Monoclonal antibody against gD protein of infectious laryngotracheitis virus, hybridoma cell line thereof and application thereof

Abstract

The present invention discloses a monoclonal antibody that specifically recognizes the gD protein of infectious laryngotracheitis virus (ILTV). The present invention also discloses a kit for detecting ILTV antibodies and the use of the monoclonal antibody in the preparation of a medicament for preventing and / or treating ILTV. The anti-ILTV monoclonal antibody of the present invention not only provides a powerful tool for studying the interaction between ILTV and host cells, but can also be used to detect ILTV, showing promising application prospects in the clinical diagnosis of ILTV and the development of vaccines.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, in particular to an anti-infectious laryngotracheitis virus monoclonal antibody, a hybridoma cell line thereof, and an infectious laryngotracheitis virus antibody blocking ELISA detection kit containing the monoclonal antibody. Background Art

[0002] Infectious laryngotracheitis (ILT) is an acute respiratory infection of chickens caused by the Avian Infectious Laryngotracheitis virus (ILTV). It is a major infectious disease threatening my country's poultry industry. It can infect chickens of all ages, but is more common in adult chickens, where symptoms are most typical. Symptoms include difficulty breathing, coughing up bloody exudates, sometimes forming pseudomembranes that block the larynx, and swelling, bleeding, and erosions of the laryngeal and tracheal mucosa. The disease can cause mortality and decreased egg production. Infection rates can exceed 90% in flocks, with mortality typically ranging from 5% to 20%. Studies have shown that viral glycoproteins play a crucial role in ILTV replication and inducing host immune responses.

[0003] my country is a major poultry producer. The sheer scale and intensive farming model present challenges for poultry disease prevention and control. Infectious laryngotracheitis (ILT) infection in chickens can lead to mortality and decreased egg production, resulting in significant economic losses for poultry companies. Vaccination is an effective means of preventing the disease, and vaccine design must be based on an understanding of the interaction between pathogens and host cells. By targeting key proteins involved in viral attachment or invasion, vaccines can achieve optimal preventive efficacy.

[0004] The mechanism of action of ILTV's surface glycoprotein gD protein in adsorption and invasion of host cells is still lacking in-depth research. It is necessary to develop a monoclonal antibody that recognizes ILTV gD protein to provide a powerful tool for further research on the interaction between ILTV and host cells and provide technical support for the development of ILTV vaccines. Summary of the Invention

[0005] The present invention aims to solve the technical problem of the lack of monoclonal antibody products for studying the interaction between infectious laryngotracheitis virus and host cells in the current market, and provides an anti-infectious laryngotracheitis virus monoclonal antibody. The monoclonal antibody can specifically recognize the gD protein of infectious laryngotracheitis virus and can be used not only to study the mechanism of action between infectious laryngotracheitis virus and host cells, but also to detect infectious laryngotracheitis virus, thereby providing an excellent product for the clinical diagnosis of infectious laryngotracheitis virus and the development of its vaccine.

[0006] In order to solve the above-mentioned technical problems, the present invention is achieved through the following technical solutions:

[0007] In one aspect of the present invention, a monoclonal antibody that specifically recognizes the gD protein of infectious laryngotracheitis virus is provided.

[0008] Preferably, the monoclonal antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO: C2024177.

[0009] The antibody subtype of the monoclonal antibody is IgG1, and the light chain is a kappa chain.

[0010] In another aspect of the present invention, a hybridoma cell line that secretes and produces the above-mentioned monoclonal antibody is also provided.

[0011] Preferably, the accession number of the hybridoma cell line is CCTCC NO: C2024177.

[0012] In another aspect of the present invention, a kit for detecting antibodies to infectious laryngotracheitis virus of chickens is provided, comprising the above-mentioned monoclonal antibody.

[0013] Preferably, the kit of the present invention further comprises an ELISA plate coated with an infectious laryngotracheitis virus antigen, a positive control serum, a negative control serum, a sample diluent, an enzyme-labeled secondary antibody, a color developing solution, and a stop solution.

