CN115925887B - African swine fever virus pA104R protein immunodominant B cell epitope, monoclonal antibody thereof and application - Google Patents
African swine fever virus pA104R protein immunodominant B cell epitope, monoclonal antibody thereof and application Download PDFInfo
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Abstract
The invention discloses an african swine fever virus pA104R protein immunodominant B cell epitope, a monoclonal antibody thereof and application thereof, and belongs to the technical field of molecular immunology. The monoclonal antibody of the African swine fever virus pA104R protein is prepared from the following components in percentage by mass: the hybridoma cell strain of C2022334 is generated, and the monoclonal antibody recognition epitope is positioned in MSTKKKPTITKQELYSL region of pA104R protein, which is the immunodominant B cell antigen epitope region of pA104R protein. The invention can be widely applied to etiology diagnosis, serology diagnosis, immunological detection and disease control of African swine fever virus, preparation of African swine fever vaccine, pathogenesis research and the like. The invention provides a new material for preparing African swine fever vaccine and diagnostic reagent and a new direction for preparing African swine fever virus-resistant medicines.
Description
Technical Field
The invention belongs to the technical field of molecular immunology, and particularly relates to an african swine fever virus pA104R protein dominant B cell antigen epitope polypeptide, a monoclonal antibody thereof and application thereof.
Background
African Swine Fever (ASF) is a hemorrhagic disease with high infectivity and is characterized by high fever, toxemia, hemorrhagic diathesis and high mortality. African Swine Fever Virus (ASFV) is the only member of the African swine fever virus genus of the African swine fever virus family and is also the only large arbovirus DNA. The disease is high in morbidity and mortality, and destructive economic loss is often caused to pig industry in affected countries, so that the disease is listed as legal report by the world animal health Organization (OIE), and the disease is also listed as an animal epidemic disease which is important to prevent in China.
Although vaccination is an ideal method for controlling most animal diseases. However, due to the complexity of viral genome and viral composition, the development of vaccines is hampered by the unclear mechanisms of immunization and infection. To date, there are no commercial vaccines and effective antiviral drugs available for preventing and controlling ASFV infections. In order to promote the research of the prevention and control technology of the disease, the research of the immunological function of important antigen proteins of ASFV is urgently needed, and the virus protein antigens with the immune protection function and the serological diagnosis value are analyzed and identified.
Disclosure of Invention
pA104R is a structural protein with histone-like characteristics encoded by African swine fever virus, and is involved in viral DNA replication, transcription, and genome packaging, a protein necessary for ASFV replication. pA104R is strongly immunogenic and is capable of inducing higher antibody levels in virally infected animals and is considered a very valuable vaccine candidate. Studies have shown that pA104R can inhibit natural immunity, and its DNA binding functional region with biological function is located in the middle-rear region of protein (Chinese patent CN114989266A, an amino acid site related to the immunosuppression of African swine fever virus pA104R protein and application thereof).
The invention aims to provide a monoclonal antibody of an African swine fever virus pA104R protein and application thereof, and provides a hybridoma cell secreting the monoclonal antibody.
The invention also aims to provide the dominant B cell epitope peptide of the African swine fever virus pA104R protein and application thereof.
The aim of the invention is achieved by the following technical scheme:
a monoclonal antibody AH7 of African swine fever virus pA104R protein is produced by hybridoma cell line AH 7. The recognition epitope of the monoclonal antibody AH7 is positioned in MSTKKKPTITKQELYSL (SEQ ID NO. 1) region of pA104R protein, the amino acid sequence of the region is highly conserved in all genotypes of African swine fever virus, and the region does not contain an immunosuppression region of the protein.
The hybridoma cell strain AH7 is preserved in China center for type culture collection (address: university of Wuhan, china) for 10 months and 25 days in 2022, and is classified and named as hybridoma cell strain AH7, and the preservation number is CCTCC NO: and C2022334.
