CN111139233B - Broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM monoclonal antibody and application thereof - Google Patents

Abstract

The invention relates to a broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibody and application thereof, belonging to the field of biotechnology. The antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1 in a sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2 in the sequence table. The present invention provides a human-mouse chimeric monoclonal antibody capable of simultaneously resisting EV71, CA16, CA10 and CA6, which is composed of a variable region of murine IgM and a constant region of human IgM. The chimeric antibody disclosed by the invention has higher affinity to EV71, CA16, CA10 and CA6, shows broad-spectrum efficient neutralization activity to EV71, CA16, CA10 and CA6, and can be used for treating hand-foot-and-mouth disease caused by A-type enteroviruses such as EV71, CA16, CA10 and CA 6.

Description

Broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibody, and a preparation method and application thereof.

Background

Enterovirus 71 (Enterovirus 71, EV71) and Coxsackie virus type A16 (Coxsackie virus A16, CA 16) have been identified as major pathogens inducing hand-foot-and-mouth disease (hand foot and mouth disease, HFMD). In recent years, studies have shown that: coxsackie virus A10 (CA 10) and Coxsackie virus A6 (CA 16, CA 6) infection caused cases of hand-foot-and-mouth disease to appear in an ascending trend. At present, inactivated EV71 vaccines which have been successfully marketed show high-efficiency protection effect against EV71 infection, but have no cross protection effect on other enteroviruses such as CA16, CA10, CA6 and the like. Meanwhile, the study shows that: after the CA16 vaccine immunizes rhesus monkeys, the neutralizing antibodies produced by the vaccine are not effective in protecting the host against viral infection. Thus, development of virus control with passive immunization against enteroviruses with broad-spectrum efficient neutralizing antibodies (broadly neutralizing antibodies, bNAbs) has become one of the new strategies for antiviral. The monoclonal antibodies reported so far are of the IgG type, have neutralizing therapeutic effects only on enteroviruses of a single type, and have no cross-protective effect. How to overcome the defects of the prior art is a problem which needs to be solved in the field of biotechnology at present.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provide a broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibody, a preparation method and application thereof. Therefore, the invention utilizes the human-mouse embedding method to modify and transform the mouse-derived antibody, thereby reducing the immunogenicity of the mouse-derived antibody to human bodies. The human-mouse chimeric antibody is formed by connecting a variable region of a mouse antibody with a constant region of a human antibody by utilizing a genetic engineering technology, and can keep the affinity and the specificity of the original antibody, reduce the reaction of the human anti-mouse antibody, prolong the half-life and improve the pharmacokinetics.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the present invention provides a method for preparing a broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 antibody hybridoma cell strain, comprising the steps of:

step (1), preparation of immunogen: purified EV71 and CA16 viruses are used as immunogens;

step (2), animal immunization: four immunizations were performed at 2 week intervals on 6-8 week old Balb/c mice with the following procedure: after primary immunization, fully mixing Freund's complete adjuvant and an equal amount of immunogen for emulsification, performing subcutaneous multipoint injection on the back; the second immunization and the third immunization, the adjuvant is replaced by Freund's incomplete adjuvant, and the method and the metering are the same as the first immunization; the fourth time of intraperitoneal injection of immunogen without adjuvant is adopted, and the injection quantity is the same as that of the first immunization; after 3 days, spleen cells of immunized mice were taken for preparation of hybridoma cells;

step (3), preparation and screening of hybridoma cell strains: and (2) carrying out cell fusion on SP2/0 cells in the logarithmic growth phase and spleen cells of the immunized mice in the step (2) under the action of PEG4000, culturing the fused hybridoma cells by using a1 XHAT selective medium, detecting cell supernatants 10-14 days after cell fusion, and screening hybridoma cell strains capable of specifically secreting broad-spectrum neutralizing antibodies against EV71, CA16, CA10 and CA6 by an enzyme-linked immunosorbent assay.