[0014] The kit adopts a blocking ELISA method to detect antibodies against infectious laryngotracheitis virus of avian origin, comprising the following steps:

[0015] ELISA plates were coated with purified infectious laryngotracheitis virus antigen;

[0016] After the serum sample to be tested reacts with the coated antigen, the above-mentioned monoclonal antibody, enzyme-labeled secondary antibody, color development solution and stop solution are added in sequence;

[0017] Read the absorbance value OD using a microplate reader 450nm , and according to the formula: blocking rate PI (%) = (average OD of negative control wells 450nm Value - Average OD of sample wells 450nm value) / average OD of negative control wells 450nm The blocking rate of the serum sample to be tested was calculated by multiplying the value by 100% to obtain the test result.

[0018] In another aspect of the present invention, the use of the above monoclonal antibody in preparing a product for diagnosing infectious laryngotracheitis is also provided.

[0019] In another aspect of the present invention, there is also provided the use of the above monoclonal antibody in the preparation of a medicament for preventing and / or treating infectious laryngotracheitis in chickens.

[0020] In another aspect of the present invention, there is also provided the use of the hybridoma cell line in the preparation of a medicament for preventing and / or treating infectious laryngotracheitis.

[0021] In another aspect of the present invention, the invention also provides the use of the above-mentioned infectious laryngotracheitis virus antibody detection kit in the preparation of a product for diagnosing infectious laryngotracheitis.

[0022] The anti-ILTV monoclonal antibody of the present invention can specifically recognize the ILTV gD protein, not only providing a powerful tool for studying the interaction between ILTV and host cells, but also can be used to detect ILTV, and has good application prospects in the clinical diagnosis of ILTV and the development of its vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Figure 1 This is a graph showing the semi-quantitative PCR identification results of ILTV in Example 1 of the present invention;

[0025] Figure 2 This is a diagram showing the hybridoma cell screening results of Example 1 of the present invention;

[0026] Figure 3 This is a diagram showing the analysis results of the gD monoclonal antibody recognizing ILTV in Example 2 of the present invention.

[0027] The hybridoma cell line 4F11 (Hybridoma cell line 4F11) of the present invention, which secretes monoclonal antibodies against the gD protein of infectious laryngotracheitis virus of chickens, has been deposited in the China Center for Type Culture Collection (CCTCC) on June 5, 2024, with the deposit number CCTCC NO: C2024177. DETAILED DESCRIPTION

[0028] To further explore the mechanism of interaction between ILTV and host cells and develop an effective ILTV vaccine, the present invention used ILTV as an immunogen and hybridoma technology to generate fusion cells. Enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) were then used to screen for two hybridoma cell lines that secrete monoclonal antibodies that specifically recognize the ILTV gD protein. Cell microneutralization assays demonstrated that the monoclonal antibodies secreted by these two hybridoma cell lines were able to neutralize ILTV, confirming that the ILTV gD protein can stimulate the animal immune system to produce antibodies with neutralizing activity.

[0029] Example 1 Preparation and identification of monoclonal antibodies against the gD protein of infectious laryngotracheitis virus

[0030] 1. Materials and Methods

[0031] 1.1 Infectious laryngotracheitis virus (ILTV) strains, cell lines, plasmids, main reagents, and experimental animals

[0032] The ILTV strain, chicken hepatoma cells (LMH), sp2 / 0 cells, and eukaryotic expression plasmid of gD protein used in this experiment were preserved by the Waterfowl Virus Disease and Animal Influenza Prevention and Control Team of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences; polyethylene glycol solution was purchased from SIGMA-ALORICH; goat anti-mouse HRP-IgG and goat anti-mouse FITC-IgG were purchased from Thermo Fisher Scientific; DMEM / F-12 (HAM) 1:1 was purchased from Biological Industries; rapid immune adjuvant was purchased from Biolong Biotechnology Co., Ltd.; and BALB / c mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.

