CN108913665B - Hybridoma cell strain secreting anti-CpHV-1 monoclonal antibody and application thereof - Google Patents
Hybridoma cell strain secreting anti-CpHV-1 monoclonal antibody and application thereof Download PDFInfo
- Publication number
- CN108913665B CN108913665B CN201810831978.0A CN201810831978A CN108913665B CN 108913665 B CN108913665 B CN 108913665B CN 201810831978 A CN201810831978 A CN 201810831978A CN 108913665 B CN108913665 B CN 108913665B
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- hybridoma cell
- cphv
- strain
- secreting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
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
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr 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
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
-
- 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
-
- 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
- C07K2317/35—Valency
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Oncology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Communicable Diseases (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a hybridoma cell strain secreting an anti-CpHV-1 monoclonal antibody and application thereof, belonging to the technical field of immunity. The invention screens out a hybridoma cell strain 4A4 from an established hybridoma cell bank secreting an anti-CpHV-1 monoclonal antibody. The mouse ascites monoclonal antibody prepared by the method has a neutralizing titer of 2 to CpHV-18. The monoclonal antibody secreted by the hybridoma cell 4A4 strain can be used for clinical treatment of CpHV-1 diseased sheep, and has the advantages of obvious curative effect, safety and no adverse side effect. The monoclonal antibody therapeutic agent prepared from the hybridoma cell 4A4 strain has no reduction of neutralization potency after being stored for two years, and has good stability. In addition, the indirect Immunofluorescence (IFA) detection method established by using the monoclonal antibody secreted by the hybridoma cell 4A4 strain as a detection antibody has better sensitivity and specificity, and can be used for differential diagnosis of CpHV-1 infection.
Description
Technical Field
The invention belongs to the technical field of immunity, and particularly relates to a hybridoma cell strain secreting anti-goat herpes virus type I (CpHV-1) monoclonal antibody.
Background
Goat herpes virus type I (Caprine herpesvirus 1, CpHV-1) is a double stranded DNA virus belonging to the genus varicella of the subfamily Physarae of the family herpesviridae. The virus infects newborn lambs and then presents with severe infection, the main symptoms are fever, conjunctivitis, eye secretion increase, dyspnea, intestinal ulcer and necrotic lesions, and the disease incidence and mortality are higher. Adult goat infection is manifested as a subclinical infection, which can result in different symptoms including respiratory diseases, fever and leukopenia, vulvovaginitis and balanoposthitis. Abortion can be induced after infection of goats pregnant for 3 to 4 months. In 1974 CpHV-1 was first isolated from neonatal lamb mordants showing enteritis in California, USA, after which the viral epidemic appeared in Europe, Atlanta and south America, seriously compromising the development of the sheep industry. The virus infection has not been reported in China, and the virus is separated from a goat nose swab sample showing serious respiratory tract infection in 2014 in the laboratory for the first time. At present, no vaccine and medicine which are approved to be on the market for the virus exist in China, so that the development of medicines for the infection of the epidemic strains in China is urgently needed.
The CpHV-1 infection diagnosis method comprises PCR detection and virus separation identification experiment. These methods are inherently practical, but have many disadvantages. The PCR method is a relatively common identification method, is used for detecting nucleic acid of virus, and is easy to generate false positive although having certain sensitivity and specificity. The virus isolation and identification experiment is to obtain a strain through cell isolation and culture, and then carry out a series of molecular biological identification, although the method is classical, the method is long in time consumption. Therefore, establishing a specific and rapid differential diagnosis method is crucial to reducing the incidence of CpHV-1 infection.
Disclosure of Invention
In order to solve the technical problem, the invention establishes a hybridoma cell bank secreting the anti-goat herpes virus type I monoclonal antibody.
Furthermore, the invention establishes a hybridoma cell bank for secreting the anti-goat herpesvirus type I monoclonal antibody, and the hybridoma cell bank stably secretes the anti-goat herpesvirus type I monoclonal antibody.
Furthermore, the invention establishes a hybridoma cell bank secreting monoclonal antibodies against goat herpesvirus type I, the hybridoma cell bank adopts CpHV-1 virus as immunogen to immunize female BALB/c mice, and the hybridoma cell bank is obtained by fusing mouse myeloma cells with the immunized female BALB/c mice.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against goat herpesvirus type i, wherein cell fusion is performed using methods including, but not limited to, PEG cell fusion.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against goat herpesvirus type i, wherein cell fusion is performed using PEG 2000.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against goat herpes virus type i, wherein the immunization is boosted prior to cell fusion.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against goat herpesvirus type i, wherein the immunization is boosted 3 days before cell fusion.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against the goat herpesvirus i, wherein the immunization is performed at least once using the ultracentrifugation goat herpesvirus i as an immunogen and the immunization is boosted before cell fusion.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against the goat herpesvirus i, wherein the immunization is performed at least once using ultracentrifugation goat herpesvirus i as an immunogen and boosting the immunization 3 days before cell fusion.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpes virus type I monoclonal antibody, wherein mouse myeloma cells are pretreated before cell fusion, so that the good growth of the mouse myeloma cells is ensured.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against goat herpesvirus type i, wherein the mouse myeloma cells are pre-treated prior to cell fusion, preferably grown to log phase.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against goat herpesvirus type i, said method comprising pre-fusion treatment or not of mouse myeloma cells with immunized BALB/c mouse spleen cells.
