Monoclonal antibodies that specifically bind coxsackievirus a6 and uses thereof

Abstract

The application discloses a monoclonal antibody specifically binding to coxsackievirus A6 and application thereof. The monoclonal antibody comprises a light chain and a heavy chain, the amino acid sequence of the complementarity determining region HCDR1 of the heavy chain is shown as SEQ ID No:1, the amino acid sequence of the complementarity determining region HCDR2 of the heavy chain is shown as SEQ ID No:4, the amino acid sequence of the complementarity determining region HCDR3 of the heavy chain is shown as SEQ ID No:7, the amino acid sequence of the complementarity determining region LCDR1 of the light chain is shown as SEQ ID No:10, the amino acid sequence of the complementarity determining region LCDR2 of the light chain is shown as GTS, and the amino acid sequence of the complementarity determining region LCDR3 of the light chain is shown as SEQ ID No:13. The monoclonal antibody has good specificity and sensitivity and has a virus neutralization capacity, and shows great potential in virus identification, diagnosis, treatment and development of multivalent vaccines.

Description

[0001] This application is a divisional application of application number 2022114909090, the original application date is November 25, 2022, and the invention is named "Monoclonal antibody specifically binding to coxsackievirus A6 and its use". TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine technology, and specifically relates to a monoclonal antibody specifically binding to coxsackievirus A6 and its use. BACKGROUND

[0003] Hand, foot, and mouth disease (HFMD) is a global infectious disease mainly in children, and is a legal class B infectious disease in China. The virus spreads quickly and has strong infectivity, and its incidence often ranks among the top three of class B infectious diseases in China. HFMD is a syndrome caused by multiple pathogens, mainly infecting children. The typical clinical symptoms include cough, fever, rash on hands, feet, mouth, and buttocks, loss of appetite, drooling, etc. Some patients only show HA symptoms, and a small number of patients may have central nervous system damage, showing listlessness, irritability, convulsions, etc. Severe cases show rapid heart rate and respiration, brain function failure, low blood pressure, or shock, etc. Atypical HFMD (aHFMD) is mainly caused by coxsackievirus A6 (CV A6), and the clinical manifestations include atypical skin rash or systemic skin rash, peeling, nail loss, and adult infection, etc.

[0004] The pathogen composition of HFMD is complex, and most of them are members of Enterovirus of Picornaviridae. Enterovirus A71 (EV A71), Coxsackievirus A (CV A) 2-10, 12, 14, 16, 21, 24, Coxsackievirus B (CV B) 1-6, Echovirus (Echo) 1, 4-7, 9, 11, 13, 18, 19, 24, 25, 30, etc. have been reported. Since the world's first report of HFMD in 1957, CV A16 and EVA71 have been the main pathogenic agents of HFMD, but with the application of EV A71 vaccine in China in recent years and the change of HFMD pathogen spectrum, CVA6 has gradually replaced EV A71 and CV A16 to become another important pathogenic agent of HFMD. CV A6 belongs to Enterovirus A group of Picornaviridae, is icosahedral spherical, has no envelope, is a single-stranded positive-strand RNA, the full-length of the genome is about 7400 bp, contains 1 open reading frame and 5' and 3' non-coding regions, P1 region encodes 4 structural proteins (VP1-VP4), P2 and P3 regions encode 7 non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D), among the 4 structural proteins, VP4 is located on the inner side of the virus particle shell and is closely connected with the virus core, VP1-VP3 are exposed on the surface of the virus particle and contain the main antigenic determinant.

[0005] There are studies analyzing the basic reproduction number R0 of CVA6 as 5.04, which is higher than that of EVA71 and CVA16, and the proportion of high fever caused by CVA6 infection is higher than that of EVA71 and CVA16, and the skin lesions are more serious, and the probability of the infection cases of the loss of the nail is also higher than that of EVA71 and CVA16. In addition, CVA6 can also cause convulsions, abnormal pupils and tachycardia and other severe symptoms, which are highly prevalent in adults. Different clinical symptoms may be related to viral gene recombination, which leads to an increase in the number of severe cases of CVA6 infection, which increases the difficulty of prevention and control. With the popularization of EVA71 vaccine, EVA71 infection of HFMD is gradually less, and CVA6 has replaced EVA71 to become the main pathogen of HFMD in China, which not only further confirms the protective effect of the vaccine, but also makes the clinical treatment of HFMD caused by CVA6 infection more difficult, and there is no effective treatment drug at present. Therefore, the preparation of passive immunization preparations with high efficiency, low price and small side effects, the research and development of multivalent combined vaccine, the development of detection kits capable of detecting enterovirus A6 serotype and the like have a very important role in preventing and controlling CVA6 serotype related HFMD. The most critical technology in these aspects is to screen a suitable monoclonal antibody, and then the monoclonal antibody can be used for disease treatment drug development, an effective vaccine antigen evaluation system is established, and clinical disease detection and diagnosis are carried out, which has great significance for the prevention and control of HFMD and the reduction of social medical and economic burden. SUMMARY

[0006] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a monoclonal antibody specifically binding to coxsackievirus A6 and uses thereof, in order to solve the problems existing in the prior art.

[0007] In order to achieve the above-mentioned purpose, the present application specifically adopts the following technical solutions.

[0008] The first aspect of the present application protects a monoclonal antibody specifically binding to coxsackievirus A6, comprising a light chain and a heavy chain, the complementarity determining region of the heavy chain comprising HCDR1, HCDR2 and HCDR3, the complementarity determining region of the light chain comprising LCDR1, LCDR2 and LCDR3,

[0009] The sequence of HCDR1 comprises any one of the amino acid sequences as shown in SEQ ID No: 1-SEQ ID No: 3;

[0010] The sequence of HCDR2 comprises any one of the amino acid sequences as shown in SEQ ID No: 4-SEQ ID No: 6;

[0011] The sequence of HCDR3 comprises an amino acid sequence of any one of the sequences shown as SEQ ID No: 7-SEQ ID No: 9;

[0012] The sequence of LCDR1 comprises an amino acid sequence of any one of the sequences shown as SEQ ID No: 10-SEQ ID No: 12;

[0013] The sequence of LCDR2 comprises an amino acid sequence of any one of the sequences shown as GTS, AAT, AVT;

[0014] The sequence of LCDR3 comprises an amino acid sequence of any one of the sequences shown as SEQ ID No: 13-SEQ ID No: 15.

[0015] In certain embodiments, the sequence of HCDR1 comprises an amino acid sequence of the sequence shown as SEQ ID No: 1, the sequence of HCDR2 comprises an amino acid sequence of the sequence shown as SEQ ID No: 4, and the sequence of HCDR3 comprises an amino acid sequence of the sequence shown as SEQ ID No: 7; the sequence of LCDR1 comprises an amino acid sequence of the sequence shown as SEQ ID No: 10, the sequence of LCDR2 comprises an amino acid sequence of the sequence shown as GTS, and the sequence of LCDR3 comprises an amino acid sequence of the sequence shown as SEQ ID No: 13.

[0016] In certain embodiments, the sequence of HCDR1 comprises an amino acid sequence of the sequence shown as SEQ ID No: 2, the sequence of HCDR2 comprises an amino acid sequence of the sequence shown as SEQ ID No: 5, and the sequence of HCDR3 comprises an amino acid sequence of the sequence shown as SEQ ID No: 8; the sequence of LCDR1 comprises an amino acid sequence of the sequence shown as SEQ ID No: 11, the sequence of LCDR2 comprises an amino acid sequence of the sequence shown as AAT, and the sequence of LCDR3 comprises an amino acid sequence of the sequence shown as SEQ ID No: 14.