[0033] 1.2 ILTV amplification, purification and concentration

[0034] ILTV was inoculated into LMH cells for viral amplification. The culture was harvested when the cytopathic effect reached 80% or higher. After repeated freeze-thawing, the culture was centrifuged at 3000 rpm at 4°C for 10 minutes, and the supernatant was collected. The virus was then purified using PEG precipitation. The virus-containing precipitate was centrifuged at 8500 rpm at 4°C for 30 minutes, and the precipitate was collected. The culture was then resuspended in an appropriate amount of PBS, sonicated, and centrifuged at 12000 rpm at 4°C for 5 minutes. The supernatant was then dialyzed and concentrated to obtain a crude, concentrated virus solution. The ILTV content in the virus solution was then assessed using semi-quantitative PCR. This concentrated virus solution was used as the immunizing antigen for monoclonal antibody preparation and as the coating antigen for fusion screening.

[0035] 1.3 ILTV Immunity and Antibody Level Detection

[0036] Crude ILTV was mixed with a rapid immunization adjuvant in equal proportions and injected intramuscularly into the calf muscles of 7-week-old female BALB / c mice, 200 μL per mouse. A second immunization was performed 21 days after the first immunization, using the same dosage and method. Antibody titers in immune sera were measured by indirect ELISA 7-8 days after the second immunization. The serum antibody titer was determined by the maximum dilution with a P / N value ≥ 3. Cell fusion was performed when the titer reached 1:100,000.

[0037] 1.4 Establishment of indirect ELISA screening method

[0038] Matrix titration was used to determine the optimal working concentrations of the coating antigen and antibody for the indirect ELISA method. The purified ILTV, mouse positive serum and negative serum were diluted in multiples, and sp2 / 0 cell culture supernatant and blank control were set up. The mouse positive serum OD 450nm The antigen and antibody dilution concentrations with values ​​above 1.0 and the largest P / N values ​​were taken as the optimal working concentrations.

[0039] 1.5 Establishment of indirect immunofluorescence screening method

[0040] LMH cells were transfected with a eukaryotic expression plasmid for ILTV gD protein and an empty plasmid pCAGGS. 18-24 hours after transfection, the cells were fixed, permeabilized, and blocked. Mouse positive and negative sera were diluted serially as primary antibodies, and FITC goat anti-mouse IgG was used as a secondary antibody. Three replicate wells were set up. Under a fluorescence microscope, cells transfected with the eukaryotic expression plasmid for gD protein showed specific fluorescence, while cells transfected with the empty plasmid pCAGGS and negative serum controls showed no fluorescence. This indicated that the immune sera contained antibodies that recognized ILTV gD protein. An indirect immunofluorescence assay for detecting antibodies against ILTV gD protein was established.

[0041] 1.6 Cell fusion

[0042] When the antibody titer of the immune serum reaches 1:100,000 or above, cell fusion is initiated. Two weeks after the last immunization, intraperitoneal pulse immunization is performed, and cell fusion is performed 3-5 days after the pulse immunization. Cells are screened and cultured using HAT selective culture medium. When cell clones have grown to 1 / 4-3 / 4 of the bottom area of ​​the well plate, the cell supernatant is analyzed using indirect ELISA and IFA.

[0043] 1.7 Preparation of Monoclonal Antibody Ascites

[0044] Ascites was prepared from 8-12 week old BALB / c mice. 0.5 mL of Freund's incomplete adjuvant was injected intraperitoneally into the mice. 7-10 days later, monoclonal antibody cell lines were injected, and 5×10 5 After the mouse abdomen was visually enlarged, ascites was collected. After overnight at 4°C, the ascites was centrifuged at 1000 rpm for 10 minutes to remove impurities such as red blood cells, and the supernatant was collected. The supernatant was then centrifuged at 12000 rpm for 10 minutes, and the clear liquid below the fat was collected, aliquoted, and stored at -80°C until further use.

