CN112501129B

CN112501129B - anti-IBDV VP2 protein monoclonal antibody combination and application thereof in identifying and detecting IBDV

Info

Publication number: CN112501129B
Application number: CN202011466765.6A
Authority: CN
Inventors: 祁小乐; 王笑梅; 高玉龙; 高立; 崔红玉; 李凯; 刘长军; 潘青; 张艳萍
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2022-12-02
Anticipated expiration: 2040-12-14
Also published as: CN112501129A

Abstract

The invention discloses a monoclonal antibody combination of anti-IBDV VP2 protein and application thereof in identifying and detecting IBDV. The monoclonal antibody combination consists of hybridoma cell strains 7D of the IBDV VP2 protein monoclonal antibody and monoclonal antibodies secreted by 25C, wherein the strain preservation numbers of the hybridoma cell strains 7D and 25C are CGMCC NO.21009 and CGMCC NO.21008 respectively. Experiments prove that 7D can identify IBDV classical virus, virulent virus and variant strains, while 25C can only identify IBDV classical virus and virulent virus and cannot identify IBDV variant strains. In addition, the invention also establishes an indirect immunofluorescence technique by utilizing the two monoclonal antibodies, and can distinguish and detect IBDV variant strains and non-variant strains (classical strains and super-virulent strains), so the invention provides a new technical means for IBDV differentiation, and has important significance for comprehensive prevention and control of IBDV.

Description

anti-IBDV VP2 protein monoclonal antibody combination and application thereof in identifying and detecting IBDV

Technical Field

The invention relates to a monoclonal antibody and application thereof in virus detection, in particular to a monoclonal antibody combination of an anti-Infectious Bursal Disease Virus (IBDV) VP2 protein and application thereof in identifying and detecting IBDV. The invention belongs to the technical field of virus detection.

Background

Infectious Bursal Disease (IBD) is an important immunosuppressive disease of chickens, the etiology of which is Infectious Bursal Disease Virus (IBDV). The damage of IBDV to the poultry industry is divided into direct and indirect damage. Direct hazard: destroy the bursa of Fabricius of central immune organs and can kill directly. Indirect hazard: the low immunity of chicken infected by IBDV causes vaccine immunity failure of other important epidemic diseases such as avian influenza, newcastle disease and the like, and is easy to cause secondary infection, influence the production performance of chicken groups and cause serious economic loss. In conclusion, IBD seriously affects the health development of the poultry industry, and its preventive situation is very severe.

The genome of IBDV consists of two segments, A and B. The full length of the segment A is about 3200bp, and the segment A encodes polyprotein (PP, VP2-VP4-VP 3) and VP5 protein, and the PP is further subjected to self-cleavage maturation into VP2, VP3 and VP4. Among them, VP2 and VP3 are important structural proteins of IBDV. VP2 is an important component constituting the viral capsid, which is located on the outermost side of the virion and is a key host protective antigen. The B segment has a total length of about 2800bp and encodes a VP1 protein with RNA-dependent RNA polymerase activity, which plays an important role in the transcription and translation of viral genes.

There are two serotypes of IBDV: serogroup I and serogroup II. The type I serum causes diseases to the chicken, and the type II serum does not cause diseases to the chicken. Serous type I IBDV is divided into classical, super-virulent and variant strains. Since the last 90 s, the world is covered by Very virulent IBDV (vvIBDV) which is mainly characterized by acute and high lethality, causing huge losses to the poultry industry. Over the last 30 years of effort, vvIBDV infections are gradually being controlled based on factors such as increased levels of feed management and widespread use of vaccines. However, in recent years there have been new changes in IBD, and atypical IBD by IBDV variants has become a new threat to poultry industry, which directly destroys the central immune organ bursa and B lymphocytes, resulting in severe immunosuppression and decreased productivity, causing severe economic losses (fanlin et al,2019 fan et al,2019,2020 xu et al, 2019.

Currently, IBDV classical, virulent and variant strains are epidemiologically in a coexisting state. However, the differential detection can only rely on gene sequencing technology, and special technology and equipment are needed.

