CN112680421A - Monoclonal antibody with broad spectrum neutralizing porcine epidemic diarrhea virus and application thereof - Google Patents
Monoclonal antibody with broad spectrum neutralizing porcine epidemic diarrhea virus and application thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody with broad spectrum neutralizing porcine epidemic diarrhea virus and application thereof. The monoclonal antibody is secreted and produced by hybridoma with the preservation number of CGMCC No. 13840. The monoclonal antibody has broad spectrum, can effectively neutralize G2 gene subtype strains such as PEDV-LNCT2, PEDV-LNSY, PEDV-Hjms and the like, and can also neutralize CV777 strain (G1 gene subtype) to lose the capability of infecting cells. In view of the biological characteristics of the monoclonal antibody and the broad-spectrum capability of neutralizing the PEDV strain, the monoclonal antibody can be used for establishing a series of PEDV diagnosis methods including blocking ELISA, capturing ELISA, indirect ELISA, competitive ELISA, colloidal gold test strips and the like, and has wide application value and research value.
Description
Technical Field
The invention relates to a monoclonal antibody, a hybridoma cell strain secreting the monoclonal antibody and application of the monoclonal antibody. In particular to a monoclonal antibody with broad spectrum neutralizing porcine epidemic diarrhea virus, a hybridoma cell strain secreting the monoclonal antibody and application thereof. The invention belongs to the field of biotechnology.
Background
Porcine Epidemic Diarrheal Virus (PEDV) is one of the important pathogens causing Diarrhea, emaciation and dehydration of piglets in the pig industry, and brings great economic loss to the pig industry. Studies have shown that PEDV has two gene subtypes, i.e., the G1 and G2 subtypes. Before 2010, the epidemic strain of PEDV predominated the subtype G1, and after 2010, the epidemic strain of PEDV predominated the subtype G2. Gene sequence analysis indicated that the major difference between PEDV G1 and G2 subtypes was in the S gene. The S gene of the G1 subtype strain consists of 4152nt and codes 1383 amino acids, while the S gene of G2 consists of 4161nt and codes 1386 amino acids, and 9 bases are added on the length of the gene. There are also large differences within the gene. The largest variation in the PEDV genome is located in the S gene, and mutations in the S gene are mainly concentrated at the N-terminus. Compared with NPL-PEDV/2013, the ORF1a/b protein, the nucleoprotein, the NS3B protein, the membrane protein and the cyst membrane protein of NPL-PEDV/2013/P10 are completely consistent at the amino acid level. While six nucleotide polymorphisms are contained in spike protein (S), resulting in amino acid position changes: F375L, T486P, D856E, a1081V, a1099S, and Y1253D. There were 2 amino acid variations in the S1 region and 4 amino acid variations in the S2 region (Lawrence et al, 2014). The coronavirus S protein can induce the generation of neutralizing antibodies, and S gene variation necessarily causes the neutralizing antibodies induced by the S protein to have difference in virus neutralization.
Therefore, the monoclonal antibody with broad spectrum neutralizing porcine epidemic diarrhea virus is obtained, and has important significance for preventing and treating the porcine epidemic diarrhea virus.
Disclosure of Invention
The invention aims to provide a monoclonal antibody capable of neutralizing porcine epidemic diarrhea virus in a broad spectrum, a hybridoma cell strain secreting the monoclonal antibody and application of the monoclonal antibody.