Further, it is preferable that in the step (2), the first immunization dose is 50ul 10 ⁷ TCID50/ml EV71 and 50ul 10 ⁷ TCID50/ml CA16。

Further, in the step (3), it is preferable that the SP2/0 cells in the logarithmic growth phase and the spleen cells of the immunized mice of the step (2) are subjected to cell fusion by the action of PEG4000, and the fused hybridoma cells are cultured by using a1 XHAT selective medium, specifically comprising the steps of:

selecting a Balb/c mouse with the age of 6-8 weeks, taking abdominal macrophages of the Balb/c mouse to prepare feeder cells; mixing SP2/0 cells in logarithmic growth phase with spleen cells of immunized mice in step (2) according to cell number ratio of 1:10, performing cell fusion with 40% polyethylene glycol 4000, adding hybridoma obtained after cell fusion into feeder cellsThe culture plate for the layered cells was prepared by using a medium containing 1 XHAT selective medium at 37℃and 5% CO ₂ Culturing under the condition.

Further, it is preferable to further include cloning of hybridoma cells, specifically: subcloning and culturing the hybridoma cell strain by limiting dilution method, continuously culturing for multiple times until the antibody in the cloning hole detects 100% positive, enlarging culturing and freezing storing.

The second aspect of the present invention provides a method for producing a broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibody, the broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 antibody hybridoma cell strain produced by the above-described method for producing a broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 antibody hybridoma cell strain; the method comprises the following steps:

extracting RNA of broad-spectrum neutralizing antibody hybridoma cell strains resisting EV71, CA16, CA10 and CA6, and performing reverse transcription to obtain cDNA; then amplifying the antibody heavy chain variable region by nested PCR, and amplifying the antibody light chain variable region by nested PCR; then, connecting an antibody variable region by using PVITRO 1-hygro-mcs-IgM/kappa connection vectors to obtain an IgM type antibody; meanwhile, PVITRO 1-hygro-mcsIgG/kappa connection carrier is adopted to connect the antibody variable region, so as to obtain an IgG antibody; then, the positive monoclonal antibody obtained by connection is transfected into CHO-S cells, and then purified, thus obtaining broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibodies and IgG type monoclonal antibodies; the DNA sequence of the PVITRO 1-hygro-mcs-IgM/kappa linking vector is shown as SEQ ID NO. 7; the DNA sequence of the PVITRO 1-hygro-mcsIgG/kappa linking vector is shown as SEQ ID NO. 8.

In a third aspect, the present invention provides the broad spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibodies and IgG type monoclonal antibodies thereof prepared by the above method for preparing broad spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibodies.

In a fourth aspect, the invention provides a broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibody, which is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1 in a sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2 in the sequence table.

In a fifth aspect, the present invention provides a DNA encoding a broad-spectrum neutralising anti-EV 71, CA16, CA10 and CA6 human-murine chimeric IgM-type monoclonal antibody as described above, characterized in that it comprises a heavy chain variable region and a light chain variable region;

the coding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 3;

the coding DNA sequence of the light chain variable region is shown as SEQ ID NO. 4.

In a sixth aspect the present invention provides an expression vector comprising a heavy chain constant region DNA sequence and a light chain constant region DNA sequence for expressing a broad spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-murine chimeric IgM-type monoclonal antibody according to claim 5;

the coding DNA sequence of the heavy chain constant region (C-Mu) is shown in SEQ ID NO. 5;

the coding DNA sequence of the light chain constant region (C-kapa) is shown in SEQ ID NO. 6.

In a seventh aspect, the invention provides a host cell comprising an expression vector as described above.

In an eighth aspect, the invention provides a pharmaceutical composition comprising an anti-PD-1 monoclonal antibody as described above and a pharmaceutically acceptable carrier.

In an eighth aspect, the present invention provides the use of a broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibody as described above in the preparation of a medicament for the treatment or prevention of human viral infections, tumors and inflammatory diseases.

The preparation method of the nucleotide molecule of the invention is a conventional preparation method in the field, and preferably comprises the following preparation methods: the nucleotide molecules encoding the monoclonal antibodies are obtained by gene cloning techniques such as PCR methods and the like, or by artificial full sequence synthesis.