Therefore, the invention prepares a hybridoma cell strain secreting the anti-IBDV VP2 protein monoclonal antibody, establishes a method for identifying and detecting IBDV variant strains and non-variant strains (classical virus and super-virulent virus) by using a monoclonal antibody mediated indirect immunofluorescence technology, and can identify and detect IBDV variant strains and non-variant strains (classical virus and super-virulent virus), so that the invention provides a new technical means for IBDV identification and has important significance for comprehensive prevention and control of IBD.

Disclosure of Invention

The invention aims to provide a monoclonal antibody for identifying and detecting IBDV and establish a corresponding indirect immunofluorescence detection method.

In order to achieve the purpose, the invention adopts the following technical means:

the invention provides a hybridoma cell strain combination for secreting anti-Infectious Bursal Disease Virus (IBDV) VP2 protein monoclonal antibody, which consists of hybridoma cell strains 7D and 25C for secreting anti-IBDV VP2 protein monoclonal antibody, wherein one hybridoma cell strain for secreting anti-IBDV VP2 protein monoclonal antibody is named as 7D and classified as hybridoma cell strain for secreting anti-infectious bursal disease virus VP2 protein monoclonal antibody, and is preserved in China General Microbiological Culture Collection Center (CGMCC), wherein the address is No. 3 of Ministry of Western West Luo No. 1 of Yangtze district in Beijing, the preservation number is CGMCC NO.21009, and the preservation time is 2020, 11 months and 5 days;

the other hybridoma cell strain secreting the anti-IBDV VP2 protein monoclonal antibody is named as 25C, is classified and named as a hybridoma cell strain secreting the anti-chicken infectious bursal disease virus VP2 protein monoclonal antibody, is preserved in China General Microbiological Culture Collection Center (CGMCC), is addressed to No. 3 of Xilu 1 of North Chen of the sunny region in Beijing, and is preserved with the preservation number of CGMCC NO.21008, and is preserved for 11 months and 5 days of 2020.

The invention further provides a monoclonal antibody combination of the Infectious Bursal Disease Virus (IBDV) VP2 protein, which consists of hybridoma cell strains 7D of the IBDV VP2 protein-resistant monoclonal antibody and monoclonal antibodies secreted by 25C.

The invention further provides application of the monoclonal antibody combination in preparing a reagent for identifying and detecting the infectious bursal disease virus.

Preferably, the infectious bursal disease virus comprises infectious bursal disease classical virus, virulent virus and variant strains.

Of these, preferred infectious bursal disease classical virus includes IBD17JL01, genBank accession no: MN604241 and MN604242, ultra virulent virus including HLJ0504, genBank accession no: GQ451330 and GQ451331, variants including SHG19, genBank accession no: MN393076 and MN393077.

Wherein, preferably, the monoclonal antibody combination is used for detecting through indirect immunofluorescence, and the hybridoma cell strain 7D secreting the anti-IBDV VP2 protein monoclonal antibody can identify IBDV classical virus, super virulent virus and variant strains; among them, hybridoma cell line 25C secreting monoclonal antibody against IBDV VP2 protein can only recognize IBDV classical virus and super-virulent virus, but not IBDV variant.

Compared with the prior art, the invention has the beneficial effects that:

infectious Bursal Disease (IBD) is an important immunosuppressive disease of chickens. Its etiology includes IBDV classical virus, super virulent virus and variant strain. The invention prepares two hybridoma cell strains 7D and 25C secreting anti-IBDV VP2 protein monoclonal antibodies, wherein the hybridoma cell strain 7D secreting anti-IBDV VP2 protein monoclonal antibodies can identify IBDV classical virus, super-strong virus and variant strains, and the hybridoma cell strain 25C secreting anti-IBDV VP2 protein monoclonal antibodies can only identify IBDV classical virus and super-strong virus and cannot identify IBDV variant strains. In addition, the invention also utilizes the two monoclonal antibodies to establish an indirect immunofluorescence technique, can distinguish and detect IBDV variant strains and non-variant strains (classical virus and super-virulent virus), and has important significance for the comprehensive prevention and control of IBD.