Further, the invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against goat herpes virus type I, wherein the pretreatment of mouse myeloma cells and immunized BALB/c mouse spleen cells comprises the step of fully and uniformly mixing the mouse myeloma cells and the immunized BALB/c mouse spleen cells in a ratio of 1:3-1: 5.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpes virus type I monoclonal antibody, wherein a culture medium preheated to 37 ℃ is added from slow to fast in the cell fusion process.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against goat herpesvirus type i, wherein a half-exchange is performed 7 days after cell fusion, and the used exchange medium is RPMI-1640 medium containing HAT and 20% FBS.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against goat herpesvirus type i, wherein the hybridoma cells are screened according to the OD value of the sample.
Further, the present invention provides a method for establishing a hybridoma cell bank secreting monoclonal antibodies against goat herpesvirus type i, wherein the hybridoma cells are screened according to the OD ratio of positive serum to negative serum.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein the hybridoma cells are screened by an enzyme-linked immunosorbent assay method.
Further, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein the hybridoma cells are screened at least once by an enzyme-linked immunosorbent assay method at least once.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein a colchicine method is adopted to detect the chromosome of a hybridoma cell strain secreting the anti-goat herpesvirus type I monoclonal antibody.
Further, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein when detecting the chromosome of a hybridoma cell strain, methanol: glacial acetic acid 3:1 was used as fixative.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein the hybridoma cells are screened by adopting an enzyme-linked immunosorbent assay method and a chromosome detection method.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein the hybridoma cells are screened by an enzyme-linked immunosorbent assay method and a colchicine chromosome detection method.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein the hybridoma cells are screened by adopting an enzyme-linked immunosorbent assay method and a chromosome detection method at least once.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein the hybridoma cells are screened at least once by adopting an enzyme-linked immunosorbent assay method and a colchicine chromosome detection method.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein hybridoma cells are screened according to a serum OD value, an enzyme-linked immunosorbent assay method and a chromosome detection method.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein hybridoma cells are screened according to the OD ratio of positive serum to negative serum, an enzyme-linked immunosorbent assay method and a chromosome detection method.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein hybridoma cells are screened according to the OD ratio of positive serum and negative serum, an enzyme-linked immunosorbent assay method and a colchicine chromosome detection method.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein hybridoma cells are screened according to the OD value of serum, at least one enzyme-linked immunosorbent assay method and a chromosome detection method.
Further, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein the specificity of the hybridoma cell is detected by adopting an indirect immunofluorescence assay.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein the stability of the hybridoma cells is continuously detected by adopting a method of multiple continuous culture passages, liquid nitrogen cryopreservation and recovery test and indirect enzyme-linked immunosorbent assay.
Further, the present invention provides a method for screening hybridoma cells secreting monoclonal antibodies against goat herpesvirus type i, wherein the hybridoma cells are screened for OD values in serum.
Further, the invention provides a screening method of hybridoma cells secreting monoclonal antibodies against goat herpes virus type I, wherein the hybridoma cells are screened according to the OD ratio of positive serum to negative serum.
Furthermore, the invention provides a screening method of hybridoma cells secreting anti-goat herpesvirus type I monoclonal antibodies, wherein the hybridoma cells are screened by an enzyme-linked immunosorbent assay method.
Furthermore, the invention provides a screening method of hybridoma cells secreting anti-goat herpesvirus type I monoclonal antibodies, wherein the hybridoma cells are screened at least once by an enzyme-linked immunosorbent assay method at least once.
Furthermore, the invention provides a screening method of hybridoma secreting anti-goat herpesvirus type I monoclonal antibody, wherein the hybridoma is screened by adopting an enzyme-linked immunosorbent assay method and a chromosome detection method.
Furthermore, the invention provides a screening method of hybridoma cells secreting anti-goat herpesvirus type I monoclonal antibodies, wherein the hybridoma cells are screened by an enzyme-linked immunosorbent assay method and a colchicine chromosome detection method.
Furthermore, the invention provides a screening method of hybridoma cells secreting anti-goat herpesvirus type I monoclonal antibodies, wherein the hybridoma cells are screened according to the OD ratio of positive serum and negative serum, an enzyme-linked immunosorbent assay method and a colchicine chromosome detection method.
Further, the invention provides a hybridoma cell strain secreting the goat herpes virus resistant type I monoclonal antibody, wherein the hybridoma cell is preserved in the China center for type culture Collection in 2018, 6 months and 26 days, the address is Wuhan university, Wuhan code 430072, the preservation number is CCTCC NO: c2018150, classification name: hybridoma cell line 4a 4.