In order to achieve the purpose, the invention adopts the following technical means:
the invention takes virus particles of a purified epidemic strain LNCT2 of Porcine Epidemic Diarrhea Virus (PEDV) as an antigen to immunize a BALB/c mouse, and obtains 1 hybridoma cell strain which stably secretes an anti-PEDV monoclonal antibody, which is named as 5F 7. Through identification, the titer of the antibody of the hybridoma cell culture supernatant and the ascites is respectively 1:1000 and 1: 100000. The subclass of 5F7 mAb is IgG2a, and the light chain is kappa type. IFA (indirect immunofluorescence assay) detection shows that the 5F7 monoclonal antibody reacts with virus; westernblot results show that the 5F7 monoclonal antibody reacts with both PEDV particles and S protein; the two results show that the epitope of the monoclonal antibody is linear epitope and is positioned between 1261aa and 1386aa of the S protein. The PEDV S protein is divided into two areas S1 and S2 according to the structure and the function, wherein the S1 area contains a receptor binding area of virus invading cells, and the area is also a key area for inducing a body to generate neutralizing antibodies; the main function of the S2 domain is to mediate membrane fusion of the virus to the cell. The epitope of the 5F7 antibody is found to be located in the S2 region by epitope identification. The antibody is a first neutralizing antibody aiming at the PEDV S2 region, and has broad spectrum. Virus neutralization experiments also show that the monoclonal antibody can effectively neutralize G2 gene subtype strains such as PEDV-LNCT2, PEDV-LNSY, PEDV-Hjms and the like, so that the strains lose the capability of infecting cells, and can also neutralize CV777 strain (G1 gene subtype). In view of the biological characteristics of the monoclonal antibody, the monoclonal antibody has the capability of neutralizing PEDV strains in a broad spectrum, can be used for establishing a series of diagnostic methods such as blocking ELISA, capturing ELISA, indirect ELISA, competitive ELISA, colloidal gold test strips and the like, and has wide application value and research value.
The hybridoma cell strain for stably secreting the monoclonal antibody against the Porcine Epidemic Diarrhea Virus (PEDV) is named as 5F7 and is classified and named as a monoclonal antibody cell strain against the Porcine Epidemic Diarrhea Virus of mice, the hybridoma cell strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the address of the microorganism research institute of China academy of sciences No. 3 of North West Lu 1 of the sunny district in Beijing, the preservation number is CGMCC No.13840, and the preservation time is 24 days 4.4.2017.
Furthermore, the invention also provides a monoclonal antibody with broad spectrum neutralizing porcine epidemic diarrhea virus, which is secreted and generated by the hybridoma cell strain.
Furthermore, the invention also provides application of the monoclonal antibody in preparation of a reagent for detecting the porcine epidemic diarrhea virus.
Preferably, the porcine epidemic diarrhea virus comprises porcine epidemic diarrhea virus of subtypes G1 and G2.
Preferably, the reagent comprises a reagent for detecting porcine epidemic diarrhea virus by blocking ELISA, capturing ELISA, indirect ELISA, competitive ELISA and colloidal gold test strip.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a neutralizing monoclonal antibody aiming at a PEDV S2 region for the first time, which has broad spectrum and can effectively neutralize G2 gene subtype strains such as PEDV-LNCT2, PEDV-LNSY, PEDV-Hjms and the like, so that the strains lose the capability of infecting cells and can also neutralize a CV777 strain (G1 gene subtype). In view of the biological characteristics of the monoclonal antibody and the broad-spectrum capability of neutralizing the PEDV strain, the monoclonal antibody can be used for establishing a series of PEDV diagnosis methods including blocking ELISA, capturing ELISA, indirect ELISA, competitive ELISA, colloidal gold test strips and the like, and has wide application value and research value.
Drawings
FIG. 1 is a schematic diagram of epitope identification of 5F7 antibody;
FIG. 2 is an electron microscope viewing of PEDV virions;
FIG. 3 shows IFA detection of 5F7 mab reacted with PEDV;
wherein LNCT2 is 5F7 monoclonal antibody reacting with cells inoculated with LNCT2 strain; CV777 is a reaction between 5F7 monoclonal antibody and cells inoculated with CV777 strain; e6 is the reaction result of 5F7 monoclonal antibody and healthy cells, and the scale in the figure is 400 nm;
FIG. 4 shows the reaction of 5F7 monoclonal antibody with PEDV protein fraction as verified by Western-blot;
wherein, 1: PEDV-LNCT 2S protein; 2: PEDV-LNCT 2; 3: PEDV-CV 777; 4: Vero-E6 cells;
FIG. 5 shows the assay of 5F7 monoclonal antibody neutralization with virus;
FIG. 6 shows epitope identification of monoclonal antibodies;
wherein, figure 1 shows the reaction result of cells transfected by eukaryotic expression plasmids expressing 1261aa to 1386aa in S protein and 5F7 monoclonal antibody; FIG. 2 shows the reaction results of healthy Vero E6 cells with 5F7 mAb, which is scaled at 200 nm.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. 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 modifications may be made without departing from the spirit and scope of the invention.