It will be appreciated by those skilled in the art that nucleotide sequences encoding the amino acid sequences of the above monoclonal antibodies may be appropriately introduced into substitutions, deletions, alterations, insertions or additions to provide a homolog of a polynucleotide or a conservative variant thereof. The homolog of the polynucleotide of the present invention or a conservative variant thereof can be obtained by substituting, deleting or adding one or more bases of the gene encoding the monoclonal antibody within a range that retains the activity of the antibody.

The expression vectors of the invention are conventional in the art and are intended to include appropriate regulatory sequences, e.g

Promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other suitable sequences

Is described. The expression vector may be a virus or plasmid, such as a suitable phage or phagemid, see, e.g., sambrook et al Molecular Cloning for further technical details: a Laboratory Manual, second edition, coldSpring Harbor Laboratory Press,1989. A number of known techniques and protocols for nucleic acid manipulation are described in Current Protocols in Molecular Biology, second edition, ausubel et al. Expression vectors in the present invention refer to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art.

The host cell of the present invention is a conventional host cell in the art, so long as the recombinant expression vector can stably and self-replicate, and the nucleotide carried can be effectively expressed. The host cell of the invention may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. The host cell preferably comprises: COS, CHO, heLa cell lines, bone marrow cell lines such as SP2/0 cell lines, NS0, sf9, sf21, DH 5. Alpha. BL21 (DE 3) or E.coli TG1, YB2/0 cell lines, etc., as well as transformed B-cells or combinations of one or more of the hybridoma cells. More preferably E.coli TG1, BL21 cells (expressing single chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full length IgG antibodies).

The expression vector is transformed into a host cell, so that the preferred recombinant expression transformant of the invention can be obtained. Wherein the conversion process is conventional in the art, preferably chemical, heat shock or electrotransformation.

The host cells used in the present invention are all of the prior art, and are commercially available, and the medium used in the culture is also various conventional media, and a person skilled in the art can empirically select a suitable medium to culture under conditions suitable for the growth of the host cells. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time. The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

The monoclonal antibodies of the invention may be used by formulating pharmaceutical compositions in any manner known in the art. Such compositions comprise the monoclonal antibody as the active ingredient, together with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the designed dosage form. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol and the like, various preservatives, lubricants, dispersants, flavoring agents. Humectants, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as desired to aid stability of the formulation or to aid in enhancing activity or its bioavailability or to impart an acceptable mouthfeel or odor in the case of oral administration, in which compositions the inhibitor may be used in the form of its original compound itself, or optionally in the form of its pharmaceutically acceptable salt, the monoclonal antibody of the invention may be administered alone, or in various combinations, as well as in combination with other therapeutic agents. The composition so formulated may be administered in any suitable manner known to those skilled in the art, as desired.

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

the invention adopts a hybridoma method to prepare a monoclonal antibody of a murine origin, but the monoclonal antibody can cause human anti-mouse antibody (HAMA) reaction, so that the treatment effect of the monoclonal antibody in human body is affected and side reaction is generated. Therefore, the invention utilizes the human-mouse embedding method to modify and transform the mouse-derived antibody, thereby reducing the immunogenicity of the mouse-derived antibody to human bodies. The human-mouse chimeric antibody is formed by connecting a variable region of a mouse antibody with a constant region of a human antibody by utilizing a genetic engineering technology, and can keep the affinity and the specificity of the original antibody, reduce the reaction of the human anti-mouse antibody, prolong the half-life and improve the pharmacokinetics.

Human-murine chimeric antibodies obtained according to the invention: 20-IgM, exhibited a high degree of broad-spectrum neutralization for EV71, CA16, CA10 and CA6 infections both in vitro (RD) and in vivo (one-day-old rats). The antibody of the invention can be used for preventing or treating EV71, CA16, CA10 and CA6 infection, and further can be possibly applied to the treatment of hand-foot-and-mouth disease caused by other enterovirus infection.