[0045] 2. Results

[0046] 2.1 Virus amplification, purification and concentration

[0047] After sedimentation and concentration, ILTV was identified by semi-quantitative PCR. Before concentration, the ILTV culture medium was diluted 1000 times to amplify a clear band ( Figure 1a), after concentration and dilution 100,000 times, obvious bands can still be amplified ( Figure 1 b), it can be seen that ILTV is concentrated 100 times per unit volume. This shows that the sedimentation purification and concentration effects are obvious. Figure 1 In the figure, M1: DNA molecular weight standard (DL2000); 1-3: semi-quantitative PCR with 10-fold dilution before ILTV concentration; 4-8: semi-quantitative PCR with 10-fold dilution after ILTV concentration.

[0048] 2.2 Titer of ILTV-immunized mouse serum

[0049] Sera from mice 8 days after the second immunization were diluted 10-fold and analyzed by indirect ELISA. The indirect ELISA conditions, determined by array titration, were: coating antigen at a 1:800 dilution, overnight coating at 4°C for 16 hours, primary and secondary antibodies incubated at 37°C for 1 hour, and HRP goat anti-mouse IgG diluted 1:4000. The ELISA results are shown in Table 1. When diluted 100,000-fold, the P / N ratios for sera from mice #1, #2, and #3 ranged from 7.898 to 15.562, with P / N values ​​≥ 3. Therefore, the titers of sera from immunized mice all reached 1:100,000, meeting the requirement for cell fusion.

[0050] Table 1 Results of indirect ELISA test on mouse serum

[0051]

[0052] 2.3 Screening of hybridoma cells

[0053] Positive cell clones were screened using the IFA method. The IFA reaction conditions were: plasmid transfection time 24 hours; 1% BSA blocking for 1 hour; primary and secondary antibodies incubated at 37°C for 1 hour; FITC goat anti-mouse IgG diluted 1:400. Figure 2 As shown, the culture supernatants of 1C7 and 4F11 produced green fluorescence in LMH cells transfected with a plasmid expressing the gD protein, but did not produce green fluorescence in LMH cells transfected with an empty plasmid expressing pCAGGS. Therefore, the present invention obtained two hybridoma cell lines that secrete anti-ILTV gD protein-specific antibodies, named 1C7 and 4F11, respectively. Hybridoma cell line 4F11 was deposited with the China Center for Type Culture Collection (CCTCC) on June 5, 2024, with the accession number CCTCCNO: C2024177. The viability of the culture of the hybridoma cell line 4F11 was tested by the CCTCC on June 11, 2024, and the result was viable.

[0054] Figure 2In the figure, a: 1C7-transfected LMH with pCAGGS-gD; b: 4F11-transfected LMH with pCAGGS-gD; c: sp2 / 0-transfected LMH with pCAGGS-gD; d: 1C7-transfected LMH with empty pCAGGS; e: 4F11-transfected LMH with empty pCAGGS; f: sp2 / 0-transfected LMH with empty pCAGGS.

[0055] Example 2 Analysis of ILTV Monoclonal Antibody Characteristics

[0056] 1. Materials and Methods

[0057] 1.1gD monoclonal antibody cell culture supernatant indirect ELISA detection

[0058] The culture supernatant of the monoclonal antibody cell line and the mouse ascites prepared with the monoclonal antibody cell line were serially diluted and their OD values ​​were determined by indirect ELISA. 450nm value.

[0059] 1.2 Cell microneutralization test

[0060] The culture supernatants of gD monoclonal antibody cells and sp2 / 0 cells and their respective ascites were diluted 2-fold using 2% FBSDMEM culture medium. At the same time, blank controls containing only culture medium were set up at 37°C and 100 TCID 50 The cells were incubated with ILTV for 2 hours. The cells were seeded on a 96-well plate with a density of 80% LMH cells, with three replicates. After 96 hours of culture, the cell lesions were observed under the condition of blank control with all lesions.