[0017] In certain embodiments, the sequence of HCDR1 comprises an amino acid sequence of the sequence shown as SEQ ID No: 3, the sequence of HCDR2 comprises an amino acid sequence of the sequence shown as SEQ ID No: 6, and the sequence of HCDR3 comprises an amino acid sequence of the sequence shown as SEQ ID No: 9; the sequence of LCDR1 comprises an amino acid sequence of the sequence shown as SEQ ID No: 12, the sequence of LCDR2 comprises an amino acid sequence of the sequence shown as AVT, and the sequence of LCDR3 comprises an amino acid sequence of the sequence shown as SEQ ID No: 15.

[0018] In certain embodiments, the heavy chain variable region of the monoclonal antibody comprises an amino acid sequence selected from the group consisting of any one of SEQ ID No: 40 to SEQ ID No: 42.

[0019] In certain embodiments, the light chain variable region of the monoclonal antibody comprises an amino acid sequence selected from the group consisting of any one of SEQ ID No: 43 to SEQ ID No: 45.

[0020] In certain embodiments, the heavy chain comprises an amino acid sequence selected from the group consisting of any one of SEQ ID No: 46 to SEQ ID No: 48.

[0021] In certain embodiments, the light chain comprises an amino acid sequence selected from the group consisting of any one of SEQ ID No: 49 to SEQ ID No: 51.

[0022] A second aspect of the present application protects a biological material associated with the monoclonal antibody as described above, said biological material comprising one or more of:

[0023] 1) a nucleotide encoding the monoclonal antibody as described above;

[0024] 2) a recombinant expression vector containing the nucleotide of 1);

[0025] 3) a bioengineered bacterium containing the nucleotide of 1), or a bioengineered bacterium containing the recombinant expression vector of 2).

[0026] In certain embodiments, in 1), the nucleotide encoding the heavy chain comprises any one of SEQ ID No. 52 to SEQ ID No. 54.

[0027] In certain embodiments, in 1), the nucleotide encoding the light chain comprises any one of SEQ ID No. 55 to SEQ ID No. 57.

[0028] A third aspect of the present application protects a pharmaceutical composition comprising the monoclonal antibody as described above and / or the biological material as described above, and a pharmaceutically acceptable carrier.

[0029] A fourth aspect of the present application protects a product comprising the monoclonal antibody as described above or the biological material as described above.

[0030] In certain embodiments, the product is selected from the group consisting of a detection reagent, a kit, a chip and a membrane strip.

[0031] Preferably, the kit further comprises a rabbit anti-CVA6 VLP polyclonal antibody serum and a HPR-labeled mouse secondary antibody.

[0032] The fifth aspect of the present application protects the use of the monoclonal antibody as described above or the biological material as described above in the preparation of a virus detection product, the preparation of a product for preventing or treating viral infection, the screening and detection of a virus vaccine or the evaluation of the activity of a virus vaccine.

[0033] In some embodiments, the virus is a Coxsackie virus.

[0034] Preferably, the virus is Coxsackie virus A6.

[0035] Preferably, the monoclonal antibody is used in the preparation of a hand-foot-mouth disease detection product, the preparation of a product for preventing or treating hand-foot-mouth disease, the screening of a hand-foot-mouth disease vaccine or the detection of the activity of a vaccine antigen or vaccine.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] 1) The present application immunizes BALB / c mice with inactivated A6 virus, and after cell fusion using B lymphocyte hybridoma technology, three hybridoma cell strains are screened, which can stably secrete specific anti-CVA6 monoclonal antibodies 1H2, 6G4 and 12C9, respectively.

[0038] 2) The monoclonal antibodies of the present application can all specifically recognize A6 virus-like particles (VLPs), but cannot recognize denatured CVA6 VLPs, suggesting that the epitopes recognized by the monoclonal antibodies are likely to be conformational epitopes.

[0039] 3) The monoclonal antibodies of the present application have a minimum detection limit of 3.9 ng / ml to 31.25 ng / ml for A6 virus-like particles, which provides a favorable theoretical basis for developing them into A6 virus detection kits and vaccine antigen quantification kits.

[0040] 4) The monoclonal antibodies of the present application show strong neutralizing activity, with a neutralization concentration of 70.4 ng / ml to 1250 ng / ml, making them reliable candidates for antiviral drugs and therapeutic monoclonal antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The figure shows the SDS-PAGE diagram of the monoclonal antibodies 1H2, 6G4 and 12C9 in Example 2 of the present application.

[0042] Figure 2 The figure shows the specificity analysis results of the monoclonal antibodies 1H2, 6G4 and 12C9 in Example 2 of the present application.

[0043] Figure 3Figure 2 shows the results of Western blot analysis of monoclonal antibodies 1H2, 6G4, and 12C9 of Example 2 of the present application.

[0044] Figure 4 Figure 3 shows the results of sandwich ELISA analysis of monoclonal antibodies 1H2, 6G4, and 12C9 of Example 3 of the present application.

[0045] Figure 5 Figure 4 shows the sequence confirmation of monoclonal antibodies 1H2, 6G4, and 12C9 of Example 5 of the present application. DETAILED DESCRIPTION

[0046] Terminology of the present application

[0047] Monoclonal antibody Refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that can occur in a few instances. Monoclonal antibodies have high specificity for a single epitope on an antigen. Monoclonal antibodies are different from polyclonal antibodies, which are a mixture of antibody molecules that recognize different epitopes on an antigen. Although traditional monoclonal antibodies are secreted by hybridoma cells, the monoclonal antibodies of the present application are not limited to this method of production. For example, the monoclonal antibodies of the present application can be obtained using hybridoma technology or using recombinant DNA technology.

[0048] Antibody Refers to any immunoglobulin, including a monoclonal, polyclonal, bispecific, or multispecific antibody that is capable of binding a specific antigen. A complete antibody comprises two heavy chains and two light chains. Each heavy chain contains a variable region and a first, second, and third constant region; each light chain contains a variable region and a constant region. The antibody is in the shape of a "Y", with the neck of the "Y" shape comprising the second and third constant regions of the two heavy chains, which are joined by disulfide bonds. Each arm of the "Y" shape comprises the first constant region and the variable region of one of the heavy chains, and the variable region and the constant region of one of the light chains. The variable regions of the light and heavy chains determine antigen binding; each chain's variable region contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the light chain (L) comprise LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain (H) comprise HCDR1, HCDR2, and HCDR3). The three CDRs are separated by framework regions (FRs). The framework regions are more conserved than the CDR regions and form a scaffolding to support the hypervariable regions. Unless otherwise specified, refers to a complete immunoglobulin, but also to fragments of an immunoglobulin (e.g., at least an immunologically active segment of an immunoglobulin molecule), such as a Fab, Fab', F(ab')2, Fv fragment, single chain antibody molecule, or a multispecific antibody formed from any fragment of an immunoglobulin molecule containing one or more CDR regions.

[0049] Epitope epitope refers to a specific chemical group present in an antigen molecule that determines the specificity of the antigen. A single antigen molecule can have one or more different epitopes, the size of which corresponds to the antigen binding site of the corresponding antibody, and each epitope has only one antigen specificity. Epitopes include linear epitopes and conformational epitopes, and antigen epitopes composed of short peptides composed of consecutive linearly arranged amino acid residues are linear epitopes, and some amino acids are not arranged continuously in sequence, but form a specific conformation in space, which is a conformational epitope.

