tumor Virus (EVTV-2) and methods of use
1. Coating antigen: diluting the ENTV-2 diseased sheep nasal solution, positive control (ENTV-2 purified virus (20 mug/mL), nasal solution samples 1-8 (ENTV-2 positive by fluorescent quantitative PCR), nasal solution samples 9-18 (ENTV-2 negative by fluorescent quantitative PCR) and blank control respectively by using a coating buffer solution, mixing the coating buffer solution and the diluted samples according to a volume ratio of 3:1 during dilution, coating 100 mug/hole at 4 ℃ for overnight, washing 5 times by using PBST washing solution, and beating to dryness;
2. closing: adding a blocking solution, 200 mu L/hole, incubating for 45min at 37 ℃, washing for 5 times by using PBST washing solution, and drying by beating;
3. adding an antibody: adding 6E4 purified monoclonal antibody (0.7 mug/hole), incubating for 45min at 37 ℃, washing 5 times by using PBST washing liquid, and beating to dry;
4. adding a secondary antibody: adding peroxidase-labeled goat anti-mouse IgG (H+L) at a dilution of 1:6000, incubating at 37 ℃ for 45min at 100 μl/well, and washing with PBST washing solution for 5 times;
5. color development: adding TMB color development solution, 100 mu L/hole, and incubating for 10min at 37 ℃;
6. and (3) terminating: adding 2% H 2 SO 4 50. Mu.L/well;
7. reading: enzyme label instrument OD 450 OD values were read.
8. And (3) result judgment: OD (optical density) 450 And if the value is more than or equal to 0.3220, determining that the EVTV-2 is positive, otherwise, determining that the EVTV-2 is negative. (the ELISA results are consistent with the fluorescent quantitative PCR results)
From the above examples, the present invention provides a hybridoma cell secreting goat local intranasal tumor virus monoclonal antibody and its application, and the hybridoma cell has a preservation name of hybridoma cell line 6E4, and the preservation date is as follows: 2021, 10, 27, deposit unit: china center for type culture Collection, accession number: CCTCC NO: C202181. The monoclonal antibody has good specificity, the antibody titer is as high as 1:819200, the purification method is simple, the purity is high, and the monoclonal antibody can be used for preparing a rapid detection kit for goat local intranasal tumor virus ELISA and the like, and is suitable for clinical rapid detection of goat local intranasal tumor virus diseases.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (3)
1. A hybridoma cell secreting goat local intranasal tumor virus monoclonal antibody, wherein the hybridoma cell has a preservation name of hybridoma cell line 6E4, and the preservation date is as follows: 2021, 10, 27, deposit unit: china center for type culture Collection, accession number: CCTCCNO: C202181.
2. A goat endemic intranasal tumor virus monoclonal antibody secreted by the hybridoma of claim 1.
3. Use of a goat local intranasal tumor virus monoclonal antibody as claimed in claim 2 in the preparation of a goat local intranasal tumor virus detection kit.
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