The african swine fever virus pA104R protein immunodominant B cell epitope is located in MSTKKKPTITKQELYSL region of pA104R protein.
An african swine fever virus pA104R protein immunodominant B cell epitope peptide having an amino acid sequence of all or part of MSTKKKPTITKQELYSL.
The monoclonal antibody AH7 of the African swine fever virus pA104R protein or the immunodominant B cell epitope peptide thereof can be used for preparing the following products: an etiology diagnosis product of ASFV, a serology diagnosis product of ASFV, an immunology detection product of ASFV, and a product for preventing and treating ASFV.
The african swine fever virus pA104R protein immunodominant B cell epitope peptide also has the following application: the anti-pA 104R protein antibody can be used for specifically detecting the immune of an organism or the anti-pA 104R protein antibody generated after infection, can be used for ASFV infection diagnosis, vaccine immune effect evaluation and can be used as various vaccine components for inducing immune pigs to generate specific antibodies.
The African swine fever virus pA104R protein immunodominant B cell epitope peptide also has the application of preparing a swine fever vaccine.
The vaccine comprises subunit vaccine, multi-epitope chimeric vaccine, DNA vaccine, mRNA vaccine, virus vector vaccine and the like.
The monoclonal antibody has the following advantages and beneficial effects:
(1) The antibody recognizes an epitope that is highly conserved in all genotypes of ASFV.
(2) Is easy for mass production; the titer is high, and the antibody titer after purification is more than 1/32,768,000.
(3) Can be widely applied to etiology diagnosis, serology diagnosis, immunological detection, disease prevention and treatment, pathogenesis research and the like of African swine fever viruses. Provides a new material for preparing diagnostic reagents and vaccines of African swine fever virus and a new direction for preparing anti-African swine fever virus medicines.
The pA104R dominant B cell epitope has the following advantages and beneficial effects:
(1) High conservation and good universality, and is highly conserved in all genotypes ASFV.
(2) The dominant B cell antigen epitope sequence is short, does not contain a natural immunosuppression region, has high application value in preparing African swine fever vaccines by combining the B cell antigen epitope polypeptide or other protein antigen dominant regions, and comprises subunit vaccines, multi-epitope chimeric vaccines, DNA vaccines, mRNA vaccines, virus vector vaccines and the like. The B cell antigen epitope polypeptide can exert the immune protection function of the protein, has no immune suppression characteristic, can realize the chimeric use of multiple antigens and realizes better protection effect.
(3) Can be widely applied to etiology diagnosis, serology diagnosis, immunological detection and disease prevention and treatment of African swine fever virus, and can be applied to preparation of African swine fever vaccine (the vaccine comprises subunit vaccine, multi-epitope chimeric vaccine, DNA vaccine, mRNA vaccine, virus vector vaccine and the like), pathogenesis research and the like. Provides a new material for preparing African swine fever vaccines and diagnostic reagents and provides a new direction for preparing African swine fever virus-resistant medicaments.
Drawings
FIG. 1 is a diagram showing SDS-PAGE results of expression purification of pA104R recombinant protein.
FIG. 2 is a graph showing the results of measuring the titers of monoclonal antibodies (AH 7, BE9, BC5, CH9, CH10, DF10 and DH 2) by the indirect ELSIA method.
FIG. 3 is a graph showing the results of Western blotting (western blot) detection of specific reactions of monoclonal antibodies (AH 7, BE9, BC5, CH9, CH10, DF10 and DH 2) with pA104R protein, vector being an empty control.
FIG. 4 is a graph showing the results of indirect immunofluorescence assay for detecting ASFV recognition by monoclonal antibodies (AH 7, BE9, BC5, CH9, CH10, DF10 and DH 2).
FIG. 5 is a graph showing the result of detecting ASFV by monoclonal antibodies (AH 7, BE 9) in an immunohistochemical assay.
FIG. 6 is a schematic representation of pA104R truncated protein.
FIG. 7 is a SDS-PAGE result of pA104R truncated protein expression, vector in the figure being an empty control.
