CN102277333B - Monoclonal antibody resisting foot and mouth disease virus, epitope identified by monoclonal antibody, as well as application of monoclonal antibody - Google Patents

Abstract

The invention discloses a monoclonal antibody resisting the foot and mouth disease virus, epitope identified by the monoclonal antibody, as well as application of the monoclonal antibody, and belongs to the field of prevention and control of the foot and mouth disease. The hybridoma cell line capable of excreting the neutralizing monoclonal antibody resisting O-type FMDV (foot and mouth disease virus) has a microbial preservation number of CGMCC (China General Microbiological Culture Collection Center) 2691. The invention also discloses amino acid sequences of linear neutralization epitope of O-type FMDV VP1 protein identified by the monoclonal antibody respectively. In-vitro neutralization tests and in-vivo animal protection tests prove that: the monoclonal antibody disclosed by the invention has excellent passive immune effect, can be applied to emergency prevention of the foot and mouth disease, and plays an excellent immune effect to the passive immunity of the foot and mouth disease.

Description

Anti-foot-and-mouth disease virus monoclonal antibody and its recognized epitope and application

This case is a divisional application of an invention patent application with the application number "200810171257.8", the invention title "anti-foot-and-mouth disease virus monoclonal antibody and its recognized antigenic epitope and application", and the publication date is "June 9, 2010". document. the

technical field

The present invention relates to monoclonal antibodies, in particular to neutralizing monoclonal antibodies against Asian type 1 FMDV and neutralizing monoclonal antibodies against O-type FMDV, and the present invention also relates to neutralizing epitopes recognized by the above-mentioned monoclonal antibodies, The present invention further relates to the application of the monoclonal antibody and the neutralizing epitope in preventing foot-and-mouth disease, belonging to the field of prevention and treatment of foot-and-mouth disease. the

Background technique

Foot and mouth disease virus (Foot and mouth disease virus, FMDV) is a member of the genus FMDV in the picornaviridae family. Foot-and-mouth disease is one of the important infectious diseases that seriously harms cloven-hoofed animals. The disease causes the death of young animals, decreased milk production, decreased meat consumption, decreased meat products, and decreased animal production performance, resulting in huge economic losses. In addition, the losses caused by trade restrictions and prohibitions are even greater, and the direct economic losses caused by this can reach tens of billions of dollars every year in the world. Since 2005, Asia1 type FMD (Asia1/JS/CHA/05, GenBank accession number: EF149009) has broken out in Jiangsu, Shandong, Gansu, Beijing, Hebei and other places in my country. Because it was first isolated in Jiangsu Province, it is called Asia1 type Jiangsu genealogy. Once the foot-and-mouth disease virus of this lineage became popular among cattle in my country, it spread rapidly and caused extremely serious losses. At the beginning of this century, the O-type foot-and-mouth disease virus pan-Asian lineage was found in China (O/Tibet/CHA/99, GenBank accession number: AJ539138) and neighboring countries and even Europe (Mason PW et al. J Gen Virol. 2003, 84 (Pt 6): 1583-93) were popular, causing huge losses to many countries. Therefore, active research on Asia1 and O foot-and-mouth disease (FMD) is of great significance to the prevention and control of FMD in my country. the

The antigenic structure of foot-and-mouth disease virus is very complex. The antigenic epitopes and antigenic sites of different serotypes, genotypes and isolates of foot-and-mouth disease virus are different, and there are extensive antigenic variations. Using monoclonal antibody escape mutant sequence analysis and cross-neutralization test, some antigenic sites of different serotypes of FMDV have been identified, and the relevant research mainly comes from O, A, C and Asia1 types of FMDV. Neutralizing antibodies play a leading role in protecting susceptible animals against foot-and-mouth disease virus infection. The antigenic sites that induce the body to produce neutralizing antibodies are mainly located on VP1 of each serotype FMDV, and the G-H loop of VP1 is the most important center of the virus. Where the neutral antigenic site is located, there are both linear epitopes and conformational epitopes in this loop. In the VP1 protein of O-type FMDV, the linear epitope of G-H loop 133-157aa and C-terminal 200-213aa constitutes an important antigenic site 1. Saiz et al. reported that FMDV A5 subtype has two neutralizing antigenic sites, one is a linear epitope located at the C-terminus of VP1, the key residue is at 198aa, and the other is located at the B-C loop of VP2, which consists of two epitopes Bit composition, including 72aa and 79aa. Santina Graziolideng further confirmed with a large number of monoclonal antibodies that there are four neutralizing antigenic sites in Asia1 type FMDV: site 1 is located at 142aa of VP1, corresponding to 144aa of O-type VP1, flanking the conserved RGD motif; site 2 Located in the B-C loop region of VP2, including multiple residues such as 67, 72, 74, 77 and 79aa, and associated with 49aa and 207aa of VP1; site 4 is located on the B-B node of VP3, the key residues are 58aa and 59aa, which is associated with site 2; site 5 is located at the C-terminus of VP3, which was first proposed as a newly discovered independent site, and 218aa is the key residue. These foreign studies use monoclonal antibody pressure screening or synthetic peptide scanning technology, which can only determine the amino acids or peptides related to the epitope, but cannot accurately determine the nature and position of the epitope. In my country, articles on neutralizing monoclonal antibodies to Asia1 and O-type FMDV have been published, but no research reports on neutralizing epitopes have been seen. Therefore, the functionality and value of these published neutralizing monoclonal antibodies are still unclear . the

Contents of the invention

One of the purposes of the present invention is to provide a hybridoma cell line that can secrete neutralizing monoclonal antibodies against Asian 1 type FMDV;

Two of the purpose of the present invention is to provide a hybridoma cell line that can secrete neutralizing monoclonal antibodies against O-type FMDV;

The third object of the present invention is to provide the neutralizing epitope amino acid sequence and the neutralizing epitope amino acid sequence of the Asian type 1 FMDV recognized by the above-mentioned neutralizing monoclonal antibody respectively;

The fourth object of the present invention is to use the above-mentioned monoclonal antibody or neutralizing epitope for diagnosis or prevention of foot-and-mouth disease. the

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

A hybridoma cell line that can secrete neutralizing monoclonal antibodies against Asian FMDV type 1, its microorganism preservation number is: CGMCC 2692; preservation date: October 8, 2008; preservation unit: China Microorganism Culture Preservation Management General Microbiology Center of the Committee; Preservation Address: Datun Road, Chaoyang District, Beijing. The neutralizing monoclonal antibody against Asian type 1 FMD secreted by the hybridoma cell line is named 3E11;

A hybridoma cell line that can secrete neutralizing monoclonal antibodies against O-type FMDV, its microorganism preservation number is: CGMCC 2691; preservation date: October 8, 2008; preservation unit: China Microorganism Culture Preservation Management Committee General Microbiology Center; Preservation Address: Datun Road, Chaoyang District, Beijing; The neutralizing monoclonal antibody against O-type FMDV secreted by the hybridoma cell line is named 8E8;

Asian type 1 FMDV conformational neutralizing epitope recognized by monoclonal antibody 3E11, the key amino acid residues of this conformational epitope are Ser ¹⁴⁰ or Ser ¹⁴¹ , Gly ¹⁴⁴ , Leu ¹⁴⁶ on the VP1 protein of Asian type 1 FMDV and Leu ¹⁴⁹ ; further preferably, the amino acid sequence of the mimic epitope of the conformational epitope is GSLXXL (SEQ ID NO: 1), wherein, X is selected from any amino acid residue;

A linear neutralizing epitope sequence in the VP1 protein of O-type FMDV recognized by monoclonal antibody 8E8, its amino acid sequence is GDLNVRT (SEQ ID NO: 2) or its derivative sequence;

Wherein, the derivative sequence is any amino acid sequence obtained by deleting and/or replacing one or more amino acids in GDLNVRT, wherein the deletion is G deletion; the replacement is selected from Any one or combination of more than one of the following 6 types: (1) G is replaced by W; (2) L is replaced by A; (3) N is replaced by Q, A or W; (4) V is replaced by I or T replacement; (5) R is replaced by L, K or A; (6) T is replaced by A; it has been proved by experiments that the above-mentioned derivative sequence obtained after deletion and/or replacement has the same performance as GDLNVRT use. the

Detailed description of the technical solution of the present invention:

The present invention immunizes BALB/c mice with purified whole virus to prepare monoclonal antibodies against Asia1 type FMDV Jiangsu lineage Asia1/YS/CHA/05 strain and O type FMDV pan sublineage O/YS/CHA/05 strain, each obtaining one Strains have strong neutralizing activity of monoclonal antibodies 3E11 and 8E8. Indirect immunofluorescence and Western blot detection showed that 3E11 recognized a conformational dominant neutralizing epitope of Asia1 type FMDV, and the monoclonal antibody did not react with a parental mutant strain (144 was mutated from G to S), so it was inferred that 144 on the VP1 protein Gly is the key amino acid of the epitope recognized by monoclonal antibody 3E11; 8E8 recognizes a broad-spectrum linear neutralizing epitope of O-type FMDV. the

The present invention further utilizes the phage display random 12 peptide library to screen the mimic epitopes recognized by the above two monoclonal antibodies, and finds that an epitope motif GSLXXL (X represents any one of 20 amino acid residues) has binding activity to the monoclonal antibody 3E11, while The mimotope motif of mAb 8E8 is GDLNVRT. the

In view of the characteristics of the two monoclonal antibodies, the site-directed mutagenesis analysis of the four conserved amino acid residues of the mimic epitope motif recognized by the monoclonal antibody 3E11 was carried out with synthetic peptides, and these amino acids were all key amino acids; Asia1/YS/ The 136-153aa sequence of the GH loop of the VP1 protein of the CHA/05 strain, the octadecapeptide contains all four key amino acids of the GSLXXL motif, and the peptide ELISA test shows that it has the same binding activity as the monoclonal antibody 3E11, thus determining the key to the conformational epitope The active amino acid residues are composed of Ser ¹⁴⁰ or Ser ¹⁴¹ , Gly ¹⁴⁴ , Leu ¹⁴⁶ and Leu ¹⁴⁹ on the VP1 protein.

The mimotope motif of monoclonal antibody 8E8 is highly homologous to the 146-152 amino acid sequence of the VP1 protein of the O/YS/CHA/05 strain, so ^{the 146} GDLQVLT ¹⁵² sequence and its truncated peptide sequence were fused with GST to express and Westernized Blot analysis confirmed that ¹⁴⁷ DLQVLT ¹⁵² is a linear B-cell epitope on the VP1 of type O FMDV; a series of site-directed mutagenesis peptides (P-8E8) were synthesized for the 146-152aa peptide (P-8E8) in the true sequence of the virus VP1 and analyzed by competition ELISA. It was shown that the amino acid residue D is the most critical for the activity of the epitope, the residues V, L ¹⁴⁸ , L ¹⁵¹ , Q and T play an important role in the activity of the epitope, and the residue T cannot be deleted.

In view of the high neutralization titer of the above two monoclonal antibodies in vitro, the present invention further uses the suckling mouse protection test to verify the immune protection effect of the above two monoclonal antibodies against FMDV infection in vivo. The results of the test show that the half protective doses (PD ₅₀ ) of monoclonal antibody _3E11 and 8E8 ascites against Asia1 _type and O type FMDV are respectively 1995 and 2371; Protection of suckling mice against lethal doses of Asia1 and O-type FMDV challenges. The percentage of body weight gain of passively immunized suckling mice after challenge increased with the increase of monoclonal antibody concentration, and with the increase of monoclonal antibody concentration, the initial death time of suckling mice was delayed and the terminal death time was moved forward.

In vitro neutralization test and in vivo animal passive immune protection test show that the 3E11 monoclonal antibody of the present invention and the 8E8 monoclonal antibody have a good passive immune effect, and these two strains of monoclonal antibodies can be applied to the emergency prevention of foot-and-mouth disease, and can be used for the prevention of foot-and-mouth disease. Passive immunization has a good immune effect; the protective antigenic epitope of the FMDV serotype recognized by these two monoclonal antibodies can be further studied or developed as a bivalent epitope vaccine for foot-and-mouth disease, which will have important significance for the immune prevention of foot-and-mouth disease . the

Description of drawings

Figure 1 ELISA method to screen phages with specific affinity for monoclonal antibody 3E11; each phage clone with the same number of plaques was added to a microwell plate coated with monoclonal antibody 3E11, and monoclonal antibody 4C6 of the same antibody subtype, containing BSA The blocking solution and the original peptide library (Phage Library, PL) were used as controls. the

Figure 2 Detection of the binding activity of the synthetic peptide P-S7 and its mutant peptides to monoclonal antibody 3E11; coated with streptavidin, adding serially diluted biotinylated short peptide P-S7 and randomly scrambled peptide P-S7- After the SCR reacts with 3E11, add HRP-labeled goat anti-mouse secondary antibody to develop color and measure the absorbance value. the

Figure 3 Detection of the binding activity of the synthetic peptide P-S7 and its mutant peptides to the monoclonal antibody 3E11; the synthetic peptide P-S7 was used to competitively inhibit the binding of the monoclonal antibody 3E11 to the viral antigen: 1ug of the viral antigen was coated, and the synthetic peptide and the monoclonal antibody 3E11 were added Master mix, and finally add goat anti-mouse enzyme-labeled secondary antibody. the

Figure 4 Detection of the binding activity of the synthetic peptide P-S7 and its mutated peptides to the monoclonal antibody 3E11; the synthetic peptide site-directed mutagenesis test identifies the key amino acids for the interaction between the P-S7 peptide and the monoclonal antibody 3E11, and the monoclonal antibody 3E11 and the unmutated short peptide The OD ₄₀₅ value of P-S7 reaction was 100%, and the binding rate of other short peptides was calculated.

Figure 5 The binding activity of the synthesized 18-aa GH cyclic octadecapeptide to monoclonal antibody 3E11; coated with streptavidin, added serially diluted biotinylated short peptide Asia1pG-H, reacted with monoclonal antibody 3E11, and added HRP labeling The goat anti-mouse secondary antibody was used to measure the OD ₄₀₅ absorbance value after color development.

Figure 6 ELISA method for screening phages with affinity to monoclonal antibody 8E8; each phage clone with the same number of plaques was added to a microwell plate coated with monoclonal antibody 8E8, and monoclonal antibody 5F7 of the same antibody subtype was blocked with 1% BSA Liquid and phage peptide library (Phage Library, PL) were used as controls. the

Figure 7 Western blot analysis of positive phage clones. the

Figure 8 SDS-PAGE (a) and Western blot analysis (b) of the epitope and its truncated peptide GST fusion protein. the

Figure 9 Competition ELISA experiment of synthetic peptides; synthetic mutant peptides are used to competitively inhibit the binding of monoclonal antibody 8E8 to the virus. the

Fig. 10 Competition ELISA experiment of synthetic peptides; the peptide concentration is 1ug/100ul, and the relative inhibition rate of each synthetic mutant peptide is calculated with the inhibition rate of P-8E8 being 100%. the

Figure 11 Competition ELISA experiment of synthetic peptides; synthetic mutant peptides are used to competitively inhibit the binding of monoclonal antibody 8E8 to the virus. the

Figure 12 Competition ELISA experiment of synthetic peptides; the peptide concentration was 1ug/100ul, and the relative inhibition rate of each synthetic mutant peptide was calculated as the inhibition rate of P-8E8 was 100%. the

Figure 13 The relationship between the concentration of monoclonal antibody 3E11 and the survival rate of suckling mice; after the suckling mice were challenged with 10LD ₅₀ Asia1/YS/CHA/05, different concentrations of monoclonal antibody 3E11 (that is, the half-protection amount of different neutralizing antibodies) led to the survival rate of suckling mice difference.

Figure 14 The relationship between the concentration of monoclonal antibody 8E8 and the survival rate of suckling mice; after the neonatal mice were challenged by 10LD ₅₀ O/YS/CHA/05, different concentrations of monoclonal antibody 8E8 (that is, the half-protection amount of different neutralizing antibodies) led to the survival rate of suckling mice difference.

Figure 15 The relationship between the concentration of monoclonal antibody 3E11 and the survival time of suckling mice; after the neonatal mice were challenged with 10LD ₅₀ Asia1/YS/CHA/05, different concentrations of monoclonal antibody 3E11 lead to differences in the death time of suckling mice.

Figure 16 The relationship between the concentration of monoclonal antibody 8E8 and the survival time of suckling mice; after the neonatal mice were challenged with 10LD ₅₀ O/YS/CHA/05, different concentrations of monoclonal antibody 8E8 lead to differences in the death time of suckling mice.

