CN112680421B - Monoclonal antibody with broad-spectrum neutralization of porcine epidemic diarrhea virus and its application - Google Patents

Abstract

The invention discloses a monoclonal antibody with broad-spectrum neutralization of porcine epidemic diarrhea virus and its application. The monoclonal antibody is secreted and produced by the hybridoma cell with the deposit number of CGMCC No.13840. The monoclonal antibody has a broad spectrum and can effectively neutralize the G2 gene subtype strains such as PEDV-LNCT2, PEDV-LNSY and PEDV-Hjms, so that it loses the ability to infect cells, and can also neutralize the CV777 strain ( G1 genotype). Given the biological properties of this monoclonal antibody and its ability to broadly neutralize PEDV strains, this monoclonal antibody can be used to establish a series of assays including blocking ELISA, capture ELISA, indirect ELISA, competitive ELISA and colloidal gold assays The PEDV diagnostic methods including paper strips and other methods have broad application value and research value.

Description

Monoclonal antibody with broad-spectrum neutralization of porcine epidemic diarrhea virus and its application

technical field

The present invention relates to a monoclonal antibody, a hybridoma cell line secreting the monoclonal antibody and its application. In particular, it relates to a monoclonal antibody with broad-spectrum neutralization of porcine epidemic diarrhea virus, a hybridoma cell line secreting the monoclonal antibody and its application. The present invention belongs to the field of biotechnology.

Background technique

Porcine Epidemic Diarrhea Virus (PEDV) is one of the important pathogens that cause diarrhea, weight loss and dehydration in piglets in the pig industry, which brings great economic losses to the pig industry. Studies have shown that PEDV has two genotypes, the G1 and G2 subtypes. Before 2010, the predominant PEDV strains were G1 subtype, and after 2010, the predominant PEDV strains were G2 subtype. Gene sequence analysis showed that the main difference between PEDV G1 and G2 subtypes was in the S gene. The S gene of the G1 subtype strain is composed of 4152nt, encoding 1383 amino acids, while the S gene of G2 is composed of 4161nt, encoding 1386 amino acids, which is an increase of 9 bases in the gene length. There are also large differences within genes. The largest variation in the PEDV genome is located in the S gene, and mutations in the S gene are mainly concentrated in the N-terminus. Compared with NPL-PEDV/2013, NPL-PEDV/2013/P10 was completely identical at the amino acid level in ORF1a/b protein, nuclear protein, NS3B, membrane protein and envelope protein. The spike protein (S) contains six nucleotide polymorphisms, resulting in amino acid site changes: F375L, T486P, D856E, A1081V, A1099S and Y1253D. There are 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 production of neutralizing antibodies, and the mutation of the S gene will inevitably lead to differences in the neutralizing antibodies induced by the S protein in neutralizing the virus.

Therefore, obtaining a monoclonal antibody with broad-spectrum neutralization of porcine epidemic diarrhea virus is of great significance for the prevention and treatment of porcine epidemic diarrhea virus.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a monoclonal antibody with broad-spectrum neutralization of porcine epidemic diarrhea virus, a hybridoma cell line secreting monoclonal antibody and its application.

In order to achieve the above object, the present invention has adopted the following technical means:

The invention uses the purified porcine epidemic diarrhea virus (PEDV) epidemic strain LNCT2 virus particles as antigens, immunizes BALB/c mice, and obtains a hybridoma cell line that stably secretes anti-PEDV monoclonal antibodies, named 5F7. It was identified that the antibody titers of the hybridoma cell culture supernatant and ascites were 1:1000 and 1:100000 respectively. The subclass of the 5F7 monoclonal antibody was IgG2a, and the light chain was κ type. IFA (indirect immunofluorescence detection) detection showed that the 5F7 mAb reacted with the virus; Western blot results showed that the 5F7 mAb reacted with PEDV particles and S protein; the above two results showed that the antigenic epitope of the mAb was linear. The epitope is located between 1261aa and 1386aa of the S protein. PEDV S protein can be divided into two regions: S1 and S2 according to structure and function. The S1 region contains the receptor-binding region of the virus invading cells, and this region is also a key region for inducing the body to produce neutralizing antibodies; the main functions of the S2 region are: Mediates membrane fusion of viruses and cells. The epitope identification found that the epitope of 5F7 antibody is located in the S2 region. The antibody is the first neutralizing antibody against the S2 region of PEDV with broad spectrum. The virus neutralization experiment also showed that the monoclonal antibody can effectively neutralize the G2 subtype strains such as PEDV-LNCT2, PEDV-LNSY and PEDV-Hjms, making it lose the ability to infect cells, and can also neutralize the CV777 strain (G1 genotype). In view of the biological properties of the monoclonal antibody, the monoclonal antibody has the ability to neutralize PEDV strains in a broad spectrum, and the antibody can be used to establish a series of blocking ELISA, capture ELISA, indirect ELISA, competition ELISA and colloidal gold test strips, etc. The diagnostic method has broad application value and research value.

