CN116284350B - Cat herpesvirus monoclonal antibody, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of medicines, and particularly relates to a feline herpesvirus monoclonal antibody, a preparation method and application thereof. The invention provides a hybridoma cell strain capable of secreting a feline herpesvirus monoclonal antibody, the preservation number of the hybridoma cell strain is CGMCC No.45346, and the preservation address is the China general microbiological culture Collection center (China Committee for culture Collection). The hybridoma cell can stably secrete the feline herpesvirus monoclonal antibody, so that the feline rhinotracheitis caused by feline herpesvirus type 1 is cured, the death rate of cats is greatly reduced, and the technical problem that no specific medicine aiming at the feline herpesvirus exists in the market at present is solved.

Description

Cat herpesvirus monoclonal antibody, and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a feline herpesvirus monoclonal antibody, a preparation method and application thereof.

Background

Feline rhinotracheitis is caused by infection with feline herpesvirus type 1 (feline herpesvirus type, fhv-1), and is also known as feline rhinotracheitis virus (feline rhinotracheitis virus) due to its major cause. FHV-1 is a linear double stranded DNA virus belonging to the herpesviridae, subfamily A herpesviridae. FHV-1 virus particles are elliptic or circular, are 20-plane symmetrical, and consist of nucleic acid, capsid, shell and envelope. The genome is about 130kb in length and contains 78 ORFs, consisting of a long unique region, an inverted repeat, a short unique region, and a terminal repeat. FHV-1 proliferates mainly in the nose, throat, trachea, bronchi and conjunctiva of cats. Mainly invading kittens, and after infection, the kittens are manifested by upper respiratory tract inflammation, keratoconjunctivitis and chronic nasosinusitis, and can invade trigeminal nerve and optic nerve; severe infection is manifested by elevated body temperature and symptoms of the upper respiratory tract, the morbidity can reach 100%, and the mortality can reach more than 50%.

At present, vaccination is the most effective method for preventing and treating the disease, clinical treatment mainly comprises symptomatic and supportive treatment, and the vaccination can reduce the incidence rate to a certain extent and improve the body health of cats.

However, since there is no specific drug for FHV-1 in the market at present, a large number of cats can not quickly and effectively recover health only by means of their own immune systems after suffering from FHV-1, so there is a lack of a drug for treating FHV infection with high specificity.

Disclosure of Invention

The invention provides a feline herpesvirus monoclonal antibody, a preparation method and application thereof, and aims to solve the technical problem that no specific medicine aiming at FHV-1 exists in the market at present in the background technology.

The technical scheme adopted by the invention is as follows:

on the one hand, the invention provides a hybridoma cell strain capable of secreting the feline herpesvirus monoclonal antibody, the preservation number of the hybridoma cell strain is CGMCC No.45346, the hybridoma cell strain is preserved in the China general microbiological culture Collection center (address is North Silu No. 1, court-Yang area of Beijing city, 3), the preservation date is 2023, 01 and 06 days, and the hybridoma cell strain is classified and named.

In another aspect, the invention provides a feline herpesvirus monoclonal antibody secreted by the hybridoma cell line described above.

In another aspect, the present invention provides a method for preparing the feline herpesvirus monoclonal antibody as described above, comprising the steps of:

s1, separating a feline herpesvirus BJT 01 strain to obtain the BJT 01 strain;

s2, amplifying the BJT 01 strain obtained in the step S1 through PCR, detecting the BJT 01 strain through gel electrophoresis, and taking the BJT 01 strain with positive detection result;

s3, purifying the BJT 01 strain positive in the step S2, immunizing a mouse, fusing spleen cells of the immunized mouse with myeloma cells of the mouse, detecting antibodies by an ELISA method, detecting positive cells to be the required hybridoma cells, and performing amplification culture or in-vivo ascites induction on the hybridoma cells positive in the screening result by using a homologous mouse to obtain the feline herpes virus monoclonal antibody.

On the basis of the technical scheme, the invention can be improved as follows.

Further, in step S2, in the PCR amplification, specific primers are used as follows:

FHV-F, the sequence of FHV-F is shown as SEQ ID NO:1 is shown in the specification;

FHV-R, the sequence of FHV-R is shown as SEQ ID NO:2 is shown in the figure;

FPV-F, the sequence of which is shown in SEQ ID NO:3 is shown in the figure;

FPV-R, the sequence of which is shown in SEQ ID NO:4 is shown in the figure;

FCV-F, the sequence of which is shown in SEQ ID NO:5 is shown in the figure;

FCV-R, the sequence of which is shown in SEQ ID NO: shown at 6.