Figure 17 The relationship between the concentration of monoclonal antibody 3E11 and the percentage of body weight growth of suckling mice; the difference in percentage of body weight growth of suckling mice caused by different concentrations of monoclonal antibody 3E11 after challenged with 10LD ₅₀ Asia1/YS/CHA/05.

Figure 18 The relationship between the concentration of monoclonal antibody 8E8 and the percentage of body weight growth of suckling mice; the difference in percentage of body weight growth of suckling mice caused by different concentrations of monoclonal antibody 8E8 after challenged with 10LD ₅₀ O/YS/CHA/05.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand 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. the

1. Materials and methods

1. Viruses, cells, strains and experimental animals

The foot-and-mouth disease virus strain Asia1/YS/CHA/05 belongs to the Asia1 type FMDV Jiangsu lineage, and the foot-and-mouth disease virus O/YS/CHA/05 strain belongs to the O-type pan-sublineage; the suckling hamster kidney cells BHK-21 and SP2/0 myeloma cells are mainly Laboratory preservation; plasmid pGEX-6p-1 and recipient strain BL21 were preserved by our laboratory; clean grade female BALB/c mice and SPF grade BALB/c suckling mice were purchased from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. the

2. Main reagents

Fusion agent PEG/DMSO (Mw, 1450), HAT salt (50x), HT salt (50x), HRP or FITC-labeled goat anti-mouse IgG, Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma; Monoclonal antibody subclass identification kits were purchased from Southern Biotech; L-glutamine and glycine were purchased from Amresco; dimethyl sulfoxide (DMSO), o-phenylenediamine (OPD), and PEG6000 were purchased from Solarbio Ph.D.-12 ^TM peptide library kit was purchased from New England Biolabs; HRP-labeled anti-M13 phage antibody was purchased from GE Healthcare; restriction enzymes BamHI, XhoI, and PstI were purchased from TaKaRa; T4DNA Ligase was purchased from New England Biolabs; high-sugar DMEM dry powder was purchased from GIBCO; imported premium fetal bovine serum (PAA) was purchased from Nalgene, Spain; 96-well cell culture plates were purchased from JET Biochemical, Canada.

3. Rat immunization and preparation of monoclonal antibodies

(1) Inoculate the Asia1 type and O type FMDV cell-adapted virus into BHK-21 cells that are overgrown with a single layer, and collect the virus after more than 75% of the cells have pathological changes. The harvested cell culture medium was repeatedly frozen and thawed three times to initially lyse the cells and release the virus. The frozen-thawed virus solution was added to TritonX-100 at 1:1000 and formaldehyde at 4:10000 to lyse and inactivate for more than 48 hours, and 1 mL of the inactivated virus solution was taken to BHK-21 cells for three generations to test whether the inactivation was complete. Centrifuge the freeze-thawed culture solution at 9000 rpm (Beckman high-speed centrifuge, JA-10 rotor) at 4°C for 90 min, recover the supernatant of the virus solution, and discard the cell pellet. Add 80g/L PEG6000 and 40g/L NaCl according to the volume of the virus liquid supernatant, stir at room temperature for 2h until completely dissolved, and precipitate overnight at 4°C. The next day, centrifuge the supernatant at 9000 rpm and 4°C for 90 min, discard the supernatant, and recover the precipitate. The pellet was gently resuspended in NET buffer at low temperature. Centrifuge the resuspended supernatant at 29,000 rpm at 4°C for 2 hours to recover the precipitate, gently resuspend the precipitate with an appropriate amount of NET buffer at low temperature, mix well, aliquot, and freeze at -70°C for future use. The purified antigen was diluted 10 times, 100 times and 1000 times with NET buffer, and the protein concentration was measured with a spectrophotometer. the

(2) Use 200 μg/200uL of purified Asia1 and O-type FMDV antigens plus an equal volume of Freund’s complete adjuvant, fully emulsify, and subcutaneously inject 6-week-old female BALB/c mice on the back; perform the second immunization after 2 weeks , the adjuvant was Freund's incomplete adjuvant; after an interval of 2 weeks, the third immunization was carried out with the same amount of antigen without adjuvant. In order to stimulate the mice to quickly produce a strong immune response, 3 days before the fusion, the combination of tail vein and spleen injection was used for booster immunization, and double the amount of antigen was injected. the

(3) cell fusion

a) Preparation of feeder layer cells: The feeder layer cells were prepared the day before cell fusion, ophthalmic scissors, ophthalmic tweezers, plates and other test equipment were sterilized by high temperature and dry heat before use, and HAT complete culture medium was used for sterility test. The eyeballs of 2 BALB/c mice were enucleated and bled, and the negative serum was separated by conventional methods. The mice were killed by cervical dislocation, soaked in 75% alcohol, and moved to the ultra-clean workbench after 10 minutes. Fix the abdomen of the mouse on the mouse frame upwards, lift the skin in the middle of the abdomen with tweezers, cut a small mouth horizontally with ophthalmic scissors, do not cut the peritoneum, and tear the skin up and down with scissors and tweezers to fully expose the peritoneum. Gently lift the peritoneum with tweezers, inject 8-10mL HAT complete culture solution drawn from a 10mL syringe into the abdominal cavity, do not puncture the viscera and intestinal tract, do not pull out the syringe, pump back and forth in the abdominal cavity 5-10 times, and then Massage the abdomen on both sides with tweezers for about 30 seconds, then perform suction, and repeat the above operation 2-3 times. Aspirate intraperitoneal fluid with a syringe. Take care to avoid mesentery and adipose tissue, so as not to block the needle. Add the peritoneal cells taken from 2 mice into 55 mL of HAT culture medium, blow and mix the cells, and distribute them in six 96-well culture plates, 100 μL/well. Place them in a 37°C, 5% _CO2 incubator.

b) Preparation of myeloma cells: 36-48 hours before fusion, the myeloma cells were expanded and cultured, so that the cells were in logarithmic growth phase. On the day of fusion, use 15mL DMEM basal medium to blow off the cells from the bottle wall and collect them in a 50mL centrifuge tube. Centrifuge at 1000rpm for 10min. Resuspend the cell pellet in 20mL DMEM basal culture medium and mix well. Take a small amount of myeloma cell suspension, trypan blue staining and counting, set aside. the

c) Preparation of immune splenocytes: before fusion, mouse eyeballs were removed to collect blood, and positive serum was prepared. The mice were killed by cervical dislocation, soaked in 75% alcohol, and placed on a clean table after 10 minutes. Aseptically open the abdominal cavity, separate the connective tissue and take out the spleen, put the spleen into a plate with sterilized nylon mesh and 15 mL of DMEM basal culture solution, and grind the spleen with the inner core of a sterilized glass syringe to make all the spleen cells pass through the mesh into the dish. Transfer the splenocyte solution into a 50mL centrifuge tube, add DMEM basal culture medium to about 30mL, and mix well. Centrifuge at 1000rpm for 8min, discard the supernatant. Suspend the cell pellet in 10mL DMEM basal culture medium and mix well. Take the cell suspension, trypan blue staining and counting, set aside. the

d) Fusion of splenocytes and myeloma cells: Preheat 65mL of HAT culture medium, 15mL of basic DMEM culture medium and 1mL of 50% PEG in a 37°C water bath, and prepare a 200mL beaker filled with 37°C water. Take 5 parts of spleen cells plus 1 part of myeloma cells to take the corresponding amount of cell suspension, add it to a 50 mL glass centrifuge tube, add DMEM basic culture medium to 30 mL, and mix well. Centrifuge at 1000rpm for 10min, discard the supernatant, and drain as much as possible. Lightly tap the bottom of the centrifuge tube with your palm to loosen the pelleted cells and make a paste. Put the centrifuge tube into a 200mL beaker filled with 37°C water, rotate the centrifuge tube evenly with one hand, and use the other hand to absorb 1mL of 37°C 50% PEG solution with a 1mL Pasteur pipette, add it within 1min, and let stand for 2min. Then, the DMEM basal culture solution was added slowly and then quickly to terminate the reaction, and the reaction was left in a water bath at 37°C for 10 minutes. Centrifuge at 1000rpm for 10min, discard the supernatant, add 65mL of HAT medium, gently blow off the cells, inoculate 0.1mL per well on six 96-well culture plates that have been cultured with feeder cells, and place in a 37°C, 5% _CO2 incubator cultivated in. After 5 days, half of the medium was changed, and after 8 days, the whole medium was changed. When the clones grew to 1/4-1/3 of the area of the bottom of the well, the supernatant was taken for detection and replaced with HT medium.

4. Antibody detection ELISA

Coat the purified viral antigen on a microwell plate, add 100uL hybridoma cell culture supernatant, incubate at 37°C for 1 hour, wash, add 1:5000 diluted HRP-labeled goat anti-mouse secondary antibody, add substrate after washing OPD was protected from light for 10 minutes to develop color, and the absorbance value was measured at a wavelength of 492 nm. the

5. Indirect Immunofluorescence

Cultivate BHK-21 cells in a microwell plate to a single layer, inoculate foot-and-mouth disease virus 4×10 ³ TCID ₅₀ /well, fix with cold absolute ethanol before CPE appears, add 50uL hybridoma cell culture supernatant, and incubate at 37°C for 40min Wash with PBS three times, add 1:200 diluted fluorescein-labeled goat anti-mouse IgG antibody, incubate at 37°C for 40 min, observe under an inverted fluorescent microscope after washing, and record the fluorescence intensity as the number of +.

6. Trace cell neutralization test

(1) Determination of virus toxicity: inoculate the virus on a monolayer of cells, add maintenance solution after adsorption at 37°C for 1 hour, and cultivate in an incubator; observe daily, and when the cytopathic pathology (CPE) reaches more than 75%, harvest the virus suspension and freeze Melt 3 times, centrifuge at 3000r/min for 10min, take the supernatant, quantitatively aliquot into 1ml vials and store at -70°C for later use. The virus selected must have relatively stable pathogenicity to cells. Take a bottle of virus stored in a -70°C refrigerator, and make a 10-fold progressive dilution of the virus on a 96-well culture plate, that is, 10 ^-1 , 10 ^-2 , 10 ^-11 ..., and the amount of virus suspension in each well is 50 μl. Make 8 wells for each dilution, add 100 cell suspensions to each well, and set 8 wells of cells as a control in the last row of each plate, and prepare the concentration of the cell suspension so that the cells grow into a single layer within 24 hours. Place the culture plate in a 5% CO ₂ incubator and culture at 37°C, observe the cytopathic changes every day from 48 to 72 hours, and record the results.

Calculate TCID ₅₀ according to the method of Reed and Muench.

Table 1

TCID ₅₀ calculation (inoculation dose 50μl)

Ig TCID ₅₀ = log of virus dilution above 50% - distance ratio x log of dilution factor

Above 50% virus dilution has a logarithm of -6, a distance scale of 0.26, and a logarithm of the dilution factor of -1. the

IgTCID ₅₀ =-6+0.26×(-1)

＝-6.3

Then TCID ₅₀ =10 ^-6.3 , 50 means that the virus is diluted by 10 ^-6.3 and inoculated with 50 μl per well, which can make half of the tissue cell tubes pathological.

(2) Neutralization test: Animal ascites contains a variety of protein components, such as complement, immunoglobulin and anti-complement antibody, which have auxiliary effects on antibodies to neutralize viruses. In order to exclude these heat-labile non-specific reaction factors, the serum or ascites used in the neutralization test must be inactivated by heating at 56°C for 30 minutes. Take the inactivated monoclonal antibody ascites, and make a series of doubling dilutions with serum-free DMEM on a 96-well micro-cell culture plate, so that the dilutions are 1:4, 1:8, and 1 :16, 1:32, 1:64, etc., the content of each well is 50 μl, and each dilution is 4 wells. Take the virus solution stored in a -70°C refrigerator, and dilute the original virus concentration to 200TCID ₅₀ , 50ul (mixed with the same amount of ascites, the virus concentration is 100TCID ₅₀ ). If the virus titer is 10 ^-6.3 , 50 μl. Therefore, the virus should be diluted 2×10 ^-4.3 . Add 50 μl of virus solution to each well, seal the lid, and place in a 37°C incubator for 1 hour. When preparing the cell suspension, the concentration is based on the monolayer growing within 24 hours: the serum and the virus are neutralized for 1 hour, and 100 μl of the cell suspension is added to each well. Place them in a 37°C incubator with 5% CO ₂ , observe and record them daily from 48 hours of cultivation, and make final judgments at 120 hours. In order to ensure the accuracy of the test results, the following controls must be set for each test. The positive and negative (sp2/0) ascites were tested in parallel with the ascites to be tested. The positive ascites control should have no cytopathic changes, while the negative ascites controls should have cytopathic changes. A virus control was set up on each plate for each test, and the virus was firstly diluted at 0.1, 1, 10, 100, 1000 TCID ₅₀ , each dilution was made into 4 wells, and 50 μl was added to each well. Then 100 μl of cell suspension per well. 0.1TCID TCID ₅₀ should not cause cytopathic, and 100TCID ₅₀ must cause cytopathic, otherwise the test cannot be established. In order to check whether the tested ascites itself has any toxic effect on the cells, it is necessary to set up the tested ascites toxicity control. That is to add low-fold dilution of the ascites to be tested (equivalent to the lowest dilution of the ascites to be tested in the neutralization test) to the tissue cells. The normal cell control refers to the cell suspension wells not inoculated with virus and the ascites fluid to be tested. The normal cell control should maintain good shape and living characteristics throughout the neutralization test. In order to avoid experimental errors caused by the culture plate itself, this control should be set up on each plate. When the virus regression test, positive, negative, normal cell control, and ascites toxicity control are all established, the judgment can be made. 100% CPE in the tested ascites hole is judged as negative, and more than 50% of the cells are protected as positive; fixed virus dilution The calculation of the results of the ascitic fluid neutralization test is to calculate the ascitic fluid dilution that can protect 50% of the cell pores from causing cytopathic changes, and this dilution is the neutralizing antibody titer of the serum. The results were calculated using Reed and Muench's method (or Karber's method).

Table 2

Calculation of neutralizing antibody titer by fixed virus-diluted serum method

Use Reed and Muench's method (or Karber method) to calculate the result

IgPD ₅₀ = log of serum dilution above 50% - distance ratio x log of dilution factor

IgPD ₅₀ =-1.5-0.5×(-0.3)=-1.35

Then PD ₅₀ =10 ^−1.36 , 50 μl

Because 10 ^-1.35 = 1/22, that is, the serum of 1:22 can protect 50% of the cells from pathological changes, and 1:22 is the neutralizing antibody titer of this serum.

7. Biopanning

The ascites was first crudely extracted by octanoic acid-ammonium sulfate method, and then purified by affinity chromatography with NAb ^TM Protein G Spin Purfiication Kit (PIERCE). After the purity was identified by SDS-PAGE, it was used for biopanning, and M13 phage was used to display random linearity. Dodecapeptide library for epitope mapping of mAbs. Biopanning was carried out according to the kit instructions: in the first round of panning, 10ug of monoclonal antibody was coated in a microwell plate, in the second and third rounds, 5ug and 1ug were respectively coated, and overnight at 4°C; after washing with 0.05% TBST, wash with 10g/ LBSA blocking, adding 1.5×10 ¹¹ pfu/100uL M13 phage displaying dodecapeptide, shaking at room temperature for 1 hour, washing with TBST for 10 times, using elution buffer to elute the bound phage at room temperature for 10 minutes, adding buffer The eluted phages were neutralized in the solution and inoculated with Escherichia coli ER2738 for amplification. The amplified phages were recovered and titered, and 1.5×10 ¹¹ pfu phages were taken to enter the next round of elutriation. In the fourth round of elutriation, the protein G capture method was used for liquid phase binding, and the amount of monoclonal antibody used was 300ng. A single phage clone was picked for amplification, its activity was identified by phage affinity capture ELISA, and finally the single-stranded DNA of the positive phage was sequenced.

8. Phage affinity capture ELISA

Coat 100ng of monoclonal antibody (100uL per well, 0.1M sodium bicarbonate pH8.6) in a 96-well polyethylene microtiter plate, overnight at 4°C, set up duplicate wells, and set up the recognition of different epitopes from the same mouse Prepared monoclonal antibody, 1% BSA blocking solution was used as control. After blocking at room temperature for 2 hours, dilute the phage clones with 0.1% TBST to 10 ¹⁰ pfu/100uL, add to each well, shake gently at room temperature for 1 hour, wash with 0.1% TBST six times, add 1:5000 dilution of HRP-labeled mouse anti-M13 For phage antibody, use the substrate ABTS[2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)] to develop color and then measure the absorbance at 405nm, and take P/N>2.1 as the criterion for judging the positive result of the sample to be tested.

9. Preparation and sequencing of phage single-stranded DNA template

After the third and fourth rounds of panning, phage clones were randomly selected and inoculated into 1 mL of 1:100 diluted ER2738 mid-log phase culture, and incubated at 37°C for 4.5 h with shaking. Centrifuge at 12000rpm for 10min, take 500uL supernatant into new tubes, add 200uL PEG/NaCl to each tube, mix well, let stand for 10min, centrifuge at 12000rpm for 10min, dissolve the precipitate in 100uL sodium iodide buffer, then add 250uL anhydrous Ethanol, let stand at room temperature for 10min, centrifuge at 12000rpm for 10min, discard the supernatant, wash the precipitate with 70% ethanol, dry it naturally, dissolve it with 30uL pure water, and then use it as a sequencing template. The sequencing was done by Shanghai Bioengineering Co., Ltd. The sequencing primer was -96gIII, and the sequence was: 5'-CCC TCA TAG TTA GCG TAA CG-3'. the

10. Peptide ELISA

Various peptides were synthesized by Beijing Zhongke Yaguang Co., Ltd. and identified by HPLC and MS analysis, with a purity of ≥95%. Biotinylation of peptides was performed using a biotinylation kit from PIERCE. Add 200ng, 100ng, 50ng, 25ng, 12.5ng, 6.25ng, 3.125ng, and 1.56ng of biotinylated peptides to the wells coated with 250ng streptavidin, add 3E11 monoclonal antibody 100ng/100ul/well after blocking, Gently shake at room temperature for 1 h, wash four times with 0.05% TBST, add 1:5000 diluted HRP-labeled goat anti-mouse secondary antibody (SIGMA), and measure absorbance at 405 nm after color development with substrate ABTS. the

11. Peptide Competitive Inhibition ELISA

Coat viral antigen 1ug/well in the microtiter plate, pre-mix the serially diluted synthetic peptide and monoclonal antibody in the blocking solution, 37°C for 1h, then overnight at 4°C, then add to each well, incubate at 37°C for 20min, wash Goat anti-mouse enzyme-labeled secondary antibody was added, and the absorbance was measured at 405nm after color development with the substrate ABTS. the

12. GST fusion expression of epitope peptide

Artificially synthesize two complementary oligonucleotide chains encoding the epitope and the truncated epitope DNA. The BamHI sticky extension end is introduced upstream, and the stop codon and XhoI sticky extension end are introduced downstream. The epitope gene is selected according to the codon preference of E. coli For codon optimization, the sequence is shown in Table 3:

Table 3 Complementary oligonucleotide chains of DNA encoding epitopes and their truncated mutants

(The boxed part is the introduced BamHI sticky end, and the underline is the introduced XhoI sticky end). the

The two complementary strands were directly annealed to form double-stranded DNA with cohesive ends, inserted between BamHI and XhoI of pGEX-6p-1, transformed into BL21 competent cells, identified recombinant plasmids with PstI digestion, and named them respectively (Table 1). Pick a single positive colony for overnight culture and sequence verification, then inoculate in fresh liquid LB medium at 1:100 dilution, shake culture at 37°C until OD _600nm = 0.6, add inducer IPTG to a final concentration of 1mmol/L, 37°C Continue to culture for 5 hours, collect the bacteria and resuspend with an appropriate amount of PBS, and the fusion proteins are named as GST-p7, GST-p7-lossG, GST-p7-lossT, GST-p7-lossLT and GST-p7-lossVLT.

13. SDS-PAGE electrophoresis and Western blot detection

SDS-PAGE electrophoresis (15% Tris-glycine) to separate GST-p7, GST-p7-lossG, GST-p7-lossT, GST-p7-lossLT and GST-p7-lossVLT fusion proteins, transfer to nitrocellulose filter membrane On (90V, 45min), block overnight at 4°C with 5% skimmed milk-PBS blocking solution, incubate with monoclonal antibody 8E8 at 37°C for 1h, react with HRP-labeled goat anti-mouse IgG for 1h at room temperature, DAB (6mg/ 10mL) for color development. the

14. FMDV LD ₅₀ detection

There are 5-7 litters of 4-day-old suckling mice, 4-6 in each litter, and 1 female mouse is used for breastfeeding. Use sterilized PBS to dilute Asia1 type and O type FMDV in 10-fold gradients to 10 ^-1 , 10 ^-2 , 10 ^-3 ......, each dilution of FMDV is subcutaneously injected into a litter of suckling mice on the back of the neck, 200ul/ At the same time, a PBS control group was set up. Observe every day, record the morbidity and death of suckling mice, and calculate the virulence of foot-and-mouth disease virus according to the Reed-Muench method.

Table 4

Calculation of LD ₅₀ (inoculation dose is 0.2ml)

Calculation of LD ₅₀ : Calculated according to Reed and Muench's method.

Logarithm of LD ₅₀ = logarithm of virus dilution above 50% + distance ratio × logarithm of dilution factor

This example is above the log -6 of the 50% virus dilution, the distance scale is 0.5, and the log of the explanatory coefficient is -1. Substitute into the above formula:

_LgLD50 ＝-6+0.5×(-1)＝-6.5

Then LD ₅₀ =10 ^-6.5 , 0.2ml, that is, the virus is diluted 10 ^-6.5 , and inoculation of 0.2ml can kill half of the suckling mice.

15. Immune protection test in suckling mice

5-8 litters of suckling mice were used in the passive protection test of Asia1 type and O-type monoclonal antibody, each litter contained 4-6 suckling mice, and 1 female mouse was used for breastfeeding. The 3E11 and 8E8 monoclonal antibody ascites were diluted with sterilized PBS to 2 times respectively, and each dilution was intraperitoneally injected into 4-6 neonatal mice, 200ul/mouse. After an interval of 1 hour, each group of suckling mice was subcutaneously injected with a lethal dose (10LD ₅₀ ) of Asia1 type and O type FMDV (200ul/mouse) on the back of the neck, and a monoclonal antibody control and a PBS control were set up at the same time. After the injection, the morbidity and death of the suckling mice were observed every day.

2. Test results

(1) Preparation of 3E11 monoclonal antibody and identification of its recognition epitope

1. Screening and identification of a monoclonal antibody against Asia1 type FMDV conformational neutralizing epitope

The culture supernatant of hybridoma cells fused with splenocytes and SP2/0 cells of immunized mice was detected by indirect ELISA and _verified by IFA. /c The ratio of mouse serum and SP2/0 cell supernatant to FMDV antigen OD ₄₉₂ value is greater than 2.1, and the immunofluorescence result of hybridoma cell supernatant to normal BHK-21 cells is negative, and it is judged as positive hybridoma cell. After screening and cloning by limiting dilution three times, three positive hybridoma cells were obtained, named 1F5, 3E11 and 4C6 respectively. The hybridoma supernatants were used to identify the immunoglobulin subclasses of the antibodies secreted by the 3 hybridoma cells. The results showed that the heavy chain type of 1F5 was IgG2b, the heavy chain type of 3E11 and 4C6 were IgG1, and the light chains of the three monoclonal antibodies were all of the κ type ( table 5). The neutralization test with the Asia1/YS/CHA/05 virus strain showed that both monoclonal antibodies 1F5 and 4C6 failed to reach a dilution ratio of 1:32, and were judged to have no neutralizing activity; while 3E11 had a strong neutralizing activity, neutralizing The titer reached 1:1024 (Table 5). We performed whole-virus Western blot analysis on each strain of mAbs, and no specific reaction bands were found. Therefore, it is believed that the obtained 3 strains of mAbs all recognize conformational epitopes.

In the study, a virus antigen mutant strain was found, whose VP1 protein was mutated from G to S at position 144, and the rest of the genome sequence was exactly the same as that of the parent strain, but the mutation of this single amino acid residue caused the monoclonal antibody 3E11 to lose reactivity with the mutant strain ( Both the IFA and the micro-cell neutralization test were verified), so it was inferred that amino acid G at position 144 of the VP1 protein is a key amino acid for the epitope recognized by the monoclonal antibody 3E11. Both monoclonal antibody 3E11 and Asia1-type FMDV Jiangsu lineage strains produced specific fluorescence, and their neutralization titers were high, indicating that this epitope is a conserved neutralizing epitope of Asia1-type FMDV. the

Table 5. Identification of biological characteristics of monoclonal antibodies against Asia1 type foot-and-mouth disease virus

2. The mimotope motif recognized by monoclonal antibody 3E11 is GSLXXL

In order to identify the conformational epitope recognized by mAb 3E11, the inventors used a phage library displaying random linear dodecapeptides to perform 4 rounds of biopanning on purified mAb 3E11, and picked 42 phages in the third and fourth rounds The clone was amplified, and its reactivity was identified by phage affinity capture ELISA. The monoclonal antibody 4C6 of the same antibody subtype prepared by the same mouse and the blocking solution BSA were set up as controls to exclude the interference of the antibody constant region and BSA, and finally obtained Eight positive phage clones targeting antibody variable regions (Figure 1), among which phage clones S7, S12, S2, S1 and S6 have higher affinity. Sequence analysis was performed on the single-stranded DNA of these positive phage clones, and 8 different 12-peptide sequences were obtained (Table 6). Sequence comparison showed that the top four phage clones with higher affinity all contained a characteristic sequence GSLXXL (X represents any one of 20 amino acid residues), which was tentatively designated as the conformational neutralizing epitope recognized by mAb 3E11 motif. There are two leucines (L) in the motif structure, and the fixed interval between them is two amino acid residues, which may play a key role in supporting the skeleton to form the epitope space structure, although the type of amino acid in the interval There is no rule to follow. Western blot analysis of each phage-positive clone and 3E11 monoclonal antibody showed no reaction bands. Combined with the results of phage ELISA detection, these four linear 12-peptides displayed on the surface of phages could simulate Form the conformational epitope recognized by mAb 3E11, but cannot be recognized in the denatured linear state. In positive phage clone S6, the first leucine (L) in the 3E11 monoclonal antibody recognition epitope motif was replaced by isoleucine (I), although both are hydrophobic aliphatic amino acids with similar properties, Only one methyl position on the side chain differs, however this substitution still results in a reduced binding ability of the peptide to 3E11 (Fig. 1). In the other three positive phage clones (S11, S18, and S10), no epitope motifs recognized by the above-mentioned 3E11 were found, and no obvious consistent sequences were found. The factors for their binding to the monoclonal antibody 3E11 are still unclear. The above studies show that the mimotope recognized by mAb 3E11 is a short peptide containing the motif GSLXXL. the

In order to further identify the binding and functionality of the GSLxxL motif, a series of mutant peptides were synthesized with the highest affinity S7 phage display peptide sequence and analyzed for binding activity. In the peptide ELISA assay, monoclonal antibody 3E11 showed dose-dependent binding to streptavidin-captured biotinylated peptide P-S7, while the amino acid scrambled biotinylated peptide (P-S7-SCR) had no binding activity ( Figure 2), which demonstrates that the binding of the mimetic peptide S7 to mAb 3E11 is sequence-specific; in a competition ELISA assay, the mAb bound to the P-S7 peptide no longer bound to the antigen, and this inhibition was shown in a dose-dependent manner , while the scrambled P-S7-SCR peptide does not have this inhibitory activity (Figure 3), which proves the specificity of the binding of the mimetic peptide S7 to 3E11 from an immunological point of view; The effect of amino acid residues on epitope activity was analyzed by ELISA with synthetic mutant peptides (Figure 4). The results showed that the four conserved amino acid residues in the mimic epitope motif were all effective for the antigen-antibody recognition of the epitope. Crucially, any of these substitutions would cause the P-S7 peptide to lose its binding activity to mAb 3E11. The above results speculate that in the presence of monoclonal antibody 3E11, these linear peptides can form a certain configuration in the solution, so that the key amino acid residues can interact with the monoclonal antibody 3E11 that recognizes the conformational epitope at a suitable spatial position. Function; the change of each key amino acid of the conformational epitope motif GSLXXL makes the peptide unable to form the corresponding configuration, thus losing the binding activity; the two amino acid residues between the two leucines of the epitope motif Although the types and properties of can be changed, the spacing remains unchanged, indicating that the spatial structure of these two leucine residues is very important for the formation of conformational epitopes of linear peptides. the

Table 6. Sequence determination of positive phage mimotope peptides

^a motif amino acid residues are bolded and underlined.

The difference between ^b -positive phage clone S6 and other motifs is that I replaces L, and the side chains between these two amino acids are similar and can be replaced in some cases.

Table 7. Synthetic peptide sequences used for site-directed mutagenesis studies

^aG →S substitution designed according to the variation of the RGD mutant strain, and amino acids in other motifs were replaced with alanine (A).

^b The 18-aa peptide (136-153aa) in the GH loop of the VP1 protein of the Asia1/YS/CHA/05 strain contains all the key amino acid residues of the 3E11 mimotope.

^c The amino acids at the mutation sites of the synthetic peptides are bolded and underlined, and the purity is ≥95% after HPLC and MS analysis.

3. G-H loop synthetic peptide and its structural simulation to determine the recognition epitope of monoclonal antibody 3E11

As mentioned above, the conformational epitope recognized by 3E11 has been positioned around the RGD in the GH loop of the viral VP1 protein with the FMDV RGD mutant, and G ¹⁴⁴ is a key amino acid residue of the epitope. Subsequently, using phage display random peptide library technology combined with synthetic peptide site-directed mutagenesis technology, the mimotope motif recognized by 3E11 was determined to be GSLXXL (Figure 1, Figure 2, Figure 3, Figure 4, Table 6, Table 7). Comparing the amino acid sequence of the motif with the original virus sequence, it was found that the sequence corresponding to this position in the GH loop was GDLXXL, not GSLXXL. Therefore, we searched for traces of S in the GH loop, and found that there were two consecutive Ss at positions 140 and 141, and synthesized 136-153aa octadecapeptide in the GH loop of Asia1/YS/CHA/05 strain VP1 protein based on this. The binding activity of the octadecapeptide GH ring (AsialpG-H) diluted serially was tested by peptide ELISA (Figure 5). The results showed that the short peptide AsialpG-H showed its binding activity to monoclonal antibody 3E11 in a dose-dependent manner, indicating that The 18-aa peptide contains all the key amino acids of the epitope recognized by the monoclonal antibody 3E11. Considering that the GH loop itself has certain mobility or flexibility, Ser ¹⁴⁰ or Ser ¹⁴¹ may be an essential amino acid for the recognition epitope of mAb 3E11; the GxLXXL structure contained in the octadecapeptide and the identified epitope motif The structure is basically consistent, and the Ser amino acid residue of the epitope may be folded close to the other three amino acid residues to form the conformational epitope. According to the above research results and analysis, the key amino acid residues of the Asia1 type FMDV conformational neutralizing epitope recognized by monoclonal antibody 3E11 are Ser ¹⁴⁰ or Ser ¹⁴¹ , Gly ¹⁴⁴ , Leu ¹⁴⁶ and Leu ¹⁴⁹ .

(2) Preparation of 8E8 monoclonal antibody and identification of its recognition epitope

1. Screening and identification of a linear neutralizing epitope monoclonal antibody against type O FMDV

FMDV O/YS/CHA/05 strain immunized mouse splenocytes were fused with SP2/0 cells, hybridoma cell culture supernatants were detected by indirect ELISA and IFA verification, and the positive judgment standard was that hybridoma cell culture supernatants were used to detect FMDV The ratio of the OD ₄₉₂ light absorption value of the antigen to the normal BALB/c mouse serum and SP2/0 cell culture supernatant to the OD ₄₉₂ value of the FMDV antigen is greater than 2.1, and the supernatant of hybridoma cells and normal BHK-21 cells do not produce fluorescence The reaction is judged as positive. Antibody-positive hybridoma cells were subcloned three times by limiting dilution method to obtain a stable antibody-secreting hybridoma cell strain, which was named 8E8. Identification of immunoglobulin subclasses showed that the heavy chain type was IgG1 and the light chain type was κ. The micro-cell neutralization test showed that 8E8 had high neutralizing activity, and the neutralizing titer in ascites was as high as 1:1024. Monoclonal antibody 8E8 was used to perform Western blot analysis on the whole virus, and an obvious specific reaction band appeared, so it was considered that monoclonal antibody 8E8 recognized a linear epitope of O-type FMDV.