Furthermore, the invention provides a chromosome detection method of a hybridoma cell strain secreting the anti-goat herpesvirus type I monoclonal antibody, which is carried out by using colchicine.
Furthermore, the invention provides a monoclonal antibody for resisting goat herpes virus type I, and the monoclonal antibody is prepared from the hybridoma cell.
Furthermore, the invention provides a monoclonal antibody for resisting goat herpes virus type I, wherein the monoclonal antibody is prepared from mouse ascites containing the hybridoma cell.
Further, the present invention provides an indirect immunofluorescence detection method, which is performed using the monoclonal antibody prepared as above.
Further, the invention provides a method for detecting the goat herpes virus type I, which is carried out by adopting the monoclonal antibody prepared by the method.
Further, the invention provides a kit for detecting goat herpes virus type I, which comprises the monoclonal antibody prepared as above.
Further, the invention provides a preparation method of the monoclonal antibody against the goat herpesvirus I, which comprises establishing a hybridoma cell bank secreting the monoclonal antibody against the goat herpesvirus I.
Further, the invention provides a preparation method of the monoclonal antibody against goat herpes virus type I, and the method is carried out by using the hybridoma cell.
Further, the present invention provides a diagnostic reagent for goat herpesvirus type i, which comprises the monoclonal antibody prepared as above.
Furthermore, the invention provides a method for establishing a hybridoma cell bank secreting the anti-goat herpesvirus type I monoclonal antibody, wherein the method adopts a goat herpesvirus type I virulent strain as an antigen to immunize a BALB/c mouse.
Further, the invention provides an anti-goat herpes virus type I monoclonal antibody for indirect immunofluorescence detection, and the monoclonal antibody is prepared by the method.
Further, the invention provides an anti-goat herpesvirus type I monoclonal antibody, and the neutralizing titer of the monoclonal antibody is not reduced after the monoclonal antibody is stored for two years.
Further, the present invention provides a therapeutic agent for goat herpesvirus type i, which comprises the monoclonal antibody prepared as above.
Further, the present invention provides a goat herpesvirus type i therapeutic agent comprising the monoclonal antibody prepared as above and a stabilizer.
Furthermore, the invention provides a hybridoma cell strain secreting the anti-goat herpesvirus I monoclonal antibody, and the neutralization titer of the hybridoma cell strain is as high as 28。
Further, the present invention provides a method for preparing a monoclonal antibody of IgG2a kappa subclass, wherein the monoclonal antibody of IgG2a kappa subclass is prepared using the hybridoma cell bank as described above.
Further, the present invention provides a method for preparing a monoclonal antibody of IgG2a kappa subclass, wherein the monoclonal antibody of IgG2a kappa subclass is prepared using hybridoma cells as described above.
Furthermore, the establishment of the hybridoma cell strain is obtained by taking a CpHV-1 virulent strain separated from domestic clinical pathological materials as an antigen, immunizing a BALB/c mouse and utilizing a CpHV-1 specific monoclonal antibody hybridoma cell bank established by a lymphocyte hybridoma technology. The mouse ascites prepared from the hybridoma cell 4A4 strain is proved to have higher neutralization titer by virus neutralization test, is prepared into a therapeutic agent, is applied to clinical treatment of CpHV-1 pathogenic animals, and has obvious effect. The mouse ascites prepared from the hybridoma cell 4A4 strain is prepared into a detection reagent which is used as a detection antibody and is applied to the indirect immunofluorescence detection of CpHV-1 infection, and the detection reagent has high sensitivity and strong specificity.
Compared with the prior art, the invention has the beneficial effects that:
1. the CpHV-1 immunogen used in the present invention is a novel CpHV-1 domestic circulating virulent strain JHA 1405 isolated from a herd of sheep exhibiting severe respiratory infections.
2. Firstly establishes a hybridoma cell bank of 89 strains secreting CpHV-1 monoclonal antibodies, screens out a hybridoma cell strain 4A4 strain, and uses the mouse ascites antibody prepared by the hybridoma cell bank to neutralize the CpHV-1 viruses to achieve the titer as high as 28。
3. The monoclonal antibody therapeutic agent prepared from the hybridoma cell strain is used for clinical treatment of CpHV-1 diseased sheep, the cure rate reaches 90%, the curative effect is obvious, and no adverse reaction exists. The neutralizing titer of the antibody is not reduced after the antibody is stored for two years at the temperature of minus 20 ℃, and the stability is good.
4. The indirect Immunofluorescence (IFA) detection method established by using the monoclonal antibody secreted by the hybridoma cell as a detection antibody has better sensitivity and specificity, and can be used for differential diagnosis of CpHV-1.
Drawings
For a better illustration of the invention, the following figures are provided, which are provided for illustration of the invention only and are not limiting of the invention:
FIG. 1 is a graph showing the results of an indirect Immunofluorescence (IFA) assay for the detection of the specificity of monoclonal antibodies for CpHV-1 according to the present invention: a: MDBK cell assay results after CpHV-1 infection, B: MDBK cell assay results after infection with border disease virus, C: and (5) detecting results of normal MDBK cells.
