CN110790838B - Monoclonal antibody for blocking EV71 infection and application thereof - Google Patents

Abstract

The invention discloses a monoclonal antibody for blocking EV71 infection and application thereof. The monoclonal antibody for blocking EV71 infection provided by the present invention is a SCARB2 monoclonal antibody. It is proved by experiments that the SCARB2 monoclonal antibody or its antigen-binding part of the present invention has the following characteristics: A) binds to human SCARB2; B) binds to human SCARB2 expressed on the cell membrane surface; C) inhibits enterovirus 71 from infecting a host expressing SCARB2 cells; D) inhibiting the replication of enterovirus type 71 in host cells expressing SCARB2; E) binding to the amino acid sequences shown in positions 144-151 and 188-198 of human SCARB2 protein. The present invention also provides derivatives such as immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of the present invention. The antibodies and derivatives provided by the invention can be used for treating and preventing diseases caused by EV71 virus infection.

Description

Monoclonal Antibody Blocking EV71 Infection and Its Application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody for blocking EV71 infection and application thereof.

Background technique

SCARB2, also known as lysosomal integral membrane protein II (LIMP-II) and 85KD lysosomal membrane sialoglycoprotein (LGP85), belongs to the scavenger receptor B subfamily together with CD36 and SCARB1. There is an uncleaved signal peptide at the N-terminus, hydrophobic amino acid fragments are expressed at the C-terminus, and the intracellular segment contains 11 N-glycosylation sites. It is a type III transmembrane glycoprotein.

SCARB2 is closely related to the occurrence and development of many diseases. SCARB2 mutations are the cause of myoclonus-renal failure syndrome (AMRF), a monogenic genetic disorder accompanied by severe myoclonic epilepsy and renal failure symptoms, which often lead to rapid death if left untreated. Secondly, SCARB2 is the receptor of β-glucocerebrosidase (β-GC), which can mediate the transfer of the latter from the endoplasmic reticulum to the lysosome. In mice with SCARB2 gene deletion, β-GC The transport of lysosomes is severely blocked, and it cannot effectively enter the lysosome, but is secreted out of the cell, causing lysosomal storage disease and Parkinson's disease. In addition, SCARB2 can also serve as a functional receptor for EV71, the main pathogen of HFMD, mediating virus entry and uncapsidation. Studies have found that the helical bundle formed by α4, α5, and α7 of SCARB2 is indispensable for the receptor to bind ligands, including β-GC and EV71 capsid protein; and this region is also involved in the formation of SCARB2 dimers, It mediates lipid transport and is critical for both the transport of β-GC and the uncapsidation of EV71.

Contents of the invention

One object of the present invention is to provide a monoclonal antibody to SCARB2 or an antigen-binding portion thereof.

The SCARB2 monoclonal antibody or its antigen-binding part provided by the present invention binds to the helical bundle formed by human SCARB2 protein α4, α5 and α7 (the helical bundle region is mainly located in the amino acid sequence shown at positions 138-190 of human SCARB2 protein), The infection of enterovirus type 71 (EV71) can be inhibited or blocked by directly competing or changing the structure of the helical bundle.

The above SCARB2 monoclonal antibody or its antigen-binding portion exhibits one or more of the following properties in A)-F):

A) binds to human SCARB2;

B) binding to human SCARB2 expressed on the cell membrane surface;

C) inhibiting or blocking enterovirus type 71 (EV71) infection of host cells expressing SCARB2;

D) inhibiting the replication of enterovirus type 71 (EV71) in host cells expressing SCARB2;

E) binding to the amino acid sequences shown in positions 144-151 and 188-198 of human SCARB2 protein;

F) Mediates endocytosis of SCARB2 into cells.

The helical bundle formed by binding to human SCARB2 protein α4, α5 and α7 is reflected in the amino acid sequences shown in positions 144-151 and 188-198 of human SCARB2 protein.

Among the above monoclonal antibodies to SCARB2,

The coding gene of the light chain variable region of the SCARB2 monoclonal antibody is the product of mouse IGKV19-93 and IGKJ2 genes or derived from the gene; the coding gene of the heavy chain variable region is mouse IGHV6-7, IGHD1- 2 and IGHJ3 gene products or derived from this gene.

Further, the light chain variable region of the SCARB2 monoclonal antibody sequentially includes the full-length or partially encoded protein or polypeptide of the mouse IGKV19-93 gene and the full-length or partially encoded protein or polypeptide of the mouse IGKJ2 gene; The heavy chain variable region of the SCARB2 monoclonal antibody sequentially includes the protein or polypeptide encoded by the full length or part of the mouse IGHV6-7 gene, the protein or polypeptide encoded by the full length or part of the mouse IGHD1-2 gene, and the product of the IGHJ3 gene or derived from this gene.

Further, the light chain variable region of the SCARB2 monoclonal antibody or its antigen-binding portion sequentially includes the protein encoded by the mouse IGKV19-93 gene and the protein encoded by the mouse IGKJ2 gene; and its heavy chain The variable region sequentially includes the protein encoded by the mouse IGHV6-7 gene, the protein encoded by the mouse IGHD1-2 gene and the protein encoded by the mouse IGHJ3 gene.

Among the above monoclonal antibodies to SCARB2,

The light chain variable region of the SCARB2 monoclonal antibody includes light chain variable region CDR1, light chain variable region CDR2 and light chain variable region CDR3; the heavy chain variable region of the SCARB2 monoclonal antibody includes heavy chain variable region Chain variable region CDR1, heavy chain variable region CDR2 and heavy chain variable region CDR3;

The amino acid sequence of the light chain variable region CDR1 is as follows (1) or (2):

(1) the amino acid sequence shown in sequence 3;

(2) an amino acid sequence having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 3;

The amino acid sequence of the light chain variable region CDR2 is as follows (3) or (4):

(3) the amino acid sequence shown in sequence 4;

(4) an amino acid sequence having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 4;

The amino acid sequence of the light chain variable region CDR3 is as follows (5) or (6):

(5) the amino acid sequence shown in sequence 5;

(6) an amino acid sequence having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 5;

The amino acid sequence of the heavy chain variable region CDR1 is as follows (7) or (8):

(7) the amino acid sequence shown in sequence 8;

(8) an amino acid sequence having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 8;

The amino acid sequence of the heavy chain variable region CDR2 is as follows (9) or (10):

(9) the amino acid sequence shown in sequence 9;

(10) an amino acid sequence having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 9;

The amino acid sequence of the heavy chain variable region CDR3 is as follows (11) or (12):

(11) the amino acid sequence shown in sequence 10;

(12) An amino acid sequence having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 10.

Among the above monoclonal antibodies to SCARB2,

The amino acid sequence of the light chain variable region of the SCARB2 monoclonal antibody is a1) or a2):

a1) the amino acid sequence shown in sequence 2;

a2) an amino acid sequence having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2;

The amino acid sequence of the heavy chain variable region of the SCARB2 monoclonal antibody is b1) or b2):

b1) the amino acid sequence shown in sequence 7;

b2) An amino acid sequence having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 7.

Among the above monoclonal antibodies to SCARB2,

The coding gene sequence of the light chain variable region of the SCARB2 monoclonal antibody is c1) or c2) or c3):

c1) DNA molecules shown in sequence 1;

c2) A cDNA molecule or a genomic DNA molecule that has 75% or more identity to the nucleotide sequence defined in c1) and encodes the light chain variable region of the above SCARB2 monoclonal antibody;

c3) a cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined by c1) or c2) under stringent conditions and encodes the light chain variable region of the monoclonal antibody to SCARB2;

The coding gene sequence of the heavy chain variable region of the SCARB2 monoclonal antibody is d1) or d2) or d3):

d1) DNA molecules shown in sequence 6;

d2) A cDNA molecule or a genomic DNA molecule that has 75% or more identity to the nucleotide sequence defined in d1) and encodes the heavy chain variable region of the monoclonal antibody to SCARB2;

d3) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the heavy chain variable region of the above SCARB2 monoclonal antibody.

Among the above monoclonal antibodies to SCARB2,

The amino acid sequence of the light chain of the SCARB2 monoclonal antibody is p1) or p2):

p1) the amino acid sequence shown in sequence 14;

p2) an amino acid sequence having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in sequence 14;

The amino acid sequence of the heavy chain of the SCARB2 monoclonal antibody is q1) or q2):

q1) the amino acid sequence shown in sequence 16;

q2) An amino acid sequence having the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 16.

Among the above monoclonal antibodies to SCARB2,

The coding gene sequence of the light chain of the monoclonal antibody of SCARB2 is m1) or m2) or m3):

m1) DNA molecules shown in sequence 13;

m2) has 75% or more identity with the nucleotide sequence defined by m1), and encodes the cDNA molecule or genomic DNA molecule of the light chain of the monoclonal antibody of SCARB2;

m3) a cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined by m1) or m2) under stringent conditions and encodes the light chain of the monoclonal antibody to SCARB2;

The coding gene sequence of the heavy chain of the monoclonal antibody of SCARB2 is n1) or n2) or n3):

n1) DNA molecules shown in sequence 15;

n2) A cDNA molecule or a genomic DNA molecule that has 75% or more identity to the nucleotide sequence defined in n1) and encodes the heavy chain of the above-mentioned SCARB2 monoclonal antibody;

n3) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined by n1) or n2) under stringent conditions and encodes the heavy chain of the monoclonal antibody to SCARB2.

Another object of the present invention is to provide the above-mentioned SCARB2 monoclonal antibody or a derivative of the antigen-binding portion thereof.

The above-mentioned SCARB2 monoclonal antibody or the derivative of the antigen-binding part thereof provided by the present invention is any one of the following (e1)-(e6):

(e1) An antagonist of enterovirus type 71 (EV71), the active ingredient of which is the above-mentioned monoclonal antibody to SCARB2 or an antigen-binding portion thereof;

(e2) a composition comprising the above-mentioned monoclonal antibody to SCARB2 or an antigen-binding portion thereof;

(e3) an immunoconjugate containing the above-mentioned SCARB2 monoclonal antibody or an antigen-binding portion thereof;

(e4) an antibody obtained by modifying and/or transforming the above-mentioned SCARB2 monoclonal antibody or its antigen-binding part;

(e5) a humanized antibody obtained by humanizing the above SCARB2 monoclonal antibody or its antigen-binding portion;

(e6) A multispecific molecule comprising the above-mentioned monoclonal antibody to SCARB2 or an antigen-binding portion thereof.

Among the above derivatives, the composition may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound, ie, antibody, immunoconjugate or bispecific molecule, may be coated in a material to protect the compound from acids and other natural conditions that would render the compound inactive.

The pharmaceutical compositions of the present invention may also contain one or more pharmaceutically acceptable salts. A pharmaceutically acceptable salt is one that retains the desired biological activity of the parent compound and does not cause any unwanted toxicological effects (see e.g. Berge, S.M. et al. (1977) J.Pharm.Sci.66:1-19 ). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, and from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids. salts derived from acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., and from nontoxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloride Salts derived from procaine, choline, diethanolamine, ethylenediamine, procaine, etc.

The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbic acid palmitate Esters, butylated hydroxyanisole (BHA), butylated hydroxytoluene (DHT), lecithin, propyl gallate, α-tocopherol, etc.; (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA) , sorbitol, tartaric acid, phosphoric acid, etc.

Compositions of the invention may be administered by one or more routes of administration using one or more methods known in the art. It will be appreciated by those skilled in the art that the route and/or manner of administration will vary depending on the desired result. Preferred routes of administration for the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion.

In the above derivatives, the immunoconjugate refers to a conjugate obtained by coupling the SCARB2 monoclonal antibody or its antigen-binding portion of the present invention with a therapeutic agent such as a cytotoxin, a drug (such as an immunosuppressant) or a radioactive toxin .

In the above derivatives, the modification is a conservative modification, which refers to an amino acid modification that will not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by techniques well known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

Among the above-mentioned derivatives, the multispecific molecule is derivatized or linked to one or more other functional molecules of the antibody of the present invention to generate two or more different binding sites and and/or multispecific molecules to which a target molecule binds. To generate multispecific molecules of the invention, antibodies of the invention may be functionally linked (e.g., by chemical conjugation, genetic fusion, covalent binding, etc.) to obtain multispecific molecules.

Still another object of the present invention is to provide biological materials related to the above-mentioned monoclonal antibody to SCARB2 or an antigen-binding portion thereof.

The biological material related to the above-mentioned SCARB2 monoclonal antibody or its antigen-binding part provided by the present invention is any one of the following A1)-A12);

A1) a nucleic acid molecule encoding a monoclonal antibody to SCARB2 or an antigen-binding portion thereof;

A2) an expression cassette containing the nucleic acid molecule of A1);

A3) a recombinant vector containing the nucleic acid molecule of A1);

A4) a recombinant vector containing the expression cassette described in A2);

A5) a recombinant microorganism containing the nucleic acid molecule of A1);

A6) a recombinant microorganism containing the expression cassette described in A2);

A7) A recombinant microorganism containing the recombinant vector described in A3);

A8) a recombinant microorganism containing the recombinant vector described in A4);

A9) a transgenic cell line containing the nucleic acid molecule of A1);

A10) a transgenic cell line containing the expression cassette described in A2);

A11) a transgenic cell line containing the recombinant vector described in A3);

A12) A transgenic cell line containing the recombinant vector described in A4).

In the above-mentioned biological material, the nucleic acid molecule encoding the above-mentioned SCARB2 monoclonal antibody or its antigen-binding part described in A1) is the coding gene sequence of the above-mentioned SCARB2 monoclonal antibody light chain and the above-mentioned SCARB2 monoclonal antibody heavy chain coding gene sequence.

Another object of the present invention is to provide the application of the above SCARB2 monoclonal antibody or its antigen-binding part or the above derivative or the above biological material in at least one of the following 1)-8):

1) Binding to human SCARB2;

2) Binding to human SCARB2 expressed on the cell membrane surface;

3) Inhibiting or blocking enterovirus 71 from infecting host cells expressing SCARB2;

4) Inhibit the replication of enterovirus type 71 in host cells expressing SCARB2;

5) binding to the amino acid sequences shown at positions 144-151 and 188-198 of human SCARB2 protein;

6) mediate SCARB2 endocytosis into cells;

7) Prepare anti-SCARB2 antibody;

8) Prepare products for treating and/or preventing diseases caused by enterovirus 71 infection.

In the above antibody or application, the binding to human SCARB2 is reflected in binding to SCARB2 monomer or SCARB2 dimer or glycosylated SCARB2;

The host cell can specifically be 293A-SCARB2 cells (293A cells overexpressing SCARB2) or human rhabdomyosarcoma cells RD.

The last object of the present invention is to provide a method for preparing anti-SCARB2 antibody.

The method for preparing anti-SCARB2 antibody provided by the invention comprises the following steps:

B1) provide:

(i) a light chain variable region sequence, which contains a CDR1 sequence selected from sequence 3, a CDR2 sequence selected from sequence 4, and a CDR3 sequence selected from sequence 5;

Or (ii) a heavy chain variable region sequence, which contains a CDR1 sequence selected from sequence 8, a CDR2 sequence selected from sequence 9, and a CDR3 sequence selected from sequence 10;

B2) altering at least one amino acid residue within at least one variable region antibody sequence selected from said light chain variable region antibody sequence and said heavy chain variable region antibody sequence, thereby producing at least one altered antibody sequence;

B3) Expressing said altered antibody sequence as a protein.

The SCARB2 monoclonal antibody of the present invention is prepared by an authoritative monoclonal antibody preparation method (for example, Paterson, H.M.V.a.Y. (Jone Wiley and Sons, Inc. New York, 1995). Production of Antibodies. Current Protocols in Immunology). The preferred animal for producing hybridomas is a mouse. The generation of hybridomas in mice is a very well established procedure. Immunization procedures and isolation techniques for immunized splenocytes for fusion are well known in the art. Fusion partners (eg, murine myeloma cells) and fusion methods are also known.

In addition to obtaining the anti-SCARB2 antibody by amplifying hybridoma cells in vivo or in vitro, the gene encoding the antibody of the present invention can also be cloned into a eukaryotic expression vector by traditional molecular cloning methods, and the antibody can be obtained by eukaryotic expression and purification.

The invention provides a monoclonal antibody for blocking EV71 infection and application thereof. The monoclonal antibody for blocking EV71 infection provided by the invention is the monoclonal antibody of II SCARB2, a member of the B subfamily of scavenger receptors. The monoclonal antibody of the present invention can inhibit the infection of enterovirus type 71 (EV71) by binding to the helical bundle formed by SCARB2 protein α4, α5 and α7. It is proved by experiments that the SCARB2 monoclonal antibody or its antigen-binding part of the present invention has the following characteristics: A) binding to human SCARB2; B) binding to human SCARB2 expressed on the cell membrane surface; C) inhibiting enterovirus type 71 (EV71) infection and expression of SCARB2 D) inhibiting the replication of enterovirus 71 (EV71) in host cells expressing SCARB2; E) binding to the amino acid sequences shown at positions 144-151 and 188-198 of human SCARB2 protein. The present invention also provides derivatives such as immunoconjugates, multispecific molecules and pharmaceutical compositions comprising the antibodies of the present invention. The antibodies and derivatives provided by the invention can be used for treating and preventing diseases caused by EV71 virus infection.

Description of drawings

Figure 1 shows a screen for antibodies that bind to human scavenger receptor subfamily B member II SCARB2.

Figure 2 shows the nucleotide sequence and amino acid sequence of the light chain variable region of SCARB2 monoclonal antibody 5C2. The black shaded area in the figure marks the CDR1 region (SEQ ID NO: 3), the CDR2 region (SEQ ID NO: 4) and the CDR3 region (SEQ ID NO: 5).

Figure 3 shows the nucleotide sequence and amino acid sequence of the heavy chain variable region of SCARB2 monoclonal antibody 5C2. The black shaded area in the figure marks the CDR1 region (SEQ ID NO: 8), the CDR2 region (SEQ ID NO: 9) and the CDR3 region (SEQ ID NO: 10).

Fig. 4 shows the comparison result of the amino acid sequence (SEQ ID NO: 2) of the light chain variable region of the SCARB2 monoclonal antibody 5C2 and the mouse amino acid sequence (SEQ ID NO: 11). The light chain variable region composition of SCARB2 monoclonal antibody 5C2: the V gene is IGKV19-93, and the J gene is IGKJ2. Shown in the figure is the comparison result of the light chain amino acid sequence of SCARB2 monoclonal antibody 5C2 and its original VJ gene amino acid sequence.

Fig. 5 shows the comparison result of the amino acid sequence (SEQ ID NO: 7) of the heavy chain variable region of the SCARB2 monoclonal antibody 5C2 and the mouse amino acid sequence (SEQ ID NO: 12). The heavy chain variable region composition of SCARB2 monoclonal antibody 5C2: the V gene is IGHV6-7, the D gene is IGHD1-2, and the J gene is IGHJ3. Shown in the figure is the comparison result of the heavy chain amino acid sequence of SCARB2 monoclonal antibody 5C2 and its original VDJ gene amino acid sequence.

Figure 6 shows that SCARB2 monoclonal antibody 5C2 binds to SCARB2 expressed on the cell membrane surface at the flow level.

Figure 7 shows that SCARB2 monoclonal antibody 5C2 binds to SCARB2 expressed on the cell membrane surface at the level of immunofluorescence.

Figure 8 shows that SCARB2 monoclonal antibody 5C2 binds to SCARB2 at the level of western blot.

Figure 9 shows that SCARB2 monoclonal antibody 5C2 can IP out two forms of SCARB2.

Figure 10 shows the binding ability of SCARB2 monoclonal antibody 5C2 antibody.

Figure 11 shows that SCARB2 monoclonal antibody 5C2 can mediate receptor endocytosis.

Figure 12 shows that SCARB2 monoclonal antibody 5C2 inhibits EV71 infection of the cell line 293A-SCARB2 overexpressing SCARB2.

Figure 13 shows that SCARB2 monoclonal antibody 5C2 inhibits EV71 from infecting host cell RD cells.

Figure 14 shows a schematic representation of chimeras of human and murine SCARB2.

Figure 15 shows that SCARB2 monoclonal antibody 5C2 binds to human SCARB2 positions 144-151 and 188-198 at the flow level.

Figure 16 shows that SCARB2 monoclonal antibody 5C2 binds to human SCARB2 positions 144-151 and 188-198 at the level of western blot.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The quantitative tests in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Example 1, Preparation of anti-human SCARB2 monoclonal antibody

1. Preparation of human scavenger receptor B subfamily member II SCARB2 antibody

1. Preparation of human SCARB2 antigen

(1) According to the sequence of human SCARB2, the following primers were designed:

SCARB2-F: GCTCTAGAATCGATGCCACCATGGGCCGATGCTGCTTCTACACG;

SCARB2-R: CCTTAATTAATTCATCCGCTGTTCCCTCATCCATGGAT.

(2) Use the cDNA of isolated human pDC (plasmacytoid dendritic cell) as a template, and use the primers designed in step (1) to carry out PCR amplification to obtain the PCR amplification product, which is the human scavenger receptor B subfamily The extracellular region, transmembrane region and intracellular segment of member II SCARB2 up to the fragment before E471.

The above PCR reaction conditions are: pre-denaturation, 98°C, 2 minutes; denaturation, 98°C, 30 seconds; annealing, 60°C, 30 seconds; extension, 72°C, 1.5 minutes; a total of 30 cycles, and finally an extension of 10 minutes.

(3) Restriction endonucleases Xba I and Pac I digest the PCR amplification product obtained in step (2) and the pEGFP-C1 vector (Clontech, Cat: #6084-1), and connect to obtain the pEGFP-human SCARB2E471 plasmid . In the pEGFP-humanSCARB2E471 plasmid, the EGFP gene is fused downstream of the SCARB2E471 sequence. When SCARB2E471 is expressed, a GFP signal can be observed.

CRL-2648^TM)中，转染48小时后，得到膜表面表达人SCARB2胞外区的小鼠L细胞。通过分析转染人SCARB2胞外区的小鼠L细胞中EGFP的表达情况确定转染效率并用于下述小鼠免疫实验。(4) Transfect the pEGFP-human SCARB2E471 plasmid into mouse L cells (ATCC, catalog number:

CRL-2648 ^TM ), after 48 hours of transfection, mouse L cells expressing the extracellular region of human SCARB2 on the membrane surface were obtained. The transfection efficiency was determined by analyzing the expression of EGFP in mouse L cells transfected with the extracellular region of human SCARB2 and used for the following mouse immunization experiments.

2. Immunization of BalB/C mice

Mice were immunized with human SCARB2 as immunogen. The specific steps are as follows: Mix 5,000,000 mouse L cells expressing the extracellular region of human SCARB2 with 20 μg CpG1826 (synthesized by TAKARA) to form a 0.5ml suspension, and immunize 6-week-old BalB/C mice by intraperitoneal injection . Immunization once a month, a total of 4 times of immunization. Finally, a booster immunization was performed using the same method, and hybridoma fusion was performed 4 days later.

3. Construction of HEK293T cell line stably expressing human SCARB2

The pEGFP-human SCARB2E471 plasmid was transfected into the HEK293T cell line using Lipofectamine2000 (Invitrogen), and the cells stably transfected with the plasmid (the plasmid contains the puro resistance gene) were obtained by puromycin selection, so that it could stably express the extracellular region of human SCARB2 and the transmembrane region (SCARB2 and GFP fusion expression) to obtain a HEK293T cell line stably expressing human SCARB2. This cell line will be used in the antibody screening experiments described below.

4. Hybridoma Fusion

Mice to be fused after immunization were sacrificed, and spleen cells were removed. By cell counting, the mouse spleen cells were mixed with the mouse myeloma cell line SP2/0 (ATCC, CRL1581) at a ratio of 1:3, and 50% PEG (Sigma) was used to fuse the cells. Add the fused cells to 60ml of RPMI medium (Cellgro, product number: 15-041-CV) containing 1X HAT (containing 10% fetal bovine serum), and add 1-2 drops per well to the 96-well cells in the culture plate. Subsequently, the hybridoma cells were cultured at 37° C. under the condition of 5% CO ₂ , and half of the medium was changed in 3 to 4 days. Antibody screening was performed about 10 days later.

5. Screening of human scavenger receptor B subfamily member II SCARB2 antibody

The day before the screening, aspirate about 100 μl of the supernatant from the 96-well cell culture plate into a 96-well U-shaped bottom culture plate, and add 100 μl of fresh RPMI medium containing 1X HAT to the original well to continue culturing the cells. Harvest the cultured HEK293T cell line stably expressing human SCARB2, add it to a 96-well U-shaped bottom culture plate, 10000 cells/200 μl/well. After centrifugation at 2200 rpm/3 minutes, the supernatant was thrown off, and 100 μl hybridoma culture supernatant was added, resuspended and incubated at 4°C for 30 minutes. After incubation, centrifuge and discard the supernatant, add 200 μl FACS buffer (PBS containing 2% FBS and 2mM EDTA) to each well, resuspend the cells and perform centrifugation, then discard the supernatant to achieve the purpose of washing. PE-labeled goat anti-mouse IgG antibody (Biolegend, Poly4053, 405307) diluted 1:400 in FACSbuffer was added to each well, and the cells were resuspended and incubated at 4°C for 30 minutes in the dark. After incubation, centrifuge the culture plate and remove the supernatant, wash the cells once as described above, and then add 200 μl FACS buffer to resuspend the cells. Finally, the cellular GFP and PE signals were analyzed using the flow cytometer Guava (Millipore). GFP and PE double-positive wells are SCARB2-binding antibody-positive wells (Figure 1).

According to the above steps, the monoclonal hybridoma cell line 5C2, which can bind to SCARB2 in the spatial orientation of the membrane surface and stably secrete the SCARB2 monoclonal antibody, was finally selected and named as the hybridoma cell line 5C2. The antibody secreted by the hybridoma cell line 5C2 was named mouse anti-human SCARB2 monoclonal antibody 5C2.

Part of the mouse anti-human SCARB2 monoclonal antibody 5C2 was sent to Tianjin Sanjian Biotechnology Co., Ltd. for FITC fluorescein labeling, and the labeled antibody was named mouse anti-human SCARB2 monoclonal antibody 5C2-FITC.

2. The sequence of mouse anti-human SCARB2 monoclonal antibody 5C2

1. Extraction of RNA

The hybridoma cell line 5C2 was lysed with Trizol (Invitrogen), and the RNA of the hybridoma cell line 5C2 was extracted. The specific operation steps of RNA extraction are as follows:

Add 200μl chloroform to every 1ml Trizol, shake well and let stand for 10 minutes; centrifuge at 13000rpm/4℃/15 minutes; absorb 400μl supernatant and add to 400μl pre-cooled isopropanol, mix well and let stand overnight at -20℃; Centrifuge at 13000rpm/4°C/15 minutes; remove the supernatant, add 70% ethanol; centrifuge at 13000rpm/4°C/10 minutes; remove the supernatant, add 70% ethanol; remove the supernatant, add 40μl water to dissolve, and obtain an RNA solution.

2. Acquisition of cDNA

The RNA obtained in step 1 was reverse-transcribed to obtain cDNA. The specific operation steps of reverse transcription are as follows: draw 16 μl of the RNA solution prepared in step 1, add 1 μl of Oligo dT (Invitrogen) with a concentration of 100 nM; react at 70°C for 5 minutes; place it on ice immediately; add 1 μl of RNase inhibitor (Takara ), 1 μl of 10 mM dNTP (Takara), 1 μl of MLV reverse transcriptase and 5 μl of 5X buffer (Promega); react at 42°C for 60 minutes; and treat at 80°C for 10 minutes.

3. PCR amplification and sequencing

Using the cDNA obtained in step 2 as a template, carry out PCR amplification using heavy chain primer F and heavy chain primer R, light chain primer F and light chain primer R, respectively, and obtain and sequence the fragments encoding the heavy chain and light chain . The primer sequences are as follows:

Light chain primer F: GAYATTGTGMTSACMCARWCTMCA;

Light chain primer R: GGATACAGTTGGTGCAGCATC;

Heavy chain primer F: SARGTNMAGCTGSAGSAGTC;

Heavy chain primer R: CTTGACCAGGCATCCTAGAGTCA;

Among them, R=a,g; Y=c,t; M=a,c; K=g,t; S=c,g; W=a,t; V=a,c,g; N=a, c, g, t.

The above PCR reaction conditions: pre-denaturation, 98°C, 2 minutes; denaturation, 98°C, 30 seconds; annealing, 54°C; extension 72°C, 1 minute; a total of 35 cycles, and finally an extension of 10 minutes.

The sequencing results are as follows:

The nucleotide and amino acid sequences of the light chain variable region of SCARB2 monoclonal antibody 5C2 are shown in Figure 2, the nucleotide sequence of the light chain variable region of SCARB2 monoclonal antibody 5C2 is sequence 1, and the light chain of SCARB2 monoclonal antibody 5C2 The variable region amino acid sequence is sequence 2; the amino acid sequence (sequence 3) shown in the 27th-32nd position of the light chain variable region amino acid sequence of SCARB2 monoclonal antibody 5C2 is named 5C2 light chain CDR1, and the SCARB2 monoclonal antibody The 50th-52nd amino acid sequence (sequence 4) of the light chain variable region amino acid sequence of the cloned antibody 5C2 is named 5C2 light chain CDR2, and the light chain variable region amino acid sequence of the SCARB2 monoclonal antibody 5C2 is The amino acid sequence shown at position 89-96 (SEQ ID NO: 5) is named as 5C2 light chain CDR3.

The nucleotide and amino acid sequences of the heavy chain variable region of SCARB2 monoclonal antibody 5C2 are shown in Figure 3, the nucleotide sequence of the heavy chain variable region of SCARB2 monoclonal antibody 5C2 is sequence 6, and the heavy chain of SCARB2 monoclonal antibody 5C2 The amino acid sequence of the variable region is sequence 7; the amino acid sequence (sequence 8) shown in the 26th-33rd position of the heavy chain variable region amino acid sequence of SCARB2 monoclonal antibody 5C2 is named as 5C2 heavy chain CDR1, and the SCARB2 monoclonal antibody The amino acid sequence (sequence 9) shown in the 51st-60th position of the amino acid sequence of the heavy chain variable region of the cloned antibody 5C2 is named 5C2 heavy chain CDR2, and the amino acid sequence of the heavy chain variable region of the SCARB2 monoclonal antibody 5C2 is The amino acid sequence shown at position 99-110 (SEQ ID NO: 10) is designated as 5C2 heavy chain CDR3.

4. Antibody sequence analysis

The nucleic acid fragment of SCARB2 monoclonal antibody 5C2 was analyzed in the antibody sequence analysis tool igBlast tool (http://www.ncbi.nlm.nih.gov/igblast/), and it was found that: V of the light chain coding gene of SCARB2 monoclonal antibody 5C2 Gene and J gene correspond to mouse IGKV19-93 gene and IGKJ2 gene respectively, the comparison result of the amino acid sequence (sequence 2) of the light chain variable region of SCARB2 monoclonal antibody 5C2 and the mouse VJ region amino acid sequence (sequence 11) is as follows As shown in Figure 4; V gene, D gene and J gene of SCARB2 monoclonal antibody 5C2 heavy chain coding gene correspond to mouse IGHV6-7 gene, IGHD1-2 gene and IGHJ3 gene respectively, the heavy chain of SCARB2 monoclonal antibody 5C2 can be The comparison result of the amino acid sequence of the variable region (SEQ ID NO: 7) and the amino acid sequence of the mouse VDJ region (SEQ ID NO: 12) is shown in FIG. 5 .

Example 2. The mouse anti-human SCARB2 monoclonal antibody of the present invention can bind to human scavenger receptor B subfamily member II SCARB2 expressed on the membrane surface at the flow cytometric level

1. Use the HEK293T cells stably expressing human scavenger receptor B subfamily member II SCARB2 obtained in Step 3 of Example 1 to use PBS solution containing 2mM EDTA (each 1L of PBS contains KH ₂ PO ₄ 0.27g, Na ₂ HPO ₄ 1.42g, NaCl 8g, KCl 0.2g, adjust the pH to 7.2-7.4, and dilute to 1L with water) to make it a single cell suspension. 2. Add the single cell suspension stably expressing human scavenger receptor B subfamily member II SCARB2 to a 96-well U-shaped bottom culture plate, 10000 cells/200 μl/well. After centrifugation at 2200 rpm/3 minutes, the supernatant was discarded and the precipitate was collected. 3. Add FACS buffer (PBS containing 2% FBS and 2mM EDTA) containing 5 μg/ml of the mouse anti-human SCARB2 monoclonal antibody 5C2 prepared in Example 1 to the precipitate obtained in step 2, and resuspend at 4°C Incubate for 30 minutes. 4. After incubation, centrifuge, discard the supernatant, collect the precipitate, add 200 μl FACS buffer to each well, resuspend the cells and perform centrifugation, discard the supernatant, collect the precipitate and wash. 5. Add PE-labeled goat anti-mouse IgG antibody (Biolegend, Poly4053, 405307) diluted 1:400 in FACS buffer to each well, resuspend the cells and incubate at 4°C for 30 minutes in the dark. After incubation, centrifuge the culture plate and remove the supernatant, wash the cells once as described above, and then add 200 μl FACS buffer to resuspend the cells. Finally, the cellular GFP and PE signals were analyzed using the flow cytometer Guava (Millipore).

The result is shown in Figure 6. Due to the fusion expression of SCARB2 and GFP, cells with high expression of GFP also have higher expression of SCARB2. Therefore, cells with high GFP expression also bind more anti-human SCARB2 antibodies, which bind more PE-labeled goat anti-mouse IgG antibodies, and thus have higher PE signals. It can be seen from the figure that the mouse anti-human SCARB2 monoclonal antibody 5C2 can bind to the human SCARB2 receptor.

Example 3. The mouse anti-human SCARB2 monoclonal antibody of the present invention can bind to human scavenger receptor B subfamily member II SCARB2 expressed on the membrane surface at the level of immunofluorescence

1. Construction of 293A cell line overexpressing SCARB2

1. Construction of pLenti-SCARB2-puro expression plasmid

The pLenti-Puro vector (Addgene Cat: #16578) was digested with two restriction enzymes, SpeI-HF and EcoRI-HF, to obtain the backbone vector. The pcDNA-SCARB2-wt plasmid was used as a template, and the SCARB2-F-F and SCARB2-F-R primers were used for PCR amplification to obtain the SCARB2 fragment (GenBank number: NM_005506.3). The backbone vector and the SCARB2 fragment were connected to obtain the pLenti-SCARB2-puro expression plasmid. The primer sequences are as follows:

SCARB2-F-F: GACTAGTGCCACCATGGGCCGATGCTGCTTCTA;

SCARB2-F-R: GGAATTCTTAGGTTCGAATGAGGGGTGCT.

2. 293A cell line overexpressing SCARB2

The pLenti-SCARB2-puro expression plasmid constructed in step 1 was transfected into 293A cells (ThermoFisher Scientific, Cat: #R70507) with calcium phosphate, and killed with a lethal dose of puromycin (Applicom, A2856-0010) after 48 hours of transfection. Untransfected cells were treated for one week, labeled with SCARB2 polyclonal antibody (R&D, 1966-LM-050), stained on ice for 30 minutes, and then stained with 1ml FACS buffer (containing 2% FBS and 2mM EDTA Wash off the primary antibody, and resuspend the cells with PE-labeled goat anti-mouse IgG antibody (Biolegend, Poly4053, 405307) diluted 1:400 in FACS buffer, and stain on ice for 30 minutes. Wash the cells once as described above, then add 1ml FACSbuffer to resuspend the cells. The 293A cells with high expression of SCARB2 on the membrane surface were sorted by FACSArial II. After continuous culture and sorting for 4 times, the expression level of SCARB2 reached more than 99%. The 293A cells with high expression of SCARB2 were expanded and frozen.

2. Mouse anti-human SCARB2 monoclonal antibody 5C2-FTIC for immunofluorescent staining of 293A cell line overexpressing SCARB2

1. Soak the coverslip with 70% alcohol, wait until the alcohol has evaporated, place it in a 24-well plate, inoculate the 293A cells that do not express SCARB2 on the membrane surface and the 293A cells that overexpress SCARB2 prepared in step 1 on the cover slip In a 24-well plate, culture overnight. 2. Aspirate the cell culture medium and wash it once with 500μL PBS. Aspirate the PBS, add 500 μL of 2.5-4% formaldehyde, react at room temperature for 15 minutes, and fix the cells. 3. Absorb formaldehyde and wash with PBS three times. Add 100 μL of mouse anti-human SCARB2 monoclonal antibody 5C2-FITC and react for 1 hour at room temperature. 4. Wash 3 times with 1xDPBS (Thermo Fisher Scientific, Cat: #14080055). 5. Wash once with 1xDPBS (Thermo Fisher Scientific, Cat: #14200166) to reduce the salt concentration. 6. Absorb the liquid on the slide to make the slide as dry as possible. Take the mounting medium into an EP tube, and add DAPI at the ratio of 0.4 μL DAPI (Vector, H-1000) to 1 drop of mounting medium. Drop the prepared mounting medium containing DAPI onto the slide, and cover the stained slide with the cell side facing down to prevent air bubbles. Fix slides to glass slides with nail polish. The slides were observed under an Olympus FV1000 laser confocal microscope.

The results are shown in Figure 7. The 293A cell line that does not express SCARB2 on the membrane surface cannot be stained by the mouse anti-human SCARB2 monoclonal antibody 5C2-FITC, while the 293A cell line (293A-hSCARB2) that expresses SCARB2 on the membrane surface can effectively bind to Mouse anti-human SCARB2 monoclonal antibody 5C2-FITC.

Example 4, the mouse anti-human SCARB2 monoclonal antibody of the present invention can bind SCARB2 at the level of western blot

1. Mouse anti-human SCARB2 monoclonal antibody 5C2 can be used for Western blot detection

1. The 293A cells (293A-SCARB2) and 293T-SCARB2-KO cells (293T-SCARB2-KO cells) overexpressing SCARB2 obtained in step 1 of Example 3 were respectively recorded in the document "Protein Cell 2017, 8(8): 590–600”) in 60 cm cell culture dishes (3×10 ⁶ cells/dish) containing complete DMEM medium (containing 10% FBS), and cultured for 48 hours at 37°C and 5% CO ₂ .

2. The product obtained in step 1 was washed twice with phosphate buffered saline (PBS), and washed with 500 μl of ice-cold lysate (100 mM Tris-HCl (pH 7.4), 140 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1 %SDS, 1% sodium deoxycholate; supplemented with 1 mM PMSF, 0.01 M DTT, Protease Inhibitor Mix Tablet (Roche)) was lysed to obtain a cell lysate. Shake the cell lysate at 4°C for 30 minutes, and clarify at 13000rpm for 20 minutes, aspirate the supernatant, add 5x loading buffer and β-ME to the supernatant, cook the sample at 100°C for 10 minutes, and obtain the treated sample.

3. Perform SDS PAGE on the processed sample obtained in step 2. The specific steps are as follows: transfer the treated sample to a nitrocellulose membrane. Then the membrane was blocked with 5% BSA, and incubated overnight with goat anti-human SCARB2 polyclonal antibody (R&D, 1966-LM-050) and SCARB2 monoclonal antibody 5C2 prepared in Example 1, respectively. After washing 3 times with TBST on the second day, the membrane was incubated with HRP-conjugated anti-goat or anti-mouse secondary antibody (Nakasugi Jinqiao, ZB-2305) for 1 hour at room temperature, and then washed 3 times with TBST . The membrane was finally exposed to the SAGECREATION Minichemi Imaging System for color development using a chemiluminescent detection kit according to the manufacturer's instructions (Millipore, WBKLS0100).

The result is shown in Figure 8. As can be seen from the figure: the goat anti-human SCARB2 polyclonal antibody (R&D, 1966-LM-050) and the mouse anti-human SCARB2 monoclonal antibody 5C2 of the present invention all detect the band of SCARB2 in 293A-SCARB2 (located at 85kD), and no band of SCARB2 was detected in 293T-SCARB2-KO. It shows that the mouse anti-human SCARB2 monoclonal antibody 5C2 of the present invention can bind SCARB2 at the level of western blotting.

Example 5. The mouse anti-human SCARB2 monoclonal antibody of the present invention can bind to the monomer, dimer or glycosylated form of SCARB2

1. Inoculate HEK 293T cells in a 60 cm cell culture dish (3×10 ⁶ cells/dish) containing DMEM complete medium (containing 10% FBS), and culture at 37°C and 5% CO ₂ for 24 hours. Then the pLenti-SCARB2-puro plasmid expressing human SCARB2 obtained in step 1 of Example 3 and the corresponding empty vector (pLenti-EF2-Nanog-Puro vector) were transfected into HEK 293T cells respectively, and the transfection steps were according to the instructions (Thermo Fisher, Lipofectamine 2000, 11668019). Then cultured at 37°C, 5% CO ₂ for 48 hours. 2. Wash the product obtained in step 1 twice with phosphate-buffered saline (PBS), and wash it with 500 μl of ice-cold lysate (100 mM Tris-HCl (pH7.4), 140 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate; supplemented with 1 mM PMSF, 0.01 M DTT, Protease Inhibitor Mix Tablet (Roche)) were lysed to obtain cell lysates. Cell lysates were shaken at 4°C for 30 minutes, clarified at 13,000 rpm for 20 minutes, and the supernatant was aspirated. 3. Divide the supernatant in step 2 into two equal parts, add 10 μg/ml isotype control antibody IgG2a (Biolegend, MOPC-173, 400223) and the mouse anti-human SCARB2 monoclonal antibody 5C2 in Example 1, respectively, Mix overnight at 4°C. 4. On the next day, add 20 μl Protein A/G to the antibody-supernatant mixture in step 3, and continue to incubate at 4°C for 4 hours. 5. Wash the Protein A/G bound to the antibody 5 times with ice-cold lysate (100mM Tris-HCl (pH7.4), 140mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), 5 times each time minute. 6. Centrifuge the mixture in step 5 at 2000rpm/min for 1 minute. Discard the supernatant, resuspend with 1x loading buffer (with β-ME added), cook the sample at 100°C for 10 minutes, and obtain the processed sample.

7. Perform SDS PAGE on the processed sample obtained in step 6. The specific steps are as follows: transfer the treated sample to a nitrocellulose membrane. Then the membrane was blocked with 5% BSA, and incubated overnight with goat anti-human SCARB2 polyclonal antibody (R&D, 1966-LM-050) and SCARB2 monoclonal antibody 5C2 prepared in Example 1, respectively. After washing 3 times with TBST on the second day, the membrane was incubated with HRP-conjugated anti-goat or anti-mouse secondary antibody (Nakasugi Jinqiao, ZB-2305) for 1 hour at room temperature, and then washed 3 times with TBST . The membrane was finally exposed to the SAGECREATION Minichemi Imaging System for color development using a chemiluminescent detection kit according to the manufacturer's instructions (Millipore, WBKLS0100).

The result is shown in Figure 9. It can be seen from the figure that the mouse anti-human SCARB2 monoclonal antibody 5C2 of the present invention can bind to two different forms of SCARB2 expressed in transfected cells: monomer and dimer or glycosylated form (85kD and 120kD) .

Example 6. Detection of the binding ability of the mouse anti-human SCARB2 monoclonal antibody of the present invention to human scavenger receptor B subfamily member IISCARB2

1. The 293A cells overexpressing SCARB2 obtained in Step 1-2 of Example 3 were treated with PBS solution containing 2mM EDTA to make it a single cell suspension.

2. Add the single cell suspension obtained in step 1 to a 96-well U-bottom culture plate, 10,000 cells/200 μl/well. After centrifugation at 2200 rpm/3 minutes, the supernatant was discarded and the precipitate was collected.

3. Add the FACS buffer (PBS containing 2% FBS and 2mM EDTA) containing the mouse anti-human SCARB2 monoclonal antibody 5C2 prepared in Example 1 to the precipitate obtained in step 2, so that the final concentrations in the system are respectively 0.001μg/ml, 0.0025μg/ml, 0.005μg/ml, 0.01μg/ml, 0.05μg/ml, 0.1μg/ml, 0.2μg/ml, 0.4μg/ml, 0.6μg/ml, 1μg/ml, 2μg/ml, 4μg/ml, 8μg/ml, after resuspension, incubate at 4°C for 30 minutes. At the same time, an equal amount of mouse IgG2a (Biolegend, MOPC-173, 400223) was used as a negative control.

4. Centrifuge after the incubation, discard the supernatant, collect the precipitate, add 200 μl FACS buffer to each well, resuspend the cells and perform centrifugation, discard the supernatant, collect the precipitate and wash.

5. Add PE-labeled goat anti-mouse IgG antibody (Biolegend, Poly4053, 405307) diluted 1:400 in FACS buffer to each well, resuspend the cells and incubate at 4°C for 30 minutes in the dark. After incubation, centrifuge the culture plate and remove the supernatant, wash the cells once as described above, and then add 200 μl FACS buffer to resuspend the cells. Finally, the cellular PE signal was analyzed using the flow cytometer Guava (Millipore).

The results are shown in Figure 10. The mouse anti-human SCARB2 monoclonal antibody 5C2 of the present invention can effectively bind SCARB2 molecules on the surface of 293A cells stably expressing human SCARB2 at a very low concentration, ie 0.01 μg/mL. And with the increase of antibody concentration, the ability of 5C2 antibody to bind SCARB2 was significantly enhanced.

Example 7, the mouse anti-human SCARB2 monoclonal antibody of the present invention mediates receptor endocytosis

1. Soak the coverslip with 70% alcohol, wait until the alcohol has evaporated, place it in a 24-well plate, inoculate the 293A cells overexpressing SCARB2 obtained in step 1 of Example 3 into a 24-well plate covered with cover fragments , cultured overnight. 2. Aspirate the cell culture medium and wash it once with 500μL PBS. 3. Add 100 μL mouse anti-human SCARB2 monoclonal antibody 5C2-FITC, and react at 4°C and 37°C for 30 minutes, 1 hour, 2 hours and 4 hours, respectively. 4. Wash the sample for 2 hours with 1xDPBS (Thermo Fisher Scientific, Cat: #14080055) for 3 times, add AF594-labeled anti-mouse IgG antibody (Invitrogen, Cat: A-11005) diluted 1:400 in FACS buffer , 4°C for 30 minutes. Samples at other times will not be processed separately. 5. All samples were washed 3 times with 1xDPBS (Thermo Fisher Scientific, Cat: #14080055). 6. Wash once with 1xDPBS (Thermo Fisher Scientific, Cat: #14080055) to reduce the salt concentration. 7. Absorb the liquid on the slide to make the slide as dry as possible. Take the mounting medium into an EP tube, and add DAPI at the ratio of 0.4 μL DAPI (Vector, H-1000) to 1 drop of mounting medium. Drop the prepared mounting medium containing DAPI onto the slide, and cover the stained slide with the cell side facing down to prevent air bubbles. Fix slides to glass slides with nail polish. The slides were observed under an Olympus FV1000 laser confocal microscope.

The results are shown in Figure 11, under the condition of staining at 4°C, regardless of the staining time, the mouse anti-human SCARB2 monoclonal antibody 5C2-FITC can only bind to the surface of 293A cells (293A-SCARB2 cells) overexpressing SCARB2; While staining at 37°C, mouse anti-human SCARB2 monoclonal antibody 5C2-FITC could enter 293A-SCARB2 cells, and with the extension of staining time, the antibody entering 293A-SCARB2 cells gradually increased. It shows that the mouse anti-human SCARB2 monoclonal antibody 5C2 of the present invention can mediate receptor endocytosis. The results of staining for 2 hours showed that some mouse anti-human SCARB2 monoclonal antibody 5C2-FITC still stayed on the 293A-SCARB2 cell membrane, indicating that at 37°C, the SCARB2 expressed by the cell would be continuously transported to the cell membrane.

Example 8. Mouse anti-human SCARB2 monoclonal antibody of the present invention inhibits enterovirus 71 type EV71 from infecting 293A-SCARB2 stable cell line

1. Mouse anti-human SCARB2 monoclonal antibody 5C2 inhibits the replication of EV71-GFP in 293A-SCARB2

1. The 293A cells overexpressing SCARB2 obtained in Step 1 of Example 3 were inoculated in a 24-well cell culture plate (1×10 ⁵ cells/well) containing DMEM complete medium (containing 10% FBS), at 37° C. Incubate overnight at 5% CO ₂ . 2. The next day, the cell supernatant was discarded, and the DMEM complete medium containing the mouse anti-human SCARB2 monoclonal antibody 5C2 prepared in Example 1 was added to a 24-well cell culture plate, so that the final concentration in the system was 0 μg /ml, 0.1μg/ml, 0.2μg/ml, 0.4μg/ml, 0.6μg/ml, 1μg/ml, 2μg/ml, 4μg/ml, 8μg/ml, incubate at 37℃, 5% _CO2 1h. At the same time, an equal amount of mouse IgG2a was used as a negative control. 3. Add 0.1MOI EV71-GFP virus to each well of step 2 (EV71-GFP virus is recorded in the document "Protein Cell 2017, 8(8):590-600"), at 37°C, 5% CO ₂ conditions Incubate for 12-14 hours to obtain post-infected cells. 4. Wash the infected cells obtained in step 3 twice with 1ml PBS solution, then digest the cells with 0.025% trypsin for 2 minutes, then add FACS buffer (PBS containing 2% FBS and 2mM EDTA), resuspend and add an equal amount Fix with 4% PFA for 20 min at room temperature. Finally, the cell GFP signal was analyzed using a flow cytometer Guava (Millipore), and the blocking efficiency was calculated. The blocking efficiency calculation formula is as follows: 1-(GFP positive cells% after adding antibodies/GFP positive cells% when not adding antibodies).

The results are shown in Figure 12A and Figure 12B, when the antibody concentration is 4 μg/mL, compared with the control group, the blocking efficiency of 5C2 reaches the highest, which is 90.2% (1-2.16%/21.98%) (2.16% means no Antibody (Mock) GFP positive cell ratio; 21.98% means GFP positive cell ratio after adding antibody). And when the antibody concentration is greater than 0.4 μg/mL, the blocking efficiency is no longer dose-dependent, that is, it reaches a plateau.

2. Mouse anti-human SCARB2 monoclonal antibody 5C2 inhibits the replication of wild-type EV71 in 293A-SCARB2

1. Virus infection and supernatant collection

(1) Inoculate the 293A cells overexpressing SCARB2 obtained in Step 1 of Example 3 into a 24-well cell culture plate (1×10 ⁵ cells/well) containing DMEM complete medium (containing 10% FBS), at 37° C. , 5% CO ₂ and cultured overnight. (2) The next day, the cell supernatant was discarded, and the DMEM complete medium containing the mouse anti-human SCARB2 monoclonal antibody 5C2 prepared in Example 1 was added in a 24-well cell culture plate, so that the final concentrations in the system were respectively 0 μg/ml or 0.25 μg/ml, incubate at 37°C, 5% CO ₂ for 1 hour. At the same time, an equal amount of mouse IgG2a was used as a negative control. (3) Add 0.1 MOI of EV71 wild-type virus to each well of step 2 (EV71 wild-type virus is described in the literature "Protein Cell2017, 8(8):590-600"), at 37°C, 5% CO ₂ After incubation for 1 h, the virus liquid was aspirated. The cells were cleared three times with 1ml PBS, and then fresh medium was added, and the cell supernatant was collected at this time and marked as 0h. (4) 16-24 hours after infection, collect the infection supernatant.

2. Viral RNA extraction

Use the kit Biospin virus RNA extraction kit to extract viral RNA. The extraction method is completed according to the kit instructions. The specific operation steps are as follows:

(1) 24 hours after the virus infected the cells, centrifuge the cell culture plate at 2000 rpm for 3 minutes, take 100 μl of the culture supernatant and add it to an RNase-free centrifuge tube, and at the same time take 100 μl of the virus stock with known titer liquid as a standard. (2) Add 300 μl of protein lysate to the virus sample to be tested, shake and mix well. (3) Add 400 μl of absolute ethanol to the mixture, vortex to mix well, and centrifuge briefly to make the liquid gather into the tube. (4) Add the mixed liquid into the column, and centrifuge at 12,000 rpm for 1 minute. (5) Discard the waste liquid, add 200 μl DNase to the column, and centrifuge at 12,000 rpm for 1 minute. (6) Discard the waste liquid, add 600 μl washing solution to the column, and centrifuge at 12,000 rpm for 1 minute. (7) Discard the waste liquid, add 250 μl washing solution to the column, and centrifuge at 12,000 rpm for 2 minutes. (8) Transfer the column to a new RNase-free centrifuge tube, add 50 μl of eluent, stand still at room temperature for 1 minute, and centrifuge at 12,000 rpm for 1 minute, and the centrifuged liquid is RNA solution.

3. Reverse transcription cDNA

The RNA obtained in step 2 was reverse transcribed, and the reverse transcription process was also carried out according to Promega's RNA reverse transcriptase M-MLV instructions. The specific operation steps are as follows:

(1) Take an RNase-free PCR tube, add 12 μl RNA and 1 μl 20mM Oligo dT. (2) Mix the above mixture and place it in a PCR instrument for denaturation at 70°C for 5 minutes, and then immediately cool down at 4°C for 1 minute, so that the Oligo dT primer and the 3' end Poly(A) of the mRNA are fully combined to prevent RNA hairpins , stem-loop and other secondary structure recovery. (3) Add the following components to the above reaction system and mix well: 1 μl of M-MLV, 4 μl of 5xBuffer, 1 μl of 10 mM dNTPs, and 1 μl of RNase inhibitor (RRI). (4) Place the well-mixed reaction system on a PCR machine, and carry out reverse transcription according to the following procedure: 42°C, 1 hour; 75°C, 10 minutes; 4°C, -. (5) Store the cDNA obtained by reverse transcription at -80°C or proceed directly to the next step.

4. Real-time PCR detection of virus titer

Take the cDNA obtained by reverse transcription in step 3, dilute it 50 times with sterile water as a template, and further dilute the diluted standard 10 times in 6-7 gradients to draw a standard curve. Prepare the reaction system, the specific dosage is as follows: Easy Taq 0.2μl, 10xbuffer 2μl, 2.5mM dNTPs 1.6μl, diluted cDNA 5μl, LG515 (10mM) 2μl, LG517 (10mM) 2μl, LG516probe (1mM) 2μl, sterile water 5.2 μl.

The PCR reaction system is as follows: first step, 95°C pre-denaturation for 5 minutes; second step, 95°C denaturation for 15 seconds, 57°C return for 15 seconds, 72°C extension for 20 seconds, a total of 45 cycles; third step, 25°C cooling 1 minute. The sequences of real-time fluorescent quantitative PCR primers are shown in Table 5.

EV71 quantitative real-time PCR primer sequence

Numbering name Sequence (5'-3') 5' mark 3' mark LG515 reverse primer TCCTCCGGCCCCTGA LG517 forward primer AATTGTCACCATAAGCAGCCA LG516 probe CGGAACCGACTACTTTGGGTGTCCGT FAM TAM

The results are shown in Figure 12C. When the antibody concentration was 0.25 μg/mL, compared with the control without antibody, the efficiency of 5C2 in blocking wild-type EV71 infection was about 75%.

Example 9. The mouse anti-human SCARB2 monoclonal antibody of the present invention binds to IISCARB2, a member of the subfamily of human scavenger receptor B, and inhibits enterovirus 71 type EV71 from infecting target cells of human rhabdomyosarcoma cells RD

1. Mouse anti-human SCARB2 monoclonal antibody 5C2 inhibits the replication of EV71-GFP in RD cells

CCL-136^TM)接种于含有DMEM完全培养基(含有10％FBS)的24孔细胞培养板中(1×10⁵细胞/孔)，在37℃、5％CO₂条件下培养过夜。2、次日，弃细胞上清，在24孔细胞培养板中加入含有实施例1制备的小鼠抗人SCARB2单克隆抗体5C2的DMEM完全培养基，使其在体系中的终浓度分别为0μg/ml、0.4μg/ml、5μg/ml、10μg/ml、20μg/ml，在37℃、5％CO₂条件下孵育1h。同时用等量的小鼠IgG2a作为阴性对照。3、向步骤2的各孔中加入0.1MOI EV71-GFP病毒(EV71-GFP病毒记载于文献“Protein Cell2017,8(8):590–600”中)，在37℃、5％CO₂条件下孵育12～14h，得到感染后细胞。4、用1mlPBS溶液洗涤步骤3获得的感染后细胞两次，然后用0.025％胰蛋白酶消化细胞2分钟，之后加入FACS buffer(含2％FBS和2mM EDTA的PBS)，重新悬浮后加入等量的4％PFA室温固定20分钟。最后，使用流式细胞仪Guava(Millipore)分析细胞GFP信号。1. Human rhabdomyosarcoma cells RD cells (ATCC,

CCL-136 ^TM ) were seeded in 24-well cell culture plates (1×10 ⁵ cells/well) containing DMEM complete medium (containing 10% FBS), and cultured overnight at 37°C and 5% CO ₂ . 2. The next day, the cell supernatant was discarded, and the DMEM complete medium containing the mouse anti-human SCARB2 monoclonal antibody 5C2 prepared in Example 1 was added to a 24-well cell culture plate, so that the final concentration in the system was 0 μg /ml, 0.4μg/ml, 5μg/ml, 10μg/ml, 20μg/ml, incubate at 37℃, 5% CO ₂ for 1h. At the same time, an equal amount of mouse IgG2a was used as a negative control. 3. Add 0.1MOI EV71-GFP virus to each well of step 2 (EV71-GFP virus is recorded in the literature "Protein Cell2017,8(8):590-600"), at 37°C, 5% CO ₂ Incubate for 12-14 hours to obtain post-infected cells. 4. Wash the infected cells obtained in step 3 twice with 1ml PBS solution, then digest the cells with 0.025% trypsin for 2 minutes, then add FACS buffer (PBS containing 2% FBS and 2mM EDTA), resuspend and add an equal amount of Fix with 4% PFA for 20 minutes at room temperature. Finally, the cellular GFP signal was analyzed using the flow cytometer Guava (Millipore).

The results are shown in Figure 13A. When the concentration of antibody 5C2 was 20 μg/mL, compared with the control group, the blocking efficiency of 5C2 was about 60%.

2. Mouse anti-human SCARB2 monoclonal antibody 5C2 inhibits the replication of wild-type EV71 in RD cells

1. Inoculate human rhabdomyosarcoma cells RD cells in a 24-well cell culture plate (1×105 cells/well) containing DMEM complete medium (containing 10% FBS), and culture overnight at 37°C and 5% CO ₂ . 2. The next day, the cell supernatant was discarded, and the DMEM complete medium containing the mouse anti-human SCARB2 monoclonal antibody 5C2 prepared in Example 1 was added to a 24-well cell culture plate, so that the final concentration in the system was 0 μg /ml or 1μg/ml, incubate for 1h at 37°C, 5% CO ₂ . At the same time, an equal amount of mouse IgG2a was used as a negative control. 3. Add 0.1 MOI of EV71 wild-type virus to each well of step 2 (EV71 wild-type virus is recorded in the document "Protein Cell 2017,8(8):590-600"), at 37°C, 5% CO ₂ conditions After incubation for 1 h, the virus liquid was aspirated. The cells were cleared three times with 1ml PBS, and then fresh medium was added, and the cell supernatant was collected at this time and marked as 0h. 4. 16-24 hours after infection, collect the infection supernatant. 5. Detect the viral infection load by the method of Step 2/3/4 of Example 8.

The results are shown in Figure 13B. When the concentration of antibody 5C2 was 1 μg/mL, compared with the control group, the blocking efficiency of 5C2 was about 50%.

Example 10. Mouse anti-human SCARB2 monoclonal antibody 5C2 of the present invention binds to human scavenger receptor B subfamily member II SCARB2 positions 144-151 and 188-198

1. Human-mouse SCARB2 chimera construction

Plasmid hSCARB2 is a vector obtained by inserting the human SCARB2 coding gene sequence (GenBank number: NM_005506.3) between the NotI and EcoRI restriction sites of the pcDNA vector (addgene, cat: #52535);

Plasmid mSCARB2 is a vector obtained by inserting the mouse SCARB2 coding gene sequence (GenBank number is NM_007644.4) between the NotI and EcoRI restriction sites of the pcDNA vector;

Human-mouse SCARB2 chimeric plasmid m144-151 is the coding gene sequence of SCARB2 (wherein, the coding gene sequence of SCARB2 amino acid 144-151 is the coding gene sequence of mouse SCARB2 amino acid 144-151, and the rest are all human SCARB2 coding genes Sequence) the vector obtained between the NotI and EcoRI restriction sites inserted into the pcDNA vector;

Human-mouse SCARB2 chimera plasmid m188-198 is the coding gene sequence of SCARB2 (wherein, the coding gene sequence of SCARB2 amino acids 188-198 is the coding gene sequence of mouse SCARB2 amino acids 188-198, and the rest are human SCARB2 coding genes Sequence) the vector obtained between the NotI and EcoRI restriction sites inserted into the pcDNA vector;

Human-mouse SCARB2 chimeric plasmid m278-288 is the coding gene sequence of SCARB2 (wherein, the coding gene sequence of SCARB2 278-288 amino acids is the coding gene sequence of mouse SCARB2 278-288 amino acids, and the rest are all human SCARB2 coding genes Sequence) the vector obtained between the NotI and EcoRI restriction sites inserted into the pcDNA vector;

Human-mouse SCARB2 chimeric plasmid m354-385 is the coding gene sequence of SCARB2 (wherein, the coding gene sequence of SCARB2 amino acid 354-385 is the coding gene sequence of mouse SCARB2 amino acid 354-385, and the rest are human SCARB2 coding genes Sequence) the vector obtained between the NotI and EcoRI restriction sites inserted into the pcDNA vector;

Human-mouse SCARB2 chimera plasmid h144-151 is the coding gene sequence of SCARB2 (wherein, the coding gene sequence of SCARB2 amino acids 144-151 is the coding gene sequence of human SCARB2 amino acids 144-151, and the rest are mouse SCARB2 coding genes Sequence) the vector obtained between the NotI and EcoRI restriction sites inserted into the pcDNA vector;

Human-mouse SCARB2 chimera plasmid h188-198 is the coding gene sequence of SCARB2 (among them, the coding gene sequence of SCARB2 amino acids 188-198 is the coding gene sequence of human SCARB2 amino acids 188-198, and the rest are mouse SCARB2 coding genes Sequence) the vector obtained between the NotI and EcoRI restriction sites inserted into the pcDNA vector;

Human-mouse SCARB2 chimeric plasmid h144-151&h188-198 is the SCARB2 coding gene sequence (wherein, the coding gene sequence of SCARB2 144-151 and 188-198 amino acids is human SCARB2 144-151 and 188-198 Amino acid coding gene sequence, the rest are the mouse SCARB2 coding gene sequence) inserted into the vector obtained between the NotI and EcoRI restriction sites of the pcDNA vector;

Human-mouse SCARB2 chimera plasmid h144-198 is the coding gene sequence of SCARB2 (wherein, the coding gene sequence of SCARB2 amino acids 144-198 is the coding gene sequence of human SCARB2 amino acids 144-198, and the rest are mouse SCARB2 coding genes sequence) was inserted into the vector obtained between the NotI and EcoRI restriction sites of the pcDNA vector.

The structure of ten human-mouse SCARB2 chimeras of the present invention is shown in Figure 14, the solid part represents the sequence of human SCARB2, and the hollow part represents the sequence of mouse SCARB2.

2. The mouse anti-human SCARB2 monoclonal antibody of the present invention binds to human scavenger receptor B subfamily member II SCARB2 at the flow cytometric level and requires residues 144-151 and 188-198 regions

1. Inoculate 293T-SCARB2-KO cells (293T-SCARB2-KO cells are described in the literature "Protein Cell2017,8(8):590-600") in 24 wells containing DMEM complete medium (containing 10% FBS) Plates (1×10 ⁵ cells/well), cultured at 37°C, 5% CO ₂ for 24 hours. Then, the human-mouse SCARB2 chimera plasmid prepared in Step 1 was transfected into HEK 293T cells respectively, and the transfection step was carried out according to the instructions (Thermo Fisher, Lipofectamine 2000, 11668019). Incubate for 48 hours at 37°C, 5% CO ₂ . 2. Use the transfected cells obtained in step 1 with PBS solution containing 2mM EDTA (each 1L of PBS contains KH ₂ PO ₄ 0.27g, Na ₂ HPO ₄ 1.42g, NaCl 8g, KCl 0.2g, adjust the pH to 7.2-7.4 , and dilute to 1L with water) to make it a single-cell suspension. 3. Add the single-cell suspension of transfected cells to a 96-well U-bottom culture plate, 10,000 cells/200 μl/well. After centrifugation at 2200 rpm/3 minutes, the supernatant was discarded and the precipitate was collected. 4. Add the mouse anti-human SCARB2 monoclonal antibody 5C2 and the isotype control antibody IgG2a containing 1 μg/ml of the mouse anti-human SCARB2 monoclonal antibody prepared in Example 1 to the precipitate obtained in step 3 respectively. (PBS containing 2% FBS and 2mM EDTA), Incubate at 4°C for 30 minutes after resuspension. 5. After incubation, centrifuge, discard the supernatant, collect the precipitate, add 200 μl FACSbuffer to each well, resuspend the cells and centrifuge, discard the supernatant, collect the precipitate and wash. 6. Add PE-labeled goat anti-mouse IgG antibody (Biolegend) diluted 1:400 in FACS buffer to each well, resuspend the cells and incubate at 4°C for 30 minutes in the dark. After incubation, the culture plate was centrifuged and the supernatant was removed, the cells were washed once as described above, and then 200 μl FACSbuffer was added to resuspend the cells. Finally, the cellular PE signal was analyzed using the flow cytometer Guava (Millipore).

The results are shown in Figure 15, the 144-151 and 188-198 regions of human SCARB2 are required for the mouse anti-human SCARB2 monoclonal antibody 5C2 of the present invention to be able to bind to human SCARB2 at the FACS level.

3. The mouse anti-human SCARB2 monoclonal antibody of the present invention needs residues 144-151 and 188-198 regions to bind to human scavenger receptor B subfamily member II SCARB2 at the level of western blot

1. Inoculate 293T-SCARB2-KO cells (293T-SCARB2-KO cells are described in the literature "Protein Cell2017,8(8):590-600") in 24 wells containing DMEM complete medium (containing 10% FBS) Plates (1×105 cells/well), cultured at 37°C, 5% CO ₂ for 24 hours. Then, the human-mouse SCARB2 chimera plasmid prepared in Step 1 was transfected into HEK 293T cells respectively, and the transfection step was carried out according to the instructions (Thermo Fisher, Lipofectamine 2000, 11668019). Incubate for 48 hours at 37°C, 5% CO ₂ . 2. Wash the product obtained in step 1 twice with phosphate-buffered saline (PBS), and wash it with 500 μl of ice-cold lysate (100 mM Tris-HCl (pH7.4), 140 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate; supplemented with 1 mM PMSF, 0.01 M DTT, Protease Inhibitor Mix Tablet (Roche)) were lysed to obtain cell lysates. The cell lysate was shaken at 4°C for 30 minutes, clarified at 13,000 rpm for 20 minutes, the supernatant was aspirated, 5x loading buffer and β-ME were added to the supernatant, and the sample was boiled at 100°C for 10 minutes to obtain the processed sample. 3. Perform SDS PAGE on the processed sample obtained in step 2. The specific steps are as follows: transfer the treated sample to a nitrocellulose membrane. Then the membrane was blocked with 5% BSA, and incubated overnight with goat anti-human SCARB2 polyclonal antibody (R&D, 1966-LM-050) and SCARB2 monoclonal antibody 5C2 prepared in Example 1, respectively. After washing 3 times with TBST on day 2, the membrane was incubated with HRP-conjugated anti-goat or anti-mouse secondary antibody (Nakasugi Jinqiao, ZB-2305) for 1 hour at room temperature, followed by washing 3 times with TBST. The membrane was finally exposed to the SAGECREATION Minichemi Imaging System for color development using a chemiluminescent detection kit according to the manufacturer's instructions (Millipore, WBKLS0100).

The results are shown in Figure 16, the mouse anti-human SCARB2 monoclonal antibody 5C2 of the present invention can bind to human SCARB2 at the level of western blot and also requires the 144-151 and 188-198 regions of human SCARB2.

sequence listing

<110>Guangdong Xuanyu Health Biotechnology Co., Ltd.

<120> Monoclonal antibody blocking EV71 infection and its application

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Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr

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Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

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His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Lys Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

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Tyr Thr Ser

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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

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Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Val Gly Ile Tyr

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Tyr Cys Thr Ser His Tyr Tyr Gly Tyr Val Pro Phe Ala Tyr Trp Gly

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Gln Gly Thr Leu Val Thr Val Ser Ala

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Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

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His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

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Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Glu Glu Lys Leu Asp Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Thr Asn Ser

20 25 30

Trp Met Asn Trp Phe Cys Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Lys Ser Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr

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Tyr Cys Thr Trp Phe Ile Thr Thr Val Val Ala Phe Ala Tyr Trp Gly

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Thr Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp

35 40 45

Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro

50 55 60

Arg Leu Leu Ile His Tyr Thr Ser Ser Thr Leu Gln Pro Gly Ile Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Lys Asp Tyr Ser Phe Ser Ile Ser

85 90 95

Asn Leu Glu Pro Glu Asp Leu Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp

100 105 110

Asn Leu Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala

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Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu

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Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

Ser Met Ser Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His

195 200 205

Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile

210 215 220

Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230

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<213>Artificial Sequence

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tgtgttgcct ctggattcac ttttagtgac tactggatga actgggtccg ccagtctcca 180

gagaaaggac tggagtgggt agcacaaatt agatacaaac cttataattc tgaaacattt 240

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gccaaaacaa cagccccatc ggtctatcca ctggcccctg tgtgtggaga tacaactggc 480

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tggaactctg gatccctgtc cagtggtgtg cacaccttcc cagctgtcct gcagtctgac 600

ctctacaccc tcagcagctc agtgactgta acctcgagca cctggcccag ccagtccatc 660

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gggcccacaa tcaagccctg tcctccatgc aaatgcccag cacctaacct cttgggtgga 780

ccatccgtct tcatcttccc tccaaagatc aaggatgtac tcatgatctc cctgagcccc 840

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gagttcaaat gcaaggtcaa caacaaagac ctcccagcgc ccatcgagag aaccatctca 1080

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atgactaaga aacaggtcac tctgacctgc atggtcacag acttcatgcc tgaagacatt 1200

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gtggaaagaa atagctactc ctgttcagtg gtccacgagg gtctgcacaa tcaccacacg 1380

actaagagct tctcccggac tccgggtaaa 1410

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<212>PRT

<213>Artificial Sequence

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Met Tyr Phe Gly Leu Ser Cys Val Phe Ile Val Phe Leu Leu Lys Gly

1 5 10 15

Val Gln Cys Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Arg Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Tyr Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Gln Ile Arg Tyr Lys Pro Tyr Asn Ser Glu Thr Phe

65 70 75 80

Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Lys Ser Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Val

100 105 110

Gly Ile Tyr Tyr Cys Thr Ser His Tyr Tyr Gly Tyr Val Pro Phe Ala

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr

130 135 140

Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly

145 150 155 160

Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro

165 170 175

Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Ser Val

195 200 205

Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val

210 215 220

Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Lys Ile Glu Pro Arg

225 230 235 240

Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp

260 265 270

Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp

275 280 285

Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn

290 295 300

Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn

305 310 315 320

Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp

325 330 335

Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro

340 345 350

Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala

355 360 365

Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys

370 375 380

Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile

385 390 395 400

Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn

405 410 415

Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys

420 425 430

Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys

435 440 445

Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe

450 455 460

Ser Arg Thr Pro Gly Lys

465 470

Claims (9)

1. A monoclonal antibody, or antigen binding portion thereof, of SCARB2, the light chain variable region of the monoclonal antibody of SCARB2 comprising a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3;

the heavy chain variable region of the monoclonal antibody of SCARB2 comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2 and a heavy chain variable region CDR3;

the amino acid sequence of the CDR1 of the light chain variable region is shown as a sequence 3;

the amino acid sequence of the CDR2 of the light chain variable region is shown as a sequence 4;

the amino acid sequence of CDR3 of the light chain variable region is shown as a sequence 5;

the amino acid sequence of CDR1 of the heavy chain variable region is shown as a sequence 8;

the amino acid sequence of CDR2 of the heavy chain variable region is shown as a sequence 9;

the amino acid sequence of CDR3 of the heavy chain variable region is shown as a sequence 10.

2. The monoclonal antibody or antigen-binding portion thereof of SCARB2 according to claim 1, characterized in that:

the amino acid sequence of the variable region of the light chain of the monoclonal antibody of SCARB2 is shown as a sequence 2;

the amino acid sequence of the heavy chain variable region of the SCARB2 monoclonal antibody is shown as a sequence 7.

3. The monoclonal antibody or antigen binding portion thereof of SCARB2 according to claim 1 or 2, characterized in that:

the coding gene sequence of the light chain variable region of the SCARB2 monoclonal antibody is shown as a sequence 1;

the coding gene sequence of the heavy chain variable region of the SCARB2 monoclonal antibody is shown as a sequence 6.

4. The monoclonal antibody for SCARB2 or a derivative of an antigen-binding portion thereof according to any one of claims 1-3, which is any one of the following (e 1) to (e 5):

(e1) An antagonist of Enterovirus 71 having as an active ingredient a monoclonal antibody or antigen-binding portion thereof against SCARB2 according to any one of claims 1 to 3;

(e2) A composition comprising a monoclonal antibody or antigen-binding portion thereof of SCARB2 according to any one of claims 1-3;

(e3) An immunoconjugate comprising the monoclonal antibody or antigen binding portion thereof of SCARB2 of any one of claims 1-3;

(e4) A humanized antibody obtained by humanizing the monoclonal antibody against SCARB2 according to any one of claims 1 to 3 or an antigen-binding portion thereof;

(e5) A multispecific molecule comprising a monoclonal antibody or antigen-binding portion thereof of SCARB2 according to any one of claims 1 to 3.

5. A biological material related to the monoclonal antibody or antigen binding portion thereof of SCARB2 according to any one of claims 1-3, which is any one of the following A1) -a 12);

a1 A nucleic acid molecule encoding a monoclonal antibody or antigen binding portion thereof of SCARB2 according to any one of claims 1-3;

a2 An expression cassette comprising the nucleic acid molecule according to A1);

a3 A recombinant vector containing the nucleic acid molecule according to A1);

a4 A recombinant vector containing the expression cassette of A2);

a5 A recombinant microorganism containing the nucleic acid molecule according to A1);

a6 A recombinant microorganism containing the expression cassette of A2);

a7 A recombinant microorganism containing the recombinant vector of A3);

a8 A recombinant microorganism containing the recombinant vector of A4);

a9 A transgenic cell line containing the nucleic acid molecule according to A1);

a10 A transgenic cell line containing the expression cassette of A2);

a11 A transgenic cell line containing the recombinant vector of A3);

a12 A transgenic cell line containing the recombinant vector of A4).

6. Use of a monoclonal antibody or antigen binding portion thereof of SCARB2 according to any of claims 1-3 or a derivative according to claim 4 or a biomaterial according to claim 5 in at least one of the following 1) -6):

1) Preparing a human SCARB 2-binding product;

2) Preparing a product for inhibiting or blocking infection of host cells expressing SCARB2 by enterovirus 71;

3) Preparing a product that inhibits replication of enterovirus type 71 in host cells expressing SCARB2;

4) Preparing a product for mediating SCARB2 endocytosis into cells;

5) The application of preparing the antibody of the SCARB2;

6) Preparing a product for treating and/or preventing diseases caused by enterovirus 71 infection.

7. Use according to claim 6, characterized in that: the human SCARB2 is human SCARB2 which is expressed on the surface of a combined cell membrane.

8. Use according to claim 6, characterized in that: the human SCARB2 is an amino acid sequence shown in 144 th-151 th and 188 th-198 th positions of the human SCARB2 protein.

9. A method of making an anti-SCARB 2 antibody comprising the steps of:

b1 Providing:

(i) A light chain variable region sequence comprising a CDR1 sequence selected from those shown in seq id No. 3, a CDR2 sequence selected from those shown in seq id No. 4, and a CDR3 sequence selected from those shown in seq id No. 5;

(ii) A heavy chain variable region sequence comprising a CDR1 sequence selected from those set forth in seq id No. 8, a CDR2 sequence selected from those set forth in seq id No. 9, and a CDR3 sequence selected from those set forth in seq id No. 10;

b2 The variable region sequence is expressed as a protein.

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