In order to determine the serotype specificity and antigen response spectrum of neutralizing monoclonal antibody 8E8, the reactivity between 8E8 and FMDV isolates was analyzed by indirect immunofluorescence test and microcellular neutralization test. The results showed that monoclonal antibody 8E8 and O-type FMDV The isolates of the three genotypes all produced specific fluorescence, and the neutralization titers were all high, indicating that this epitope widely exists in various genotype strains of O-type FMDV, and is a conserved neutralizing expression of O-type FMDV. On the contrary, monoclonal antibody 8E8 had no immunofluorescence reaction with Asia1 type FMDV, nor could it neutralize Asia1 type FMDV strain, indicating that the epitope recognized by monoclonal antibody 8E8 did not exist in Asia1 type isolates. In view of the important role of the neutralizing monoclonal antibody in the study of the biological characteristics of FMDV, the present invention uses a phage display random peptide library for biopanning to identify the neutralizing epitope recognized by the monoclonal antibody 8E8. the

2. The mimotope motif recognized by monoclonal antibody 8E8 is GDLNVRT

Purified mAb 8E8 was subjected to 4 rounds of biopanning with a phage library displaying random linear 12 peptides. After the third and fourth rounds of panning, a total of 24 phage clones were selected for amplification, and their reactivity was detected by phage affinity capture ELISA. At the same time, monoclonal antibody 5F7 of the same antibody subtype prepared by the same mouse was set up, containing 0.1 The PBS blocking solution of %BSA and the Phage Library (PL) of the phage peptide library were used as controls (Figure 6). After excluding the phage clones that combined with the antibody constant region and BSA, 7 positive phage clones specific to the variable region of mAb 8E8 were finally obtained. Through single-strand DNA sequence analysis, 6 different phage-displayed 12-peptide sequences were obtained, one of which repeated once (Table 8). The Western blot detection of the phage clones of these six sequences showed that the monoclonal antibody 8E8 had a binding reaction with each phage clone (Figure 7), thus confirming that the epitope recognized by the monoclonal antibody 8E8 is a linear epitope. the

Sequence comparison of these 6 12 peptides (Table 8) shows that the amino acid residue G (Gly) appears 4 times in the 6 sequences; the residue D (Asp) appears in all 6 sequences, and it is speculated that it is a monoclonal antibody A key amino acid residue of the 8E8 recognition epitope; residue L (Leu) appeared 5 times in 6 sequences; residue N (Asn) also appeared 5 times in 6 sequences, and phage P23 displayed a Q(Gln) with the same amide side chain, in theory, Q can replace N without affecting the chemical properties of epitope amino acids; residue V(Val) appears 3 times in 6 sequences, and phage P14 is at this position An I(Ile) with a similar side chain can generally be replaced without affecting its function; the residue R(Arg) appears 3 times in the 6 sequences, and K(Lys) with similar properties appears twice at this position, Therefore, the amino acid residue at this position is designated as R; the residue T (Thr) is also an amino acid residue displayed in all six sequences. Based on the above analysis, it is believed that a characteristic sequence with GDLNVRT as the motif is the mimotope recognized by mAb 8E8. the

Table 8 Phage display peptide sequence combined with monoclonal antibody 8E8

^a The 140-160 amino acid sequence of O-type O/YS/CHA/05 strain FMDV VP1.

^b The conserved amino acid residues of the mAb 8E8 recognition motif contained in each phage display peptide are marked with shading.

^3. The true epitope recognized by monoclonal antibody 8E8 is ¹⁴⁷ D L Q V L T ¹⁵²

Monoclonal antibody 8E8 has strong neutralizing activity, so the epitope it recognizes should be located at the neutralizing antigenic site of the virus. Among the identified neutralizing antigenic sites of O-type FMD virus, the most important ones are located in the GH loop of VP1 protein. Accordingly, we compared the 6 phage display sequences with the amino acid sequence of the VP1 protein of the O/YS/CHA/05 strain, and found that the mimotope motif GDLNVRT displayed by the phage was highly correlated with ^{the 146} GDLQVLT ¹⁵² sequence in the viral GH loop. At the same time, we further analyzed the amino acid arrangement of the 7-peptide mimotope (Table 8) and found that G was replaced by W in the mimotope displayed by the P1 phage clone, and the mimetic peptide displayed by the P3 phage was from Starting from D ¹⁴⁷ , there is a vacancy at G ¹⁴⁶ , but both have high levels of binding activity to mAb 8E8 (Figure 6), so we speculate that ^{the 147} DLQVLT ¹⁵² 6 peptide on the VP1 protein is the virus recognized by mAb 8E8 The smallest unit of an epitope motif. To test this hypothesis, we fused ¹⁴⁶ GDLQVLT ¹⁵² , ¹⁴⁷ DLQVLT ¹⁵² , ¹⁴⁶ GDLQVL ¹⁵¹ , ¹⁴⁶ GDLQV ¹⁵⁰ and ¹⁴⁶ GDLQ ¹⁴⁹ sequences with GST in E. coli to obtain GST-p7, GST-p7-lossG, GST- SDS-PAGE electrophoresis and Western blot activity detection of p7-lossT, GST-p7-lossLT and GST-p7-lossVLT (Figure 8) showed that monoclonal antibody 8E8 specifically combined with GST-p7 and GST-p7-lossG, It does not react with other fusion peptides and GST control. These results show that G ¹⁴⁶ is not required for the recognition and binding of the epitope to the monoclonal antibody 8E8, but T ¹⁵² is necessary for the activity of the epitope, thus confirming that ¹⁴⁷ D L Q V L T ¹⁵² is the shortest sequence recognized by the monoclonal antibody 8E8, It is a linear neutralizing epitope of O-type FMDV VP1 protein.

4. Key amino acids of the epitope recognized by monoclonal antibody 8E8

In order to further understand the role of each amino acid residue in the recognition epitope of monoclonal antibody 8E8 in the process of binding to monoclonal antibody, a series of site-directed mutagenesis peptides were synthesized for the 146-152aa peptide (P-8E8) in the true sequence of the viral VP1 protein , P-8E8 was used as a positive control, and its random scrambled peptide (P-8E8-SCR) was used as a negative control, see Table 9 for details. Site-directed mutagenesis of peptides is to replace P-8E8 with two hydrophobic amino acids and one hydrophilic amino acid respectively. The hydrophobic amino acids include alanine (A) with a shorter The chain is tryptophan (W) of aromatic group; the amino acid of hydrophilic nature is arginine (R). The binding activity of these mutated peptides to monoclonal antibody 8E8 was determined by competitive ELISA method, and the OD ₄₀₅ absorbance value was used to draw the curve (Figure 9, Figure 11). When the concentration of the synthetic peptide was 1ug/100ul, the inhibition rate of P-8E8 is 100%, calculate the inhibition rate (Fig. 10, Fig. 12) of each mutant peptide relative to P-8E8 against the O-type FMDV antigen, evaluate these amino acid residues by calculating the change of the inhibition rate of each mutant peptide at this concentration role in epitope activity.

The comparative analysis of the 6 mimetic peptide sequences displayed by phage shows that the amino acid residues D, Q and T are relatively conserved in the motif of the epitope recognized by mAb 8E8 (Table 8). In order to further analyze the role of these three amino acid residues in the composition of the 8E8 recognition epitope, we first synthesized three mutant peptides P-8E8-D-A, P-8E8-Q-A and P-8E8-T-A (Table 9) , replacing these three conserved residues with A(Ala), respectively. The results of competition ELISA (Figure 9) showed that the monoclonal antibody 8E8 combined with the synthetic peptide P-8E8 no longer combined with the O-type FMDV antigen, and this inhibitory effect was in a dose-dependent manner, while the mutant peptide P-8E8-D-A and random scrambled Peptide P-8E8-SCR also had little inhibitory activity. This indicates that amino acid residue D of the 8E8 recognition epitope is absolutely required for antigen-antibody recognition and/or binding. The mutant peptides P-8E8-Q-A and P-8E8-T-A still combined with the monoclonal antibody (Figure 9), but the inhibition rate of both decreased (Figure 10), and the inhibitory rate of the mutant peptide P-8E8-Q-A (46.4% ) is greater than that of P-8E8-T-A (75.4%), indicating that both residues Q and T are important amino acids of this epitope. Although alanine (A) can mimic most of the functions of threonine (T), resulting in the mutant peptide P-8E8-T-A still binding to mAb 8E8, deletion mutation analysis (Figure 8) showed that after T was deleted The activity of the epitope was completely lost, indicating that the presence of residue T or other residues at this position in the epitope is required for the activity of the epitope. the

