CN115819568B

CN115819568B - Enterovirus A71 monoclonal antibody and application thereof

Info

Publication number: CN115819568B
Application number: CN202211451008.0A
Authority: CN
Inventors: 刘庆伟; 王晓黎; 阮丽珠; 杨义; 边金; 赵亚楠; 刘艳; 赵胜涛
Original assignee: Huasong Shanghai Biomedical Technology Co ltd
Current assignee: Huasong Shanghai Biomedical Technology Co ltd
Priority date: 2022-11-18
Filing date: 2022-11-18
Publication date: 2024-06-25
Anticipated expiration: 2042-11-18
Also published as: CN115819568A

Abstract

The application relates to the field of antibodies, in particular to an enterovirus A71 monoclonal antibody and application thereof. The A71 monoclonal antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 sequences; wherein the sequence of HCDR1 comprises the sequence shown in SEQ ID No. 1; the sequence of HCDR2 includes the sequence shown in SEQ ID No. 2; the sequence of HCDR3 includes the sequence shown in SEQ ID No. 3; the sequence of LCDR1 includes the sequence shown in SEQ ID No. 4; the sequence of LCDR2 includes the sequence shown in SEQ ID No. 5; the sequence of LCDR3 includes the sequence shown in SEQ ID No. 6. Through screening detection, a monoclonal antibody capable of specifically binding to A71 is obtained: 1G10.1G10 is not only a powerful and effective tool for detection, but also a reliable candidate of diagnostic reagents and therapeutic humanized monoclonal antibodies, A71 vaccine screening and qualitative and quantitative detection and detection reagents of vaccine antigens, and an antibody reference for evaluating the activity of the A71 vaccine.

Description

Enterovirus A71 monoclonal antibody and application thereof

Technical Field

The application relates to the field of antibodies, in particular to an enterovirus A71 monoclonal antibody and application thereof.

Background

Hand-foot-and-mouth disease (HFMD) is a syndrome caused by multiple pathogens, mainly infected people are children, clinically, the disease is usually manifested as fever, eruption of hands, feet, mouth, buttocks and the like, and aseptic meningitis and the like can be caused even death when the disease is serious. The etiologic agents of HFMD are complex and are most members of the enterovirus genus of the family MicroRNA, enterovirus group 71 (EnterovirusA, EV A71), coxsackie virus group A (CoxsackievirusA, CV A) types 2-10, 12, 14, 16, 21, 24, coxsackie virus group B (CoxsackievirusB, CV B) types 1-6, and Epstein-Barr virus (Echovirus) types 1,4-7,9, 11, 13, 18, 19, 24, 25, 30, etc. have been reported. Severe HFMD and related deaths are mainly caused by EV a71 infection, which has been shown to account for 70.03% and 92.23% of cases of HFMD related severe and fatal infections, respectively.

Enterovirus type 71 (EV a 71) is one of the more thoroughly studied viruses, EV a71 was first isolated in 1969 from stool specimens of infants with symptoms of central nervous system infection in california. In 1998, EV A71 was first isolated from Shenzhen HFMD infant specimens in China. In 2007, EV a71 infection-related diseases are widely prevalent in China. The virus particles are extremely small and have the diameter of 20-30 nm and are in an icosahedron stereo symmetrical spherical structure. The genome is about 7500bp long, with only one open reading frame flanked by 5 'and 3' non-coding regions. The viral particle capsid comprises 60 subunits, each assembled from 4 capsid proteins into a pentameric-like structure. VP4 is embedded inside the outer shell of the virion and tightly connected with the viral core, and other three structural proteins are exposed on the surface of the virion.