FIG. 2 is a graph showing the results of detection of a bovine kidney cell line (MDBK) cell sample using an indirect Immunofluorescence (IFA) method according to the present invention: a: MDBK cell assay results after CpHV-1 infection, B: and (5) detecting results of normal MDBK cells.
Detailed Description
The invention will be further described with reference to specific embodiments and drawings attached to the description, but the invention is not limited thereto.
EXAMPLE 1 establishment of monoclonal antibody hybridoma cell line
1. Preparation of CpHV-1 antigen
The inventor separates a novel CpHV-1 domestic epidemic virulent strain JSHA1405 (Helifei, goat herpes virus I type vaccine strain) from a herd showing severe respiratory tract infection and application thereof]Jiangsu: CN107893056A, 2018-04-10), the strain has larger variation with the foreign epidemic strain, only regarding gB gene, the foreign epidemic strain encodes 919 amino acids, while the domestic epidemic strain JHA 1405gB gene encodes 920 amino acids, and mutation of 11 amino acid sites occurs. The strains were inoculated at 0.01-0.1MOI with 70% confluent MDBK cells (purchased from China institute for veterinary medicine) and incubated at 37 deg.C and 5% CO2Culturing in a cell culture box in a dark place, collecting cell culture 48h after inoculation, repeatedly freezing and thawing for 3 times, centrifuging to obtain supernatant, ultracentrifuging at 40000 g for 2h, discarding supernatant, and dissolving precipitate with appropriate amount of PBS overnight.
2. Animal immunization
The CpHV-1 super-ionovirus was used as an immunogen to immunize female BALB/c mice (purchased from the center of comparative medicine, university of Yangzhou) 6-8 weeks old at multiple sites subcutaneously on the back for 3 times, with 2 weeks between each immunization. The first immunization was performed using 100. mu.L of the virus mixed with an equal volume of Freund's complete adjuvant (purchased from Sigma) and the last two immunizations were performed using 100. mu.L of the virus mixed with an equal volume of Freund's incomplete adjuvant (purchased from Sigma). Collecting blood after three times of immunization of mice after 14d tail breaking, separating serum, measuring the antibody titer of the obtained antiserum by adopting an indirect ELISA method, and selecting the serum antibody titer more than 106The mice were boosted 3d before the cell fusion experiment by intraperitoneal injection with the virus without adjuvant (100. mu.L/mouse).
3. Cell fusion
The PEG cell fusion method is adopted. Taking mouse bone marrowMixing tumor cell (SP2/0) and spleen cell of BALB/c mouse at a ratio of 1:3-1:5, centrifuging at 25 deg.C for 5min at 2000rpm, discarding supernatant, flicking tube bottom with palm to make cells loose, preheating in 37 deg.C water bath, adding 37 deg.C PEG within 1min20000.8mL (purchased from Sigma) was gently shaken while adding, and then 12mL of serum-free RPMI-1640 medium preheated to 37 ℃ was added to each of 1mL/min, 2mL/min, 3mL/min, and 3mL/min within 5min, left to stand at 37 ℃ for 10min, centrifuged at 2000rpm for 5min at 25 ℃, the supernatant was discarded, and 20% Fetal Bovine Serum (FBS) (purchased from Beijing Quanyujin Biotechnology Co., Ltd.) and HAT (purchased from Sigma) were added to resuspend the mixture in a 96-well plate containing feeder cells, and the mixture was aliquoted in a 5% CO-free 96-well plate2And (5) culturing in a cell culture box. After 7 days of culture, half of the medium was replaced with RPMI-1640 medium containing HAT and 20% FBS, and when the fused cells grew to 1/10-1/5 of the bottom area of the well of the 96-well plate, the supernatant was taken for antibody detection.
4. Screening of hybridoma cells
Coating the enzyme label plate with 500-fold diluted CpHV-1 super-detached virus by using 0.05mol/L of carbonate buffer solution with pH9.6 as a coating solution at the temperature of 4 ℃ overnight, washing for 3 times by using PBST (Poly-p-phenylene benzobisoxazole) for 5min each time, and patting dry; each well was blocked with PBST containing 1% BSA (purchased from Sigma) at 200. mu.L/well, left at 37 ℃ for 2h, washed 3 times with PBST for 5min each, and blotted dry; adding the fusion cell supernatant, 1:1000 diluted immune mouse positive serum and 1:1000 diluted mouse negative serum into corresponding holes, performing action at 37 ℃ for 1h at a rate of 100 mu L/hole, washing for 3 times by PBST (PBST), and patting to dry for 5min each time; adding Horse Radish Peroxidase (HRP) labeled goat anti-mouse IgG (purchased from Beijing all-style gold Biotechnology Co., Ltd.) diluted at 1:4000, reacting at 37 deg.C for 1h with 100 μ L/well, washing with PBST for 3 times (5 min each time), and patting to dry; adding TMB substrate, 100 μ L/hole, and displaying at room temperature in dark for 10 min; the reaction was stopped by adding 50. mu.L of 2mol/L sulfuric acid per well. OD determination by enzyme-linked immunosorbent assay450nmThe values were zeroed for blank control, P is the value of each well, N is the OD of the negative serum450nmValue, OD of negative serum450nmThe value is less than or equal to 0.1, and the OD of the positive reference serum450nmValue and OD of negative reference serum450nmThe ratio of the values is more than or equal to 2.1, namely negativeAnd on the premise that positive control is established, detecting holes with the P/N being more than or equal to 2.1 are judged to be positive, detecting again after 2-3d, and cloning hybridoma cells with positive detection results in two times.