FIG. 8 shows the truncated pA104R proteins recognized by the western blot identification monoclonal antibodies (AH 7, BE9, BC5, CH9, CH10, DF10 and DH 2), vector being an empty control.
FIG. 9 is a graph showing the result of western blot detection of pA104R truncated protein in response to African swine fever positive porcine serum, in which Vector is an empty control.
FIG. 10 shows the sequence alignment of the dominant B cell epitope of pA104R in ASFV of different genotypes.
Preservation information
Hybridoma cell line AH7, date of preservation: 2022, 10, 25, deposit unit: china center for type culture Collection (address: university of Wuhan, china), class name: hybridoma cell strain AH7, preservation number CCTCC NO: and C2022334.
Detailed Description
The following examples are provided to further illustrate the present invention and should not be construed as limiting the invention, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the invention are intended to be equivalent substitutes.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
TABLE 1 primers used in the examples below
Example 1 construction of pA104R expression plasmid and expression purification of recombinant protein
Primers A104R-F and A104R-R were synthesized based on the popular ASFV gene type II SY18 strain (GenBank: MH 766894) in China. Amplifying A104R gene by using ASFV inactivated nucleic acid as a template, inserting the ASFV inactivated nucleic acid between restriction enzyme BamHI and XhoI cleavage sites of pPET-30a vector to obtain pET-30a-A104R plasmid, transforming the recombinant plasmid into competent cells BL21 (DE 3), inoculating positive bacterial liquid into LB liquid culture medium containing antibiotics according to a ratio of 1:100, placing the LB liquid culture medium into a constant temperature shaking culture medium at 37 ℃ for 2-3h, and culturing until OD is obtained 600 The value is between 0.5 and 0.6, IPTG is added to the mixture to make the final concentration to be 0.8mM, and the mixture is placed in a shaking table at the constant temperature of 16 ℃ for shake culture for 16 to 20 hours.
After the induction, cells were collected using PBS Buffer (137mM NaCl,2.7mM KCl,10mM Na) 2 HPO 4 ，2mM KH 2 PO 4 ，ddH 2 O is fixed to volume to 1L), pressure crushing is carried out after three times of washing, and after centrifugation, the crushed supernatant liquid is collected and purified by using Ni-NTA resin for affinity chromatography.
SDS-PAGE gel analysis of purified pA104R protein: 10-15% of separation gel and 5% of concentrated gel are prepared, tris glycine electrophoresis buffer solution is added to operate at 80V, the voltage is adjusted to 120V when bromophenol blue indicates that the separation gel is entered, and the target strip is stopped when the target strip is migrated to a proper position. End of electrophoresis the gel was stained in coomassie brilliant blue, followed by a decolorizing analysis. The results are shown in FIG. 1, indicating successful expression and purification of pA 104R.
EXAMPLE 2 animal immunization and preparation of pA104R monoclonal antibody
The obtained pA104R recombinant protein is used as an antigen, the protein and Freund's complete adjuvant are fully emulsified by using the same volume, 100 mug of the protein is subcutaneously injected at a plurality of points at the nape of the neck of a female BALB/c mouse of 5 weeks old for one immunization, then the same amount of the protein emulsified by Freund's incomplete adjuvant is respectively subjected to two or three immunization, and each immunization interval is 14 days.