Drawings

FIG. 1 is a diagram of PCR gel electrophoresis of the heavy chain variable region of an antibody;

FIG. 2 is a diagram of PCR gel electrophoresis of the light chain variable region of an antibody;

FIG. 3 is an amino acid sequence analysis of an antibody; wherein A is a heavy chain sequence of a 20-IgM monoclonal antibody and a gene targeting result diagram; b is a 20-IgM monoclonal antibody light chain sequence and a gene targeting result diagram; c is a 20-IgM monoclonal antibody structure simulation diagram;

FIG. 4 is a graph showing the results of the identification of the size and purity of antibody molecules; wherein, ladder: protein molecular standard; 20-IgG (non-reducing): a non-reduced state 20-IgG;20-IgM (non-reducing): a non-reduced state 20-IgM;20-IgG (reducing): reduced state 20-IgG;20-IgM (reducing): reduced state 20-IgM;

FIG. 5 is a graph showing the identification result of the cross recognition of antibodies and enteroviruses of different types;

FIG. 6 is a graph of antibody affinity assay results; wherein ligant Concentration is ligand concentration, fnorm [ mill ] is response value; (a): 20-IgM was assayed for affinity with EV71, CA16, CA10, CA6, respectively; (b): 20-IgG was assayed for affinity with EV71, CA16, CA10, CA6, respectively.

FIG. 7 is a graph of the identification results of neutralizing epitopes of antibodies; a: identifying EV71 capsid protein by 20-IgM and 20-IgG; b: identifying CA16 capsid protein by 20-IgM and 20-IgG; c: identifying EV71 capsid protein by 20-IgM and 20-IgG; d: identifying CA6 capsid protein by 20-IgM and 20-IgG; E-G: ELISA method for identifying antigen peptide recognized by 20-IgM and 20-IgG; H-I: an antigen peptide inhibition test identifies that the 20-IgM and the 20-IgG bind to the antigen peptide; j: ELISA method for identifying key amino acid sites of 20-IgM and 20-IgG; k:20-IgM and 20-IgG recognize EV71 amino acid sites; l:20-IgM and 20-IgG recognize CA16 amino acid sites; m:20-IgM and 20-IgG recognize CA6 amino acid sites; n:20-IgM and 20-IgG recognize CA10 amino acid sites;

FIG. 8 is a diagram of antibody neutralizing effector cytopathy;

FIG. 9 is a graph of antibody neutralization potency assay results; upper left: IC50 and IC80 determination of EV71 by 20-IgM and 20-IgG; upper right: IC50 and IC80 assays for CA16 for 20-IgM and 20-IgG; left lower: IC50 and IC80 assays for CA10 with 20-IgM and 20-IgG; the right lower: IC50 and IC80 of CA6 were measured for 20-IgM and 20-IgG. IC50 (half maximal inhibitory concentration, half-effective inhibitory concentration;

FIG. 10 is a graph showing the dose response of antibodies to infected rats; wherein, the day post-infection is the number of Days after infection; percent survivinal as a Percent of survival; body weight change is the change in body weight. A: clinical symptoms in mice in the infected group and in the antibody group; b: changes in survival rates of mice in EV 71-infected groups and in 20-IgM-treated groups; c: weight change in mice in EV 71-infected and 20-IgM-treated groups; d: : changes in survival rates in mice from the CA16 infected group and the 20-IgM treated group; e: : body weight changes in mice in the CA 16-infected and 20-IgM-treated groups;

FIG. 11 is a plasmid map of the pVITRO 1-hygro-mcs-IgM/kappa expression vector;

FIG. 12 is a plasmid map of the pVITRO 1-hygro-mcs-IgG/kappa expression vector.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.

Unless otherwise indicated herein, percentages refer to percentages by volume and ratios refer to ratios by volume.

