CN115819568A - Enterovirus A71 monoclonal antibody and application - Google Patents

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

Description

Enterovirus A71 monoclonal antibody and application

Technical Field

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

Background

Hand-foot-mouth disease (HFMD) is a global infectious disease mainly caused by acute infection of children and is a legal class C infectious disease in China. The virus has high transmission speed and strong infection capacity, and the number of the diseases is usually in the first three of the third infectious diseases in China. Is a syndrome caused by multiple pathogens, the main infected people are children, clinically, the symptoms are fever, eruption on hands, feet, mouths, buttocks and other parts, and aseptic meningitis and the like can be caused in serious cases, and even death can be caused. The pathogenic composition of HFMD is complex, and is mostly a member of enterovirus genus of picornaviridae, and enterovirus type A (Enterovirus A71, EV A71), coxsackievirus type A (CoxsackievirusA, CV A) 2-10, 12, 14, 16, 21, 24, coxsackievirus type B (CoxsackievirusB, CV B) 1-6, and Echovirus (Echovirus, echo) 1,4-7,9, 11, 13, 18, 19, 24, 25, 30, etc. are 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 HFMD-related severe and fatal cases, respectively.

Enterovirus type 71 (EV a 71) is a well-studied class of viruses, EV a71 was first isolated in 1969 from stool specimens of infants with symptoms of central nervous system infections in california, usa. In 1998, EV A71 is firstly separated from Shenzhen HFMD infant specimens in China. Since 2007, diseases associated with EV A71 infection have been widely prevalent in China. The virus particles are extremely small, have the diameter of 20-30 nm and are in an icosahedron three-dimensional symmetrical spherical structure. The genome is approximately 7500bp in length, and has only one open reading frame flanked by 5 'and 3' noncoding regions. The viral particle capsid comprises 60 subunits, each of which is assembled into a pentamer-like structure from 4 capsid proteins. VP4 is embedded inside the viral particle shell and tightly linked to the viral core, and the other three structural proteins are exposed on the surface of the viral particle.

The hand-foot-and-mouth disease is self-limiting, most of the hand-foot-and-mouth diseases can be cured in eruption period, and no specific medicine is available for treating the hand-foot-and-mouth disease in a targeted manner at present. Therefore, general treatment and etiological treatment are usually adopted clinically. In 2016, 3 kinds of EV A71 inactivated vaccines in China are approved by the national food and drug administration to be used in the market, then the number of hand-foot-and-mouth disease cases and the composition ratio caused by EV A71 infection are further reduced, the composition ratio of other EVs is increased by 16.07% on average, and disease monitoring data show that the application of the EV A71 inactivated vaccines effectively reduces the hand-foot-and-mouth disease morbidity, severe illness and death caused by EV A71, but has 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, the prevalence of other EVs causing the hand-foot-and-mouth disease is gradually increased, and the prevalence of CV A16 is also enhanced in 2017-2019, so that the hand-foot-and-mouth disease is still an infectious disease with serious harm, and the research and the development of multivalent combined vaccines are urgent and are an inevitable trend, so that the hand-foot-and-mouth disease can be fundamentally prevented and controlled.

The detection of the antibody in the vaccine development process is an indispensable tool for detecting the epitope in the vaccine development process so as to ensure that the detected antigen is the active epitope antigen. The development of monoclonal antibodies with strong specificity, high purity, high homogeneity and high neutralizing activity is required because good detection antibodies can promote the development process. In addition, monoclonal antibody medicines can be developed based on virus capsid protein, and the monoclonal antibody can be combined with the virus capsid protein to block virus adsorption or shelling process, thereby inhibiting virus replication. At present, no antiviral drug for hand-foot-and-mouth disease is approved by FDA, and a long path is needed for the successful development of a novel specific drug, so that the development of monoclonal antibody lays a foundation for drug research and development.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present application aims to provide an enterovirus a71 monoclonal antibody and applications thereof, which are used to solve the problems in the prior art.

To achieve the above and other related objects, the present application provides, in a first aspect, an enterovirus a71 monoclonal antibody, which 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 content of the first and second substances,

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

In any of the embodiments herein, 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 the FR4 comprises a sequence shown as SEQ ID No. 10.

In any embodiment of the present 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 FR2 comprises a sequence shown in 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 the FR4 comprises a sequence shown as SEQ ID No. 14.

In any embodiment of the present 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 present 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 a sequence shown as SEQ ID No. 18.

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

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

In a fourth aspect, the present invention provides a host cell comprising said expression vector or genome into which said nucleotide molecule has been integrated.

The fifth aspect of the present application provides the use of the monoclonal antibody or the nucleotide molecule or the expression vector or the host cell in the preparation of a virus detection product, a product for preventing or treating virus infection, or in screening and detecting a virus vaccine and evaluating vaccine activity.

In any of the embodiments herein, the virus is an enterovirus, preferably, enterovirus 71; more preferably, the monoclonal antibody is used for preparing hand-foot-and-mouth disease detection products, products for preventing or treating hand-foot-and-mouth disease, or antibody reference products for screening hand-foot-and-mouth disease vaccines, qualitative and quantitative detection and detection of vaccine antigens or activity evaluation of vaccines.

Compared with the prior art, the beneficial effect of this application is:

1. the monoclonal antibody provided by the application can specifically recognize the A71 virus-like particle, but cannot recognize the denatured virus-like particle, and the epitope recognized by the monoclonal antibody is suggested to be possibly a conformational epitope.

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

3. The monoclonal antibody provided by the application shows strong neutralizing activity, wherein the neutralizing concentration is 0.032 mu g/ml, and the monoclonal antibody is a reliable candidate for a diagnostic reagent and a therapeutic humanized monoclonal antibody and can also be used as an antibody reference for evaluating the activity of a vaccine.

Drawings

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

FIG. 2 shows a schematic diagram of monoclonal antibody-specific analysis.

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

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

FIG. 5 shows the sequence of 1G10 monoclonal antibody.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the present invention is further described below with reference to the following embodiments. It should be understood that the examples are for purposes of illustration only and are not intended to limit the scope of the present application. Unless otherwise indicated, the test methods used in the following examples are all conventional and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.

The inventors of the present application have found an enterovirus a71 monoclonal antibody and applications thereof through a large number of research studies, and have completed the present application on this basis.

As used herein, "monoclonal antibody" means an antibody molecule, a preparation of antibodies, having a common heavy chain amino acid sequence and a common light chain amino acid sequence, as opposed to a "polyclonal" antibody preparation containing a mixture of antibodies of different amino acid sequences. The antibodies useful 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 it was produced.

Monoclonal antibodies can be produced by several known techniques, such as phage technology, bacterial, yeast or ribosome display, and the classical methods exemplified by hybridoma-derived antibodies. 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 made by the hybridoma method (first proposed by Kohler et al, nature, 256. Monoclonal antibodies can also be isolated from phage antibody libraries using techniques such as those described by Clackson et al, nather,352, 624-628 (1991) and Marks et al, mol.biol., 222.

The term "variable" as used herein means that certain portions of the variable regions of 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 segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called the Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are substantially in a p-fold configuration, connected by three CDRs forming a concatemeric loop, and in some cases may form part of a p-fold structure. The CDRs in each chain are held closely together by the FR regions and form the antigen binding site of an antibody with the CDRs of the other chain, and the constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, e.g., participation in antibody-dependent cytotoxicity of the antibody.

In one aspect, the present application provides an enterovirus a71 monoclonal antibody, wherein 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 the HCDR1 comprises a 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 sequences of mab 1G10 provided herein are shown in table 1.

TABLE 1 monoclonal antibody CDR sequences

The application provides an enterovirus A71 monoclonal antibody, wherein the heavy chain variable region further comprises framework regions FR1-FR4. The amino acid sequence of FR1 comprises a sequence shown in 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 FR3 comprises a sequence shown in SEQ ID No. 9. The amino acid sequence of the FR4 comprises a sequence shown as SEQ ID No. 10. The sequence of the framework region of the single-antibody heavy chain variable region numbered 1G10 provided herein is shown in Table 2.

TABLE 2 Single-antibody heavy chain variable region framework region sequences

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

TABLE 3 monoclonal antibody light chain variable region framework region sequences

In the enterovirus a71 monoclonal antibody provided herein, the heavy chain further comprises a signal peptide. The heavy chain signal peptide comprises a sequence shown in SEQ ID No. 15. The light chain also includes a signal peptide. The light chain signal peptide comprises a sequence shown as SEQ ID No. 16. The sequence of the signal peptide of the monoclonal antibody numbered 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 present 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 obtained by PCR amplification, recombinant methods, or synthetic methods. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, 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,single underlineThe part is a signal peptide sequence, and the part is a signal peptide sequence,

is a sequence of a variable region, and the variable region is a sequence of a variable region,

constant region sequence). The sequence is as follows:

ATGAACCTCGGGTTCTGCTTGATTTTCCTTGTCCTCGTTTTAAAAGGTGTCCAGTGT

(SEQ ID No:19)。

the light chain nucleotide sequence of the monoclonal antibody is shown as SEQ ID No:20 (wherein,single underlineThe part is a signal peptide sequence, and the part is a signal peptide sequence,

is variableThe sequence of the region(s),

constant region sequence). The sequence is as follows:

ATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGT

(SEQ ID No:20)。

in another aspect, the present application provides an expression vector comprising the 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. The expression vector of the present invention is generally referred to various commercially available expression vectors well known in the art, and may be, for example, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.

In another aspect, the present application provides a host cell comprising said expression vector or genome into which said nucleotide molecule has been integrated. "host cell" any cell suitable for expression of an expression vector can be used as a host cell, e.g., 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 present application provides a use of the monoclonal antibody or the nucleotide molecule or the expression vector or the host cell in the preparation of a product for virus detection, in the preparation of a product for preventing or treating virus infection, or in screening and detecting a virus vaccine and evaluating the activity of the vaccine.

In the uses provided herein, the virus detection product can 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. Vaccine compositions the virus-like particles of the invention can be administered directly to an individual using known methods. These vaccines are typically administered using the same route of administration as conventional vaccines and/or mimicking the route of infection by pathogens. Routes of administration of the vaccine composition include: intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral, or other parenteral routes of administration. If desired, the routes of administration may be combined, 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 uses provided herein, the virus is an enterovirus, preferably, enterovirus 71; more preferably, the monoclonal antibody is used for preparing hand-foot-and-mouth disease detection products, products for preventing or treating hand-foot-and-mouth disease, antibody reference products for screening hand-foot-and-mouth disease vaccines, qualitative and quantitative detection and detection of vaccine antigens or activity evaluation of vaccines, and the like.

In the vaccine screening process, in order to ensure that the antigen to be detected is an active epitope antigen, a monoclonal antibody with high sensitivity, high specificity and high neutralizing activity is required, and in a specific embodiment of the present application, the sensitivity can be minimally detected by, for example, a sandwich Elisa assay. The specificity may be, for example, by detecting the specific binding of the monoclonal antibody to the antigen by Western blot assay. The neutralizing activity may be determined, for example, by neutralization assays. Qualitative and quantitative detection of the vaccine antigens can be obtained, for example, by sandwich Elisa experiments.

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

The 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 the pichia pastoris expression system (see patent CN 114836444). At week 0, 6-week-old female Balb/c mice were immunized subcutaneously after mixing 50 μ g of EV a71VLP with 40 μ g of Freund's Complete Adjuvant (FCA); at 1 week, 50 μ g of EV A71VLP was mixed with 40 μ g of aluminum adjuvant and subjected to intraperitoneal immunization; at 2 weeks, 50 μ g of EV a71 VLPs emulsified with 40 μ g of FIA adjuvant followed by subcutaneous multipoint immunization; at 3 weeks, 50 μ g of EV A71VLP was mixed with 40 μ g of aluminum adjuvant and immunized intraperitoneally. After the last immunization, tail vein injection of 5 μ g of EV A71VLP was performed for boosting.

3 days after the tail vein of the mouse was boosted, spleen cells of the mouse were fused with myeloma cells SP2/0 using PEG1450 to prepare hybridoma cells. After 1-2 weeks of hybridoma cell culture, screening for specific secretion of antibodies against EV A71VLP using enzyme-linked immunosorbent assay and neutralization assay. The enzyme-linked immunosorbent assay comprises the following steps: coating EV A71VLP on a 96-well enzyme label plate (200 ng/well), and incubating overnight at 4 ℃; blocking with 5% skim milk-containing PBST, adding 50. Mu.l hybridoma culture medium per well, and incubating at 37 ℃ for 2 hours; then, the cells were incubated with HRP-labeled secondary antibody for 1 hour, and finally, a color reaction was performed to read the absorbance at OD450nm. The neutralization test comprises the following steps: mixing 50 μ l hybridoma culture solution with 100TCID50/50 μ l enterovirus A71 (see patent CN 114836444), and adding 5% CO ₂ Incubate at 37 ℃ for 2h in an incubator. RD cell working solution (1.5X 105/ml) is suspended and added into a 96-well plate after incubation, 100 mu l/well and 5% CO ₂ The culture was carried out in an incubator at 35 ℃ for 7 days. CPE was observed.

Finally, monoclonal antibodies numbered 1G10 and 1H10 were selected based on their binding ability to A71VLP and their neutralizing activity, wherein 1G10 monoclonal antibody had both good binding ability and neutralizing activity, and the identification information is shown in Table 6.

TABLE 6 hybridoma cell line identification of secreted monoclonal antibodies

Hybridoma cell line numbering	Heavy chain	Light chain	Binding capacity to A71VLP	Neutralizing Activity #
					1G10	IgG2a	kappa	+++	+
1H10	IgG1	kappa	+++	-

The samples used for the analysis were all 50. Mu.l hybridoma culture cell supernatant.

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

# +: has neutralizing activity; -, no neutralizing activity

Example 2 specificity analysis of anti-A71 monoclonal antibodies

Antibody purification: female Balb/c mice were intraperitoneally injected with 500. Mu.l liquid paraffin and one week later 80 ten thousand hybridoma cells per mouse. After 1-2 weeks, ascites is collected with a needle, centrifuged at 4000rpm for 10min, the upper layer of oil and the lower layer of precipitate are removed, and clarified ascites is taken for antibody purification. Purified antibodies were obtained by purifying ascites fluid using an iProtein G Purose 4Fast Flow affinity column (thousand pure organisms) according to the instructions.

The ELISA method comprises the following steps: coating a 96-well enzyme label plate (200 ng/well) with A6 or A10 or A16 or A71VLP, and incubating overnight at 4 ℃; adding 5% skimmed milk PBST, and sealing at 37 deg.C for 1 hr; adding a detection monoclonal antibody, and incubating for 2 hours at 37 ℃; then, HRP-labeled secondary antibody was added, and the mixture was incubated for 1 hour, and finally the absorbance OD450nm was read.

Protein experiments: mixing the protein sample with loading buffer solution of polypropylene gel electrophoresis (SDS-PAGE), boiling for 5min, and separating the protein sample by 12% polyacrylamide gel. Protein bands were visualized by Coomassie blue staining or proteins were transferred to PVDF membranes for western blot analysis. The monoclonal antibody concentration was 1ug/ml, the rabbit anti-VP 0 polyclonal antibody 1 was diluted for use at 1000, 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 the A71 mAb purified from the ascites fluid. As shown in FIG. 1, mAbs 1G10 and 1H10 each show two major bands, of sizes of about 55kDa and 25kDa, corresponding to the heavy and light chains, respectively. The reactivity of the monoclonal antibody with different antigens was tested by ELISA method, including CV A6 VLP, CV A10VLP (see patent CN114836444 for preparation method), CV A16 VLP and EV A71 VLP. As shown in fig. 2, both 1G10 and 1H10 mabs can specifically recognize EV a71 VLPs, but not CV A6 VLPs, CV a10 VLPs and CV a16 VLPs. As shown in fig. 3, the binding of mab and CV A6 VLP, CV a10VLP, CV a16 VLP and EV a71VLP was analyzed by Western blot, and 1G10 mab could not recognize denatured CV A6 VLP, CV a10VLP, CV a16 VLP and EV a71VLP, suggesting that the epitope recognized by 1G10 may be a conformational epitope, and 1H10 could specifically recognize denatured EV a71VLP, indicating that the epitope recognized by the antibody is a linear epitope.

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

Sandwich ELISA: the rabbit anti-EV a71VLP polyclonal antiserum 1 was diluted at 8000 and coated with 96 enzyme-labeled plates (100 μ l/well) and incubated overnight at 4 ℃; adding PBST containing 5% skimmed milk, sealing at 37 deg.C for 1 hr, adding EV A71VLP, and incubating at 37 deg.C for 2 hr; 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 the absorbance OD450nm was read.

The monoclonal antibodies were tested for minimal detection of disease EV A71VLP by sandwich ELISA (positive when OD450 > 0.15). As shown in FIG. 4, both the 1G10 and 1H10 mabs were sensitive to detection of Enterovirus A71 VLPs with minimal detection of 3.125ng/ml and 25ng/ml, respectively, suggesting that they may be used in diagnosis of A71 infection.

Example 4 detection of neutralizing Activity

Adding the antibody of example 2 into a 96-well dilution plate containing 2% FBS DMEM, blowing, uniformly mixing, performing 5-fold gradient dilution downwards, performing 8 gradient dilutions, adding 50 ul of 8 antibodies with gradient concentration of 5000ng/50 ul-0.064 ng/50 ul into a 96-well culture plate, and setting 2 multiple wells for each dilution; 100TCID 50/50. Mu.l of enterovirus A71 was taken. Adding the working solution into the diluted positive antibody of the corresponding 96-well plate, fully and uniformly mixing, and adding 5% CO ₂ Incubate at 37 ℃ for 2h in an incubator. RD cell working solution (1.5X 10) ⁵ Ml) was suspended in a 96-well plate after incubation, 100. Mu.l/well, 5% CO ₂ The culture was carried out in an incubator at 35 ℃ for 7 days. CPE was observed.

The neutralizing activity of the 1G10 and 1H10 monoclonal antibodies is detected through a neutralization test, and the result shows that 1H10 has no neutralizing activity, while 1G10 has strong potential neutralizing activity on A71, wherein the neutralizing concentration is 0.032 mug/ml, and the method is suggested to be used for developing antiviral drugs of enterovirus A71.

Example 5 Gene sequence analysis of 1G10 monoclonal antibody

Total RNA was extracted from cells of the hybridoma cell line of example 1 using Trizol reagent, and full-length genes of heavy and light chains were amplified according to the instruction of 5' RACE kit.

The heavy chain nucleotide sequence of the obtained 1G10 monoclonal antibody is shown as SEQ ID No. 19, and the light chain nucleotide sequence is shown as SEQ ID No. 20.

The 1G10 single-antibody heavy chain variable region and light chain variable region sequences were further analyzed, and the 1G10 single-antibody 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 monoclonal antibody light chain variable region amino acids are as follows (underlined heavy chain CDR regions):

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLNSGVP SRFSGSGSGTDYSLTISNLEEEDIATYFCQQGNTAWTFGGGTKLEIK(SEQ ID No:22)。

the light chain variable region described above belongs to IGKV10 subgroup.