Further, in step S3, the BJS01 strain positive in step S2 is purified and then immunized at least once in the course of immunization.

Further, the BJT 01 strain positive in the step S2 is purified, and the mice are immunized at least three times during the immunization.

Further, the expansion culture method comprises the following steps: culturing a large amount of hybridoma cells by adopting a bioreactor fermentation culture method, collecting the cultured hybridoma cells, centrifuging, and taking the supernatant or carrying out ultrafiltration concentration on the collected cell supernatant;

the method for inducing abdominal water in vivo by the syngeneic mice comprises the following steps: selecting more than 20g of BALB/c mice, and injecting 0.5mL of sterilized liquid paraffin into each abdominal cavity; after 14-18 daysMice were inoculated intraperitoneally with hybridoma cells (0.5-1X 10) ⁶ Individual cells/cell); starting from 7 days after cell implantation, observing the abdomen of the mouse every day, after the abdomen of the mouse is obviously enlarged, puncturing the abdominal cavity by a 9-gauge needle, collecting ascites, centrifuging the ascites at 3000r/min for 10min, removing grease and sediment, and collecting supernatant.

In another aspect, the invention also provides a pharmaceutical composition comprising a feline herpesvirus monoclonal antibody as described above and a pharmaceutically acceptable carrier and/or excipient.

In another aspect, the invention also provides the use of a feline herpesvirus monoclonal antibody as described above in the manufacture of a medicament for treating a feline rhinotracheitis infection.

The hybridoma cell strain disclosed by the invention has the beneficial effects that the monoclonal antibody of the feline herpesvirus can be stably secreted, so that the feline rhinotracheitis caused by the feline herpesvirus type 1 can be cured, and the death rate of cats can be greatly reduced. Solves the technical problem that no specific medicine aiming at the feline herpesvirus exists in the market at present. The feline herpesvirus monoclonal antibody of the invention has high purity, good specificity and good neutralization titer. The preparation method can effectively obtain hybridoma cells, so that a large number of feline herpesvirus monoclonal antibodies can be prepared, and the success rate and the yield of the feline herpesvirus monoclonal antibodies are improved.

Drawings

FIG. 1 shows the CPE produced on CRFK cells by the isolated virus of example 1 of the present invention;

FIG. 2 is a gel electrophoresis chart in example 1 of the present invention;

FIG. 3 is a SDS-PAGE electrophoresis of example 1 of the present invention;

Detailed Description

I. Correlation definition

In light of the foregoing disclosure, many other modifications, substitutions, or alterations are also possible in the form of modifications, substitutions, or alterations without departing from the spirit and scope of this disclosure.

Medicament or pharmaceutical composition

The feline herpesvirus monoclonal antibodies of the present disclosure, together with one or more excipients such as adjuvants, carriers or diluents, can be placed in the form of pharmaceutical compositions, unit dose (unit dosages) or dosage forms (dosage forms). The pharmaceutical compositions may be in solid dosage forms (e.g., powders, granules, pellets, coated or uncoated tablets or filled capsules) or liquid dosage forms (e.g., solutions, suspensions, emulsions or filled capsules) or semi-solid dosage forms (e.g., gels, creams and ointments). The dissolution and release characteristics of one or more active ingredients of a pharmaceutical dosage form may vary from seconds to months.

The "drug" or "pharmaceutical composition" is designed for use in mice and humans and can be administered via all routes of administration. Preferred routes of administration are injection, oral, pulmonary, nasal, rectal, parenteral. Such pharmaceutical compositions and unit dosage forms thereof may contain conventional or novel ingredients, with or without additional active compounds or ingredients, in conventional or particular proportions, and such unit dosage forms may contain any suitable effective amount of the active ingredient to be employed commensurate with the intended daily dosage range.

The term "carrier" as applied to the pharmaceutical compositions of the present disclosure relates to a diluent, adjuvant or excipient with which the active compound is administered.

Therapeutic methods and pharmaceutical formulations

Whether by oral, injection, rectal or parenteral (including intravenous and subcutaneous) or in some cases even by topical routes, the feline herpesvirus monoclonal antibodies of the present disclosure may be administered, or together with one or more drugs (acceptable excipients, carriers or diluents), particularly preferably in the form of their pharmaceutical compositions, to a subject in need thereof, e.g., a living mouse, human, in an effective amount for treating, reducing or ameliorating, alleviating or eliminating an indication or disorder susceptible thereto or an indication or disorder set forth elsewhere in the present disclosure.