[0050] HFMD is an important disease that endangers the health of children around the world. At present, the HFMD pathogen spectrum in many places in China has changed, and coxsackievirus A6 (CV A6) has gradually occupied a dominant position. The clinical treatment of HFMD caused by CV A6 infection is more difficult, and there is no effective treatment drug at present. Therefore, the development of detection tools and antiviral drugs for CV A6 has important significance for the monitoring and treatment of CV A6 infection.

[0051] In order to achieve the above purpose, the present application uses CV A6 inactivated virus as immunogen, and prepares A6 specific monoclonal antibody by hybridoma technology. After screening and detection, three monoclonal antibodies capable of specifically binding A6 virus-like particles (VLP) are obtained: 1H2, 6G4 and 12C9, and on this basis, the present application is completed.

[0052] The first aspect of the present application protects a monoclonal antibody specifically binding coxsackievirus A6, comprising a light chain and a heavy chain, the complementarity determining region of the heavy chain comprising HCDR1, HCDR2 and HCDR3, the complementarity determining region of the light chain comprising LCDR1, LCDR2 and LCDR3,

[0053] The sequence of HCDR1 comprises any one of the amino acid sequences as shown in SEQ ID No: 1-SEQ ID No: 3;

[0054] The sequence of HCDR2 comprises any one of the amino acid sequences as shown in SEQ ID No: 4-SEQ ID No: 6;

[0055] The sequence of HCDR3 comprises any one of the amino acid sequences as shown in SEQ ID No: 7-SEQ ID No: 9;

[0056] The sequence of LCDR1 comprises any one of the amino acid sequences as shown in SEQ ID No: 10-SEQ ID No: 12;

[0057] The sequence of LCDR2 comprises any one of the amino acid sequences as shown in GTS, AAT, AVT;

[0058] The sequence of HCDR1 includes any one of the amino acid sequences as shown in SEQ ID No: 1-SEQ ID No: 3.

[0059] In the present application, the monoclonal antibody can also be obtained by genetic engineering recombination technology. The DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody are isolated by PCR amplification using nucleic acid primers specific to the heavy chain and light chain genes of the monoclonal antibody. The obtained DNA molecules are inserted into an expression vector, and then transfected into host cells such as E. coli cells, simian COS, CHO cells, or other myeloma cells that do not produce immunoglobulins. The transfected host cells are cultured under specific conditions and express the target antibody.

[0060] The sequences of HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of the present application are shown in Table 1.

[0061] Table 1

[0062]

[0063] Preferably, the sequence of HCDR1 includes an amino acid sequence as shown in SEQ ID No: 1, the sequence of HCDR2 includes an amino acid sequence as shown in SEQ ID No: 4, and the sequence of HCDR3 includes an amino acid sequence as shown in SEQ ID No: 7; the sequence of LCDR1 includes an amino acid sequence as shown in SEQ ID No: 10, the sequence of LCDR2 includes an amino acid sequence as shown in GTS, and the sequence of LCDR3 includes an amino acid sequence as shown in SEQ ID No: 13.

[0064] Preferably, the sequence of HCDR1 includes an amino acid sequence as shown in SEQ ID No: 2, the sequence of HCDR2 includes an amino acid sequence as shown in SEQ ID No: 5, and the sequence of HCDR3 includes an amino acid sequence as shown in SEQ ID No: 8; the sequence of LCDR1 includes an amino acid sequence as shown in SEQ ID No: 11, the sequence of LCDR2 includes an amino acid sequence as shown in AAT, and the sequence of LCDR3 includes an amino acid sequence as shown in SEQ ID No: 14.

[0065] Preferably, the sequence of HCDR1 comprises an amino acid sequence as set forth in SEQ ID No: 3, the sequence of HCDR2 comprises an amino acid sequence as set forth in SEQ ID No: 6, the sequence of HCDR3 comprises an amino acid sequence as set forth in SEQ ID No: 9; the sequence of LCDR1 comprises an amino acid sequence as set forth in SEQ ID No: 12, the sequence of LCDR2 comprises an amino acid sequence as set forth in AVT, and the sequence of LCDR3 comprises an amino acid sequence as set forth in SEQ ID No: 15.

[0066] In the present application, the heavy chain variable region further comprises framework regions FR1-FR4. The amino acid sequence of FR1 comprises any one of the sequences as set forth in SEQ ID No: 16-SEQ ID No: 18, the amino acid sequence of FR2 comprises any one of the sequences as set forth in SEQ ID No: 19-SEQ ID No: 21, the amino acid sequence of FR3 comprises any one of the sequences as set forth in SEQ ID No: 22-SEQ ID No: 24, and the amino acid sequence of FR4 comprises any one of the sequences as set forth in SEQ ID No: 25-SEQ ID No: 27. The sequences of the framework regions FR1-FR4 of the heavy chain variable region are shown in Table 2.

[0067] Table 2

[0068]

[0069] Preferably, the amino acid sequence of FR1 comprises the sequence as set forth in SEQ ID No: 16, the amino acid sequence of FR2 comprises the sequence as set forth in SEQ ID No: 19, the amino acid sequence of FR3 comprises the sequence as set forth in SEQ ID No: 22, and the amino acid sequence of FR4 comprises the sequence as set forth in SEQ ID No: 25.

[0070] Preferably, the amino acid sequence of FR1 comprises the sequence as set forth in SEQ ID No: 17, the amino acid sequence of FR2 comprises the sequence as set forth in SEQ ID No: 20, the amino acid sequence of FR3 comprises the sequence as set forth in SEQ ID No: 23, and the amino acid sequence of FR4 comprises the sequence as set forth in SEQ ID No: 26.

[0071] Preferably, the amino acid sequence of FR1 comprises the sequence as set forth in SEQ ID No: 18, the amino acid sequence of FR2 comprises the sequence as set forth in SEQ ID No: 21, the amino acid sequence of FR3 comprises the sequence as set forth in SEQ ID No: 24, and the amino acid sequence of FR4 comprises the sequence as set forth in SEQ ID No: 27.

[0072] In the present application, the light chain variable region further comprises framework regions FR1-FR4. The amino acid sequence of FR1 comprises any one of the sequences shown as SEQ ID No: 28-SEQ ID No: 30, the amino acid sequence of FR2 comprises any one of the sequences shown as SEQ ID No: 31-SEQ ID No: 33, the amino acid sequence of FR3 comprises any one of the sequences shown as SEQ ID No: 34-SEQ ID No: 36, and the amino acid sequence of FR4 comprises any one of the sequences shown as SEQ ID No: 37-SEQ ID No: 39. The sequences of the framework regions FR1-FR4 of the light chain variable region are shown in Table 3.

[0073] Table 3

[0074]

[0075] Preferably, the amino acid sequence of FR1 comprises the sequence shown as SEQ ID No: 28, the amino acid sequence of FR2 comprises the sequence shown as SEQ ID No: 31, the amino acid sequence of FR3 comprises the sequence shown as SEQ ID No: 34, and the amino acid sequence of FR4 comprises the sequence shown as SEQ ID No: 37.