Analysis by indirect ELISA: with a coating solution (1.59 g NaCO) 3 ，2.93g NaHCO 3 Adding a proper amount of ddH 2 O was fixed to 1L after solubilization) the antigen was diluted to the appropriate concentration and ELISA plates were coated at 100 μl/well, refrigerator overnight at 4 ℃. The antigen solution was discarded, and PBST (PBS containing 0.1% Tween-20) was washed 3 times with 200. Mu.L of each well and gently swirled at room temperature for 5min. And (5) removing the liquid in the hole as much as possible. A 5% skim milk lock in PBS was used to lock overnight at 4 ℃. The blocking solution was removed, washed 3 times with PBST, and the immunized mouse serum and the blank mouse serum were diluted with PBS in a gradient, and added to ELISA reaction plates at a concentration of 100. Mu.L per well, respectively, for 1 hour at 37 ℃. Serum was discarded, washed 3 times with PBST, and goat anti-mouse HRP-IgG enzyme-labeled secondary antibody diluted 1/8000 was added, 100. Mu.L/well, and incubated at 37℃for 30min. And discarding the enzyme-labeled secondary antibody, washing with PBST for 3 times, and finally sequentially drying the liquid in the hole as much as possible. Adding TMB color development liquid, and developing for 10min at room temperature in dark place.
After determining that the mice produce higher antibodies, adopting spleen lymphocyte hybridoma technology to fuse spleen cells and myeloma cells of the immunized mice, cloning and screening by 4-5 rounds of limiting dilution method to obtain 7 hybridoma cells with good growth vigor and good secretion specificity aiming at pA104R antibodies, which are respectively named as AH7, BE9, BC5, CH9, CH10, DF10 and DH2.
7 hybridoma cells (2X 10) 6 And collecting the mouse ascites monoclonal antibody 7-10 days after injecting the mouse abdominal cavity. The 7-strain antibody titer was determined by the indirect ELSIA method. As a result, as shown in FIG. 2, the monoclonal antibodies of strain 7 have high antibody titers, wherein the AH7 titer is the highest, and the titer exceeds 1/32,768,000.
Hybridoma cell line AH7 was deposited with China center for type culture collection (address: university of Wuhan, china) for 10 months and 25 days in 2022, and was designated as hybridoma cell line AH7, with a deposit number of CCTCC NO: C2022334.
EXAMPLE 3 characterization of monoclonal antibodies
HEK293T cells were plated into cell culture dishes, and when the cells grew to 80%, plasmid pCAGGS-HA-A104R (see Chinese patent CN114989266A for construction) and pCAGGS-HA empty vector control were transfected into the cells, and plasmid transfection was performed using jetPRIME Versatile DNA/siRNA transfection reagent of Polyplus Transfection. After 24h transfection, the medium was discarded, a cell lysate containing protease inhibitors and phosphatase inhibitors was added to the cells, after sufficient lysis on ice, the lysate was scraped off gently with a cell scraper and transferred to a 1.5mL centrifuge tube, and the supernatant was collected as a cellular protein sample by centrifugation at 15000g for 10min at 4 ℃ and subjected to western immunoblotting: after completion of SDS-PAGE gel electrophoresis, the gel and PVDF membrane were loaded into an electrophoresis tank according to the instructions of a Bio-RAD electrophoresis apparatus. The transfer condition is a constant current 330mA transfer for 1h. The PVDF membrane onto which the protein was transferred was blocked in TBST (containing 5% BSA) at room temperature for 2 hours, followed by incubation with 7 monoclonal antibodies as an antibody, and development was performed using a chemiluminescent imaging system. The results are shown in FIG. 3, which shows that 7 monoclonal antibodies can specifically react with pA104R protein.
WSL cells were plated into cell culture dishes and ASFV was infected when the cells grew to 80%, and an indirect immunofluorescence experiment was performed 72h after infection: the supernatant was discarded, and pre-chilled PBS was added to wash 1-2 times. 4% paraformaldehyde was added for fixation for 20min. After washing, pre-chilled methanol was added for 15min. After washing, 5% BSA (diluted with PBS) was added and blocked for 1h. After washing, the antibody was added for 1h, washed 3 times, and after incubation for 5min with DAPI, washed again, and then observed under a fluorescence microscope. As a result, only AH7 and BE9 were able to recognize the native virus in the 7-strain monoclonal antibody as shown in FIG. 4.