1. Preparation method of EV71, CA16, CA10 and CA6 resistant double-effect hybridoma cell strain

1) Immunogen preparation: purified EV71 and CA16 viruses are used as immunogens;

2) Animal immunization: four immunizations were performed at 2 week intervals on 6-8 week old Balb/c mice with the following procedure: after primary immunization, freund's complete adjuvant and equivalent immunogen are fully mixed and emulsified, the back subcutaneous multipoint injection is carried out, 100ul 10 ⁷ TCID50/ml; the second immunization and the third immunization, the adjuvant is replaced by Freund's incomplete adjuvant, and the method and the metering are the same as the first immunization; the fourth time of intraperitoneal injection of immunogen without adjuvant is adopted, and the injection quantity is the same as that of the first immunization; after 3 days, spleen cells of immunized mice were taken for preparation of hybridoma cells;

3) Preparation of hybridoma cell lines: the method specifically comprises the following steps: the spleen cells of immunized mice were taken 2 days before cell fusion for preparation of hybridoma cells. Selecting a Balb/c mouse with the age of 6-8 weeks, taking abdominal macrophages to prepare feeder cells, and enabling the cell concentration to be 10 ⁵ Adding 96-well plate at a volume of 100 μl/mL, placing at 37deg.C with 5% CO ₂ Culturing under the condition for standby. SP2/0 cells (purchased from ATCC) in logarithmic growth phase were mixed with spleen cells of immunized mice of step 2) in a cell number ratio of 1:10, and hybridoma cells obtained by fusing 45% polyethylene glycol 4000 (PEG) (purchased from sigma) cells were added to a 96-well plate containing feeder cells, and a culture medium containing 1 XHAT selective medium [50 XHAT (purchased from sigma), 20% (V/V) fetal bovine serum, 1640 medium (purchased from Corning) was used]At 37℃with 5% CO ₂ Culturing under the condition.

4) The hybridoma cell strain is screened, and specifically: culturing the hybridoma obtained in the step 3) under the condition of containing the 1 XHAT selective medium for 10-14 days, and replacing half amount of the 1 XHAT complete medium every 4 days. After 12 days of culture, hybridoma cell supernatants were subjected to an indirect ELISA to initially screen for hybridoma cells capable of specifically secreting anti-EV 71, CA16, CA10 and CA 6. The method comprises the following specific steps: EV71 or CA16 coats the ELISA plate, a blank control is arranged, a culture supernatant is taken as a primary antibody, SP2/0 cell culture supernatant is taken as a negative control, a secondary antibody is HRP-marked goat anti-mouse IgG (1:6000) (Thermo Scientific), after color development of TMB (Solarbio) is terminated, an ELISA plate instrument detects an absorbance value (A450 value) at 450nm, holes with a height of Yu Yinxing, which is more than 2.1 times of the A450 value, are taken as positive cell holes, and subcloning of the next step is carried out.

5) Cloning of hybridoma cells, specifically: selecting positive cell well obtained by screening in step 4), cloning cells in the positive well by limiting dilution method, sucking 200 μl of cells per well into a sample-adding tank, and culturing with 1×HT complete medium [ (50×HT (purchased from sigma), 20% (V/V) fetal bovine serum, 1640 medium (purchased from Corning)]After dilution to 20mL, the mixture was uniformly added to a new 96-well plate at 200. Mu.L per well at 37℃in 5% CO ₂ After 5 days of culture, indirect ELISA detection is carried out again, cell holes which are more than 2.1 times higher than the negative control A450 value and have good cell growth state are selected again, the cells are cloned to another 96-well plate, 1 XHAT selective culture medium is replaced for culture, and three to four rounds of culture are repeated until 100% of antibodies in the cloning holes are detected positive. Selecting cells with good growth state, culturing with common complete culture medium [20% (V/V) fetal bovine serum 1640 medium (purchased from Corning)]StepwiseAnd (5) performing expansion culture and freezing in liquid nitrogen.

In the subcloning culture screening process, ELISA plates are respectively coated with EV71, CA16, CA10 and CA6, hybridoma fine culture supernatants are taken for indirect ELISA after sealing, blank control is set, SP2/0 cell culture supernatant is used as negative control, goat anti-mouse IgG (1:6000) marked by HRP is used as secondary antibody, monoclonal cells capable of simultaneously identifying EV71 and CA16 are screened according to the readings of A450 values of the two ELISA plates, and further subcloning and amplification culture are carried out on hybridoma cell strains.