5. Cloning of hybridoma cells
The viable cells in the positive wells were first stained and counted with trypan blue, diluted to 100 cells/10 mL medium in 20% FBS-containing RPMI-1640 medium, the diluted cell suspension was added to a 96-well cell culture plate at 100. mu.L/well and placed at 37 ℃ in 5% CO2Culturing in a cell culture box, observing the formation of clone cells under a microscope after 4-5 days, recording the growth hole of only a single clone, taking out cell supernatant when 8-9 days, and carrying out ELISA detection. Selecting positive monoclonal cells, cloning for more than 3 times until all cell well supernatants are positive, and detecting OD in each well450nmThe values are closer. And (3) carrying out expanded culture on the cloned CpHV-1 specific monoclonal antibody hybridoma cell strain and freezing and storing. Through cell fusion, screening, cloning and identification for a plurality of times, a 89 hybridoma cell bank which stably secretes CpHV-1 monoclonal antibody is finally established.
EXAMPLE 2 preparation of ascites
Injecting 10-12 weeks old BALB/c mice (purchased from the university of Yangzhou, comparative medicine center) 0.5 mL/mouse intraperitoneally with sterilized liquid paraffin, and injecting hybridoma cell strain into the abdominal cavity of each mouse 0.2mL (containing 2 × 10 cells) 7 days later6-3×106Individual hybridoma cells), ascites of the mouse with obvious abdominal bulge is collected after 7-10 days, and centrifugation is carried out at 5000rpm for 10 min. Collecting supernatant, subpackaging, and storing at-20 deg.C for use.
Example 3 virus neutralization assay
Ascites prepared by 89 established hybridoma cell lines stably secreting CpHV-1 monoclonal antibodies are respectively subjected to CpHV-1 neutralization tests, and monoclonal antibody hybridoma cell lines with high secretion neutralization potency are further screened. First, TCID of CpHV-1 JSAE 1405 strain was measured50. MDBK cells were digested and plated in 96-well cell plates using a method of fixing virus dilution antibodies. The ascites of mice with 10 times serial dilution of each monoclonal antibody and the equivalent antibody respectivelyIntegrating 200TCID50The CpHV-1 suspension is mixed uniformly and acted at 37 ℃ for 1h, 0.1mL of the virus-antibody suspension is inoculated into the 96-well cell plate, a CpHV-1 and normal MDBK cell control is set up and placed at 37 ℃ and 5% CO2Culturing in a cell culture box, and observing the result after 2 d. Among 89 hybridoma cell lines of CpHV-1 specific monoclonal antibodies, mouse ascites prepared from hybridoma cell line 4A4 strain has the highest neutralization titer on CpHV-1, and the neutralization titer is 28。
EXAMPLE 4 biological Properties of hybridoma cell line 4A4
1. Chromosome analysis of hybridoma cell lines
The hybridoma cells were chromosome-counted by giemsa staining. Respectively culturing SP2/0 myeloma cell and positive hybridoma cell, growing to logarithmic phase, adding colchicine into cell bottle to make its final concentration be 0.1 μ g/mL, and placing into cell culture box for further culture for 4-5 h. Blowing up the cells by 5mL of 0.075mol/L KCL hypotonic solution preheated at 37 ℃, uniformly mixing, placing in a 37 ℃ incubator for 30min, adding 1mL of newly prepared fixing solution (methanol: glacial acetic acid is 3:1) while dropwise adding, uniformly mixing, and centrifuging at 1000rpm for 10 min. Discarding the supernatant to leave cell precipitate, blowing up the cells with 5mL of fixative, acting at 37 ℃ for 30min, centrifuging at 1000rpm for 10min, and repeating the above operation once. The cell pellet is suspended and mixed evenly by 1mL of fixing solution, 1 drop of suspension is absorbed, dropped on a pre-frozen glass slide, laid on the glass slide and dried naturally. Staining with fresh Jiemsa staining solution for 10min, washing with tap water, air drying, and observing under microscope. The number of chromosomes of the hybridoma cell strain is 96, the number of chromosomes of myeloma cells is 62, and the number of chromosomes of mouse spleen cells is 40, so that the obtained hybridoma cell strain is proved to be a result of fusion of the two cells.