[0076] Preferably, the amino acid sequence of FR1 comprises the sequence shown as SEQ ID No: 29, the amino acid sequence of FR2 comprises the sequence shown as SEQ ID No: 32, the amino acid sequence of FR3 comprises the sequence shown as SEQ ID No: 35, and the amino acid sequence of FR4 comprises the sequence shown as SEQ ID No: 38.

[0077] Preferably, the amino acid sequence of FR1 comprises the sequence shown as SEQ ID No: 30, the amino acid sequence of FR2 comprises the sequence shown as SEQ ID No: 33, the amino acid sequence of FR3 comprises the sequence shown as SEQ ID No: 36, and the amino acid sequence of FR4 comprises the sequence shown as SEQ ID No: 39.

[0078] In the present application, the heavy chain variable region of the monoclonal antibody comprises any one or more of the sequences shown as SEQ ID No: 40-SEQ ID No: 42. The sequence of the heavy chain variable region is shown in Table 4, wherein The underlined part is a CDR region, and the italicized part is a framework region.

[0079] In the present application, the light chain variable region of the monoclonal antibody comprises any one or more of the sequences shown as SEQ ID No: 43-SEQ ID No: 45. The sequence of the light chain variable region is shown in Table 4, wherein The underlined portions are CDR regions, and the italicized portions are framework regions.

[0080] Table 4

[0081]

[0082] Preferably, the monoclonal antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No: 40, and a light chain variable region having an amino acid sequence as set forth in SEQ ID No: 43.

[0083] Preferably, the monoclonal antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No: 41, and a light chain variable region having an amino acid sequence as set forth in SEQ ID No: 44.

[0084] Preferably, the monoclonal antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No: 42, and a light chain variable region having an amino acid sequence as set forth in SEQ ID No: 45.

[0085] In the present application, the heavy chain comprises any one or more of the sequences having an amino acid sequence as set forth in SEQ ID No: 46 to SEQ ID No: 48. The sequence of the heavy chain is shown in Table 5, wherein underlined The underlined portion is a signal peptide sequence, and the italicized portion is a variable region sequence, is a constant region sequence.

[0086] In the present application, the light chain comprises any one or more of the sequences having an amino acid sequence as set forth in SEQ ID No: 49 to SEQ ID No: 51. The sequence of the light chain is shown in Table 5, wherein underlined The underlined portion is a signal peptide sequence, and the italicized portion is a variable region sequence, is a constant region sequence.

[0087] Table 5

[0088]

[0089]

[0090] Preferably, the amino acid sequence of the heavy chain is as set forth in SEQ ID No: 46, and the amino acid sequence of the light chain is as set forth in SEQ ID No: 49.

[0091] Preferably, the amino acid sequence of the heavy chain is as set forth in SEQ ID No: 47, and the amino acid sequence of the light chain is as set forth in SEQ ID No: 50.

[0092] Preferably, the amino acid sequence of the heavy chain is as set forth in SEQ ID No: 48, and the amino acid sequence of the light chain is as set forth in SEQ ID No: 51.

[0093] To the above purpose, another aspect of the present application provides a biological material related to the monoclonal antibody as described above, the biological material including one or more of the following:

[0094] 1) a nucleotide encoding the monoclonal antibody as described above;

[0095] 2) a recombinant expression vector containing the nucleotide of 1);

[0096] 3) a bioengineered bacterium containing the nucleotide of 1), or a bioengineered bacterium containing the recombinant expression vector of 2).

[0097] In the present application, in 1), the nucleotide encoding the heavy chain includes any one of the sequences as set forth in SEQ ID No. 52 to SEQ ID No. 54.

[0098] Heavy chain nucleotide sequence (wherein, the single underlined portion is a signal peptide sequence, the italicized portion is a variable region sequence, and the dotted underlined portion is a constant region sequence):

[0099]

[0100]

[0101] Heavy chain nucleotide sequence (wherein, the single underlined portion is a signal peptide sequence, the italicized portion is a variable region sequence, and the dotted underlined portion is a constant region sequence):

[0102]

[0103] Heavy chain nucleotide sequence (wherein, underlined the single underlined portion is a signal peptide sequence, the italicized portion is a variable region sequence, and the dotted underlined portion is a constant region sequence):

[0104]

[0105] In the present application, in 1), the nucleotide encoding the light chain includes any one of the sequences as set forth in SEQ ID No. 55 to SEQ ID No. 57.

[0106] Light chain nucleotide sequence (wherein, underlined the single underlined portion is a signal peptide sequence, the italicized portion is a variable region sequence, and the dotted underlined portion is a constant region sequence):

[0107] ​​

[0108] Light chain nucleotide sequence (wherein, part is signal peptide sequence, italic part is variable region sequence, is constant region sequence):

[0109]

[0110] Light chain nucleotide sequence (wherein, underlined part is signal peptide sequence, italic part is variable region sequence, is constant region sequence):

[0111]

[0112]

[0113] Preferably, the monoclonal antibody comprises a heavy chain of nucleotide sequence as set forth in SEQ ID No: 52 and a light chain of nucleotide sequence as set forth in SEQ ID No: 55.

[0114] Preferably, the monoclonal antibody comprises a heavy chain of nucleotide sequence as set forth in SEQ ID No: 53 and a light chain of nucleotide sequence as set forth in SEQ ID No: 56.

[0115] Preferably, the monoclonal antibody comprises a heavy chain of nucleotide sequence as set forth in SEQ ID No: 54 and a light chain of nucleotide sequence as set forth in SEQ ID No: 57.

[0116] In the present application, in 2), the recombinant expression vector can be constructed by inserting the nucleotide into a multiple cloning site of an expression vector. The expression vector can be transformed, transduced or transfected into a host cell, so that the genetic material elements carried by the expression vector can be expressed in the host cell. The recombinant expression vector can be a viral vector or a non-viral vector. For example, the non-viral vector includes a plasmid, a phagemid, a cosmid, an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1-derived artificial chromosome (PAC), a bacteriophage such as a lambda phage or a M13 phage, and an animal virus, etc. The viral vector includes a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpes virus (such as a herpes simplex virus), a poxvirus, a baculovirus, a papillomavirus, a papovavirus (such as SV40). The vector can contain various elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene. In addition, the vector can also contain a replication initiation site. The vector can also include components to assist its entry into the cell, including but not limited to a viral particle, a liposome or a protein coat.

[0117] In the present application, in 3), the bioengineered bacteria are the bacteria into which the recombinant expression vector or the genome described above is introduced or the bacteria in which the nucleotide described above is integrated into the genome. Any cell suitable for expression of the expression vector can be used as the bioengineered bacteria. For example, the bioengineered bacteria can be a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a mammalian cell. The bioengineered bacteria include many cell types such as a prokaryotic cell such as Escherichia coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as a S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast cell, a CHO cell, a COS cell, a NSO cell, a HeLa cell, a BHK cell, a HEK 293 cell or a human cell.

[0118] To achieve the above object, another aspect of the present application provides a pharmaceutical composition comprising the monoclonal antibody described above and / or the biomaterial described above, and a pharmaceutically acceptable carrier.

[0119] In the present application, the pharmaceutically acceptable carrier refers to a carrier that does not induce an allergic reaction or other discomfort in the patient to whom it is administered. The pharmaceutically acceptable carrier includes, for example, one or more of water, physiological saline, a phosphate buffer, dextrose, glycerol, ethanol and other similar substances, and a combination thereof.