The hand-foot-and-mouth disease has self-limiting property, most of the hand-foot-and-mouth disease can be cured in the eruption period, and no specific medicine is available at present for treating the hand-foot-and-mouth disease in a targeted manner. Therefore, the general treatment and the etiology treatment are adopted clinically. In 2016, 3 EV A71 inactivated vaccines in China are approved by the national food and drug administration to be used on the market, then the number of cases and the composition ratio of hand-foot-and-mouth disease caused by EV A71 infection are further reduced, the composition ratio of other EVs is increased by 16.07% compared with the year average, and disease monitoring data show that the application of the EV A71 vaccine effectively reduces the morbidity, the severe symptoms and the death of the hand-foot-and-mouth disease caused by EV A71, but has very little effect on preventing the hand-foot-and-mouth disease caused by different enteroviruses because cross protection antibodies cannot be generated. Therefore, the incidence rate of the hand-foot-and-mouth disease is not obviously reduced, other EV (electric vehicle) epidemic of the hand-foot-and-mouth disease is gradually increased, and the CV A16 epidemic is also enhanced in 2017-2019, so that the hand-foot-and-mouth disease is still an infectious disease with serious harm, the development of multivalent combined vaccines is urgent, and the development of multivalent combined vaccines is a necessary trend, so that the hand-foot-and-mouth disease can be fundamentally prevented and controlled.

The detection of antibodies in the development process of vaccines is an indispensable tool for the detection of epitopes in the development process of vaccines, so as to ensure that the detected antigens are active epitope antigens. Good detection antibodies can promote the development process, so that monoclonal antibodies with strong specificity, high purity, high homogeneity and high neutralization activity need to be developed. In addition, monoclonal antibody can be combined with the virus capsid protein to block the virus adsorption or uncoating process, thereby inhibiting the virus replication. At present, no antiviral drug for hand-foot-and-mouth disease is approved by FDA, and a long path is required for successful development of novel specific drugs, so that the development of monoclonal antibodies lays a foundation for drug development.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present application is directed to providing an enterovirus a71 monoclonal antibody and application thereof, which are used for solving the problems in the prior art.

To achieve the above and other related objects, a first aspect of the present application provides an enterovirus a71 monoclonal antibody comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 sequences; wherein,

The sequence of HCDR1 includes the sequence shown in SEQ ID No. 1; the sequence of HCDR2 includes the sequence shown in SEQ ID No. 2; the sequence of HCDR3 includes the sequence shown in SEQ ID No. 3; the sequence of LCDR1 includes the sequence shown in SEQ ID No. 4; the sequence of LCDR2 includes the sequence shown in SEQ ID No. 5; the sequence of LCDR3 includes the sequence shown in SEQ ID No. 6.

In any embodiment of the application, the heavy chain variable region further comprises framework regions FR1-FR4; the amino acid sequence of the FR1 comprises a sequence shown in SEQ ID No. 7; the amino acid sequence of the FR2 comprises a sequence shown in SEQ ID No. 8; the amino acid sequence of the FR3 comprises a sequence shown in SEQ ID No. 9; the amino acid sequence of FR4 comprises the sequence shown in SEQ ID No. 10.

In any embodiment of the application, the light chain variable region further comprises framework regions FR1-FR4; the amino acid sequence of the FR1 comprises a sequence shown in SEQ ID No. 11; the amino acid sequence of the FR2 comprises a sequence shown in SEQ ID No. 12; the amino acid sequence of the FR3 comprises a sequence shown in SEQ ID No. 13; the amino acid sequence of FR4 comprises the sequence shown in SEQ ID No. 14.

In any embodiment of the application, the heavy chain further comprises a signal peptide; the heavy chain signal peptide comprises a sequence shown as SEQ ID No. 15;

And/or, the light chain further comprises a signal peptide; the light chain signal peptide comprises a sequence shown as SEQ ID No. 16.

In any embodiment of the application, the amino acid sequence of the heavy chain comprises the sequence shown in SEQ ID No. 17;

And/or the amino acid sequence of the light chain comprises the sequence shown in SEQ ID No. 18.

In a second aspect, the application provides a nucleotide molecule encoding said monoclonal antibody.

In a third aspect the application provides an expression vector comprising said nucleotide molecule.

In a fourth aspect, the application provides a host cell comprising said expression vector or genome having said nucleotide molecule integrated therein.

In a fifth aspect, the application provides the use of said monoclonal antibody or said nucleotide molecule or said expression vector or said host cell in the preparation of a virus detection product, in the preparation of a product for the prevention or treatment of a virus infection, or in the screening and detection of a virus vaccine and in the evaluation of vaccine activity.

In any embodiment of the application, the virus is an enterovirus, preferably enterovirus type 71; more preferably, the monoclonal antibody is used for preparing a hand-foot-and-mouth disease detection product, a hand-foot-and-mouth disease prevention or treatment product, or an antibody reference product for screening hand-foot-and-mouth disease vaccines, qualitative and quantitative detection and verification of vaccine antigens or evaluation of vaccine activity.

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

1. the monoclonal antibody provided by the application can specifically recognize the A71 virus-like particle, but cannot recognize the denatured virus-like particle, which suggests that the epitope recognized by the monoclonal antibody may be a conformational epitope.

2. The monoclonal antibody provided by the application has a minimum detection limit of 3.125ng on A71 virus-like particles, and is a powerful and effective detection tool.

3. The monoclonal antibody provided by the application has strong neutralization activity, the neutralization concentration is 0.032 mug/ml, and the monoclonal antibody is a reliable candidate for diagnostic reagents and therapeutic humanized monoclonal antibodies, and can also be used as an antibody reference product for evaluating vaccine activity.

Drawings

FIG. 1 shows a schematic diagram of SDS-PAGE analysis of monoclonal antibodies.

FIG. 2 shows a schematic diagram of a monoclonal antibody specificity assay.

FIG. 3 shows a schematic diagram of Western blot analysis of monoclonal antibodies.

FIG. 4 shows a schematic diagram of sandwich ELISA assay monoclonal antibodies.

FIG. 5 shows a schematic diagram of sequence validation of 1G10 mab.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application will be further described with reference to examples. It is to be understood that the examples are provided for the purpose of illustrating the application and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.

Through a great deal of research and study, the inventor of the application discovers an enterovirus A71 monoclonal antibody and application thereof, and the application is completed on the basis.

As used herein, "monoclonal antibody" means a preparation of antibody molecules, antibodies, having a common heavy chain amino acid sequence and a common light chain amino acid sequence, as opposed to a "polyclonal" antibody preparation, which contains a mixture of antibodies of different amino acid sequences. The antibodies used in the present invention are derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, rather than the method by which they are produced.

Monoclonal antibodies can be produced by several known techniques, such as phage technology, bacterial, yeast or ribosome display, as well as classical methods exemplified by antibodies derived from hybridomas. The term (monoclonal) thus refers to all antibodies derived from one nucleic acid clone. Monoclonal antibodies can be obtained by various methods well known to those skilled in the art. For example, monoclonal antibodies can be produced by the hybridoma method (first proposed by Kohler et al, nature,256:495 (1975)) or by the recombinant DNA method (U.S. Pat. No. 4,481,967). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, by Clackson et al Nather,352:624-628 (1991) and Marks et al, mol. Biol.,222:581-597 (1991).

The term "variable" as used herein means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called the Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are in a substantially p-folded configuration, connected by three CDRs forming the linker loop, and in some cases form part of a p-folded structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody, and the constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity.

In one aspect, the application provides an enterovirus A71 monoclonal antibody, wherein the A71 monoclonal antibody comprises heavy chain variable regions of HCDR1, HCDR2 and HCDR3 sequences and light chain variable regions containing LCDR1, LCDR2 and LCDR3 sequences. Wherein the sequence of HCDR1 comprises the sequence shown in SEQ ID No. 1. The sequence of HCDR2 includes the sequence shown in SEQ ID No. 2. The sequence of HCDR3 includes the sequence shown in SEQ ID No. 3. The sequence of LCDR1 includes the sequence shown in SEQ ID No. 4. The sequence of LCDR2 includes the sequence shown in SEQ ID No. 5. The sequence of LCDR3 includes the sequence shown in SEQ ID No. 6. The CDR sequence of the monoclonal antibody No. 1G10 provided by the application is shown in table 1.

TABLE 1 monoclonal antibody CDR sequences

In the enterovirus A71 monoclonal antibody provided by the application, the heavy chain variable region further comprises framework regions FR1-FR4. The amino acid sequence of the FR1 comprises a sequence shown as SEQ ID No. 7. The amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No. 8. The amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No. 9. The amino acid sequence of FR4 comprises the sequence shown in SEQ ID No. 10. The sequence of the framework region of the heavy chain variable region of the monoclonal antibody No. 1G10 provided by the application is shown in table 2.

TABLE 2 heavy chain variable region framework region sequences

In the enterovirus A71 monoclonal antibody provided by the application, the light chain variable region further comprises framework regions FR1-FR4. The amino acid sequence of the FR1 comprises a sequence shown as SEQ ID No. 11. The amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No. 12. The amino acid sequence of the FR3 comprises a sequence shown as SEQ ID No. 13. The amino acid sequence of FR4 comprises the sequence shown in SEQ ID No. 14. The sequence of the framework region of the light chain variable region of the monoclonal antibody No. 1G10 provided by the application is shown in Table 3.

TABLE 3 light chain variable region framework region sequences for monoclonal antibodies

The application provides an enterovirus A71 monoclonal antibody, wherein the heavy chain also comprises a signal peptide. The heavy chain signal peptide comprises a sequence shown as SEQ ID No. 15. The light chain further comprises a signal peptide. The light chain signal peptide comprises a sequence shown as SEQ ID No. 16. The signal peptide sequence of the monoclonal antibody No. 1G10 provided by the application is shown in Table 4.

TABLE 4 monoclonal antibody Signal peptide sequences

	Amino acid sequence	SEQ ID NO：
			Heavy chain signal peptide	MNLGFCLIFLVLVLKGVQC	15
Light chain signal peptide	MSSAQFLGLLLLCFQGTRC	16

In the enterovirus A71 monoclonal antibody provided by the application, the amino acid sequence of the heavy chain comprises a sequence shown as SEQ ID No. 17. The amino acid sequence of the light chain comprises a sequence shown as SEQ ID No. 18. The sequence is as follows:

MNLGFCLIFLVLVLKGVQCEVKLVESGGGLVKPGGSLKLSCAASGFTFSSYVMSWVRQTPEKRLEWVASISNGGSTFYPDSVKGRLTISRDNARNILYLQMSSLRSEDTAMYYCARAYGNDWYFDVWGAGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID No:17).

MSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLNSGVPSRFSGSGSGTDYSLTISNLEEEDIATYFCQQGNTAWTFGGGTKLEIKRADAAPTVSIFPPSSDQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID No:18).

In another aspect, the application provides a nucleotide molecule encoding said monoclonal antibody. The full-length nucleotide sequence of the monoclonal antibody of the present application or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

The heavy chain nucleotide sequence of the monoclonal antibody is shown as SEQ ID No. 19 (wherein, the single underlined part is a signal peptide sequence, the italic part is a variable region sequence, and the dotted line is a constant region sequence). The sequence is as follows:

The light chain nucleotide sequence of the monoclonal antibody is shown as SEQ ID No. 20 (wherein, the single underlined part is a signal peptide sequence, the italic part is a variable region sequence, and the dotted line is a constant region sequence). The sequence is as follows:

In another aspect, the application provides an expression vector comprising said nucleotide molecule. An "expression vector" refers to a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Expression vectors in the present application generally refer to a variety of commercially available expression vectors well known in the art, and may be, for example, bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

In another aspect, the application provides a host cell comprising said expression vector or genome having said nucleotide molecule integrated therein. "host cell" any cell suitable for expression of an expression vector may be used as a host cell, e.g., the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

In another aspect, the application provides the use of said monoclonal antibody or said nucleotide molecule or said expression vector or said host cell in the preparation of a virus detection product, in the preparation of a product for the prevention or treatment of a virus infection, or in the screening and detection of a virus vaccine and in the evaluation of vaccine activity.

In the use of the application, the virus detection product may be, for example, a vaccine composition. The vaccine composition may be monovalent (containing only one virus-like particle) or multivalent (containing multiple virus-like particles). The vaccine composition can be prepared into various conventional dosage forms, such as: injection, granule, tablet, pill, suppository, capsule, suspension, spray, etc. The vaccine composition may be administered directly to an individual using known methods for the virus-like particles of the application. These vaccines are typically administered by the same route of administration as conventional vaccines and/or by a route that mimics pathogen infection. The route of administration of the vaccine composition includes: intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral or other parenteral routes of administration. The routes of administration may be combined, if desired, or adjusted according to the disease condition. The vaccine composition may be administered in a single dose or in multiple doses, and may include administration of booster doses to elicit and/or maintain immunity.

In the application, the virus is enterovirus, preferably enterovirus 71; more preferably, the monoclonal antibody is used for preparing hand-foot-and-mouth disease detection products, preparing products for preventing or treating hand-foot-and-mouth disease, or antibody reference products for screening hand-foot-and-mouth disease vaccines, qualitatively and quantitatively detecting vaccine antigens or evaluating vaccine activity and the like.

In vaccine screening, to ensure that the antigen detected is an active epitope antigen, a highly sensitive, highly specific and highly neutralizing antibody is required, which in one embodiment of the application can be minimally detected, for example, by a sandwich Elisa assay. The specificity can be detected by, for example, western blot experiments to detect the specific binding of the monoclonal antibody to the antigen. The neutralization activity can be determined, for example, by neutralization experiments. Qualitative and quantitative detection of the vaccine antigens can be obtained by a sandwich Elisa experiment, for example.

The application is further illustrated by the following examples, which are not intended to limit the scope of the application.

Information on the reagents described below is shown in Table 5.

TABLE 5 reagent information

Example 1 selection of hybridoma cells secreting enterovirus A71-specific antibodies

EV a71 virus-like particles (VLPs) were prepared by expression using pichia expression system (see patent CN 114836444). At week 0, 50 μg of EV A71 VLP was mixed with 40 μg of Freund's Complete Adjuvant (FCA) and then 6 week old female Balb/c mice were immunized subcutaneously; at 1 week, 50 μg of EV A71 VLP was mixed with 40 μg of aluminum adjuvant and was subjected to intraperitoneal immunization; at 2 weeks, 50 μg of EV a71 VLP was emulsified with 40 μg of FIA adjuvant for subcutaneous multipoint immunization; at 3 weeks, 50 μg of EV A71 VLP was mixed with 40 μg of aluminum adjuvant and was subjected to intraperitoneal immunization. After the last immunization, 5 μg of EV a71 VLP was injected into the tail vein for booster immunization.

After 3 days of mouse tail vein boosting, hybridoma cells were prepared by fusing mouse spleen cells with myeloma cells SP2/0 using PEG 1450. After 1-2 weeks of hybridoma cell culture, antibodies specifically secreting EV a71 VLPs were screened using an enzyme-linked immunosorbent assay and neutralization assay. The ELISA test steps are as follows: EV a71 VLPs were coated on 96-well elisa plates (200 ng/well), incubated overnight at 4 ℃; after blocking with PBST containing 5% skimmed milk, 50. Mu.l of hybridoma culture solution was added to each well and incubated at 37℃for 2 hours; then, the incubation was performed with HRP-labeled secondary antibody for 1 hour, and finally, a color reaction was performed to read the absorbance at OD450 nm. The neutralization test steps are as follows: after 50. Mu.l of the hybridoma culture broth was thoroughly mixed with 100TCID 50/50. Mu.l of enterovirus A71 (see patent CN 114836444), the mixture was placed in a 5% CO ₂ incubator and incubated at 37℃for 2 hours. RD cell working fluid (1.5X105/ml) is suspended and added to the 96-well plate after incubation, 100. Mu.l/well, and cultured in a 5% CO ₂ incubator at 35℃for 7 days. CPE was observed.

Finally, monoclonal antibodies numbered 1G10 and 1H10 were selected according to their binding capacity and neutralizing activity to A71 VLPs, wherein the 1G10 monoclonal antibodies had both good binding capacity and neutralizing activity, and the identification information is shown in Table 6.

TABLE 6 identification of monoclonal antibody secreting hybridoma cell lines

Hybridoma cell line numbering	Heavy chain	Light chain	Ability to bind a71VLP	Neutralization Activity ^#
					1G10	IgG2a	kappa	+++	+
1H10	IgG1	kappa	+++	-

The samples used for the analysis were 50. Mu.l of hybridoma culture cell supernatants.

*,+：OD450＞0.15；++：OD450＞0.3；+++：OD450＞0.5。

#, +: Has neutralizing activity; -, no neutralization activity

EXAMPLE 2 specificity analysis of anti-A71 mab

Antibody purification: female Balb/c mice were intraperitoneally injected with 500. Mu.l liquid paraffin oil, and 80 ten thousand hybridoma cells per mouse intraperitoneally after one week. After 1-2 weeks, collecting ascites with needle, centrifuging at 4000rpm for 10min, removing upper layer oil and lower layer precipitate, and purifying clarified ascites with antibody. According to the specification, the ascites fluid was purified using iProtein G Purose Fast Flow affinity column (thousand pure organisms) to obtain purified antibodies.

ELISA method: coating 96-well ELISA plates (200 ng/well) with A6 or A10 or A16 or A71 VLPs, and incubating overnight at 4 ℃; adding 5% skimmed milk PBST, sealing at 37deg.C for 1 hr; adding detection monoclonal antibody, and incubating for 2 hours at 37 ℃; HRP-labeled secondary antibody was then added and incubated for 1 hour, and finally absorbance OD450nm was read.

Protein experiment: the protein samples were mixed with a polypropylene gel electrophoresis (SDS-PAGE) loading buffer, boiled for 5min, and the protein samples were separated by a 12% polyacrylamide gel. Protein bands were visualized by coomassie blue staining or proteins were transferred to PVDF membranes for western blot analysis. Monoclonal antibody concentration 1ug/ml, rabbit anti-VP 0 polyclonal antibody 1:1000 was diluted for use, followed by incubation with HPR-labeled secondary antibody, and finally recorded with a luminescence image analyzer.

The results show that: SDS-PAGE identifies the purity and integrity of A71 mab purified from ascites. As shown in FIG. 1, each of the monoclonal antibodies 1G10 and 1H10 shows two major bands, which are about 55kDa and 25kDa in size, respectively, corresponding to heavy and light chains, respectively. Reactivity of the monoclonal antibodies with different antigens was examined by ELISA methods, including CV A6 VLP, CV a10VLP (see patent CN114836444 for preparation methods), CV a16 VLP and EV a71 VLP. As shown in fig. 2, both 1G10 and 1H10 mab can specifically recognize EV a71 VLP, but cannot recognize CV A6 VLP, CV a10VLP, and CV a16 VLP. Binding of the mab to CV A6 VLP, CV a10VLP, CV a16 VLP and EV a71 VLP was analyzed by Western blot, and as shown in fig. 3, 1G10 mab failed to recognize the denatured CV A6 VLP, CV a10VLP, CV a16 VLP and EV a71 VLP, suggesting that the epitope recognized by 1G10 may be conformational epitope, while 1H10 may specifically recognize the denatured EV a71 VLP, indicating that the epitope recognized by the antibody is linear epitope.

Example 3 detection of enterovirus A71 Virus-like particles by Sandwich ELISA

Sandwich ELISA: rabbit anti-EV A71 VLP polyclonal serum is diluted 1:8000 and then coated with 96 ELISA plates (100 μl/well), and incubated overnight at 4deg.C; after blocking for 1 hour at 37℃with PBST containing 5% skim milk, EV A71 VLP was added and incubated for 2 hours at 37 ℃; then, the antibody of example 2 (1.2 ng/. Mu.l) was added and incubated at 37℃for 2 hours; incubation was then performed with HPR-labeled murine secondary antibody, and finally absorbance OD450nm was read.

The minimal detection limit of the primary antibody against the diseased EV A71 VLPs was determined by sandwich ELISA (positive when OD450 > 0.15). As shown in FIG. 4, both 1G10 and 1H10 antibodies sensitively detected enterovirus A71 VLPs, with minimum detection limits of 3.125ng/ml and 25ng/ml, respectively, suggesting that they can be used for diagnosis of A71 infection.

Example 4 neutralization Activity assay

Adding the antibody of the example 2 into a 96-well dilution plate containing 2% FBSDMEM, blowing and mixing uniformly, carrying out 5-time gradient dilution downwards, carrying out 8 gradient dilutions altogether, adding 50 μl of the antibody with the gradient concentration of 5000ng/50 μl to 0.064ng/50 μl into the 96-well culture plate, and setting 2 multiple wells for each dilution; 100TCID50/50 μl enterovirus A71 was taken. The working solution is added into the positive antibody diluted by the corresponding 96-well plate, and after being fully and evenly mixed, the positive antibody is put into a 5% CO ₂ incubator for incubation for 2 hours at 37 ℃. RD cell working fluid (1.5X10 ⁵/ml) was suspended in 96-well plates after incubation, 100. Mu.l/well, 5% CO ₂ incubator at 35℃for 7 days. CPE was observed.

The neutralization activity of the 1G10 monoclonal antibodies and the 1H10 monoclonal antibodies is detected through a neutralization test, and the result shows that the 1H10 has no neutralization activity, and the 1G10 has stronger potential neutralization activity on the A71, wherein the neutralization concentration is 0.032 mug/ml, which suggests that the monoclonal antibodies can be used for the development of antiviral drugs of enterovirus A71.

EXAMPLE 5 Gene sequence analysis of 1G10 monoclonal antibody

The cells of the hybridoma cell line of example 1 were subjected to extraction of total RNA with Trizol reagent, and heavy and light chain full-length genes were amplified according to the 5' RACE kit instructions.

The obtained 1G10 monoclonal antibody has a heavy chain nucleotide sequence shown as SEQ ID No. 19 and a light chain nucleotide sequence shown as SEQ ID No. 20.

Further analysis of the 1G10 mab heavy chain variable region and light chain variable region sequences, the 1G10 mab heavy chain variable region amino acids were as follows (underlined as heavy chain CDR regions):

EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYVMSWVRQTPEKRLEWVASISNGGSTF YPDSVKGRLTISRDNARNILYLQMSSLRSEDTAMYYCARAYGNDWYFDVWGAGTTVTVSS(SEQ ID No:21).

The heavy chain variable region described above belongs to the IGHV5 subgroup.

The 1G10 mab light chain variable region amino acids are as follows (underlined as heavy chain CDR regions):

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLNSGVP SRFSGSGSGTDYSLTISNLEEEDIATYFCQQGNTAWTFGGGTKLEIK(SEQ ID No:22).

The light chain variable region belongs to IGKV10 subgroup.

EXAMPLE 6 recombinant expression and identification of monoclonal antibody genes

The coding sequences of the heavy chain and the light chain are respectively inserted between pcDNA3.3 (thermo) EcoRI and XhoI multiple cloning sites by adopting a homologous recombination mode, expression vectors pcDNA3.3-1G10-H and pcDNA3.3-1G10-L are constructed, pcDNA3.3-1G10-H and pcDNA3.3-1G10-L are co-transfected by adopting a liposome method, culture supernatants are collected for analysis after 3d, and the expression of antibodies in the culture supernatants is determined by adopting an enzyme-linked immunosorbent assay method.

As shown in fig. 5, cell supernatants expressing the 1G10 mab sequences had high binding signals to a71 VLPs and the OD450nm gradually decreased with increasing dilution; while the supernatant of control cells not transfected with the relevant plasmid did not bind a signal, the result indicated that the amplified and expressed sequence was indeed the gene for 1G10 mab.

In the research, the EV A71VLP is used for immunizing a BALB/c mouse, a B lymphocyte hybridoma technology is adopted for cell fusion, hybridoma cell strains capable of stably secreting specific anti-EV A71 monoclonal antibodies are obtained through screening, the specific anti-EV A71 monoclonal antibodies are obtained, and the antibodies are detected by using Western blot, elisa, in-vitro neutralization and other technical means. The reactivity of the monoclonal antibodies with different antigens was detected by an indirect Elisa method, which shows that both the 1G10 and 1H10 monoclonal antibodies can specifically recognize EV A71 VLPs, but not CV A6 VLPs, CV A10 VLPs and CV A16 VLPs, indicating that the antibodies have good specificity. Western blot results show that the 1G10 mab cannot recognize the denatured EV A71VLP, CV A6 VLP, CV A10 VLP and CV A16 VLP, suggesting that the recognized epitope may be a conformational epitope; while 1H10 can recognize the denatured EV a71VLP, indicating that the epitope recognized by the antibody is a linear epitope. The sandwich Elisa result shows that the minimum detection limit of the monoclonal antibody 1G10 to the EV A71VLP is 3.125ng, which provides a favorable theoretical basis for developing the monoclonal antibody into an A71 virus detection kit and a vaccine antigen quantification kit. The neutralization test result shows that the neutralization concentration of the 1G10 monoclonal antibody is 0.032 mug/ml, and the neutralizing activity of the neutralizing antibody against the virus in vitro is stronger, which indicates that the neutralizing antibody can be used for virus identification and development of antiviral drugs, and can be used for research and development of therapeutic monoclonal antibodies.

In summary, the 1G10 and 1H10 antibodies have good specificity and sensitivity, wherein 1G10 also has virus neutralization capability, so that the antibodies can be used as a detection tool in a laboratory, and have great potential in virus identification, diagnosis, treatment and multivalent vaccine development.

The above embodiments are merely illustrative of the principles of the present application and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that all equivalent modifications and variations of the application be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. An enterovirus a71 monoclonal antibody, the a71 monoclonal antibody comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 sequences; wherein,

The sequence of HCDR1 is the sequence shown in SEQ ID No. 1; the sequence of HCDR2 is the sequence shown in SEQ ID No. 2; the sequence of HCDR3 is the sequence shown in SEQ ID No. 3; the sequence of LCDR1 is the sequence shown in SEQ ID No. 4;

the sequence of LCDR2 is the sequence shown in SEQ ID No. 5; the sequence of LCDR3 is shown in SEQ ID No. 6.

2. The monoclonal antibody of claim 1, wherein the heavy chain variable region further comprises framework regions FR1-FR4; the amino acid sequence of the FR1 comprises a sequence shown in SEQ ID No. 7; the amino acid sequence of the FR2 comprises a sequence shown in SEQ ID No. 8; the amino acid sequence of the FR3 comprises a sequence shown in SEQ ID No. 9; the amino acid sequence of FR4 comprises the sequence shown in SEQ ID No. 10.

3. The monoclonal antibody of claim 1, wherein the light chain variable region further comprises framework regions FR1-FR4; the amino acid sequence of the FR1 comprises a sequence shown in SEQ ID No. 11; the amino acid sequence of the FR2 comprises a sequence shown as SEQ ID No. 12; the amino acid sequence of the FR3 comprises a sequence shown in SEQ ID No. 13; the amino acid sequence of FR4 comprises the sequence shown in SEQ ID No. 14.

4. The monoclonal antibody of claim 1, wherein the heavy chain further comprises a signal peptide; the heavy chain signal peptide comprises a sequence shown as SEQ ID No. 15;

5. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain comprises the sequence set forth in SEQ ID No. 17;

6. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-5.

7. An expression vector comprising the nucleic acid molecule of claim 6.

8. A host cell comprising the expression vector of claim 7 or the nucleic acid molecule of claim 6 integrated into the genome.

9. Use of a monoclonal antibody according to any one of claims 1 to 5 or a nucleic acid molecule according to claim 6 or an expression vector according to claim 7 or a host cell according to claim 8 for the preparation of an enterovirus 71-type test product, for the preparation of a product for the prophylaxis or treatment of enterovirus 71-type infections, or for enterovirus 71-type vaccine screening and detection and for evaluation of vaccine activity.

10. The use according to claim 9, wherein the monoclonal antibody is used for preparing a hand-foot-and-mouth disease detection product, a hand-foot-and-mouth disease prevention or treatment product, or an antibody reference product for hand-foot-and-mouth disease vaccine screening and qualitative and quantitative detection and detection of vaccine antigens or vaccine activity evaluation.