[0061] 1.3gD monoclonal antibody recognition of ILTV analysis

[0062] LMH cells were passaged into 96-well plates and infected with a specific amount of ILTV. When 50% of the cells became pathological, the cells were fixed, permeabilized, and blocked. gD monoclonal antibody cell culture supernatant was used as the primary antibody, and FITC goat anti-mouse IgG was used as the secondary antibody. Three replicates were set up, and a control group containing sp2 / 0 culture supernatant was also established. Observation was performed under a fluorescence microscope.

[0063] 1.4gD monoclonal antibody subclass identification

[0064] The subclass identification of monoclonal antibodies was performed according to the instructions of the antibody subclass identification kit produced by Southern Biotech. The experimental conditions were the same as those in 1.4 of Example 1. Conventional indirect ELISA method was used for detection. The secondary antibodies used were HRP-labeled κ and λ, IgA, IgM, IgG1, IgG 2a , IgG 2b , IgG3.

[0065] 2. Results

[0066] 2.1 Indirect ELISA detection of monoclonal antibody cell culture supernatant

[0067] The culture supernatants of monoclonal antibody-producing cells were diluted 10-fold and assayed according to the ELISA procedure. The results are shown in Table 2. The P / N ratio for the culture supernatants of 1C7 and 4F11 cells was ≥2.0 at the original solution level; it was <2.0 at all other dilutions. In the ascites fluid antibody titer assay, the P / N ratio for 1C7 ascites fluid was ≥2.0 at a 10-fold dilution level and <2.0 at all other dilutions. The P / N ratio for 4F11 ascites fluid was ≥2.0 at a 10-fold and 100-fold dilution level and <2.0 at all other dilutions.

[0068] Table 2 Indirect ELISA detection of monoclonal antibody cell culture supernatant

[0069]

[0070] 2.2 Cell microneutralization test

[0071] To test the neutralizing activity of gD monoclonal antibody antibodies in cell culture supernatants and mouse ascites, a cell microneutralization assay was performed. The results are shown in Table 3. In the cell culture supernatant, 1C7 showed no cytopathic effect at an 8-fold dilution, but exhibited cytopathic effects at dilutions from 16 to 1024. 4F11 showed no cytopathic effect at both 8- and 16-fold dilutions, but exhibited cytopathic effects at dilutions from 32 to 1024. In mouse ascites, 1C7 showed no cytopathic effect at dilutions from 8 to 128, but exhibited cytopathic effects at dilutions from 256 to 1024. 4F11 showed no cytopathic effect at dilutions from 8 to 256, but exhibited cytopathic effects at dilutions from 512 to 1024. Therefore, the antibodies in the cell culture supernatants of 1C7 and 4F11 and in the mouse ascites exhibited neutralizing activity.

[0072] Table 3 Cell microneutralization test

[0073]

[0074] Note: - represents wells without diseased cells, and + represents wells with diseased cells; the number of - represents the number of wells without diseased cells, and the number of + represents the number of wells with diseased cells.

[0075] 2.3gD monoclonal antibody recognition of ILTV analysis

[0076] LMH cells were infected with ILTV, fixed, permeabilized and blocked, and then indirect immunofluorescence assay was performed. Figure 3As shown, the culture supernatants of hybridoma cells 1C7 and 4F11 exhibited green fluorescence in LMH cells infected with ILTV but not in LMH cells not infected with ILTV. The culture supernatant of sp2 / 0 cells exhibited no green fluorescence in LMH cells infected or not infected with ILTV. Therefore, monoclonal antibodies against the gD protein can specifically bind to LMH cells infected with ILTV and can be used to identify and characterize ILTV.

[0077] Figure 3 In the figure, a: 1C7-ILTV-infected LMH; b: 4F11-ILTV-infected LMH; c: sp2 / 0-ILTV-infected LMH; d: 1C7-blank LMH; e: 4F11-blank LMH; f: sp2 / 0-blank LMH.

[0078] 2.4gD monoclonal antibody subclass identification

[0079] The subclasses of antibodies secreted by hybridoma lines 1C7 and 4F11 were identified. The results are shown in Table 4. When 1C7 and 4F11 were detected with HRP-κ as the secondary antibody, the corresponding OD 450 The values ​​were 0.61 and 0.388 respectively; when HRP-λ was used as the secondary antibody for detection, the corresponding OD 450 The values ​​were 0.167 and 0.206 respectively. When 1C7 and 4F11 were detected with HRP-IgG1 as the secondary antibody, the corresponding OD 450 The values ​​were 0.74 and 0.683, respectively, with HRP-IgA, HRP-IgM, and HRP-IgG 2a 、HRP-IgG 2b 、When HRP-IgG3 is used as the secondary antibody for detection, the corresponding OD 450 The values ​​ranged from 0.143 to 0.277. Therefore, both monoclonal antibodies were IgG1 subtypes and their light chains were κ chains.

[0080] Table 4 gD monoclonal antibody subclass identification

[0081]

[0082] Example 3 ILTV gD monoclonal antibody blocking ELISA test

[0083] 1. ILTV-gD monoclonal antibody blocking ELISA test

[0084] In order to determine the blocking effect of monoclonal antibody cell lines 1C7 and 4F11 secreting antibodies, a blocking ELISA test was performed. First, the chicken negative and positive serum were diluted 10 times, and 100 μL per well was added to the enzyme-labeled plate and incubated for 1 hour. Then, the gD monoclonal antibody cell culture supernatant was used as the primary antibody and HRP goat anti-mouse IgG was used as the secondary antibody. The conventional blocking ELISA method was used to detect and read the OD 450 The blocking rate PI was calculated by the value of negative serum well OD 450 Value-positive serum well OD 450 =OD450 value of negative serum well) / OD450 value of negative serum well] × 100%. Monoclonal antibody cell-prepared mouse ascites blocking ELISA test: the cell culture supernatant was replaced with 100-fold diluted mouse ascites.

[0085] 2. ILTV-gD monoclonal antibody blocking ELISA test results

[0086] To determine the blocking efficacy of antibodies secreted by hybridoma cell lines 1C7 and 4F11, blocking ELISA assays were performed. The results are shown in Table 5. The blocking rates for 1C7 and 4F11 cell culture supernatants were 5% and 3%, respectively, and those for 1C7 and 4F11 mouse ascites (100-fold dilution) were 3% and 8%, respectively.

[0087] Table 5 ILTV-gD monoclonal antibody blocking ELISA test

[0088]

[0089] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A monoclonal antibody that specifically recognizes the gD protein of avian infectious laryngotracheitis virus, wherein the monoclonal antibody is produced by a hybridoma cell line with a deposit number of CCTCC NO: C2024177.

2. A hybridoma cell line that secretes and produces the monoclonal antibody according to claim 1, wherein the deposit number of the hybridoma cell line is CCTCC NO: C2024177.

3. A kit for detecting antibodies to infectious laryngotracheitis virus, characterized in that: Comprising the monoclonal antibody according to claim 1.

4. The infectious laryngotracheitis virus antibody detection kit according to claim 3, characterized in that: The invention also comprises an ELISA enzyme-labeled plate coated with an infectious laryngotracheitis virus antigen, a positive control serum, a negative control serum, a sample diluent, an enzyme-labeled secondary antibody, a color developing solution, and a stop solution.

5. Use of the monoclonal antibody according to claim 1 in the preparation of a product for diagnosing infectious laryngotracheitis.

6. Use of the monoclonal antibody according to claim 1 in the preparation of a medicament for treating infectious laryngotracheitis.

7. Use of the hybridoma cell line according to claim 2 in the preparation of a medicament for treating infectious laryngotracheitis.

8. Use of the infectious laryngotracheitis virus antibody detection kit according to claim 3 or 4 in the preparation of a product for diagnosing infectious laryngotracheitis.

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