[0120] In the present application, the pharmaceutically acceptable carrier can further include a trace amount of an auxiliary substance that can improve the shelf life or efficacy of the antibody, such as a humectant or an emulsifier, a preservative or a buffer.

[0121] For the above purpose, another aspect of the present application provides a product comprising the monoclonal antibody as described above and / or the biological material as described above.

[0122] In the present application, the product is selected from the group consisting of detection reagent, kit, chip and membrane strip.

[0123] In the present application, the product can generally be used for the diagnosis of coxsackievirus A6 as a biomarker.

[0124] Preferably, the kit further comprises rabbit anti-CVA6 VLP polyclonal antibody serum and HPR-labeled mouse secondary antibody. The kit further comprises enzyme-labeled plate, coating solution, blocking solution, dilution solution, color developing solution, washing solution, termination solution, etc.

[0125] For the above purpose, another aspect of the present application provides the use of the monoclonal antibody as described above or the biological material as described above in the preparation of a virus detection product, the preparation of a product for preventing or treating viral infection, the screening and detection of a virus vaccine or the evaluation of the activity of a virus vaccine.

[0126] In the present application, the virus is coxsackievirus. Preferably, the virus is coxsackievirus A6.

[0127] In the present application, the monoclonal antibody is used in the preparation of a hand-foot-mouth disease detection product, the preparation of a product for preventing or treating hand-foot-mouth disease, the screening of a hand-foot-mouth disease vaccine or the detection of vaccine antigen for qualitative and quantitative determination or the detection of vaccine activity.

[0128] The monoclonal antibodies 1H2, 6G4 and 12C9 of the present application can specifically recognize CVA6, but cannot recognize CVA10, CVA16 and EVA71, nor can they recognize denatured CVA6, CVA10, CVA16 and EVA71 through Elisa and Western blot analysis. Then, through sandwich Elisa analysis, the minimum detection limit of CVA6 is 3.9 ng / mL, 31.25 ng / mL and 15.625 ng / mL, respectively. Further, through neutralization experiment analysis, the neutralization concentration of CVA6 is 192 ng / mL, 1250 ng / mL and 70.4 ng / mL, respectively. In summary, the monoclonal antibodies obtained in the present application have high specificity, high sensitivity and high titer for CVA6 virus (CVA6). The monoclonal antibodies of the present application can be used in the research and development, production and quality control of CVA6 virus vaccine. For example, they can be used for screening virus vaccine in research and development, and as reference for evaluating the activity of vaccine in quality control. The monoclonal antibodies of the present application can be prepared into products for detecting CVA6 virus, such as detection reagents, kits, membrane strips or chips, but are not limited to detection reagents, kits, membrane strips or chips. Any product that can detect CVA6 virus falls within the protection scope of the present application.

[0129] In the process of vaccine screening, in order to ensure that the detected antigen is an active epitope antigen, a detection monoclonal antibody with high sensitivity, high specificity and high neutralization activity is needed. In a specific embodiment of the present application, the sensitivity can be obtained by the minimum detection limit of sandwich Elisa experiment. The specificity can be obtained by the specific binding of monoclonal antibody and antigen through Western blot experiment. The neutralization activity can be obtained by neutralization experiment. The qualitative and quantitative detection of vaccine antigen can be obtained by sandwich Elisa experiment.

[0130] The following specific embodiments illustrate the embodiments of the present application. Those skilled in the art can easily understand other advantages and effects of the present application from the disclosure of the present application.

[0131] Before further describing the specific embodiments of the present application, it should be understood that the protection scope of the present application is not limited to the following specific embodiments. It should also be understood that the terms used in the embodiments of the present application are for describing the specific embodiments, but not for limiting the protection scope of the present application. The test methods in the following examples, if not specified, are usually carried out under conventional conditions, or under the conditions recommended by the manufacturers.

[0132] When the embodiments give a numerical range, it should be understood that, unless otherwise specified by the present application, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by a person skilled in the art. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material of the prior art similar or equivalent to the methods, devices and materials described in the embodiments of the present application can also be used to implement the present application according to the mastery of the prior art by a person skilled in the art and the description of the present application.

[0133] The preparation of A6 VLP, A10 VLP, A16 VLP, A71 VLP and rabbit anti-EV A71 VLP polyclonal serum in the embodiments of the present application is described in patent CN114836444.

[0134] The information of the mouse secondary antibody and the 5'RACE kit in the embodiments of the present application is shown in Table 4.

[0135] Table 4 Reagent Information

[0136] Name Manufacturer Catalogue number Mouse secondary antibody Sigma A0168-1 ml 5' RACE kit Invitrogen 18374058

[0137] Example 1 Preparation of hybridoma cell strain and monoclonal antibody

[0138] In this embodiment, the hybridoma cell is prepared and the monoclonal antibody is obtained, which includes the following steps:

[0139] 1.1 Preparation of antigen and immunization of mouse

[0140] 1.1.1 Preparation of antigen:

[0141] We synthesized the full-length sequence of A6 virus (GenBank: KR706309.1) and obtained A6 virus through reverse genetics; the A6 virus was cultured in large quantities using RD (human rhabdomyosarcoma cells), and the cultured virus was inactivated using beta-propiolactone. The inactivated virus was purified by ultracentrifugation (the ultracentrifugation method can be found in patent CN114836444), and finally the A6 inactivated virus was obtained.

[0142] 1.1.2 Immunization of mice with antigen

[0143] 1 μg of A6 inactivated virus was mixed with 60 μg of aluminum adjuvant, and the mixed preparation was used to immunize 6-week-old female Balb / c mice through intraperitoneal injection. The immunization was performed three times, and the dose of each immunization was 1 μg of the mixed preparation of A6 inactivated virus and 60 μg of aluminum adjuvant, which was injected intraperitoneally. The interval between the two immunizations was 1 week. Ten days after the last immunization, 5 μg of A6 inactivated virus was injected into the tail vein for booster immunization.

[0144] 1.2 Preparation and screening of hybridoma cell lines

[0145] Three days after the booster immunization of the mouse tail vein, the mouse spleen cells were fused with the myeloma cells SP2 / 0 using PEG1450 to prepare hybridoma cells.

[0146] After 8 days of hybridoma cell culture, the hybridoma culture solution was taken and screened for specific secretion of hybridoma cell lines against CVA6 VLPs using enzyme-linked immunosorbent assay and neutralization test.

[0147] The steps of enzyme-linked immunosorbent assay are as follows: CVA6 VLPs were coated on a 96-well enzyme-labeled plate (200 ng / well) and incubated at 4°C overnight; after blocking with PBST containing 5% skimmed milk, 50 μL of hybridoma culture solution was added to each well and incubated at 37°C for 2 hours; then HRP-labeled secondary antibody was incubated for 1 hour, and finally color development reaction was performed and the OD450nm absorbance value was read.

[0148] The steps of neutralization test are as follows: 50 μL of hybridoma culture solution was mixed with 100 TCID50 / 50 μL of coxsackievirus A6 (see patent CN114836444) and incubated at 37°C for 2 hours in a 5% CO2incubator. RD cell working solution (1.5 x 10 5 / mL) was added to the incubated 96-well plate, 100 μL / well, and incubated at 35°C for 7 days in a 5% CO2incubator. CPE was observed.

[0149] According to the binding ability to A6 VLPs and neutralization activity, three hybridoma cell lines were screened, and the identification information is shown in Table 6.

[0150] Table 6 Identification of hybridoma cell lines secreting monoclonal antibodies

[0151] Hybridoma cell line Heavy chain Light chain Binding ability to A6 VLP Neutralizing activity # ]] 1H2 IgGl kappa +++ + 6G4 IgG2a kappa +++ + 12C9 IgG2b kappa +++ +

[0152] The samples used for analysis were all 50 μL of hybridoma culture cell supernatant.

[0153] * Neutralization activity, + represents OD450>0.15; ++ represents OD450>0.3; +++ represents OD450>0.5.

[0154] # Neutralization activity, + represents neutralization activity; - represents no neutralization activity.

[0155] 1.3 Preparation of mouse ascites produced by hybridoma and antibody purification

[0156] Each female Balb / c mouse (10 weeks old) was injected intraperitoneally with 500 μl of liquid paraffin oil, and one week later, the mice were divided into three groups, one group of mice was injected intraperitoneally with 800,000 hybridoma cell strains 1H2 obtained in step 1.2, one group of mice was injected intraperitoneally with 800,000 hybridoma cell strains 6G4 obtained in step 1.2, and one group of mice was injected intraperitoneally with 800,000 hybridoma cell strains 12C9 obtained in step 1.2.

[0157] After intraperitoneal injection for 1-2 weeks, the ascites of each mouse was collected with a needle, and the ascites was centrifuged at 4000 rpm for 10 min to remove the upper oil and the lower precipitate, and the clear ascites was taken for antibody purification. According to the instructions, the ascites was purified by iProtein GPurose 4Fast Flow affinity column (Qianpure Biotechnology) to obtain three purified monoclonal antibodies 1H2, 6G4 and 12C9.

[0158] Example 2 Specificity study of monoclonal antibodies

[0159] In this example, the purified monoclonal antibodies obtained in Example 1 were used for specificity analysis, including the following:

[0160] 2.1 Polyacrylamide gel electrophoresis

[0161] The polyacrylamide gel electrophoresis (SDS-PAGE) method was used to identify the purity and integrity of the purified monoclonal antibodies obtained in Example 1.

[0162] Including the following: after mixing the monoclonal antibody with the SDS-PAG loading buffer, boiling treatment for 5 min, the protein sample was separated by 12% polyacrylamide gel, and the results are shown in Figure 1 .

[0163] As shown in Figure 1 , the monoclonal antibodies 1H2, 6G4 and 12C9 all showed two bands, with sizes of about 55 kDa and 25 kDa, corresponding to heavy chain and light chain.

[0164] 2.2 ELISA identification

[0165] Then, the Elisa method was used to detect the reaction activity of monoclonal antibodies 1H2, 6G4 and 12C9 with different antigens, including CV A6 VLP, CV A10 VLP, CV A16 VLP and EV A71 VLP.

[0166] The hybridoma culture fluid obtained in step 1.2 of Example 1 was screened by enzyme-linked immunosorbent assay (ELISA) to form the test sample. A6, A10, A16, and A71 VLPs were coated on a 96-well ELISA plate (200 ng / well) and incubated at 4°C overnight. 5% skim milk in PBST was added and blocked at 37°C for 1 hour. The test sample was added and incubated at 37°C for 2 hours. HRP-labeled secondary antibody was then added and incubated for 1 hour. The absorbance at OD450nm was read. The results are shown in Table 1. Figure 2 .

[0167] like Figure 2 As shown, monoclonal antibodies 1H2, 6G4, and 12C9 can specifically recognize CV A6 VLP, but cannot recognize CVA10 VLP, CV A16 VLP, and EV A71 VLP.

[0168] 2.3 Western blot analysis

[0169] Western blot analysis was performed to determine the binding of purified monoclonal antibodies 1H2, 6G4, and 12C9 to CV A6 VLP, CVA10 VLP, CV A16 VLP, and EV A71 VLP, respectively.

[0170] Protein bands were visualized by Coomassie Brilliant Blue staining or transferred to PVDF membrane for Western blot analysis. Monoclonal antibody concentration was 5 μg / mL, rabbit anti-VP3 polyclonal antibody was diluted 1:1000, then incubated with HPR-labeled secondary antibody, and finally recorded using a luminescence image analyzer. The results are shown in Figure 3 .

[0171] from Figure 3 As shown, the three monoclonal antibodies could not recognize the denatured CV A6 VLP, CV A10 VLP, CVA16 VLP and EV A71 VLP, suggesting that the recognized epitope may be a conformational epitope.

[0172] Example 3 Detection of Viruses by Monoclonal Antibodies and Detection Kit

[0173] In this example, the purified monoclonal antibody obtained in Example 1 was used to detect Coxsackievirus CV A6 VLP, including the following:

[0174] 3.1 Sandwich Elisa assay

[0175] The minimum detection limit of the purified monoclonal antibody against CV A6 VLP was determined by sandwich ELISA method (when OD450nm>0.15, it was judged as positive).

[0176] The following steps are included: dilute rabbit anti-CVA6 VLP polyclonal serum 1:8000 to coat 96 enzyme-labeled plates (100 μL / well), incubate at 4°C overnight; add 5% skim milk PBST, block at 37°C for 1 hour, then add CVA6 VLP, incubate at 37°C for 2 hours; then, add the monoclonal antibodies purified in Example 1 (10 ng / μL), incubate at 37°C for 2 hours; then, incubate with HPR-labeled mouse secondary antibody, finally read the absorbance value OD450nm, and the results are shown in Figure 4 .

[0177] From Figure 4 it can be seen that the monoclonal antibodies 1H2, 6G4 and 12C9 can sensitively detect CVA6 VLP, and the minimum detection limits are 3.9 ng / ml, 31.25 ng / ml and 15.625 ng / ml, respectively, indicating that the monoclonal antibodies 1H2, 6G4 and 12C9 can be used for the diagnosis of CVA6 infection.

[0178] 3.2 Kit

[0179] The kit includes:

[0180] An enzyme-labeled plate;

[0181] Detection antibody reagents: rabbit anti-CVA6 VLP polyclonal serum and HPR-labeled mouse secondary antibody;

[0182] Antibody reagents: monoclonal antibodies 1H2, 6G4 and 12C9;

[0183] Blocking solution: 5% skim milk PBST.

[0184] Example 4 Neutralization experiment

[0185] In this example, the neutralization activity of the purified monoclonal antibodies 1H2, 6G4 and 12C9 obtained in Example 1 on CVA6 was investigated by a neutralization test.

[0186] The purified monoclonal antibodies 1H2, 6G4 and 12C9 are added into 2% FBSDMEM 96-hole dilution plates respectively, mixed by blowing, and then diluted by multiple gradient. 50 μL of each of eight gradient concentrations of the antibody dilutions is added into 96-hole culture plates, wherein 1H2 is diluted by two times, the gradient range is 312 ng / 50 μL to 2.4 ng / 50 μL; 6G4 is diluted by two times, the gradient range is 1000 ng / 50 μL to 7.8 ng / 50 μL; 12C9 is diluted by five times, the gradient range is 2200 ng / 50 μL to 0.028 ng / 50 μL, two duplicate holes are set for each dilution. 100 TCID50 / 50 μL of coxsackie virus A6 virus working solution is added into the corresponding 96-hole plates with the diluted positive antibodies, mixed thoroughly, and then placed into a 5% CO2 incubator for 2 h of incubation at 37°C. The RD cell working solution (1.5 x 10 5 / mL) is suspended and added into the 96-hole plates after incubation, 100 μL / hole, 5% CO2 incubator, 35°C culture for 7 days, and CPE is observed.

[0187] The results show that the monoclonal antibodies 1H2, 6G4 and 12C9 all have strong potential neutralization activity to coxsackie virus A6, the neutralization concentrations are 192 ng / mL, 1250 ng / mL and 70.4 ng / mL respectively, which indicates that the monoclonal antibodies 1H2, 6G4 and 12C9 can be used for the development of anti-virus drugs or the reference of vaccine activity evaluation of coxsackie virus A6.

[0188] Example 5: Gene sequence analysis, recombinant expression and identification of the monoclonal antibody

[0189] In this embodiment, the RNA of the hybridoma cell strain obtained in Example 1 is extracted, and the full-length genes of the heavy chain and the light chain are amplified. The process includes the following steps.

[0190] 5.1 RNA extraction

[0191] The total RNA of the hybridoma cell strains 1H2, 6G4 and 12C9 is extracted by Trizol reagent, and the full-length genes of the heavy chain and the light chain are amplified according to the instructions of the 5' RACE kit.

[0192] The nucleotide sequence of the heavy chain of the monoclonal antibody 1H2 includes SEQ ID No: 52, the nucleotide sequence of the light chain of the monoclonal antibody 1H2 includes SEQ ID No: 55; the amino acid sequence of the heavy chain of the monoclonal antibody 1H2 includes SEQ ID No: 46, and the amino acid sequence of the light chain of the monoclonal antibody 1H2 includes SEQ ID No: 49.

[0193] The nucleotide sequence of the heavy chain of the monoclonal antibody 1H2 is as follows: underlined partSignal peptide sequence, italicized portion is variable region sequence, Constant region sequence):

[0194]

[0195] 1H2 monoclonal antibody light chain nucleotide sequence (wherein, underlined part Signal peptide sequence, italicized portion is variable region sequence, Constant region sequence):

[0196]

[0197] 1H2 monoclonal antibody heavy chain amino acid sequence (wherein, underlined part Signal peptide sequence, italicized portion is variable region sequence, Constant region sequence):

[0198]

[0199] 1H2 monoclonal antibody light chain amino acid sequence (wherein, underlined part Signal peptide sequence, italicized portion is variable region sequence, Constant region sequence):

[0200]

[0201] Further analysis of the heavy chain variable region and light chain variable region sequence of monoclonal antibody 1H2, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1H2 includes as shown in SEQ ID No: 40, and the amino acid sequence of the light chain variable region includes as shown in SEQ ID No: 43.

[0202] 1H2 monoclonal antibody heavy chain variable region amino acid sequence (wherein, The heavy chain CDR region, italicized portion is the framework region):

[0203]

[0204] The above heavy chain variable region belongs to IGHV1 subgroup.

[0205] 1H2 monoclonal antibody light chain variable region nucleotide sequence (wherein, The light chain CDR region, italicized portion is the framework region):

[0206]

[0207] The above light chain variable region belongs to IGKV4 subgroup.

[0208] The nucleotide sequence of the heavy chain of the monoclonal antibody 6G4 comprises SEQ ID No: 53, the nucleotide sequence of the light chain of the monoclonal antibody 6G4 comprises SEQ ID No: 56; the amino acid sequence of the heavy chain of the monoclonal antibody 6G4 comprises SEQ ID No: 47, and the amino acid sequence of the light chain of the monoclonal antibody 1H2 comprises SEQ ID No: 50.

[0209] The nucleotide sequence of the heavy chain of the monoclonal antibody 6G4 comprises SEQ ID No: 53, the nucleotide sequence of the light chain of the monoclonal antibody 6G4 comprises SEQ ID No: 56; the amino acid sequence of the heavy chain of the monoclonal antibody 6G4 comprises SEQ ID No: 47, and the amino acid sequence of the light chain of the monoclonal antibody 1H2 comprises SEQ ID No: 50. underlined part The italic part is the variable region sequence, and the underlined part is the constant region sequence. The italic part is the variable region sequence, and the underlined part is the constant region sequence.

[0210]

[0211] The nucleotide sequence of the heavy chain of the monoclonal antibody 6G4 comprises SEQ ID No: 53, the nucleotide sequence of the light chain of the monoclonal antibody 6G4 comprises SEQ ID No: 56; the amino acid sequence of the heavy chain of the monoclonal antibody 6G4 comprises SEQ ID No: 47, and the amino acid sequence of the light chain of the monoclonal antibody 1H2 comprises SEQ ID No: 50. underlined part The italic part is the variable region sequence, and the underlined part is the constant region sequence. The italic part is the variable region sequence, and the underlined part is the constant region sequence.

[0212]

[0213] The nucleotide sequence of the heavy chain of the monoclonal antibody 6G4 comprises SEQ ID No: 53, the nucleotide sequence of the light chain of the monoclonal antibody 6G4 comprises SEQ ID No: 56; the amino acid sequence of the heavy chain of the monoclonal antibody 6G4 comprises SEQ ID No: 47, and the amino acid sequence of the light chain of the monoclonal antibody 1H2 comprises SEQ ID No: 50. underlined part The italic part is the variable region sequence, and the underlined part is the constant region sequence. The italic part is the variable region sequence, and the underlined part is the constant region sequence.

[0214]

[0215] The nucleotide sequence of the heavy chain of the monoclonal antibody 6G4 comprises SEQ ID No: 53, the nucleotide sequence of the light chain of the monoclonal antibody 6G4 comprises SEQ ID No: 56; the amino acid sequence of the heavy chain of the monoclonal antibody 6G4 comprises SEQ ID No: 47, and the amino acid sequence of the light chain of the monoclonal antibody 1H2 comprises SEQ ID No: 50. underlined part The italic part is the variable region sequence, and the underlined part is the constant region sequence. The italic part is the variable region sequence, and the underlined part is the constant region sequence.

[0216]

[0217] Further analysis of the heavy chain variable region and light chain variable region sequences of the monoclonal antibody 6G4 shows that the amino acid sequence of the heavy chain variable region of the monoclonal antibody 6G4 comprises SEQ ID No: 41, and the amino acid sequence of the light chain variable region comprises SEQ ID No: 44.

[0218] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6G4 is as follows: The underlined part is the heavy chain CDR region, and the italic part is the framework region.

[0219]

[0220] The above heavy chain variable region belongs to the IGHV1 subgroup.

[0221] The amino acids of the light chain variable region of 6G4 monoclonal antibody are as follows ( The parts in italics are the framework regions):

[0222]

[0223] The above light chain variable region belongs to the IGKV12 subgroup.

[0224] The nucleotide sequence of the heavy chain of monoclonal antibody 12C9 includes SEQ ID No: 54, and the nucleotide sequence of the light chain of monoclonal antibody 6G4 includes SEQ ID No: 57; the nucleotide sequence of the heavy chain of monoclonal antibody 12C9 includes SEQ ID No: 48, and the nucleotide sequence of the light chain of monoclonal antibody 6G4 includes SEQ ID No: 51.

[0225] 12C9 monoclonal antibody heavy chain nucleotide sequence (wherein, underlined part is the signal peptide sequence, the italic part is the variable region sequence, is the constant region sequence):

[0226]

[0227] 12C9 monoclonal antibody light chain nucleotide sequence (wherein, underlined part is the signal peptide sequence, the italic part is the variable region sequence, is the constant region sequence):

[0228]

[0229] 12C9 monoclonal antibody heavy chain amino acid sequence (wherein, underlined part is the signal peptide sequence, the italic part is the variable region sequence, is the constant region sequence):

[0230]

[0231] 12C9 monoclonal antibody light chain amino acid sequence (wherein, underlined part is the signal peptide sequence, the italic part is the variable region sequence, is the constant region sequence):

[0232]

[0233] Further analysis of the heavy chain variable region and light chain variable region sequence of monoclonal antibody 12C9, the amino acid sequence of the heavy chain variable region of monoclonal antibody 12C9 includes SEQ ID No: 42, and the amino acid sequence of the light chain variable region includes SEQ ID No: 45.

[0234] The amino acid of the heavy chain variable region of 12C9 monoclonal antibody is as follows The underlined part is the heavy chain CDR region, and the italic part is the framework region):

[0235]

[0236] The above heavy chain variable region belongs to IGHV1 subgroup.

[0237] The amino acid of the light chain variable region of 12C9 monoclonal antibody is as follows underlined part The underlined part is the light chain CDR region, and the italic part is the framework region):

[0238]

[0239] The above light chain variable region belongs to IGKV12 subgroup.

[0240] 5.2 Construction of recombinant expression

[0241] Then, the heavy chain and light chain genes amplified in step 4.1 in this embodiment were respectively cloned into the eukaryotic expression vector pcDNA3.3(thermo) EcoRI and XhoI multiple cloning sites by homologous recombination, and positive clones were screened and sequenced to construct eukaryotic expression vectors pcDNA3.3-1H2-H and pcDNA3.3-1H2-L, pcDNA3.3-6G4-H and pcDNA3.3-6G4-L, pcDNA3.3-12C9-H and pcDNA3.3-12C9-L.

[0242] 5.3 Identification of recombinant expression of monoclonal antibody gene

[0243] In order to verify whether the cloned 1H2, 6G4, 12C9 monoclonal antibody genes are correct, pcDNA3.3-1H2-H and pcDNA3.3-1H2-L, pcDNA3.3-6G4-H and pcDNA3.3-6G4-L, pcDNA3.3-12C9-H and pcDNA3.3-12C9-L were respectively co-transfected into 293T cells by liposome method, and the culture supernatant was collected after 3 days for analysis. The presence of specific A6 VLP binding antibodies in the cell supernatant was detected by Elisa method, and the results are shown in Figure 5 .

[0244] From Figure 5It can be seen that the supernatant of the cells expressing the sequences of the 1H2, 6G4 and 12C9 monoclonal antibodies has a high binding signal with the A6 VLP, and the OD450nm gradually decreases with the increase of the dilution degree; the supernatant of the control cells without transfection of the related plasmid has no binding signal, which indicates that the amplified and expressed sequences are indeed the genes of the monoclonal antibodies 1H2, 6G4 and 12C9.

[0245] In the present application, BALB / c mice are immunized with inactivated A6 virus, and after cell fusion by B lymphocyte hybridoma technology, three hybridoma cell strains capable of stably secreting specific anti-CVA6 monoclonal antibodies are screened, and specific anti-CVA6 monoclonal antibodies are obtained. The antibodies are detected by Western blot, Elisa, in vitro neutralization and other technical means. The reactivity of the monoclonal antibodies with different antigens is detected by indirect Elisa method, which shows that the monoclonal antibodies 1H2, 6G4 and 12C9 can specifically recognize CVA6 VLP, but cannot recognize EVA71 VLP, CVA10 VLP and CVA16 VLP, indicating that these antibodies have good specificity. The Western blot results show that the three monoclonal antibodies cannot recognize denatured CVA6 VLP, suggesting that the recognized epitopes may be conformational epitopes. The sandwich Elisa results show that the monoclonal antibodies 1H2, 6G4 and 12C9 can sensitively detect CVA6 VLP, and the minimum detection limits are 3.9 ng / ml, 31.25 ng / ml and 15.625 ng / ml respectively, which provides a favorable theoretical basis for the development of the monoclonal antibodies into A6 virus detection kits and vaccine antigen quantitative kits. In addition, the neutralization abilities of the monoclonal antibodies 1H2, 6G4 and 12C9 to CVA6 are quite different, and the neutralization concentrations are 192 ng / ml, 1250 ng / ml and 70.4 ng / ml respectively, indicating the difference in recognition of neutralizing epitopes, suggesting that the antibodies can be used for virus identification and development of antiviral drugs and therapeutic monoclonal antibodies.

[0246] In summary, the 1H2, 6G4 and 12C9 antibodies have good specificity, sensitivity and virus neutralization ability, and can not only be used as a useful detection tool in the laboratory, but also show great potential in virus identification, diagnosis, treatment and development of multivalent vaccines.

[0247] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. A monoclonal antibody that specifically binds to Coxsackievirus A6, comprising a light chain and a heavy chain, characterized in that: The amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown in SEQ ID No: 1; The amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown in SEQ ID No: 4; The amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown in SEQ ID No: 7; The amino acid sequence of the complementarity determining region LCDR1 of the light chain is shown in SEQ ID No: 10; The amino acid sequence of the complementarity determining region LCDR2 of the light chain is GTS; The amino acid sequence of the complementarity determining region LCDR3 of the light chain is shown in SEQ ID No:

13.

2. The monoclonal antibody according to claim 1, wherein The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID No: 40; And / or, the amino acid sequence of the variable region of the light chain is shown in SEQ ID No:

43.

3. The monoclonal antibody according to claim 2, wherein The amino acid sequence of the heavy chain is shown in SEQ ID No: 46; And / or, the amino acid sequence of the light chain is shown in SEQ ID No:

49.

4. A biomaterial related to the monoclonal antibody according to any one of claims 1 to 3, characterized in that: The biological material is one or more of the following: 1) A nucleotide sequence encoding the monoclonal antibody according to any one of claims 1 to 3; 2) a recombinant expression vector containing the nucleotide sequence described in 1); 3) A bioengineered bacterium having the nucleotide sequence described in 1) integrated into its genome, or a bioengineered bacterium containing the recombinant expression vector described in 2).

5. The biomaterial according to claim 4, wherein 1), the nucleotide sequence encoding the heavy chain is shown as SEQ ID No. 52; And / or, in 1), the nucleotide sequence encoding the light chain is shown as SEQ ID No.

55.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the monoclonal antibody according to any one of claims 1 to 3 and / or the biomaterial according to any one of claims 4 to 5, and a pharmaceutically acceptable carrier.

7. A product, characterized in that The product comprises the monoclonal antibody according to any one of claims 1 to 3 or the biological material according to any one of claims 4 to 5.

8. The product according to claim 7, characterized in that The product is selected from detection reagents, test kits, chips and membrane strips.

9. Use of the monoclonal antibody according to any one of claims 1 to 3 or the biomaterial according to any one of claims 4 to 5 in the preparation of a virus detection product, a product for preventing or treating viral infection, or for virus vaccine screening and detection or virus vaccine activity evaluation, wherein the virus is Sarkozy virus A6.

10. The use according to claim 9, characterized in that The monoclonal antibody is used to prepare hand, foot and mouth disease detection products, prepare products for preventing or treating hand, foot and mouth disease, screen hand, foot and mouth disease vaccines, or perform qualitative and quantitative detection of hand, foot and mouth disease vaccine antigens or detect the activity of hand, foot and mouth disease vaccines.

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