The term "treating" as used herein means alleviating or alleviating at least one symptom of a disease in a subject, and within the meaning of the present disclosure, the term "treating" also refers to inhibiting, delaying onset (i.e., the early phase of the clinical manifestation of the disease), and/or reducing the risk of developing or worsening the disease.

The medicaments or pharmaceutical compositions of the present disclosure can be administered orally, topically, parenterally, or mucosally (e.g., buccally, by inhalation, or rectally) in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. It is generally desirable to use the oral route. The active agent may be administered orally in the form of capsules, tablets, etc. (see Remington: the Science and Practice of Pharmacy,20th Edition).

For oral administration in the form of a tablet or capsule, the active pharmaceutical ingredient may be combined with non-toxic, pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol, and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate, and the like); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate), coloring and flavoring agents, gelatin, sweetening agents, natural and synthetic gums (e.g., acacia, tragacanth or alginates), buffer salts, carboxymethylcellulose, polyethylene glycol, waxes and the like. For oral administration in liquid form, the pharmaceutical component may be combined with a non-toxic, pharmaceutically acceptable inert carrier (e.g., ethanol, glycerol, water), an anti-settling agent (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), an emulsifying agent (e.g., lecithin or acacia), a non-aqueous carrier (e.g., almond oil, oil esters, ethanol, or fractionated vegetable oil), a preservative (e.g., methyl or propyl p-hydroxybenzoate, or sorbic acid), and the like. Stabilizers such as antioxidants (BHA, BHT, propyl citrate, sodium ascorbate, citric acid) may also be added to stabilize the dosage form.

The medicaments or pharmaceutical compositions of the present disclosure may be administered parenterally, i.e. by intravenous (i.v.), intraventricular (i.c.v.), subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.), subcutaneous (s.d.), or intradermal (i.d.), by direct injection, via bolus injection or continuous infusion, for example. Formulations for injection may be presented in unit dosage form, for example, in ampules or multi-dose containers with added preservative. The compositions may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, in the form of excipients (vehicles), and may contain formulatory agents such as anti-settling agents, stabilisers and/or dispersants. Alternatively, the active ingredient may be reconstituted in powder form with a suitable carrier (e.g. sterile pyrogen-free water) prior to use.

The medicaments or pharmaceutical compositions of the present disclosure may also be formulated for rectal administration, for example, as suppositories or retention enemas (e.g., containing conventional suppository bases such as cocoa butter or other glycerides).

The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.

Example 1

A method for preparing a feline herpesvirus monoclonal antibody, comprising the steps of:

s1, separating a feline herpesvirus BJT 01 strain to obtain the BJT 01 strain;

s2, amplifying the BJT 01 strain obtained in the step S1 through PCR, detecting the BJT 01 strain through gel electrophoresis, and taking the BJT 01 strain with positive detection result;

s3, purifying the BJT 01 strain positive in the step S2, immunizing a mouse, fusing spleen cells of the immunized mouse with myeloma cells of the mouse, detecting antibodies by an ELISA method, detecting positive cells to be the required hybridoma cells, and performing amplification culture or in-vivo ascites induction on the hybridoma cells positive in the screening result by using a homologous mouse to obtain the feline herpes virus monoclonal antibody.

Specifically, hybridoma cells and feline herpesvirus monoclonal antibodies were prepared using the following steps:

1. isolation and identification of feline herpesvirus BJT 01 strain

1. Materials and methods

1.1, disease Material and cells

The disease material is derived from eye and nose swabs of a pet suspected to be infected with FHV cat in the Beijing certain pet hospital; CRFK cells were passaged and stored by beijing blende biotechnology limited responsible company.

1.2 reagents

DMEM medium, available from Gibco company; bovine serum, purchased from the lan civil engineering limited; trypsin, available from Hyclone company; viral genome DNA/RNA extraction kit, RNA reverse transcription kit, purchased from beijing full gold biotechnology limited; PCR and RT-PCR amplification kits were purchased from Takara Bio Inc.

1.3 isolation and identification of BJTS 01 Strain

Taking the eye and nose swabs which are subjected to PCR identification FHV positive, fully shaking and uniformly mixing, centrifuging for 8-10 minutes at 8000r/min, and taking the supernatant to be subjected to filtration sterilization at 0.22 mu m. Then inoculating to single layer CRFK cell, adding DMEM culture solution containing 2% bovine serum, standing at 37deg.C and containing 5% CO ₂ And (5) culturing and observing in an incubator. When Cytopathy (CPE) reaches 80%, freezing and thawing for 2 times, harvesting virus liquid, and passaging according to the method.