EXAMPLE 6 recombinant expression and characterization of monoclonal antibody genes

The coding sequences of the heavy chain and the light chain are respectively inserted between the EcoRI and XhoI multiple cloning sites of pcDNA3.3 (thermo) by adopting a homologous recombination mode, expression vectors pcDNA3.3-1G10-H and pcDNA3.3-1G10-L are constructed, the pcDNA3.3-1G10-H and pcDNA3.3-1G10-L are co-transfected to 293T cells by utilizing a liposome method, culture supernatant is collected for analysis after 3d, and the expression of the antibody in the culture supernatant is determined by adopting an enzyme-linked immunosorbent assay method.

As shown in FIG. 5, the cell supernatant expressing the 1G10 monoclonal antibody sequence has high binding signal with A71VLP, and the OD450nm gradually decreases with the increase of dilution; while the supernatant of control cells not transfected with the relevant plasmid did not bind the signal, indicating that the amplified and expressed sequence was indeed the gene of 1G10 mAb.

In the research, an EV A71VLP is used for immunizing a BALB/c mouse, a B lymphocyte hybridoma technology is adopted for cell fusion, a hybridoma cell strain capable of stably secreting a specific anti-EV A71 monoclonal antibody is obtained by screening, and the antibody is detected by using technical means such as Western blot, elisa, in vitro neutralization and the like. The reactivity of the monoclonal antibody with different antigens is detected by an indirect Elisa method, and the monoclonal antibodies 1G10 and 1H10 can specifically recognize EV A71VLP but cannot recognize CV A6 VLP, CV A10VLP and CV A16 VLP, which indicates that the antibody has good specificity. The Western blot result shows that 1G10 monoclonal antibody can not recognize denatured EV A71VLP, CV A6 VLP, CV A10VLP and CV A16 VLP, and the recognized epitope is possible to be a conformational epitope; while 1H10 can recognize denatured EV a71 VLPs, 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 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 antibody has strong in-vitro virus neutralization activity, which suggests that the 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 conclusion, the 1G10 and 1H10 antibodies have good specificity and sensitivity, wherein the 1G10 also has virus neutralization capacity, so that the antibodies not only can be used as a detection tool which is useful in a laboratory, but also show great potential in the aspects of virus identification, diagnosis, treatment and multivalent vaccine development.

The foregoing embodiments are merely illustrative of the principles of the present invention and its efficacy, and are not intended to limit the present application. Any person skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present application.

Claims (10)

1. An enterovirus a71 monoclonal antibody, said 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 comprises the sequence shown in SEQ ID No. 1; the sequence of HCDR2 comprises the sequence shown in SEQ ID No. 2; the sequence of HCDR3 comprises the sequence shown in SEQ ID No. 3; the sequence of LCDR1 comprises a sequence shown as SEQ ID No. 4; the sequence of LCDR2 comprises the sequence shown in SEQ ID No. 5; the sequence of LCDR3 includes the sequence 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 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 the FR4 comprises a sequence shown as 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 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 a 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;

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

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;

and/or the amino acid sequence of the light chain comprises a sequence shown as SEQ ID No. 18.

6. A nucleotide molecule encoding the monoclonal antibody according to any one of claims 1 to 5.

7. An expression vector comprising the nucleotide molecule of claim 6.

8. A host cell comprising the expression vector of claim 7 or having the nucleotide molecule of claim 6 integrated into its genome.

9. Use of the monoclonal antibody according to any one of claims 1 to 5 or the nucleotide molecule according to claim 6 or the expression vector according to claim 7 or the host cell according to claim 8 for the preparation of a product for viral detection, for the preparation of a product for the prevention or treatment of viral infection, or for screening and detection of viral vaccines and for the evaluation of vaccine activity.

10. Use according to claim 9, wherein the virus is an enterovirus, preferably enterovirus type 71; more preferably, the monoclonal antibody is used for preparing hand-foot-and-mouth disease detection products, products for preventing or treating hand-foot-and-mouth disease, or antibody reference products for screening hand-foot-and-mouth disease vaccines, qualitative and quantitative detection and detection of vaccine antigens or activity evaluation of vaccines.

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