Drawings

FIG. 1 shows the results of indirect immunofluorescence detection (scale: 400 um).

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.

EXAMPLE 1 preparation of monoclonal antibodies against IBDV VP2 protein

1. Materials and methods

1.1 viruses, cells and mice

Infectious Bursal Disease Virus (IBDV) super virulent strain Gx strain and attenuated strain Gt strain thereof, marek's Disease Virus (MDV), subgroup J avian leukosis virus (ALV-J) and reticuloendotheliosis virus (REV) are identified and preserved by the avian immunosuppressive disease team (hereinafter referred to as the laboratory) of Harbin veterinary research institute of Chinese academy of agricultural sciences. SP2/0 myeloma cells and DF1 cells were stored in the laboratory. BALB/C mice were purchased from Experimental animals center of Harbin veterinary institute, national academy of agricultural sciences.

1.2 Primary reagents and consumables

Freund's complete adjuvant, freund's incomplete adjuvant, polyethylene glycol (PEG), FITC label fluorescence anti-mouse second antibody, subclass identification kit for Sigma product; the fetal bovine serum is an AusBian product; DMEM is Thermo Scientific product; HAT and HT are Invitrigen products. The constant temperature incubator is a product of Thermo Scientific company; the clean bench is a product of Beijing Dong gang Harr apparatus manufacturing company Limited; the inverted fluorescence microscope is a product of Zeiss corporation.

1.3 immunization

A6-week-old Balb/c magnetic mouse is immunized by the purified IBDV super virulent strain Gx. Once immunization, the immunogen was mixed and emulsified with equal volume of Freund's complete adjuvant, and injected intraperitoneally into mice at a dose of 100. Mu.g/mouse. Following interval 14d, a second immunization was performed, the adjuvant was exchanged for Freund's incomplete adjuvant, the immunization route and the dose were identical. After interval 14d, a third immunization was performed, and the immunization was performed with a second immunization. 3 days before cell fusion, boosting immunization was performed, and the immunogen without adjuvant was injected via tail vein of mice at a dose of 100. Mu.g/mouse.

1.4 preparation of feeder cells

After cervical dislocation, balb/c mice aged 8 weeks were sacrificed and soaked in 75% alcohol for 10min for sterilization. Fixing the abdomen of the patient on an anatomical plate in an ultra-clean bench, cutting the skin, exposing the peritoneum, sucking 5mL of cell culture solution by using a 10mL syringe, injecting the cell culture solution into the abdominal cavity, massaging the two abdomens for 30 seconds, and then sucking back the liquid in the abdominal cavity by using the syringe. The peritoneal fluid was centrifuged at 1500rpm for 10min and the supernatant discarded. Resuspend cells in HAT medium, and load into 96-well cell culture plates at 100. Mu.L/well (about 10. Mu.L/well) ⁵ Individual cells). Culturing in 37 deg.C incubator.

1.5 preparation of myeloma cells

Myeloma cells were expanded 48h before cell fusion, and the cells were in the logarithmic growth phase. During fusion, the cells were blown up, centrifuged at 1000rpm for 10min, and the supernatant was discarded. The cells were resuspended and mixed with DMEM medium and counted with trypan blue stain for use.

1.6 spleen cell preparation of immunized mice

The immunized Balb/c mice were sacrificed by cervical dislocation and soaked in 75% alcohol for 10min to sterilize. Spleens were removed aseptically and washed once in dishes containing l0mL DMEM medium. The spleen is transferred into another plate containing 10mL of DMEM culture solution, the DMEM culture solution in the plate is sucked by an injector and injected into the spleen of the mouse, and the splenocytes are blown out. The spleen cell suspension in the plate was collected in a 50mL centrifuge tube, centrifuged at 1000rpm for 10min, and the supernatant was discarded. Cells were resuspended in 10mL DMEM medium and counted with trypan blue staining for use.