6) Hybridoma antibody type identification: the type of antibody was identified using Mouse Monoclonal Antibody Isotyping Kit (Roche), see instructions.

2. Human-mouse chimeric monoclonal antibody preparation

1) RNA extraction: by means of TRNzol-A ⁺ (TIANGEN) extraction of RNA from hybridoma cells selected in 1 and recognizing EV71 and CA16 at the same time, the number of cells was 10 ⁵ 。

2) Reverse transcription to obtain cDNA: the RNA obtained in 2.1 was reverse transcribed into cDNA using PrimeScript II 1st Strand cDNA Synthesis Kit, see kit (Takara, dalia).

3) Nested PCR amplification of antibody heavy chain variable region: the nested PCR amplification system was 54ul total:

I-5 ^TM 2x High-Fidelity Master Mix(Tsingke,China)27ul；

4ul of the cDNA obtained in step 2);

VH Forward (2 pM per primer concentration): each 1ul, 4 total, 4ul;

VH Reverse (2 pM per primer concentration): each 1ul, 19 total, 19ul.

The amplification procedure was: pre-denaturation at 98 ℃ for 1 min; denaturation at 98℃for 10 sec, renaturation at 65℃for 10 sec, extension at 72℃for 10 sec, and cycle 35 times; extension was carried out at 72℃for 5 minutes. The results are shown in FIG. 1.

4) Nested PCR amplification of antibody light chain variable region: nested PCR amplification system 50ul:

I-5 ^TM 2x High-Fidelity Master Mix(Tsingke,China)25ul；

4ul of the cDNA obtained in step 2);

VL (κ) Forward (2 pM per primer concentration): each 1ul, 4 total, 4ul;

VL (kappa) Reverse (2 pM per primer concentration): each 1ul, 17 total, 17ul.

The amplification method comprises the following steps: pre-denaturation at 98 ℃ for 1 min; denaturation at 98℃for 10 seconds, renaturation at 60℃for 10 seconds, extension at 72℃for 10 seconds, and cycle 35 times; extension was carried out at 72℃for 5 minutes. The results are shown in FIG. 2.

5) Identification of nested PCR products by agarose gel electrophoresis: sample loading: marker (DL 2000/DL 1000), VH, VL; 1% agarose gel, 100mA,20min was used. The results are shown in FIGS. 1 and 2.

6) Antibody variable region sequencing and sequence analysis: the gel recovered product of step 5) was sent to Tsingke for sequencing, and the sequencing result was compared with the analysis sequence by IgBLAST. The results show that: the amino acid sequence of heavy chain complementarity determining region 3 (complementarity determining region, CDR 3) of the 20-IgM antibody is: ala Arg Gly Gly Asn Tyr Tyr Tyr Ala Met Asp Tyr. The remaining sequence results are shown in FIG. 3.

7) And (3) connecting a carrier: step 6) identifying the subtype of the strain antibody as IgM type, wherein the carrier is PVITRO 1-hygro-mcs-IgM/kappa; the vector is synthesized by Tsingke company, the specific sequence is shown in SEQ ID NO.7, and the map is shown in FIG. 11. Meanwhile, connecting an antibody variable region to PVITRO 1-hygro-mcsIgG/kappa, and constructing an IgG type monoclonal antibody thereof; the vector is synthesized by Tsingke company, the specific sequence is shown in SEQ ID NO.8 and the map is shown in FIG. 12. DNA Ligation Kit Ver 2.1.1 (Takara, dalia) ligation was used. The connection system is as follows: solution I10 ul, PVITRO 1-hygro-mcs-IgM/kappa (figure 11) or PVITRO1-hygro-mcs IgG/kappa (figure 12) and VH/VL are connected according to the mol ratio of 3:1; firstly, connecting VH and PVITRO 1-hygro-mcs-IgM/kappa according to the mol ratio of 3:1, and then adding VL, wherein the mol ratio of VL to PVITRO 1-hygro-mcs-IgM/kappa is 3:1, so that a first connecting product is obtained; similarly, after the VH and PVITRO 1-hygro-mcsIgG/kappa are connected according to the mol ratio of 3:1, VL is added, and the mol ratio of the VL to PVITRO 1-hygro-mcsIgG/kappa is 3:1, so that a second connection product is obtained;

the connection method comprises the following steps: 16 ℃ for 30min.