Example 1 screening and identification of monoclonal antibody having broad-spectrum neutralizing property against porcine epidemic diarrhea virus
1 materials and methods
1.1 cells, test animals, antibodies and reagents
SP2/0 and Vero-E6 cell lines were maintained by the pig digestive tract infectious disease Innovation team of Harbin veterinary institute, national academy of agricultural sciences; RPMI1640 medium (Gibco); HAT and HT medium, PEG (MW4000), Freund's complete adjuvant, Freund's incomplete adjuvant were purchased from Sigma; balb/c mice were purchased from the laboratory animal center.
1.2 cultivation and purification of PEDV
1.2.1 culture of PEDV-LNCT2 Strain
After the Vero-E6 cells grew to form a monolayer, the nutrient solution was discarded, and the cell was washed three times with PBS (phosphate buffered saline, pH7.2) to inoculate the PEDV-LNCT2 strain. The virus was inoculated at a 5% concentration, and pancreatin was added to the virus at a final concentration of 10. mu.g/ml. Observing cell pathological changes, freezing and storing the virus in a-20 refrigerator when 80% -90% of cells have pathological changes, and collecting the virus for later use.
1.2.2 purification of PEDV
Centrifuging the PEDV-LNCT2 virus solution for 5min at 5000g for removing cell debris, centrifuging for 2h at 100000g by an ultracentrifuge, discarding the supernatant, and collecting the precipitate. The pellet was purified by sucrose gradient density centrifugation to set a total of 5 sucrose concentrations, 20%, 30%, 40%, 50% and 60%. After 100000g of a rotor of an ultracentrifuge SW32 is centrifuged for 2h, a sample between 50 percent and 60 percent of sucrose layers is collected, the sample is centrifuged for 2h by 100000g of a rotor of an SW32 for desugarization, supernatant is discarded, and precipitate is collected. And precipitating to obtain purified virus particles.
1.3 preparation of anti-PEDV monoclonal antibodies
1.3.1 immunization of BALB/c mice
Purified PEDV-LNCT2 virions were mixed with equal amounts of Freund's adjuvant and inoculated intraperitoneally with 5 6-week-old BALB/c mice at an antigen dose of 100. mu.g each. The same dose of Freund's incomplete adjuvant-treated antigen was injected 2 weeks later. 3d before cell fusion, the cells can be fused by using an equal amount of antigen without adjuvant (200 mug each) for intraperitoneal injection for 1 time.
1.3.2 establishment of monoclonal antibody screening method
The indirect IFA method is established according to a conventional method. After forming a monolayer by Vero-E6 cells, inoculating PEDV-LNCT2, observing cytopathic effect after 24h, fixing the cells by 4% paraformaldehyde when 50% of the cells have pathological changes, and storing at 4 ℃ to-20 ℃ for later use. Mouse negative serum, positive serum or hybridoma cells to be detected later are used as a detection sample, goat anti-mouse IgG (heavy chain + light chain) marked by Alexa Flur 488 is used as a secondary antibody, reaction is carried out for 45min, observation is carried out under an inverted fluorescence microscope, and a cell hole with the positive cells is recorded.