On the basis of the above site-directed mutagenesis analysis, the present invention further synthesized 6 mutant peptides, including P-8E8-GW, P-8E8-VW, P-8E8-QW, P-8E8-L ¹⁴⁸ -A, P- 8E8-L ¹⁵¹ -A and P-8E8-L ¹⁵¹ -R (Table 9). The results of the competition ELISA (Figure 11) showed that the mutant peptide P-8E8-GW, like P-8E8, combined with the monoclonal antibody in a dose-dependent manner. This result combined with the above-mentioned ^G146 deletion mutation analysis (Figure 8) showed conclusively that, The activity of this epitope does not require the presence of glycine (G). The mutant peptide P-8E8-VW has almost no inhibitory activity, and its inhibitory rate is the same as that of the random scrambled peptide P-8E8-SCR at a level close to zero; however, when screening the mimic peptides (Table 8), V The frequency of occurrence of V is only 3/6, and I, which is similar in nature to V, appears once (1/6), while T, which is different in nature from V, appears twice (2/6), which suggests that V is important in this epitope Although the epitope activity completely disappears after replacing the amino acid residues with W, the substitution with I and T has no significant impact on the epitope activity, indicating that V, unlike D residues, is not absolutely required for this epitope. The mutated peptide P-8E8-QW, like P-8E8-QA, also retained some activity to bind to the monoclonal antibody, and the inhibition rates of the two peptides were 39.2% and 46.4%, respectively. Although the mutated peptides P-8E8-L ¹⁴⁸ -A and P-8E8-L ¹⁵¹ -A still retained the binding activity to monoclonal antibody 8E8 (Figure 11), the inhibition rate of the two decreased significantly (Figure 12), and the mutated peptide P -8E8-L ¹⁴⁸ -A was 32.3% and P-8E8-L ¹⁵¹ -A was 30.9%, indicating that residues L ¹⁴⁸ and L ¹⁵¹ are also important amino acids of this epitope like Q. Compared with the mutant peptides of residues L ¹⁴⁸ and L ¹⁵¹ , the inhibitory rates (46.4%, 39.2% and 75.4%) of the mutant peptides P-8E8-QA, P-8E8-QW and P-8E8-TA were higher (Fig. 10 , Figure 12), indicating that the impact of amino acid residues Q (whether replaced by A or W) and T on the epitope activity in this epitope is smaller than that of amino acid residues L ¹⁴⁸ and L ¹⁵¹ , two hydrophobic amino acid residues The binding energies of L ¹⁴⁸ and L ¹⁵¹ to the mAb in the epitope are greater than those provided by the two hydrophilic amino acid residues Q and T. The mutated peptide P-8E8-L ¹⁵¹ -R can mimic all the functions of P-8E8 and bind to the monoclonal antibody, indicating that R can completely replace L ¹⁵¹ in the mimotope motif recognized by monoclonal antibody 8E8. In summary, the recognized epitope of monoclonal antibody 8E8 identified in the present invention is hexapeptide ¹⁴⁷ DLQVLT ¹⁵² , wherein residue D ¹⁴⁷ is a key amino acid, residues V, L ¹⁴⁸ , L ¹⁵¹ , Q ¹⁴⁹ and T ¹⁵² are important amino acids, and the The amino acid residue at position ^T152 of the epitope is essential for epitope activity.

Table 9 is used for the synthetic peptide of the present invention and its amino acid sequence

^a 146-152aa peptide of VP1 of O/YS/CHA/05 strain.

^b Amino acids at mutation sites are bolded and underlined, and some residues are replaced with alanine (A), tryptophan (W) and arginine (R).

(3) Immunoprotective properties of 3E11 and 8E8 monoclonal antibodies in vitro and in vivo

1. Determination of the challenge dose of suckling mice

After the monolayer BHK-21 cells were inoculated with the virus, the 96-well culture plate inoculated with the virus was observed three times a day, and measured after 3-5 days, the TCID ₅₀ of the Asia1 type FMDV Asia/YS/CHA/05 strain was 7.0, and the O type FMDV O/YS The TCID ₅₀ of /CHA/05 strain was 7.25. These two strains were serially diluted 10-fold with sterilized PBS to 10 ^-5 , 10 ^-6 , 10 ^-7 and 10 ^-8 respectively. For Asia/YS/CHA/05 and O/YS/CHA/ The median lethal dose (LD ₅₀ ) of strain 05 was determined, and the negative control group was injected with sterilized PBS, and the mice that died within 24 hours were regarded as accidental death after inoculation. Thus determined, the LD ₅₀ of the Asia1 type FMDV Asia/YS/CHA/05 strain and the O type FMDV O/YS/CHA/05 strain were 10 ⁻⁶ /0.2ml and 10 ⁻⁷ /0.2ml (Table 10, 11 ). In order to select the appropriate Asia1 type and O type FMDV challenge dose, use the above test results as the basis, select the highest dilution of Asia1 type and O type FMDV that can make all suckling mice die as the challenge dose of suckling mice, thus determine The most suitable challenge dosage for suckling mice for these two strains is 10LD ₅₀ /200ul/mouse.

Table 10 Determination of Asia1/YS/CHA/05LD ₅₀ Table 11 Determination of O/YS/CHA/05LD ₅₀

2. In vitro neutralization activity of monoclonal antibodies 3E11 and 8E8

The half-protection level of neutralizing antibody of monoclonal antibody was detected by micro-neutralization test. The virus control, ascites control and cell control set up in the experiment were all normal, indicating that the experiment was established. The results show that Asia1 type FMDV monoclonal antibody 3E11 and O type FMDV monoclonal antibody 8E8 have good neutralizing activity, and the half protective dose (PD ₅₀ ) of monoclonal antibody ascites neutralizing antibody that protects 50% of BHK-21 cells from producing CPE is respectively 1995 and 2371.

3. Immunoprotective effects of monoclonal antibodies 3E11 and 8E8 on suckling mice

The immune protection efficacy of monoclonal antibodies 3E11 and 8E8 was verified in suckling mice, and the death of suckling mice was observed every day after challenge. The suckling mice in the PBS control group died normally, and the suckling mice in the monoclonal antibody control group showed no abnormalities. In the monoclonal antibody 3E11 experimental group, the concentration of monoclonal antibody in each group was 2, 4, 8, 16, 31 and 62PD ₅₀ respectively, and the challenge dose of Asia1 type FMDV in each group of suckling mice was 10LD ₅₀ , the test results (Figure 13) It shows that the protection rate of suckling mice increases gradually with the increase of monoclonal antibody concentration, and when the monoclonal antibody 3E11 is diluted to 8 times the half protection dose of neutralizing antibody (8PD ₅₀ ), it can completely protect suckling mice from the lethal dose Asia1 type FMDV attack. In the monoclonal antibody 8E8 experimental group, the monoclonal antibody concentrations of each group were 3, 5, 11 and 21PD ₅₀ , respectively, and the challenge dose of O-type FMDV in each group of suckling mice was also 10LD ₅₀ . The test results are shown in Figure 14. The monoclonal antibody concentration and milk There is a positive correlation between the protection rates of mice. When the concentration of monoclonal antibody 8E8 is 11PD ₅₀ , it can completely protect suckling mice from the lethal dose of O-FMDV challenge.

The use of different monoclonal antibody concentrations has different effects on the survival time of suckling mice. With the increase of monoclonal antibody concentration, the initial death time of suckling mice is delayed and the terminal death time is moved forward. When the concentration of monoclonal antibody 3E11 is 2PD ₅₀ , the initial death time and terminal death time of suckling mice are respectively 42h and 128h after the virus challenge; when the concentration of monoclonal antibody 3E11 is 4PD ₅₀ , the initial death time and terminal death time The times were 55h and 68h after the virus was challenged respectively ( FIG. 15 ). In the monoclonal antibody 8E8 test group, when the monoclonal antibody concentration was 3PD ₅₀ , the initial death time and terminal death time of the suckling mice were respectively 72h and 117h after the virus challenge; when the monoclonal antibody concentration was 5PD ₅₀ , the neonatal mice died at the beginning The time and terminal death time were respectively 84h and 96h after virus challenge ( FIG. 16 ). The concentration of monoclonal antibody has a certain proportional relationship with the percentage of body weight growth of suckling mice (Figures 17 and 18). High, the protective effect on suckling mice is enhanced, and the weight growth of suckling mice tends to be normal.

Claims (3)

1. A hybridoma cell line that can secrete neutralizing monoclonal antibodies against type O foot-and-mouth disease virus, its microorganism preservation number is: CGMCC No: 2691. 2. A monoclonal antibody secreted by the hybridoma cell line of claim 1. 3. The monoclonal antibody of claim 2 is used in the preparation of reagents or medicines for diagnosing, preventing or treating foot-and-mouth disease.

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