The results of immunohistochemical experiments performed on the embedded tissue samples of african swine fever by the marchand peano fly biotechnology company are shown in fig. 5, which shows that antibodies AH7 and BE9 can recognize ASFV, but the AH7 effect is significantly better than BE9. Thus monoclonal antibody AH7 is capable of diagnosing african swine fever infection as a tissue immunofluorescence or immunohistochemical antibody specific recognition.
EXAMPLE 4 identification of monoclonal antibody-recognizing epitopes
And (3) adopting a heavy truncated protein expression series pA104R protein antigen truncated fragment and carrying out western blotting to identify the monoclonal antibody epitope. Four truncated proteins S1-S4 were designed based on the amino acid sequence of pA104R protein, and these truncated proteins overlapped with each other and covered the full length of pA104R protein, as shown in FIG. 6. Wherein, the amino acid sequence of S1 is: MSTKKKPTITKQELYSLVAADTQLNKALIERIFTSQQKIIQNALKHNQEVII; the amino acid sequence of S2 is: VAADTQLNKALIERIFTSQQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPAR; the amino acid sequence of S3 is: QQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPE; the amino acid sequence of S4 is: PPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPEHKAVKIRALKPVHDMLN.
Amplifying the full length of the A104R gene and truncated fragments S1-S4 by using pET30a-A104R plasmid as a template and primers such as GST-S1-F/R in table 1, inserting the primers between restriction enzyme BamHI and XhoI cleavage sites of pGEX-6p-1 vector to obtain A104R truncated fragment recombinant plasmid, transforming the recombinant plasmid into competent cells BL21 (DE 3), inoculating positive bacterial liquid into LB liquid culture medium containing antibiotics according to a ratio of 1:100, placing the LB liquid culture medium into a constant temperature shaking culture medium at 37 ℃ for 2-3h, and culturing until OD 600 The value is between 0.5 and 0.6, IPTG is added to the final concentration of 0.8mM, and the mixture is placed on a shaking table at the constant temperature of 37 ℃ for shake culture for 4 to 6 hours, and then SDS-PAGE is carried out for electrophoresis analysis. The results are shown in FIG. 7, indicating successful expression of pA104R truncated protein. And identifying the epitope recognized by 7 monoclonal antibodies through western blot. As shown in FIG. 8, the results indicate that each of the 7 monoclonal antibodies recognizes only the truncated fragment S1 and does not recognize S2, S3, S4. Thus the monoclonal antibody recognizes the unique region of the fragment S1, which is likely to be pA104R, which is a potential B cell epitope.
For further validation, western blot experiments were performed using four clinically isolated african swine fever positive porcine sera as primary antibodies, as shown in fig. 9, and the results showed that all swine positives also reacted strongly with S1 only, further proving that the unique region of S1 protein was pA104R as the dominant B cell epitope.
In addition, the epitope recognized by the monoclonal antibody was found to be highly conserved in all genotypes ASFV by comparing the amino acid sequence of the NCBI existing 159 pA104R protein, including genotype I, II, IV, VIII, IX, XX, XXII and part of the indeterminate genotype strain, and the partial sequence alignment was as shown in fig. 10.
Claims (5)
1. A monoclonal antibody of african swine fever virus pA104R protein, characterized in that: the preservation number is CCTCC NO: hybridoma cell line of C2022334.
2. A hybridoma cell line producing the monoclonal antibody of claim 1, characterized in that: the preservation number is CCTCC NO: C2022334.
3. use of a monoclonal antibody according to claim 1, characterized in that: the application is the application in preparing at least one of the following products: an etiology diagnosis product of ASFV, a serology diagnosis product of ASFV, an immunology detection product of ASFV, and a product for preventing and treating ASFV.
4. An african swine fever virus pA104R epitope peptide, characterized in that: the amino acid sequence is MSTKKKPTITKQELYSL.
5. The use of the epitope peptide according to claim 4, wherein: the application is the application in preparing at least one of the following products: etiology diagnosis product of ASFV, serology diagnosis product of ASFV, immunology detection product of ASFV.
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