2. Secreted antibody stability assay
Continuously culturing and subculturing the obtained hybridoma cell 4A4 strain for 50 times, freezing and storing by liquid nitrogen and recovering, and continuously detecting the antibody titer in the hybridoma cell culture supernatant to 10 by using a CpHV-1 antibody indirect ELISA method4The hybridoma cell line 4A4 is proved to be capable of continuously and stably secreting anti-CpHV-1 monoclonalA diabody.
3 subtype determination of monoclonal antibodies
The subclass of the monoclonal antibody secreted by the hybridoma cell 4A4 strain was determined using a monoclonal antibody subclass identification kit (purchased from Thermo Fisher scientific Co., Ltd.), and it was found that the subclass of the monoclonal antibody was IgG2a K.
4 potency assay of monoclonal antibodies
The titer of hybridoma cell culture supernatant and mouse ascites is measured by indirect ELISA, and the result shows that the titer of hybridoma cell 4A4 culture supernatant ELISA is 104Ascites ELISA titer 1012Virus neutralization titer in ascites is 28。
5 specificity of monoclonal antibody
5.1 Indirect immunofluorescence test monoclonal antibody specificity
Experiments were performed in 48-well cell culture plates. Respectively inoculating MDBK cells with CpHV-1JSHA1405 strain and Border Disease Virus (BDV) (Levinra et al, Detection of Border Disease Virus (BDV) in mineral flowers and deficiency of dialearrhea in easter China, virology Journal,2013,10:80.) and culturing for 24h, then discarding the cell culture solution, washing 3 times with a serum-free culture medium, adding 200 muL/well of-20 ℃ precooled absolute ethanol into the cell culture well, fixing for 30min at 4 ℃, washing 3 times with PBS and beating to dry; adding hybridoma cell culture supernatant, incubating at 37 deg.C for 1h with 200 μ L/well, washing with PBS for 3 times, and patting to dry; a200-fold dilution of FITC-labeled goat anti-mouse IgG antibody (purchased from Strobilanthes Wuhan, Drehd bioengineering, Ltd.) was added thereto at 200. mu.L/well, incubated at 37 ℃ for 30min, washed 3 times with PBS, and observed under a fluorescence microscope. Under a fluorescence microscope, the monoclonal antibody can react with CpHV-1 infected MDBK cells to generate fluorescence (shown in figure 1-A), but does not react with BDV infected MDBK cells and normal MDBK cells (shown in figures 1-B and 1-C), and the specific reaction of the monoclonal antibody with CpHV-1 is proved.
5.2 Indirect ELISA for detecting the specificity of monoclonal antibodies
MDBK cell antigen was diluted (500-fold) with 0.05mol/L pH9.6 carbonate buffer as coating solution, coated on ELISA plate at 100. mu.L/well, and coated at 4 ℃At night; PBST washing for 3 times, each time for 5min, patting dry; blocking each well with 200. mu.L/well of phosphate Tween buffer (PBST) containing 1% BSA (from Sigma), standing at 37 ℃ for 2h, washing 3 times with PBST, 5min each time, patting dry; adding hybridoma cell supernatant, 1:1000 diluted immune mouse positive serum and 1:1000 diluted mouse negative serum into corresponding holes, reacting at 37 deg.C for 1h, washing with PBST for 3 times (5 min each time), and patting to dry; adding HRP-labeled goat anti-mouse IgG (purchased from Beijing all-purpose gold Biotechnology Co., Ltd.) diluted at 1:4000 into the mixture, allowing the mixture to act at a temperature of 100 mu L/well for 1h at 37 ℃, washing the mixture with PBST for 3 times, each time for 5min, and finally drying the mixture by patting the mixture dry; adding tetramethyl benzidine (TMB) substrate, 100 μ L/hole, and displaying at room temperature in dark for 10 min; the reaction was stopped by adding 50. mu.L of 2mol/L sulfuric acid per well. OD determination by enzyme-linked immunosorbent assay450nmValue, OD of hybridoma cell supernatant and mouse negative serum450nmValues were all less than 0.1, showing good specificity.
EXAMPLE 5 preparation, testing and use of monoclonal antibody therapeutics
1. Preparation of monoclonal antibody therapeutics
Sterilized liquid paraffin was intraperitoneally injected into 10-12 week-old BALB/c mice (purchased from the university of Yangzhou, comparative medicine center) at a rate of 0.5 mL/mouse, and after 7 days, hybridoma cell strain 4A4 was injected into the abdominal cavity of each mouse, each injection was 2X 106-3×106And taking ascites of the mouse with obviously swollen abdomen 7-10 days after each hybridoma cell, and centrifuging at 5000rpm for 10 min. Collecting ascites of cloned antibody in the above list, treating at 56 deg.C for 30min, filtering, sterilizing, diluting with sterile PBS, adding appropriate stabilizer, packaging, and storing at-20 deg.C.