1.7 fusion of cells

Mixing the prepared SP2/0 cells and the mouse spleen cells in a 50mL centrifuge tube according to the proportion of 1-1. Centrifuge horizontally at 27 ℃ and 1000rpm for 10min and discard the supernatant. The centrifuge tube is tapped with the palm to thoroughly loosen and homogenize the precipitated cells. Placing the centrifugal tube in a water bath at 37 ℃ for heat preservation; dropwise adding 1mL of preheated PEG1450 into a centrifuge tube (the adding is finished within about 1min, and cannot be too fast or too slow); the tube was left to stand for 1min. And (3) dropwise adding a preheated DMEM cell culture solution into the centrifugal tube, and finally, fixing the volume to 40mL. Centrifuging at 800rpm for about 10min at room temperature, and discarding the supernatant. The cells were resuspended in 50mL HAT medium, added to feeder cells-containing cell culture plates, 100. Mu.L/well, and cultured in a cell incubator. After 5d, the HAT medium was replaced with 50% medium. After 7d, HAT medium was replaced with HT medium. At 10 days, the hybridoma cell supernatants were aspirated and their antibody titers were determined using IBDV VP2 protein-coated ELISA plates (prepared in this laboratory). Selection of OD ₄₅₀ The wells with the highest values and the largest P/N were positive wells.

1.8 subcloning and screening of fusion cells

The antibody secreting positive hybridoma cells were subcloned by limiting dilution. The hybridoma cells to be cloned are blown up into a suspension and counted. Cells were diluted in HT medium and added to 96 well cell culture plates at 100. Mu.L per well, i.e., 1.5/well. Culturing in 37 deg.C incubator for 5-8 days. Strictly selecting the cell hole of a single clone, carrying out antibody detection on the supernatant, and taking a positive clone for amplification culture. After the expanded culture, the next cloning is carried out according to the method until the positive rate reaches 100 percent. Hybridoma cells were thus subcloned 3 times. Cells which still show antibody positivity after three times of subcloning are transferred from a 96-well cell culture plate to a 24-well cell culture plate, a 6-well cell culture plate and a cell culture bottle for gradual expansion culture. After the cells grow full, the cells are put into the medium for culture in the same way, and after the cells grow full, the cells are transferred into a cell culture bottle for enlarged culture. One part of the cells is frozen and the other part of the cells is used for preparing ascites.

1.9 preparation of ascites monoclonal antibody

3 healthy Balb/c male mice of 8 weeks old are taken, and are used after being injected with 0.5mL of sterilized paraffin oil in the abdominal cavity for 10-15 days. Blowing positive hybridoma cells in logarithmic growth phase with PBS to obtain suspension, injecting into abdominal cavity of mouse, adjusting cell number to 106/mL, and injecting into abdominal cavity of each mouse 0.5mL (about 5 × 10) ⁵ One cell). Collecting ascites after one week, centrifuging at 10000rpm for l0min, discarding upper layer fat, paraffin oil and lower layer precipitate, carefully removing middle layer light yellow clear liquid to obtain monoclonal antibody ascites, and storing at-20 deg.C.

1.10 identification of monoclonal antibodies

1.10.1 monoclonal antibody specificity identification

Infecting IBDV Gt strain with DF1 cell cultured in 96-well plate, and performing indirect immunofluorescence detection with antibody to be detected after 24 h. Meanwhile, marek's Disease Virus (MDV), subgroup J avian leukosis virus (ALV-J), reticuloendotheliosis virus (REV) infection control and uninfected cell control are set.

1.10.2 monoclonal antibody titer determination

The titer of the monoclonal antibody ascites is determined by indirect ELISA. The specific judgment standard is as follows: the OD value of the negative serum is less than 0.2, the OD value of the positive serum is more than 1.0, the maximum dilution multiple of the ascites when the OD value of the detected sample is more than 0.2, and the OD value of the P/N is more than 2.1 is the ELISA titer of the antibody in the ascites.

1.10.3 monoclonal antibody immunoglobulin subclass identification

The subclass of the obtained monoclonal antibody was identified using an antibody subclass identification kit manufactured by Sigma. Coating subclass identifying reagent (goat anti-mouse subclass antibody) 100. Mu.L per well, 37 deg.C 1h. The wells were decanted, washed 3 times with PBST, and 100. Mu.L of blocking solution was added to each well for 1h at 37 ℃. The well was drained and washed 3 times with PBST, 100. Mu.L of sample to be tested was added to each well, and the temperature was 37 ℃ for 1h. The well was drained and washed 3 times with PBST, then HRP-labeled goat anti-mouse IgG was added, 30min at 37 ℃ and substrate was added for color development.

2. As a result, the

Get together10 hybridoma cell strains capable of secreting anti-IBDV VP2 protein monoclonal antibodies are obtained, wherein two strains are named as 7D and 25C, and the secreted monoclonal antibodies are also named as corresponding cell strains. Indirect immunofluorescence assay showed that the obtained monoclonal antibodies all specifically recognized IBDV. The ELISA titer of the antibody in the ascites is not less than 5.0 multiplied by 10 ⁶ . The subclass of monoclonal antibodies was identified as IgG1 type, kappa chain.

The hybridoma cell strain 7D secreting the anti-IBDV VP2 protein monoclonal antibody is preserved in China General Microbiological Culture Collection Center (CGMCC), is No. 3 of Xilu No. 1 of Beijing city, south China institute of microbiology, has a preservation number of CGMCC NO.21009, and has a preservation time of 2020 year 11 and 5 days of 11 months.

The hybridoma cell line 25C secreting the anti-IBDV VP2 protein monoclonal antibody is preserved in China General Microbiological Culture Collection Center (CGMCC), china General Microbiological Culture Collection Center, no. 3 of Ministry No. 1 of Western West Lu, china academy of sciences, the China institute of microbiology, the preservation number is CGMCC NO.21008, and the preservation time is 11 months and 5 days in 2020.

EXAMPLE 2 differential detection of IBDV variants by monoclonal antibodies 7D and 25C

1 materials and methods

1.1 monoclonal antibodies, cells and viruses

anti-IBDV VP2 protein monoclonal antibodies 7D (CGMCC NO. 21009) and 25C (CGMCC NO. 21008) were prepared as described in example 1. The chicken B lymphocyte cell line DT40 was maintained by the laboratory. Representative strains IBDV classical virus IBDV 17JL01 (GenBank accession numbers: MN604241 and MN 604242), representative strains of ultra-virulent virus HLJ0504 (GenBank accession numbers: GQ451330 and GQ 451331) and representative strains of variant strain SHG19 (GenBank accession numbers: MN393076 and MN 393077) were isolated and identified in the laboratory.

1.2 Main reagents, consumables and instruments

The cell culture solution is DMEM containing 10% fetal calf serum, and the cell maintenance solution is DMEM containing 2% fetal calf serum. The fetal bovine serum is an AusBian product; DMEM is Thermo Scientific product; FITC-labeled fluorescent anti-mouse secondary antibody is a Sigma product. The constant temperature incubator is a product of Thermo Scientific company; the clean bench is a product of Beijing Dong Bihaer Instrument manufacturing company Limited; the inverted fluorescence microscope is a product of Zeiss corporation.

1.3 differential detection of IBDV variants

A pair of anti-IBDV VP2 protein monoclonal antibodies 7D and 25C are used for carrying out identification detection on IBDV classical virus representative strain IBD17JL01, super virulent virus representative strain HLJ0504 and variant strain representative strain SHG19 based on an indirect immunofluorescence technique. The specific operation steps are as follows:

(1) DT40 cells were fed to 96-well plates.

(2) When the cells are 80% full of the bottom of the well, 200TCID is added ₅₀ The IBDV classical virus representative strain IBD17JL01, the super virulent virus representative strain HLJ0504 and the variant strain SHG19 of (1) infect DT40 cells respectively, and infect two cell wells respectively. Wells without virus infection were also set as blanks.

(3) After incubation for 1h at 37 ℃ in a cell incubator, the inoculated venom was discarded, the cells were washed 3 times with PBS and the cell maintenance solution was replaced.

(4) After incubation for 24h in a 37 ℃ cell incubator, the cell culture fluid was discarded, and a suitable amount of 4% paraformaldehyde was added to fix the cells at room temperature for 20min.

(5) Paraformaldehyde is discarded, cells are washed 3 times with PBS, 100ul of diluted anti-IBDV hypervirulent VP2 protein monoclonal antibodies 7D and 25C are added to two wells infected with the same virus, respectively, and incubated in a wet box at 37 ℃ for 1h.

(6) The mab was discarded, cells were washed 3 times with PBS, 100ul FITC-labeled anti-mouse fluorescent secondary antibody (1 diluted 200) was added, and incubated in a wet box at 37 ℃ for 1h in the dark.

(7) The fluorescent secondary antibody was discarded, and the cells were washed 3 times with PBS, and finally 100. Mu.L of PBS was added to each well and observed under an inverted fluorescence microscope. Those with green fluorescence signals were positive wells.

2 results and discussion

The indirect immunofluorescence detection result shows that the anti-IBDV VP2 protein monoclonal antibody 7D can identify IBDV classical virus, super-virulent virus and variant strains; the anti-IBDV VP2 protein monoclonal antibody 25C can only recognize IBDV classical and super-virulent viruses, but cannot recognize IBDV variant strains (FIG. 1). In addition to these three representative strains, other strains were tested in this study and all gave the same results. This demonstrates that the combination of 7D and 25C anti-IBDV VP2 protein monoclonal antibodies can identify IBDV variants as well as non-variants.

Claims

1. The hybridoma cell strain combination for secreting the anti-Infectious Bursal Disease Virus (IBDV) VP2 protein monoclonal antibody is characterized by consisting of hybridoma cell strains 7D and 25C for secreting the anti-IBDV VP2 protein monoclonal antibody, wherein the hybridoma cell strains 7D for secreting the anti-IBDV VP2 protein monoclonal antibody are preserved in China General Microbiological Culture Collection Center (CGMCC), the address of which is No. 3 of Beijing western institute of China, institute of microbiology, the preservation number is CGMCC NO.21009, and the preservation time is 11 months and 5 days 2020;

hybridoma cell line 25C secreting monoclonal antibody against IBDVVP2 protein was deposited at the China General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center, CGMCC), the accession number is CGMCC NO.21008, the preservation time is 2020, 11 months and 5 days.

2. The monoclonal antibody combination of anti-Infectious Bursal Disease Virus (IBDV) VP2 protein, which is characterized in that the monoclonal antibody combination consists of the monoclonal antibodies secreted by hybridoma cell strains 7D and 25C of the anti-IBDV VP2 protein monoclonal antibody of claim 1.

3. Use of the monoclonal antibody combination of claim 2 for the preparation of a reagent for the differential detection of infectious bursal disease virus.

4. The use of claim 3, wherein said infectious bursal disease virus is selected from the group consisting of infectious bursal disease virus, virulent strain, and variant strain.

5. The use of claim 4, wherein the infectious bursal disease classical virus comprises IBD17JL01, genBank accession No.: MN604241 and MN604242, ultra virulent virus including HLJ0504, genBank accession no: GQ451330 and GQ451331, variants including SHG19, genBank accession no: MN393076 and MN393077.

6. The use according to claim 4 or 5, wherein the monoclonal antibody combination is used for indirect immunofluorescence assay, wherein hybridoma cell line 7D, which secretes monoclonal antibodies against the IBDV VP2 protein, is recognized by IBDV classical, virulent and variant strains; among them, hybridoma cell line 25C secreting monoclonal antibody against IBDV VP2 protein can only recognize IBDV classical virus and super-virulent virus, but not IBDV variant.