1.3.3 cell fusion and establishment of Positive hybridoma cell lines
Immunized mice splenocytes were combined with SP2/0 cells according to 5: mixing at a ratio of 1, adding PEG, fusing, and culturing at 37 deg.C in 96-well culture plate with CO2Culturing in an incubator. When the culture solution of the hybridoma cells starts to turn yellow or the hybridoma cells grow to 1/10 of the area of the bottom of the well, the supernatant is aspirated, and positive hybridoma cells are screened by the IFA method. Positive wells were subcloned 3 consecutive times by limiting dilution until all monoclonal wells were positive. And (4) performing expanded culture on the finally obtained monoclonal, establishing strains, and freezing and storing.
1.3.4 preparation of monoclonal antibody ascites and determination of potency
Injecting sterilized paraffin oil (0.5ml each) into abdominal cavity of BALB/c mouse of 8 weeks old, and injecting hybridoma cells with good growth state after 7d at about 5 × 105One mice was injected intraperitoneally with BALB/c. And (3) after about 7 days, the abdomen of the mouse begins to expand obviously, ascites is repeatedly extracted, the mouse is centrifuged at 2000r/min for 10min to remove cell components and other precipitates, and supernatant is collected. Purifying, adding 0.02% sodium azide, packaging, and storing at-70 deg.C. Ascites fluid was diluted incrementally with PBS and titers of mabs were determined by IFA.
1.3.5 Western-blot analysis of monoclonal antibodies
Transferring cell cultures inoculated with PEDV-LNCT2 strain, CV777 strain and LNCT 2S protein to an NC membrane by a semi-dry method under a non-reduction SDS-PAGE method, sealing the NC membrane by 2% skim milk, reacting with 1:10000 monoclonal antibody ascites for 2h, washing for 3 times, adding IRDye 800-labeled goat anti-mouse IgG (ROCKLAND Co.) diluted by 1:10000, reacting for 1h, washing for 3 times, and scanning for imaging.
Subtype identification of monoclonal antibody at 1.3.6
The monoclonal antibodies obtained in the above experiments were subjected to subtype identification according to the instructions of the subtype identification kit.
1.3.7 determination of stability of monoclonal antibody secreted by Positive hybridoma cell line
And (3) recovering the cryopreserved positive hybridoma cells for 3 months, culturing the cells in a 24-hole cell plate, and detecting whether the supernatant is positive to judge whether the recovered positive cells have the capacity of secreting the antibody. Hybridoma cells with a positive rate of 100% after 3 subcloning were subjected to in vitro continuous subculture for 3 months, and cell supernatants were examined 1 time every 2 weeks.
1.3.8 determination of neutralizing Activity of monoclonal antibodies
The monoclonal antibody ascites fluid was filtered through a 0.22 μm filter and purified by protein G purification column. The purified antibody is diluted to 1mg/ml and then diluted 2 times by using RPMI1640 culture solution; PEDV-LNCT2, PEDV-CV777, PEDV-LNSY and PEDV-Hjms four strains of virus were diluted to 200 TCIDs per 100. mu.L of virus solution50Mixing virus solution and antibody in equal volume, reacting at 37 deg.C for 1h, adding pancreatin with final concentration of 10 μ g/ml, inoculating cells, placing in cell culture box for 5 days, and observing cytopathic condition.
1.3.9 epitope identification of monoclonal antibodies
In order to identify the epitope recognized by 5F7, the expression gene of PEDV S protein is truncated as shown in FIG. 1, the truncated gene is inserted into pCDNA3.1 vector to construct eukaryotic expression plasmid, and 293T cell is transfected by successfully constructed plasmid and verified by IFA.
2 results
2.1 purification of PEDV-LNCT2 virions
The virus particles purified by sucrose gradient density are mainly concentrated between 50-60% layers of sucrose through identification, the observation result of a sample through an electron microscope is shown in figure 2, the complete PEDV-LNCT2 virus particles can be seen, and the concentration of the virus particles is measured to be 0.56mg/ml through an absorption photometer.
2.2 preparation of anti-PEDV monoclonal antibodies
2.2.1 establishment of monoclonal antibody hybridoma cell lines
Purified PEDV-LNCT2 virus particles are used as immunogen to immunize BALB/c mice, fused hybridoma cells are subjected to IFA screening positive cloning and then are subjected to subcloning for 3 times to obtain 1 hybridoma cell strain which stably secretes specific antibodies, the hybridoma cell strain is respectively named as 5F7, and the preservation number is CGMCC No. 13840.
2.2.2 IFA analysis of monoclonal antibodies
IFA detection shows that the supernatant of the 5F7 monoclonal antibody can react with cells inoculated with PEDV-LNCT2 and CV777 strains and does not react with healthy Vero-E6, and the result is shown in figure 3.
2.2.3 Western-blot analysis of monoclonal antibodies
The reaction condition of the monoclonal antibody and PEDV is verified by Western-blot test, and the result is shown in figure 4, which shows that the 5F7 monoclonal antibody has specific reaction bands with PEDV-LNCT2 strain, CV777 strain and LNCT 2S protein; no reaction with healthy Vero-E6 cells was observed, indicating that the target protein recognized by 5F7 is the S protein.
2.2.4 preparation of monoclonal antibody ascites
Adopting BALB/c female mouse of 8 weeks old, injecting sterilized paraffin oil (0.5 mL/mouse) through abdominal cavity, injecting hybridoma cells 1 × 10 after 7d5And (4) extracting ascites of each mouse after the abdomen of the mouse is enlarged, centrifuging at 3000r/min for 10min, and taking supernatant. The titer of the antibody of the supernatant and the ascites is detected by an ELISA method. The monoclonal antibody subclass was identified using Mouse MonoAb-ID Kit (HRP), and the procedures were performed according to the Kit instructions.
2.2.5 monoclonal antibody subtype identification
The monoclonal antibody prepared in the test is subjected to subtype identification. The result shows that the 5F7 monoclonal antibody is an IgG2a subtype, and the light chain is a kappa chain.
2.2.6 monoclonal antibody potency assay
IFA detects the titer of 5F7 monoclonal antibody cell supernatant and ascites. The result shows that the 5F7 monoclonal antibody cell supernatant antibody titer is higher than 1:1000, ascites antibody titer higher than 1: 100000.
2.2.7 stability determination of monoclonal antibody secretion from fusion cell line
Recovering the frozen cell strain, culturing in a 24-hole culture plate, detecting cell supernatant by indirect ELISA, and obtaining a result OD405The value is still high. Cells were passaged for 3 months and supernatants were assayed 1 time every 2 weeks and showed OD405The value was stable.
2.2.8 monoclonal antibody neutralization potency assay
As shown in FIG. 5, PEDV-LNCT2, PEDV-LNSY, PEDV-Hjms, and PEDV-CV777 strains were all neutralized by 5F7 mAb, 5F7 mAb was added to the mixture of 1: the 80-fold dilution can completely neutralize four strains of PEDV-LNCT2, PEDV-LNSY, PEDV-Hjms and PEDV-CV 777.
2.2.9 epitope identification of monoclonal antibody
The epitope recognized by the 5F7 monoclonal antibody is between 1261aa and 1386aa by IFA identification. The results are shown in FIGS. 1 and 6.
Claims (5)
1. A hybridoma cell strain capable of stably secreting PEDV (Porcine Epidemic diarrheum Virus) monoclonal antibody is named as 5F7, and is preserved in China general microbiological culture collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.13840 and the preservation time of 2017, 4 months and 24 days.
2. A monoclonal antibody having broad spectrum neutralizing activity against porcine epidemic diarrhea virus, wherein said monoclonal antibody is secreted and produced by the hybridoma cell line of claim 1.
3. The use of the monoclonal antibody of claim 2 in the preparation of a reagent for detecting porcine epidemic diarrhea virus.
4. The use of claim 3, wherein the porcine epidemic diarrhea virus comprises porcine epidemic diarrhea virus subtypes G1 and G2.
5. The use of claim 3, wherein the reagents comprise reagents for the detection of porcine epidemic diarrhea virus by blocking ELISA, capture ELISA, indirect ELISA, competition ELISA and colloidal gold test strips.
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