2. Efficacy testing of monoclonal antibody therapeutics
Selecting 2-3 month-old healthy goats of which CpHV-1 antigen and antibody detection are negative, randomly dividing the goats into a plurality of groups, infecting the goats with the CpHV-1 JSAE 1405 strain, injecting monoclonal antibody therapeutic agents with different doses into muscles after infection, observing clinical symptoms, measuring body temperature and detecting CpHV-1 toxin expulsion every day. A control group without monoclonal antibody therapeutic agent injection is set in the experiment at the same time. The results show that: the sheep in the monoclonal antibody treatment group have no CpHV-1 clinical symptoms, the body temperature is normal, and the CpHV-1 detection is negative; the control group showed CpHV-1 clinical symptoms, the body temperature was elevated and CpHV-1 was detected as positive.
3. Safety testing of monoclonal antibody therapeutics
In order to examine the safety of the preparation, 5 times of therapeutic dose was injected intramuscularly into test sheep, and the body temperature of the test sheep was measured daily, and the mental state and diet performance were observed. The result shows that the test sheep have no adverse side effect.
4. Stability testing of monoclonal antibody therapeutics
The subpackaged monoclonal antibody therapeutic agent is respectively preserved at 37 ℃, 4 ℃ and-20 ℃, and after different preservation time, a sample is taken for virus neutralization test, and the neutralization titer is measured. The results show that: the therapeutic agent has a neutralizing potency of 2 after being stored at-20 deg.C for two years8。
5. Clinical application of monoclonal antibody therapeutic agent
The prepared monoclonal antibody therapeutic agent is used for treating CpHV-1 infected sheep in a farm, and the cure rate can reach 90% by adopting an intramuscular injection method.
Example 6 establishment of Indirect immunofluorescence method (IFA)
1. Preparation of IFA detection antibody
Sterilized liquid paraffin was intraperitoneally injected into 10-12 week-old BALB/c mice (purchased from the university of Yangzhou, comparative medicine center) at a rate of 0.5 mL/mouse, and after 7 days, hybridoma cell strain 4A4 was injected into the abdominal cavity of each mouse, each injection was 2X 106-3×106And taking ascites of the mouse with obviously swollen abdomen 7-10 days after each hybridoma cell, and centrifuging at 5000rpm for 10 min. Collecting ascites of the cloned antibody in the list, treating at 56 deg.C for 30min, filtering, sterilizing, diluting with sterile PBS 500 times, adding appropriate stabilizer, packaging, and storing at-20 deg.C.
2. IFA detection of CpHV-1 infected MDBK cell samples
The experiments were performed in 96-well cell culture plates. Inoculating the CpHV-1 JSAH 1405 strain into MDBK cells, culturing for 24h, discarding cell culture solution, washing with PBS for 3 times, adding 100 mu L/hole of-20 ℃ precooled absolute ethyl alcohol into cell culture holes, fixing for 30min at 4 ℃, washing with PBS for 3 times, and patting to dry; adding IFA detection antibody, incubating at 37 ℃ for 1h at a concentration of 100 mu L/well, washing with PBS for 3 times, and patting dry; a200-fold dilution of FITC-labeled goat anti-mouse IgG antibody (purchased from Strobilanthes Wuhan, Drehd bioengineering, Ltd.) was added thereto at 100. mu.L/well, incubated at 37 ℃ for 30min, washed 3 times with PBS, and observed under a fluorescence microscope. Under a fluorescence microscope, the IFA detection antibody can react with CpHV-1 infected MDBK cells to generate green fluorescence (figure 2-A), but does not fluoresce with normal MDBK cells (figure 2-B), and the IFA detection antibody is proved to be capable of effectively detecting CpHV-1 infection and has better sensitivity and specificity.
It should be emphasized that the above-described embodiments of the present invention are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiments of the disclosure without departing from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.
Claims (6)
1. A hybridoma cell 4A4 strain secreting anti-goat herpesvirus type I monoclonal antibody is characterized in that the hybridoma cell is preserved in China center for type culture Collection in 2018, 6 and 26 months, and the preservation number is CCTCC NO: C2018150.
2. an anti-goat herpesvirus i monoclonal antibody secreted from hybridoma cell line 4a4 of claim 1.