A hybridoma cell line of the present invention that stably secretes an anti-porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV) monoclonal antibody is named 5F7, and is classified as a mouse anti-porcine epidemic diarrhea virus monoclonal antibody cell line , the hybridoma cell line is preserved in the China General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center, CGMCC), and the address is in the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing , the preservation number is CGMCCNo.13840, and the preservation time is April 24, 2017.

Further, the present invention also proposes a monoclonal antibody with broad-spectrum neutralization of porcine epidemic diarrhea virus, which is secreted and produced by the hybridoma cell strain described above in the present invention.

Further, the present invention also proposes the application of the monoclonal antibody in preparing a reagent for detecting porcine epidemic diarrhea virus.

Wherein, preferably, the porcine epidemic diarrhea virus includes G1 and G2 subtype porcine epidemic diarrhea virus.

Among them, preferably, the reagents include reagents for blocking ELISA, capture ELISA, indirect ELISA, competitive ELISA and colloidal gold test strips to detect porcine epidemic diarrhea virus.

Compared with the prior art, the beneficial effects of the present invention are:

The present invention proposes for the first time a neutralizing monoclonal antibody against the S2 region of PEDV. The monoclonal antibody has a broad spectrum and can effectively neutralize the G2 gene subtype strains such as PEDV-LNCT2, PEDV-LNSY and PEDV-Hjms. , it lost the ability to infect cells, and it was also able to neutralize the CV777 strain (G1 genotype). Given the biological properties of this monoclonal antibody and its ability to broadly neutralize PEDV strains, this monoclonal antibody can be used to establish a series of assays including blocking ELISA, capture ELISA, indirect ELISA, competitive ELISA and colloidal gold assays PEDV diagnostic methods including paper strips and other methods have broad application value and research value.

Description of drawings

Fig. 1 is a schematic diagram of 5F7 antibody epitope identification;

Fig. 2 is electron microscope observation PEDV virus particle;

Fig. 3 is IFA detects the reaction of 5F7 monoclonal antibody and PEDV;

Among them, LNCT2 is the reaction between 5F7 monoclonal antibody and cells inoculated with LNCT2 strain; CV777 is the 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 400nm;

Figure 4 shows the reaction between 5F7 mAb and PEDV protein components verified by Western-blot;

Among them, 1: PEDV-LNCT2 S protein; 2: PEDV-LNCT2; 3: PEDV-CV777; 4: Vero-E6 cells;

Fig. 5 is the experimental determination of 5F7 monoclonal antibody and virus neutralization;

Fig. 6 is the antigenic epitope identification of monoclonal antibody;

Among them, picture 1 shows the reaction results of cells transfected with eukaryotic expression plasmids expressing S protein from 1261aa to 1386aa and 5F7 monoclonal antibody; picture 2 shows the reaction results of healthy Vero E6 cells and 5F7 monoclonal antibody. The scale bar is 200 nm.

Detailed ways

The present invention will be further described below with reference to specific embodiments, and the advantages and characteristics of the present invention will become clearer with the description. However, these examples are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Screening and identification of monoclonal antibodies with broad-spectrum neutralization of porcine epidemic diarrhea virus

1 Materials and methods

1.1 Cells, experimental animals, antibodies and reagents

SP2/0 and Vero-E6 cell lines were preserved by the Innovation Team of Porcine Digestive Tract Infectious Diseases, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; RPMI1640 medium (Gibco); HAT and HT medium, PEG (MW4000), Freund's complete adjuvant, Incomplete Freund's adjuvant was purchased from Sigma Company; Balb/c mice were purchased from the Laboratory Animal Center of our institute.

1.2 Culture and purification of PEDV

1.2.1 Culture of PEDV-LNCT2 strain

After Vero-E6 cells became confluent to form a monolayer, the nutrient solution was discarded, washed three times with PBS (phosphate buffered saline, pH 7.2), and inoculated with PEDV-LNCT2 strain. The virus inoculation dose was inoculated at a concentration of 5%, and the virus was inoculated with trypsin at the same time, and the final concentration of the trypsin was 10 μg/ml. Observation of cytopathic changes, when 80% to 90% of cells become diseased, freeze the virus in a -20 refrigerator, collect the virus, and use it for later use.

1.2.2 Purification of PEDV

After the PEDV-LNCT2 virus solution was centrifuged at 5000g for 5min to remove cell debris, it was centrifuged at 100000g for 2h with an ultracentrifuge, the supernatant was discarded, and the precipitate was collected. The pellet was purified by sucrose gradient density centrifugation, and a total of 5 sucrose concentrations were set, 20%, 30%, 40%, 50% and 60%. After centrifugation for 2 hours at 100,000g with a SW32 rotor of an ultracentrifuge, the samples between the 50%-60% sucrose layer were collected. The samples were centrifuged with a SW32 rotor at 100,000g for 2 hours for desugaring treatment, and the supernatant was discarded to collect the precipitate. The pellet is the purified virion.

1.3 Preparation of anti-PEDV monoclonal antibody

1.3.1 Immunization of BALB/c mice

The purified PEDV-LNCT2 virus particles were mixed with the same amount of Freund's adjuvant, and then 5 BALB/c mice aged 6 weeks were intraperitoneally inoculated, and the dose of antigen was 100 μg in each mouse. The same dose of incomplete Freund's adjuvant-treated antigen was injected 2 weeks later. Three days before the cell fusion, the same amount of antigen without adjuvant (200μg per mouse) was injected intraperitoneally to boost the immunization once, and then the cell fusion could be carried out.

1.3.2 Establishment of a screening method for monoclonal antibodies

The indirect IFA method was established according to the conventional method. After Vero-E6 cells formed a monolayer, they were inoculated with PEDV-LNCT2, and cytopathic changes were observed 24 hours later. When 50% of the cells showed pathological changes, cells were fixed with 4% paraformaldehyde and stored at -20°C at 4°C for later use. Mouse negative serum, positive serum or hybridoma cells to be tested in the future were used as samples, and goat anti-mouse IgG (heavy chain + light chain) labeled with Alexa Flur 488 was used as secondary antibody, and observed under an inverted fluorescence microscope after 45 minutes of reaction , the wells with positive cells were recorded.

1.3.3 Cell fusion and establishment of positive hybridoma cell lines

The immunized mouse splenocytes and SP2/0 cells were mixed at a ratio of 5:1 and then fused with PEG, and cultured in a 96-well culture plate at 37°C in a CO ₂ incubator. When the culture medium of hybridoma cells begins 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 IFA method. Positive wells were subcloned three times in a row by limiting dilution until all monoclonal wells were positive. The final obtained monoclonal was expanded and cultured to establish a strain and cryopreserved.

1.3.4 Preparation of monoclonal antibody ascites and determination of titer

8-week-old BALB/c mice were injected intraperitoneally with sterile paraffin oil (0.5 ml each), and 7d later, about 5×10 ⁵ hybridoma cells were injected intraperitoneally into BALB/c mice. Around 7d, the abdomen of the mice began to swell obviously, and the ascites was repeatedly extracted, and the cells and other precipitates were removed by centrifugation at 2000 r/min for 10 min, and the supernatant was collected. After purification, add 0.02% sodium azide, divide the package, and store at -70°C. The ascites fluid was diluted with PBS and the titer of mAb was determined by IFA.

1.3.5 Western-blot analysis of monoclonal antibodies

Under the non-reducing SDS-PAGE method, the cell cultures inoculated with PEDV-LNCT2 strain, CV777 strain and LNCT2 S protein were transferred to NC membrane by semi-dry method. After the NC membrane was blocked with 2% skim milk, it was mixed with 1:10000 mAb was treated with ascites for 2 hours, washed 3 times, added IRDye800-labeled goat anti-mouse IgG (ROCKLAND Co.) diluted 1:10000 for 1 hour, washed 3 times, and scanned for imaging.

1.3.6 Subtype identification Monoclonal antibody subtype identification

The monoclonal antibodies obtained in the above experiments were subtyped according to the operating instructions of the subtype identification kit.

1.3.7 Determination of the stability of monoclonal antibodies secreted by positive hybridoma cell lines

The positive hybridoma cells that have been frozen for 3 months are recovered, cultured in a 24-well cell plate, and whether the supernatant is positive is detected to determine whether the recovered positive cells have the ability to secrete antibodies. Hybridoma cells with a positive rate of 100% after 3 subcloning were continuously subcultured in vitro for 3 months, and the cell supernatant was detected every 2 weeks.

1.3.8 Determination of Neutralizing Activity of Monoclonal Antibody

The monoclonal antibody ascites was filtered through a 0.22 μm membrane and purified with a protein G purification column. The purified antibody was diluted to 1 mg/ml, and then diluted 2-fold with RPMI1640 medium; the four strains of PEDV-LNCT2, PEDV-CV777, PEDV-LNSY and PEDV-Hjms were diluted to contain 200 TCID ₅₀ per 100 μL of virus solution , the virus solution and antibody were mixed in equal volume, reacted at 37°C for 1 h, added trypsin, the final concentration of trypsin was 10 μg/ml, inoculated with cells, placed in a cell incubator for 5 days, and observed cytopathic conditions.

1.3.9 Epitope identification of monoclonal antibody

In order to identify the antigenic epitope recognized by 5F7, the expression gene of PEDV S protein was truncated as shown in Figure 1, and the truncated gene was inserted into the pCDNA3.1 vector to construct a eukaryotic expression plasmid, and the successfully constructed plasmid was transfected 293T cells and validated with IFA.

2 results

2.1 Purification of PEDV-LNCT2 virions

It has been identified that the virions after sucrose gradient density purification are mainly concentrated in the 50%-60% layer of sucrose. The results of electron microscope observation of the samples are shown in Figure 2. The complete PEDV-LNCT2 virions can be seen. The concentration is 0.56 mg/ml.

2.2 Preparation of anti-PEDV monoclonal antibody

2.2.1 Establishment of monoclonal antibody hybridoma cell line

The purified PEDV-LNCT2 virus particles were used as the immunogen to immunize BALB/c mice, and the fused hybridoma cells were screened for positive clones by IFA, and the positive wells were subcloned three times to obtain a hybridoma cell line that stably secretes specific antibodies. , respectively named 5F7, and the deposit number is CGMCC No.13840.

2.2.2 IFA analysis of monoclonal antibodies

IFA test found that the supernatant of 5F7 mAb could react with cells inoculated with PEDV-LNCT2 and CV777 strains, but not with healthy Vero-E6. The results are shown in Figure 3.

2.2.3 Western-blot analysis of monoclonal antibodies

Western-blot test was used to verify the reaction between the monoclonal antibody and PEDV. The results are shown in Figure 4, indicating that the 5F7 monoclonal antibody has specific reaction bands with PEDV-LNCT2 strain and CV777 strain and LNCT2 S protein; -E6 cells did not respond, indicating that the target protein recognized by 5F7 is the S protein.

2.2.4 Monoclonal antibody ascites preparation

Eight-week-old BALB/c female mice were used to inject sterile paraffin oil (0.5 mL/mice) intraperitoneally first, and then 7 days later, intraperitoneal injection of 1×10 ⁵ hybridoma cells/mice were used. The supernatant was collected by centrifugation at 3000 r/min for 10 min. Antibody titers in the supernatant and ascites were detected by ELISA. The Mouse MonoAb-ID Kit (HRP) was used to identify monoclonal antibody subclasses, and the operation was carried out according to the kit instructions.

2.2.5 Monoclonal antibody subtype identification

The monoclonal antibodies prepared in this experiment were subtyped. The results showed that the 5F7 mAb was of IgG2a subtype, and the light chain was κ chain.

2.2.6 Monoclonal antibody titer determination

The titer of 5F7 mAb cell supernatant and ascites was detected by IFA. The results showed that the antibody titer of 5F7 monoclonal antibody cell supernatant was higher than 1:1000, and the ascites antibody titer was higher than 1:100000.

2.2.7 Stability determination of the monoclonal antibody secreted by the fusion cell line

The cryopreserved cell line was recovered and cultured in a 24-well culture plate, and the cell supernatant was detected by indirect ELISA, and the OD ₄₀₅ value was still high. The cells were passaged for 3 months, and the supernatant was tested every 2 weeks, and the results showed that the OD ₄₀₅ value was stable.

2.2.8 Monoclonal antibody neutralization titer determination

As shown in Figure 5, PEDV-LNCT2, PEDV-LNSY, PEDV-Hjms and PEDV-CV777 strains were all neutralized by 5F7 mAb, and 5F7 mAb could completely neutralize PEDV-LNCT2, Four strains of PEDV-LNSY, PEDV-Hjms and PEDV-CV777.

2.2.9 Epitope identification of monoclonal antibody

According to IFA identification, the epitope recognized by 5F7 mAb is located between 1261aa and 1386aa. The results are shown in Figures 1 and 6.

Claims (5)

1. A hybridoma cell line that stably secretes anti-porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV) monoclonal antibody, named 5F7, the hybridoma cell line is preserved in the General Committee of the China Microorganism Culture Collection. Microbiology Center, the preservation number is CGMCC No.13840, and the preservation time is April 24, 2017. 2 . A monoclonal antibody capable of broadly neutralizing porcine epidemic diarrhea virus, wherein the monoclonal antibody is secreted and produced by the hybridoma cell line of claim 1 . 3. The application of the monoclonal antibody of claim 2 in the preparation of a reagent for detecting porcine epidemic diarrhea virus. 4. The use according to claim 3, wherein the porcine epidemic diarrhea virus comprises porcine epidemic diarrhea virus of G1 and G2 subtypes. 5. application as claimed in claim 3 is characterized in that, described reagent comprises the reagent that is used for blocking ELISA, capture ELISA, indirect ELISA, competition ELISA and colloidal gold test strip to detect porcine epidemic diarrhea virus.

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