2. Results

2.1 cytopathy

The cells were inoculated into CRFK cells, and the results shown in FIG. 1 were observed under a microscope, wherein the cells in FIG. 1A were normal CRFK cells, and the CPE produced by BJT 01 strain in CRFK cells in FIG. 1B were observed under a microscope, and the cells in FIG. 1B were observed to be in the form of circles, abscission, etc. The BJT 01 strain is separated through preliminary judgment.

2.2 PCR identification

PCR amplification was performed using BJT 01 strain nucleic acid as template with FHV, FPV, FCV specific primers, and the primer information is shown in Table 1 below:

TABLE 1 primer information

Then, detecting products amplified by the FHV, FPV, FCV specific primers respectively through gel electrophoresis to obtain a gel electrophoresis diagram shown in figure 2, wherein in figure 2, FHV is identified (1: BJT 01 strain virus liquid; 2: negative control; 3: positive control); FPV identification (4: BJT 01 strain virus liquid; 5: negative control; 6: positive control); FCV identification (7: BJT 01 strain virus liquid; 8: negative control; 9: positive control).

The electrophoresis result shows that only FHV is positive, which indicates that one FHV strain is successfully separated, and formally named BJT 01.

2. Preparation and biological characteristic identification of feline herpesvirus monoclonal antibody

1. Material

1.1, toxic seed

The feline herpesvirus BJT 01 strain was isolated, identified and stored by Beijing blende biotechnology limited liability company; cat parvovirus HBX05 strain isolated, identified and stored by beijing bledeli biotechnology limited liability company; the feline calicivirus BJH13 strain is isolated, identified and stored by Beijing blende biotechnology limited liability company;

1.2 cells

SP2/0 myeloma cells, CRFK cells, were passaged and stored by Beijing Boledeli Biotechnology Limited.

1.3 reagents

DMEM medium, available from Gibco company; bovine serum, purchased from the lan civil engineering limited; viral genome DNA/RNA extraction kit, RNA reverse transcription kit, purchased from beijing full gold biotechnology limited; PCR and RT-PCR amplification kits, purchased from Takara Bio-engineering (Dalian) Inc.; freund's Complete Adjuvant (FCA), freund's Incomplete Adjuvant (FIA), polyethylene glycol fusion agent (PEG 2000), HAT, HT medium, mouse monoclonal antibody typing kit, purchased from Sigma company; horseradish peroxidase-labeled goat anti-mouse IgG (h+l), purchased from shanghai bi yun biotechnology limited; protein A Antibody Purification Kit from abcam corporation; BCA protein concentration assay kit, purchased from beijing solebao technologies.

1.4, laboratory animals

BALB/c mice, females, were purchased from St Bei Fu (Beijing) Biotechnology Co., ltd.

2. Method of

2.1 preparation of hybridoma cells

2.1.1 antigen preparation

2.1.1.1 preparation of liquid for treating feline herpesvirus

Inoculating the strain BJT 01 virus liquid onto CRFK cells, and standing at 37deg.C with 5% CO ₂ Culturing in incubator, harvesting when cytopathy reaches about 80%, freezing and thawing for 2 times, harvesting virus solution, and storing at-70deg.C.

2.1.1.2 viral purification

After removing cell debris by centrifuging the virus liquid at 4 ℃ for 30 minutes at 8000r/min, collecting the supernatant 40000r/min of the virus liquid, ultracentrifugating for 3 hours, collecting the precipitate, and re-suspending the precipitate with 1% of the original volume of PBS for later use.

Preparing sucrose solutions with mass concentrations of 60%, 50%, 40%, 30% and 20%, sequentially adding into 5ml centrifuge tubes from bottom to top, adding 0.8ml of sucrose with each concentration, adding 1ml of resuspended virus, ultracentrifugating at 4deg.C and 40000r/min for 3h, collecting purified virus strips as antigens, and preserving at-70deg.C for later use; TCID50 was calculated by the Reed-Muench method and protein content was measured by the BCA method.

2.1.2 immunization of mice

10 female BALB/c mice of 6-8 weeks of age were selected. Diluting the purified FHV BJT 01 strain to 1mg/ml with PBS, emulsifying with equal volume Freund's complete adjuvant, and injecting into abdominal cavity, 0.3 ml/dose; after the first, the second and third phases are carried out after the 2 nd and 4 th weeks are respectively emulsified by purified virus and equal volume Freund incomplete adjuvant; blood is collected weekly after three-phase, ELISA method is used for measuring serum titer, when ELISA titer reaches 1:8000, FHV BJT 01 strain diluted to 1mg/ml is taken for injection into mice for enhancing immunity, and 0.15 ml/mouse is used for enhancing immunity; after 3 days, the spleens of mice were taken for fusion.

2.1.3 cell fusion

2.1.3.1 preparation of splenocytes from immunized mice

Sterile taking out spleen of immunized mice on day 3 after enhancing immunity, placing into a plate containing DMEM culture medium, gently rinsing, and removing attached connective tissue and fat; shearing spleen with scissors, placing on 200 mesh copper net, grinding spleen with plunger, washing with DMEM culture medium to make spleen cells completely pass through mesh and enter into solution, transferring spleen cell solution into centrifuge tube, centrifuging at 1000r/min for 5 min, and discarding supernatant; centrifuging and washing cells once by the same method; then, the cells are resuspended in DMEM medium, and the cell suspension is taken to obtain the spleen cells of the immunized mice, and the spleen cells are counted for later use.

2.1.3.2 preparation of SP2/0 myeloma cells

Expanding and culturing SP2/0 myeloma cells at 75cm in separate bottles 36-48 h before fusion ² Inside the cell flask. On the day of fusion, SP2/0 myeloma cells with good morphology and logarithmic growth are selected, gently blown off from the bottle wall, collected in a centrifuge tube, centrifuged at 1000r/min for 5 minutes, and resuspended in a proper amount of DMEM medium to obtain SP2/0 myeloma cells for cell fusion.

2.1.3.3 preparation of feeder cells

On the same day of cell fusion, taking blood from the eyeballs of the immunized BALB/c mice (8-12 weeks old), killing cervical dislocation, soaking in 75% alcohol for 5 minutes, placing in a wax disc of an ultra-clean workbench, and fixing the abdomen upwards; lifting the skin of the BALB/c mouse by using sterile forceps, carefully cutting off the skin of the abdomen, separating the skin from the peritoneum, and fully exposing the peritoneum; sucking a proper amount of DMEM culture medium by using a sterile injector, injecting the DMEM culture medium into the abdominal cavity, massaging the abdominal cavity, fully mixing, extracting liquid in the abdominal cavity, adding the liquid into a sterile centrifuge tube, and repeatedly flushing for 2-3 times; the resulting liquid was centrifuged at 1000r/min for 5 minutes, and the supernatant was discarded. Adding a proper amount of DMEM culture medium into the sediment, re-suspending cells, counting the cell suspension, suspending the cells by using a culture solution containing HAT, and adjusting the cell concentration to be 1-2 multiplied by 10 ⁵ Adding the mixture into 96-well cell culture plates (100 mu l/well), placing the plates at 37 ℃ and 5% CO ₂ Culturing in incubator。

2.1.3.4 cell fusion

Mixing spleen cells of immunized mice and SP2/0 cells in a logarithmic phase in a centrifuge tube according to the proportion of 5-10:1, centrifuging for 8 minutes at 1000r/min, discarding the supernatant, lightly flicking the bottom of the tube, and loosening and uniformly forming precipitated cells into paste; 1ml of 50% PEG2000 (pH 8.0) solution preheated to 37 ℃ is sucked and slowly dripped into the mixture for fusion; adding 25ml of DMEM culture medium into the centrifuge tube to terminate the fusion; centrifuging the fused cell sap at 1000r/min for 5 minutes, discarding the supernatant, and re-suspending the cell sap with HAT-containing culture solution preheated in a water bath at 37 ℃;100 μl/well of 96-well culture plate with feeder cells added, and placing at 37deg.C and 5% CO ₂ Is cultured in an incubator of (a). The growth of cells was recorded daily, and on day 4 after fusion, half-volume medium was changed with 1% HAT and continued culture with half-volume medium change with 1% HT for about 7-10 days.

2.1.4 cloning of Positive hybridoma cells

After half liquid exchange is adopted, the growth condition of cells is observed every day, so that the growth of cloned cells is seen, when a cell colony grows to 1/3 of the bottom of a hole, cell supernatant is taken, antibody detection is carried out by an ELISA method, hybridoma cells with positive detection results are cloned in time, otherwise, positive hybridoma cells are competitively inhibited, cells which do not secrete antibodies inhibit the growth of positive hybridoma cells, so that the hybridoma cells which secrete specific antibodies are lost, and cloning is an important step for obtaining a single cell population and pure and uniform monoclonal antibodies. Thus, cloning can be used to ensure long-term stable secretion of monoclonal antibodies by hybridoma cells. In this example, the cells were cloned by limiting dilution as follows:

(1) Feeder cells obtained according to the above procedure were prepared 1 day in advance.

(2) The cells were selected for cloning in a single cell colony with good cell status, and were aspirated into a new 24-well cell culture plate using a pipette and washed as clean as possible.

(3) The cells were counted at 3 cells per well and incubated in 96-well cell culture plates.

(4) Cells were observed, when 1/3 of the cell was growing to the bottom of the well, ELISA was performed on the cell supernatant, the presence or absence of specific antibodies in the supernatant was detected, and the corresponding cell wells were labeled to detect the positive rate.

(5) The cell state is also selected to be better, and single cell colony is cloned next time, so that the cloned cells should be frozen in time to avoid pollution of the cells.

(6) After 3 clones, if the positive rate of the cell supernatant reaches 100%, the positive hybridoma cell strain can be determined to stably secrete the monoclonal antibody. If the positive rate is less than 100%, cloning is continued until the positive rate reaches 100%, and positive holes are subjected to expansion culture.

2.2 Mass preparation and purification of Mao herpesvirus monoclonal antibodies

And (3) preparing the cat herpesvirus monoclonal antibody by adopting an amplification culture method or a syngeneic mouse in-vivo ascites induction method on the screened positive hybridoma cell strain.

Cell expansion culture method for preparing feline herpesvirus monoclonal antibody: culturing hybridoma cells in large quantity by adopting a bioreactor fermentation culture method, collecting culture hybridoma cell supernatant, centrifuging for 10min at 3000r/min, and collecting the supernatant or performing ultrafiltration concentration on the collected cell supernatant.

Preparation of ascites with syngeneic mouse monoclonal antibody: selecting more than 20g (8-10 weeks old) BALB/c mice, and injecting 0.5mL of sterilized liquid paraffin into each abdominal cavity; after 14-18 d, the mice were inoculated with hybridoma cells (0.5-1X 10) ⁶ Individual cells/cell); after the implantation of cells, the abdomen of the mouse is observed every day, after the abdomen of the mouse is obviously enlarged, the abdominal cavity is punctured by a 9-gauge needle head, ascites is collected, generally, the ascites can be continuously collected for 3 to 5 times, the ascites is centrifuged for 10 minutes at 3000r/min, grease and sediment are removed, supernatant fluid is collected, and the antibody titer is measured.

Purifying: purifying according to Protein A Antibody Purification Kit, dialyzing in dialysis bag with molecular weight cut-off of 30kDa, and dialyzing the purified antibody into PBS.

2.3 determination of secretion stability of Mao herpesvirus monoclonal antibody

Continuously transferring the hybridoma cells to P20 generation, freezing in liquid nitrogen for 3, 6, 9 and 12 months, recovering, centrifuging the culture solution when the cells grow fully, and taking the supernatant, and measuring according to the 'neutralization test method' of the appendix of the current 'Chinese animal pharmacopoeia'.

2.4 identification of biological Properties of Mao herpesvirus monoclonal antibodies

2.4.1 subtype identification of feline herpesvirus monoclonal antibodies

The identification is carried out according to the specification of a mouse monoclonal antibody typing kit.

2.4.2 specific identification of feline herpesvirus monoclonal antibodies

Respectively diluting feline herpesvirus (BJT 01 strain), feline parvovirus (HBX 05 strain) and feline calicivirus (BJT 13 strain) with DMEM cell culture solution containing 4% bovine serum to 200 TCID ₅₀ 0.1ml, mixing with hybridoma cell (2H 12) secreted cat herpesvirus monoclonal antibody in equal volume, neutralizing at 37deg.C for 1 hr, inoculating into 96-well cell culture plate (sample neutralized with FHV: onto CRFK cells grown into monolayer; sample neutralized with FPV: 100 μl of F81 cell suspension containing 2% bovine serum per well; 3 sample neutralized with FCV: onto F81 cells grown into monolayer; 4 wells, 100 μl per well); a normal cell control and a virus control (virus (200 TCID) ₅₀ 0.1 ml) was mixed with an equal volume of DMEM medium, neutralized at 37℃for 1 hour) 4 wells each; placing at 37deg.C, containing 5% CO ₂ Culturing and observing for 4-7 days in an incubator.

2.4.3 chromosome number analysis of hybridoma cells

Taking hybridoma cells in logarithmic growth phase, adding colchicine into culture solution according to 0.1 mug/ml, placing at 37 ℃ and 5% CO ₂ Culturing in a cell incubator for 6-10 hours; after the cells are blown off by a DMEM culture medium, centrifuging at 1000r/min for 10 minutes, discarding the supernatant, adding 5ml of a preheated 0.075mol/L KCl solution at 37 ℃, blowing and mixing uniformly, and processing at 37 ℃ for 30 minutes; adding fresh acetic acid/methanol (1:3) fixing solution 1ml into cell suspension, and stirring to obtain fine powderThe cell surface is slightly fixed to prevent cell adhesion and agglomeration; 1000 Centrifuging at r/min for 10min, discarding supernatant, adding 5-10 ml of fixing solution, gently blowing uniformly, standing at room temperature for 30 min, repeatedly fixing for 3 times, adding 5ml of fixing solution, mixing uniformly, and standing at 4 ℃ overnight; the next day, part of supernatant is discarded, 0.5-1 ml of fixing solution is blown to resuspend cells, 1-2 drops of cell suspension are dripped on a precooled glass slide, and the glass slide is dried at room temperature; after staining with the giemsa staining solution, cells with well-dispersed chromosomes, no overlap and no loss were selected under a microscope for observation and analysis, and the chromosome number was recorded.

2.4.4 identification of the purity of Mao herpesvirus monoclonal antibodies

Purified feline herpesmab was identified for purity by SDS-PAGE.

3. Results

3.1 determination of the Virus content and the protein content after purification

The BJT 01 strain virus liquid is purified, and the virus content is 107.6TCID ₅₀ /ml. Protein content was determined by BCA method, and the content of the purified viral protein was 2.8mg/ml.

3.2 screening results of Positive hybridoma cell lines

After spleen cells and hybridoma cells of the immunized mice are fused, 1 monoclonal antibody hybridoma cell strain which stably secretes anti-FHV is obtained through HAT/HT selective culture, screening identification and subcloning, and is named as 2H12. And subjecting it to strain preservation.

3.3 results of measurement of secretion stability of Mao herpesvirus monoclonal antibody

And respectively taking the cell supernatants of the P5, P10, P15 and P20 generation hybridomas, freezing and storing the cell supernatants in liquid nitrogen for 3, 6, 9 and 12 months, and then carrying out neutralization titer determination on the resuscitated cell culture supernatants of the hybridomas. The results show that the hybridoma cells continuously transmit to the generation P20, the neutralization titer is 1:126-159, the hybridoma cells are preserved in liquid nitrogen for 12 months, the antibody titer is 1:126-159, the hybridoma cells are stable in antibody secretion, and the results are shown in Table 2 and Table 3.

TABLE 2 results of hybridoma cell supernatant neutralization titers at different generations

TABLE 3 determination of the neutralization titers of hybridoma cell supernatants at different storage times in liquid nitrogen for the generation 2H12P20

3.4, results of identification of feline herpesvirus monoclonal antibodies and subclasses

The subtype of the feline herpesvirus monoclonal antibody identified by the mouse monoclonal antibody typing kit is IgG2a, and the results are shown in Table 4.

TABLE 4 monoclonal antibody subclass identification results

3.5 results of specific identification of feline herpesvirus monoclonal antibodies

The feline herpesvirus monoclonal antibody secreted by the 2H12 cells does not appear CPE with FHV neutralization wells, CPE with FPV neutralization wells, CPE with FCV neutralization wells; normal cell control wells have no CPE present and virus control wells have CPE present; the results show that the feline herpesvirus monoclonal antibody is a specific antibody against FHV.

3.6, chromosome number analysis of hybridoma cells

The number of spleen cell chromosomes of a normal BALB/c mouse is 40, and the number of myeloma cell chromosomes is 60-70, so that the number of cell chromosomes after fusion is between 100 and 110. The test results showed that the chromosome number of the 2H12 hybridoma cells varied within this range, and an average of 103 was consistent with the expected number.

3.7, results of determination of ascites neutralization titers in mice

The neutralization test method is used for measuring, and the specific experimental operation process is as follows: firstly, local skin of the lower abdomen of a BALB/c mouse is dehaired, 75% alcohol cotton ball is disinfected and anesthetized, then the skin is carefully lifted by using a sterile hemostatic forceps, and a right hand small needle or a puncture trocar is gently and vertically penetrated along the middle line of the lower abdomen close to the abdominal wall and cannot be penetrated too deeply, so that viscera injury is avoided. Then the needle head has a falling feel, the puncture needle is proved to enter the abdominal cavity, the ascites can be naturally dropped out when the ascites is more, the needle head can be slightly rotated to suck back when the ascites is less, and if the ascites flows out, the needle head and the position of the injector are immediately fixed for continuous suction. Finally, after the collection is completed, the puncture part is pressed by the dry cotton ball, and the needle head is pulled out. Through detection, the neutralization titer of the ascites FHV is about 1:17800, which meets the requirements.

3.8 identification of the purity of the Mao herpesvirus monoclonal antibody

After purification by protein A affinity chromatography and anion-cation exchange, SDS-PAGE was performed, with a heavy chain molecular weight of about 52kD and a light chain molecular weight of about 26kD, see FIG. 3.

Example 2

The feline herpesvirus monoclonal antibody obtained in example 1 was taken and subjected to the following efficacy study:

3. research on curative effect of feline herpesvirus monoclonal antibody on feline herpesvirus virulent artificial infected cats

1. Material

1.1, toxic seed

The feline herpesvirus BJT 01 strain was isolated, identified and stored in the laboratory.

1.2, test agent

The 2H12 feline herpesvirus monoclonal antibody injection (neutralizing antibody titer is 1:1024, and the characters, pH value, sterility, mycoplasma and exogenous virus are all qualified) is prepared by the laboratory.

1.3, laboratory animals

Cats of 2-3 months of age (FHV, FPV, FCV antigen negative, FHV neutralizing antibody titer no higher than 1:2) were purchased from a solid security pet market.

2. Method of

2.1 attack of toxin

FHV BJT 01 strain (virus content is 104.7TCID50/ml) is adopted to attack 12 cats by dripping 1ml of nose, and clinical symptoms such as mental state, appetite, sneeze or respiratory masa, eye and nose secretion and the like are recorded every day.

2.2 grouping

According to the morbidity criteria, 10 cats were selected and randomly grouped into 2 groups of 5 cats/group. The first group is a mab-treated group; the second group is an adjuvant therapy group.

The disease criteria are:

any 1 item (3) and (1) or (2) appear simultaneously, namely the pathogenesis is judged.

(1) Depression and anorexia;

(2) sneeze;

(3) serous secretions (clear transparent, watery) or purulent secretions (white or milky yellow, opalescent) appear in the eyes, nose.

2.3 treatment of

Mab treatment group: the injection of the feline herpesvirus monoclonal antibody is injected intramuscularly at a dose of 1mL/kg, simultaneously with the injection of ceftiofur sodium at a dose of 5mg/kg, kanamycin is injected subcutaneously at a dose of 0.2mL/kg, 1 time a day for 5 days.

Adjuvant treatment group: ceftiofur sodium is subcutaneously injected at a dose of 5mg/kg, kanamycin is subcutaneously injected at 0.2ml/kg 1 time a day for 5 days.

3. Results

On day 14 after the challenge, the cure rate of the mab treatment group was 60% (3/5), and the cure rate of the adjuvant treatment group tested cats was 20% (1/5).

The cure standard is: (1) normal spirit and appetite, and no depression or anorexia; (2) does not sneeze; (3) no new ocular and nasal secretions appear. Meets the clinical manifestations, namely, is judged to be cure.

TABLE 5 statistical forms of cat morbidity and treatment status for each group

Note that: "P" represents Post Challenge (Post Challenge) and the number is the number of days Post Challenge. "/" indicates no such item.

The test result shows that the cure rate of the monoclonal antibody treatment group is 40% higher than that of the auxiliary treatment group, and the feline herpesvirus monoclonal antibody injection has better treatment effect on test cats infected with FHV.

In the description of the present specification, the terms "one embodiment," "some embodiments," "particular embodiments," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (3)

1. A hybridoma cell strain capable of secreting a feline herpesvirus monoclonal antibody is characterized in that the preservation number of the hybridoma cell strain is CGMCC No.45346, and the preservation address is China general microbiological culture Collection center.

2. A feline herpesvirus monoclonal antibody secreted by the hybridoma cell line of claim 1.

3. Use of a feline herpesvirus monoclonal antibody according to claim 2 in the manufacture of a medicament for treating a feline rhinotracheitis infection.