3. A goat herpesvirus i therapeutic comprising the monoclonal antibody of claim 2.
4. The goat herpesvirus i therapeutic agent as in claim 3, further comprising a stabilizer.
5. A kit for detecting goat herpesvirus type i, comprising the monoclonal antibody of claim 2.
6. A method for producing a monoclonal antibody against goat herpesvirus type I, which comprises using the hybridoma cell line 4A4 according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810831978.0A CN108913665B (en) | 2018-07-26 | 2018-07-26 | Hybridoma cell strain secreting anti-CpHV-1 monoclonal antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810831978.0A CN108913665B (en) | 2018-07-26 | 2018-07-26 | Hybridoma cell strain secreting anti-CpHV-1 monoclonal antibody and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108913665A CN108913665A (en) | 2018-11-30 |
| CN108913665B true CN108913665B (en) | 2021-06-22 |
Family
ID=64418349
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810831978.0A Active CN108913665B (en) | 2018-07-26 | 2018-07-26 | Hybridoma cell strain secreting anti-CpHV-1 monoclonal antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108913665B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838678A (en) * | 2016-04-11 | 2016-08-10 | 江苏省农业科学院 | Hybridoma cell strain capable of secreting CPIV3 antibody and ELISA kit |
| CN107893056A (en) * | 2017-12-13 | 2018-04-10 | 江苏省农业科学院 | The type vaccine strain of bovid herpesvirus 5 I and its application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107406834B (en) * | 2015-02-11 | 2021-03-16 | 生物工程大学精神物质实验室 | Retargeted herpesviruses with glycoprotein H fusions |
-
2018
- 2018-07-26 CN CN201810831978.0A patent/CN108913665B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838678A (en) * | 2016-04-11 | 2016-08-10 | 江苏省农业科学院 | Hybridoma cell strain capable of secreting CPIV3 antibody and ELISA kit |
| CN107893056A (en) * | 2017-12-13 | 2018-04-10 | 江苏省农业科学院 | The type vaccine strain of bovid herpesvirus 5 I and its application |
Non-Patent Citations (4)
| Title |
|---|
| Analysis of antibody response in goats to caprine herpesvirus 1;Maria Tempesta 等;《Biologicals》;20050926;第33卷;第283-287页 * |
| First Description of Infection of Caprine Herpesvirus 1(CpHV-1) in Goats in Mainland France;Florence Suavet 等;《pathogens》;20160206;第5卷;第1-13页 * |
| Improved antigenic methods for differential diagnosis of bovine, caprine, and cervine alphaherpesviruses related to bovine herpesvirus 1;Véronique Keuser 等;《J Clin Microbiol.》;20040331;第42卷(第3期);第1229页左栏最后1段至第1230页右栏最后1段，表1-2 * |
| 自然与试验感染的山羊疱疹病毒Ⅰ型特异性IgG的亚类;Mariarosaria Marinaro 等;《中国畜牧兽医》;20091120;第36卷(第11期);第148页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108913665A (en) | 2018-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107034197B (en) | Canine influenza virus monoclonal antibody hybridoma cell strain F112 and application thereof | |
| CN107937352B (en) | Colloidal gold immunochromatographic test strip for detecting peste des petits ruminants virus H protein antibody | |
| CN110777121A (en) | Monoclonal antibody hybridoma cell 3B5 strain secreting anti-canine distemper virus H protein | |
| CN109810948B (en) | Hybridoma cell strain of monoclonal antibody against African swine fever virus K205R protein and antibody secreted by hybridoma cell strain | |
| Feng et al. | Virus-like particles produced in pichia pastoris induce protective immune responses against coxsackievirus A16 in mice | |
| CN110373393A (en) | The monoclonal antibody hybridoma cell strain 1H6 of infectivity resistant bursal disease poison VP2 albumen | |
| CN111413499B (en) | Indirect immunofluorescence kit for detecting avian adenovirus I group | |
| CN108913665B (en) | Hybridoma cell strain secreting anti-CpHV-1 monoclonal antibody and application thereof | |
| Wu et al. | Development, characterization of monoclonal antibodies specific for the ORF25 membrane protein of Cyprinid herpesvirus 2 and their applications in immunodiagnosis and neutralization of virus infection | |
| Zhang et al. | A double antibody sandwich enzyme-linked immunosorbent assay for detection of soft-shelled turtle iridovirus antigens | |
| CN108866008B (en) | Monoclonal antibody for resisting koi herpesvirus, cell strain and application thereof | |
| CN105296507B (en) | Lassa fever virus-like particle, preparation method and application thereof | |
| US11767356B1 (en) | Canine parvovirus nanobody CPV-VHH-E3 and application thereof | |
| CN107557347A (en) | New virus sample particle, its preparation method and the application of enterovirns type 71 | |
| WO2023201833A1 (en) | Anti-sars-cov-2 broad-spectrum neutralizing monoclonal antibody, and hybridoma cell strain, detection kit and use thereof | |
| CN108017714B (en) | Monoclonal antibody of II-type carp herpesvirus ORF92 protein and application thereof | |
| CN111777684A (en) | Preparation method and application of antibody induced by coronavirus tandem epitope protein | |
| CN109932504B (en) | Kit for detecting mandarin fish rhabdovirus | |
| CN113929773A (en) | anti-SARS-CoV-2S 1-RBD monoclonal antibody and its application | |
| CN105749267A (en) | Marking vaccine and serological identification method for porcine reproductive and respiratory syndrome high in pathogenicity | |
| CN114231496B (en) | Salmonella equine abortus competition ELISA antibody detection kit and application thereof | |
| CN110804097A (en) | Method for simultaneously preparing two monoclonal antibodies | |
| CN114457041B (en) | Monoclonal antibody of Marek's disease virus gB protein, preparation method and application | |
| CN112501129B (en) | anti-IBDV VP2 protein monoclonal antibody combination and application thereof in identifying and detecting IBDV | |
| Ding et al. | Preparation and characterisation of monoclonal antibodies against the N protein of the SHpd/2012 strain of porcine epidemic diarrhoea virus. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |