Preparation method and application of feline herpesvirus strain, plaque purification and monoclonal antibody

Abstract

The invention discloses a preparation method and application of a feline herpesvirus strain and plaque purification and monoclonal antibody. The preparation method comprises the following steps: collecting a sample collection solution of a mouth-nose swab of a cat clinically suffering from feline infectious rhinotracheitis; after separating and identifying the sample collection liquid, filtering and inoculating cat kidney cells (CRFK), and performing plaque purification to obtain a high-purity virus strain; the monoclonal antibody aiming at the virus particles is prepared by precipitating the virus particles through sucrose and inactivating the virus particles, so that the monoclonal antibody with good specificity is obtained. The monoclonal antibody aiming at the feline herpes virus is prepared and obtained by adopting a hybridoma method, and the antibody has relatively good specificity, can be used for detecting clinical pathogens and has relatively good application value.

Description

Technical Field

[0001] The invention belongs to the field of preparation of feline herpes virus cloned antibodies, and specifically relates to a feline herpes virus strain and plaque purification and a preparation method and application of a monoclonal antibody. Background Art

[0002] With the improvement of people's living standards and the accelerated aging of the population, the number of companion animals raised has soared in recent years. However, viral infectious diseases pose a serious threat to the health of cats.

[0003] Healthy cats can be infected by contact with sick cats infected with FHV-1, asymptomatic infected cats, or items used by sick cats; or they can be easily infected with FHV-1 due to sudden changes in living environment, such as foster care, change of residence, transportation, entering a multi-cat environment, or going to the hospital. In shelters or group-raised cats, unvaccinated cats are particularly prone to respiratory disease outbreaks. Acutely infected cats often have symptoms such as fever, loss of appetite, lethargy, and sneezing. If the condition worsens, conjunctivitis, large amounts of nasal and eye secretions, oral ulcers, pneumonia, and other symptoms may occur. FHV-1 is prone to latent infection in the trigeminal ganglion, which greatly increases the difficulty of diagnosis. Feline viral rhinotracheitis has a high prevalence and is highly contagious, and there is currently no effective treatment. Therefore, for this type of infectious disease, it is necessary to make a correct diagnosis as soon as possible and isolate it in time to prevent the spread of the disease.

[0004] Vaccination is considered the most effective measure to prevent FHV-1 infection in cats. The currently used FHV-1 vaccine cannot completely prevent infection with virulent FHV-1. In order to better protect the health of pets, it is urgent to isolate more prevalent new strains for screening vaccine strains. Therefore, a feline herpes virus strain and a method for preparing plaque purification and monoclonal antibodies and their application are needed. Summary of the invention

[0005] The purpose of the present invention is to provide a feline herpes virus strain and a plaque purification and monoclonal antibody preparation method and application.

[0006] The present invention is achieved through the following technical solutions:

[0007] In one aspect, a feline herpes virus strain, the strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on August 10, 2021, with the deposit number: CGMCC No.21727

[0008] Furthermore, the nucleotide sequence of the strain is shown in SEQ ID No.1.

[0009] In one aspect, the present invention comprises the steps of:

[0010] Sample collection fluid was collected from oral and nasal swabs of cats with clinical feline infectious rhinotracheitis;

[0011] After the sample collection fluid is separated and identified, it is filtered and inoculated into cat kidney cells to obtain a high-purity virus strain through plaque purification;

[0012] Virus particles were inactivated by precipitating them with sucrose, and monoclonal antibodies against virus particles were prepared. Monoclonal antibodies against feline herpes virus were prepared and obtained by hybridoma method.

[0013] Furthermore, the separation and identification method includes extracting nucleic acid from the sample treatment liquid, performing PCR detection to determine that the target band is consistent with the expected size, filtering the sample treatment liquid through a 0.2μM filter, adding the filtrate to the monolayer cultured CRFK cells, supplementing DMEM culture medium to the T25 cell bottle until the liquid completely covers the cell layer, and then placing the cell bottle in a 37°C, 5% CO2 incubator. After adsorption for 90 minutes, the culture medium is discarded, and DMEM containing 2% FBS is added. The cells are cultured in a 37°C, 5% CO2 incubator for 1 to 3 days, and the cells are observed every 12 hours for cytopathic effects. After 70% to 80% obvious cytopathic effects appear, the cell culture is collected, and after repeated freezing and thawing for 3 times, it is stored at -80°C for use.

[0014] Furthermore, the virus was purified by double-layer agar method, and CRFK was cultured at 7×10 5 / mL inoculated culture cells grow to a monolayer; discard the growth medium, wash twice with PBS, add 10-fold gradient diluted virus liquid, adsorb for 90 minutes in a 37°C, 5% CO2 incubator, and shake once every 30 minutes; after the virus adsorbs for 90 minutes, discard the virus liquid, wash twice with PBS, add 2mL of the first layer of covering liquid, wait for it to solidify, and culture it upside down in a 37°C, 5% CO2 incubator, and observe the cell pathological changes every 12 hours;

[0015] After the first layer of agar covering solution is spread on the plate, when the cells show obvious lesions, 2 mL of the second layer of agar covering solution is added, and the plate is placed in an incubator at 37°C and 5% CO2 for upright cultivation. The plate is observed every 3 hours, and the plaques are picked out after they are formed.

[0016] Further, the first layer of agar covering solution includes preparing a 4% low melting point agarose solution with PBS, sterilizing it with high pressure, and placing it in a 40-50°C water bath for use, preparing 2×DMEM containing 5% FBS, mixing the two in a ratio of 1:1, and placing it in a 40-50°C water bath for use, and the preparation of the second layer of agar covering solution includes preparing a 4% low melting point agarose solution with PBS, sterilizing it with high pressure, and placing it in a 40-50°C water bath for use, preparing 2×DMEM containing 5% FBS, mixing the two in a ratio of 1:1, adding 0.01% neutral red staining solution, and placing it in a 40-50°C water bath for use;

[0017] Furthermore, the purified virus particles were inactivated by formaldehyde, and the immune titer was evaluated by ELISA experiment. The mice with the highest titer were selected, and 50 μg of inactivated pathogens were injected intraperitoneally three days before the mice were subjected to the cell fusion experiment for final booster immunization.

[0018] Furthermore, mouse spleen cells and SP20 cells were taken and cell fusion was carried out by PEG method. After the fusion, the cells were screened and cultured with semi-solid culture medium, and 200 μL / well 20% newborn calf IMDM culture medium was added for one screening. The plates were coated with inactivated feline herpes virus, and the selected monoclonal clones were screened for a second time by ELISA method to obtain 13 positive hybridoma cells. The positive cell lines were respectively coated with inactivated feline herpes virus, and screened three times by ELISA method to obtain positive hybridoma cell lines.

[0019] Furthermore, the purified virus was verified by animal regression experiments.

[0020] On the other hand, the method for purifying feline herpesvirus plaques and preparing monoclonal antibodies is used in the detection field to obtain monoclonal antibodies against viral rhinotracheitis pathogens.

[0021] Compared with the prior art, the present invention adopts the above technical solution, and the biggest feature is:

[0022] Compared with the prior art, the present invention has the following effects:

[0023] The invention adopts a hybridoma method to prepare and obtain a monoclonal antibody against feline herpes virus. The antibody has good specificity, can be used for the detection of clinical pathogens, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the formation of plaques in CRFK cells infected by FHV of the present invention;

[0025] Figure 2 This is a schematic diagram of observing FHV virus particles using a transmission electron microscope according to the present invention;

[0026] Figure 3 This is a schematic diagram of clinical symptoms of the virus regression experiment of the experimental animal cat of the present invention;

[0027] A: Clinical symptoms of experimental cats 2 days after infection with the virus; B: Clinical symptoms of experimental cats 3 days after infection with the virus;

[0028] Figure 4 This is a schematic diagram of the fluorescent immunoassay for detecting monoclonal antibodies of the present invention;

[0029] Figure 5This is a schematic diagram of the monoclonal antibody for protein imprint detection of the present invention;

[0030] in Figure 5 A represents monoclonal antibody No. 9, and B represents monoclonal antibody No. 10; DETAILED DESCRIPTION

[0031] The technical scheme of the present invention is further described below in conjunction with embodiments and comparative examples, but they should not be construed as limiting the present invention:

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] A feline herpesvirus strain, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 10, 2021, with the deposit number: CGMCC No.21727, the nucleotide sequence of the strain is as shown in SEQ ID No.1, the biological material (strain) referred to is: FHV-BJ813, and the classification name is: feline herpesvirus type 1.

[0034] Example 1 Isolation and Identification of Feline Herpes Virus

[0035] Take oral and nasal swabs from cats with upper respiratory diseases, soak the swabs in 300μl PBS, take 150μl sample treatment solution to extract nucleic acids, perform PCR detection, and the target band is consistent with the expected size. The remaining 150μl sample treatment solution is filtered through a 0.2μM filter, and the filtrate is added to the monolayer cultured CRFK cells. DMEM culture medium is supplemented in the T25 cell bottle until the liquid completely covers the cell layer, and then the cell bottle is placed in a 37°C, 5% CO2 incubator. After adsorption for 90 minutes, the culture medium is discarded, and DMEM containing 2% FBS is added. The cells are cultured in a 37°C, 5% CO2 incubator for 1 to 3 days, and the cells are observed every 12 hours for cell lesions such as drawing, rounding, and shedding. After 70% to 80% obvious cell lesions appear, the cell culture is collected, and after repeated freezing and thawing 3 times, it is stored at -80°C for use.

[0036] Example 2 Plaque Purification of Feline Herpes Virus and Observation of Virus Particles by Transmission Electron Microscopy

[0037] (1) The virus was purified by double-layer agar method. CRFK was plated at 7×10 5 / mL was inoculated into a 6-well plate and the cells were cultured to grow into a monolayer; the growth medium was discarded, the cells were washed twice with PBS, 200μL of 10-fold gradient diluted virus solution was added, and adsorbed in a 37℃, 5% CO2 incubator for 90min, and shaken every 30min; the first layer of agar covering solution was prepared: 4% low melting point agarose solution was prepared with PBS, after high pressure sterilization, it was placed in a 40-50℃ water bath for use, 2×DMEM containing 5% FBS was prepared, the two were mixed in a 1:1 ratio, and then placed in a 40-50℃ water bath for use. After 90 minutes of virus adsorption, discard the virus solution, wash twice with PBS, add 2mL of the first layer of covering solution to each well, and incubate it upside down at 37℃, 5% CO2 incubator after solidification, and observe the cell pathological changes every 12 hours; prepare the second layer of agar covering solution: prepare 4% low melting point agarose solution with PBS, sterilize it by high pressure, and place it in a 40-50℃ water bath for use, prepare 2×DMEM containing 5% FBS, mix the two in a 1:1 ratio, add 0.01% neutral red staining solution, and place it in a 40-50℃ water bath for use; after the first layer of agar covering solution is spread on the plate, when the cells show obvious pathological changes, add 2mL of the second layer of agar covering solution to each well, place the 6-well plate in a 37℃, 5% CO2 incubator for upright culture, observe it every 3 hours, and pick the plaques after the formation of plaques ( Figure 1 ). Repeat this operation 3 times.

[0038] (2) The purified virus was cultured to the sixth generation, and after expansion culture, it was repeatedly frozen and thawed three times. 200 mL of virus solution was collected and centrifuged at 4°C, 5,000 g for 45 min to remove cell debris; the SW28 ultracentrifuge tube was cleaned with 75% ethanol in advance, air-dried in a biosafety cabinet and ultraviolet disinfected for 30 min, the supernatant was collected and divided into centrifuge tubes, centrifuged at 4°C, 80,000 g for 1 h, the supernatant was discarded, and the droplets on the tube wall were absorbed with filter paper; 500 μL of PBS, use a yellow gun tip with a sharpened tip to gently blow and suspend the virus; use PBS to prepare a 20% to 50% sucrose solution; use a long syringe to add 20% to 50% continuous gradient sucrose to the SW41 centrifuge tube (first add the lightest, then the densest, 5 ml of each concentration); add the virus solution to the top of the sucrose, centrifuge at 10,000g for 1 hour, use a spotlight to illuminate vertically from the top or bottom of the centrifuge tube in a dark room, mark the position of the virus band, gently aspirate the virus solution, drop 5μL of the sample on the copper mesh, let it stand for 1 minute, blot it with filter paper, drop 5μL of PBS, blot it with filter paper, repeat once, add phosphotungstic acid to stain for 1 minute, and blot it with filter paper. Virus particles of 120 to 180nm were observed by electron microscopy ( Figure 2 ).

[0039] Example 3 Animal regression experiment

[0040] The purified virus was used for animal regression experiments. Six healthy cats were randomly divided into two groups. The experimental group was inoculated with 500 μL of cell virus liquid by eye and nose drops, and the control group was inoculated with 500 μL of DMEM cell culture medium. The control group and the infected group were cultured in isolation, and the clinical symptoms and death of the experimental cats were observed. The results showed that the experimental cats were depressed on the second day, with a small amount of eye secretions. On the third day, a large amount of nasal and eye secretions began to appear in the eyes ( Figure 3 ).

[0041] Example 4 Monoclonal Antibody Preparation

[0042] (I) Mouse immunization: The purified virus particles were inactivated by formaldehyde and used to immunize three SPF-grade Balb / c female mice at 60 μg / mouse; mice were boosted 2, 4, and 6 weeks after the first immunization at 30 μg / mouse. Blood was collected from the eye sockets 1 week after the last booster immunization, and the immune titer was evaluated by ELISA. The mice with the highest titer were selected, and 50 μg of inactivated pathogens were injected intraperitoneally for the final booster immunization three days before the cell fusion experiment.

[0043] (II) Cell fusion, monoclonal selection, and three-time screening of monoclonal cells: Mouse spleen cells and SP20 cells were taken and cell fusion was performed using the PEG method. After fusion, the cells were screened and cultured using a semi-solid medium (containing HAT). Monoclonal cells were selected and cultured in a 96-well cell culture plate (previously plated with thymocytes,

[0044] 100 μL / well). Discard all the supernatant of monoclonal cells in the 96-well culture plate and add 200 μL / well

[0045] The first screening was performed by adding 20% ​​newborn calf IMDM medium (containing HT) to the wells. The plates were coated with inactivated feline herpes virus, and the selected monoclones were screened by ELISA method for the second time, and 13 positive hybridoma cells were obtained. The 13 positive cell lines were coated with inactivated feline herpes virus, and the ELISA method was used for the third screening, and 9 positive hybridoma cell lines were obtained.

[0046] (III) Monoclonal subtype identification: The 9 positive cell lines screened out were subjected to subtype identification, and finally 4 IgG positive hybridoma cell lines were obtained.

[0047]

[0048] Example 5 Monoclonal Antibody Specificity Detection (I) Indirect Immunofluorescence (IFA) detection of the reactivity of monoclonal antibodies with FHV-1.

[0049] The several monoclonal cell lines screened were expanded and cultured, and the cell supernatant was collected. The whole virus protein of FHV-1 was used as the antigen, and the collected monoclonal antibodies were diluted 1:100 times as the primary antibody. Alexa Fluor TM 488 goat anti-mouse fluorescent secondary antibody was used as the secondary antibody, and a negative control was set up. The CRFK cell samples infected with FHV-1 were subjected to IFA identification according to conventional methods.

[0050] (II) Western Blot to detect the reactivity of monoclonal antibodies with FHV-1. Several monoclonal cell lines obtained by screening were expanded and cultured, and the cell supernatants were collected. The whole virus protein of FHV-1 was used as the antigen, and the collected monoclonal antibodies were diluted 1:2000 as the primary antibody. HRP-labeled goat anti-mouse IgG was used as the secondary antibody. A negative control was set up, and the CRFK cell samples infected with FHV-1 were identified by WB according to the conventional method.

[0051] IFA results showed that two of the hybridoma cell lines screened (strains 7 and 9) were able to react with CRFK cells infected with FHV-1 and produce specific green fluorescence ( Figure 4 ). WB results showed that both hybridoma cells (strains 11 and 13) could recognize the FHV-1 virus strain ( Figure 5 ).

[0052] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A feline herpes virus strain, characterized in that The strain was deposited in the General Microbiology Center of China Culture Collection Administration on August 10, 2021, with the deposit number: CGMCC No.21727.

2. A feline herpes virus strain according to claim 1, characterized in that The nucleotide sequence of the recombinant attenuated strain is shown in SEQ ID No.

1.

3. A method for purifying feline herpesvirus plaques and preparing monoclonal antibodies, for collecting the feline herpesvirus strain according to claim 1 or 2, characterized in that: Sample collection fluid was collected from oral and nasal swabs of cats with clinical feline infectious rhinotracheitis; After the sample collection fluid is separated and identified, it is filtered and inoculated into cat kidney cells to obtain a high-purity virus strain through plaque purification; Virus particles were inactivated by precipitating them with sucrose, and monoclonal antibodies against virus particles were prepared. Monoclonal antibodies against feline herpes virus were prepared and obtained by hybridoma method.

4. A method for purifying feline herpesvirus plaques and preparing monoclonal antibodies according to claim 3, characterized in that: The separation and identification method comprises the following steps: extracting nucleic acid from a sample treatment solution, performing PCR detection to determine whether the target band is consistent with the expected size, filtering the sample treatment solution through a 0.2 μM filter, adding the filtrate to a monolayer cultured CRFK cell, supplementing a DMEM culture medium to a T25 cell bottle until the liquid completely covers the cell layer, and then placing the cell bottle in a 37°C, 5% CO2 incubator, adsorbing for 90 minutes, discarding the culture solution, adding DMEM containing 2% FBS, and culturing in a 37°C, 5% CO2 incubator for 1 to 3 days, observing the cells every 12 hours to see if cytopathic effects occur, collecting the cell culture after 70% to 80% obvious cytopathic effects occur, freezing and thawing repeatedly for 3 times, and storing at -80°C for later use.

5. A method for purifying feline herpesvirus plaques and preparing monoclonal antibodies according to claim 3, characterized in that: The virus was purified by double-layer agar method. CRFK was plated at 7×10 5 / mL inoculated culture cells grow to a monolayer; discard the growth medium, wash twice with PBS, add 10-fold gradient diluted virus liquid, adsorb for 90 minutes in a 37°C, 5% CO2 incubator, and shake once every 30 minutes; after the virus adsorbs for 90 minutes, discard the virus liquid, wash twice with PBS, add 2mL of the first layer of covering liquid, wait for it to solidify, and culture it upside down in a 37°C, 5% CO2 incubator, and observe the cell pathological changes every 12 hours; After the first layer of agar covering solution is spread on the plate, when the cells show obvious lesions, add 2 mL of the second layer of agar covering solution, place the plate in an incubator at 37°C and 5% CO2 and culture it upright. Observe it every 3 hours and pick out the plaques after they are formed.

6. A method for purifying feline herpesvirus plaques and preparing monoclonal antibodies according to claim 5, characterized in that: The first layer of agar covering solution includes preparing a 4% low melting point agarose solution with PBS, sterilizing it with high pressure, and placing it in a water bath at 40-50°C for use, preparing 2×DMEM containing 5% FBS, mixing the two in a ratio of 1:1, and placing it in a water bath at 40-50°C for use. The preparation of the second layer of agar covering solution includes preparing a 4% low melting point agarose solution with PBS, sterilizing it with high pressure, and placing it in a water bath at 40-50°C for use, preparing 2×DMEM containing 5% FBS, mixing the two in a ratio of 1:1, adding 0.01% neutral red staining solution, and placing it in a water bath at 40-50°C for use.

7. A method for purifying feline herpesvirus plaques and preparing monoclonal antibodies according to claim 3, characterized in that: The purified virus particles were inactivated by formaldehyde and the immune titer was evaluated by ELISA experiment. The mice with the highest titer were selected. Three days before the mouse cell fusion experiment, 50 μg of inactivated pathogens were injected intraperitoneally for the final booster immunization.

8. A method for purifying feline herpesvirus plaques and preparing monoclonal antibodies according to claim 3, characterized in that: Mouse spleen cells and SP20 cells were taken, and cell fusion was performed by PEG method. After the fusion, the cells were screened and cultured with semi-solid medium, and 200 μL / well of 20% newborn calf IMDM culture medium was added for one screening. Inactivated feline herpes virus was used to coat the plate, and the selected monoclonal clones were screened for a second time by ELISA method to obtain 13 positive hybridoma cell lines. The positive cell lines were respectively coated with inactivated feline herpes virus, and the ELISA method was used for three screenings to obtain positive hybridoma cell lines.

9. A method for purifying feline herpesvirus plaques and preparing monoclonal antibodies according to claim 3, characterized in that: The purified virus was verified by animal regression experiments.

10. A method for purifying feline herpesvirus plaques and preparing monoclonal antibodies as described in any one of claims 3 to 9, wherein the monoclonal antibodies against viral rhinotracheitis pathogens are obtained and used in the detection field.

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