The ligation product was transformed by addition of E.coli DH 5. Alpha: ice-bath for 1h at 42 ℃ for 90s for 2min; 100ul of antibiotic-free LB [ Tryptone (Tryptone) was added: 10g,Yeast Extract (yeast extract): 5g, naCl (sodium chloride): 10g, adding water to prepare 1L, and shake culturing for 1h at 180rpm; and (5) coating a plate.

8) Positive monoclonal antibody identification: after single colony amplification culture, the sequence was sequenced by Tsingke, and the sequencing result was analyzed by IgBLAST alignment.

9) Antibody expression: after successful construction of the antibody plasmid, it was transfected into FreeStyle ^TM CHO-S cells, freeStyle as transfection reagent ^TM MAX Reagent (Thermo Scientific, USA). The transfection method is shown in the specification.

10 Antibody purification: cell culture supernatants were purified using a protein G affinity pre-cartridge following transfection.

TABLE 1 PCR primers

3. Antibody characterization

1) Molecular weight and purity identification: non-reducing and reducing 10% SDS-PAGE gel electrophoresis was used for identification. Reduction state: the sample and 5 XSDS-PAGE protein loading buffer [250mM Tris-HCl (pH 6.8), 10% (W/V) SDS,0.5% (W/V) BPB,50% (V/V) glycerol, 5% (W/V) beta-mercaptoethanol ] were mixed in a volume ratio of 4:1, and after the sample was added, the final concentration of the buffer was 1×, and heated at 95℃for 10 minutes to sufficiently denature the protein. Non-reduction state: samples were mixed with 5 XSDS-PAGE protein loading buffer [250mM Tris-HCl (pH 6.8) 0.5% (W/V) BPB,50% (V/V) glycerol ] at a ratio of 4:1 by volume, and the final buffer concentration was 1X after addition of the samples. And (3) cooling to room temperature, and directly loading the protein sample into an SDS-PAGE gel loading hole. 80V for 30min, 150V for 50 min. The results show that: in the unreduced state: the molecular weight of 20-IgM is about 850kDa, and the molecular weight of 20-IgG is 150kDa; in the reduced state: 20-IgG sees a heavy chain of 55kDa and a light chain of 25 kDa; 20-IgM can see a heavy chain of about 80kDa and a light chain of 25 kDa. See fig. 4.

2) Identification of binding characteristics: ELISA method. The ELISA plate is coated with EV71 and CA16 respectively, the purified antibody is used as a primary antibody, the HRP-labeled antibody is used as a secondary antibody (donkey anti-human IgG), and after TMB color development is terminated, the ELISA plate detects the absorbance value (A450 value) at 450 nm. The results show that: both 20-IgM and 20-IgG recognize EV71, CA16, CA10 and CA6 simultaneously; see fig. 5.

3) Affinity identification: microphoresis (microscale thermophoresis, MST). EV71, CA16, CA10 and CA6 are respectively marked by Monolith NT Protein Labeling Kit RED, and the marking method is shown in the specification. TBST [20mM Tris (pH 7.5), 50mM NaCl,5mM beta-mercaptoethanol, 0.05% Tween-20and1% DMSO was used]Multiple dilution antibody (dilution multiple of 2 ¹ ～2 ¹⁶ Total of 16 concentrations), 10ul of diluted antibody and 10ul of labeled virus solution were mixed, and standard capillaries (Monolith TM standard-treated capillaries) for MST were added, and the equilibrium dissociation constant Kd of the antibody was calculated by affinity binding analysis. The results show that: 20-IgM has strong binding capacity to EV71, CA16, CA10 and CA6, and affinity constants (Kd) are respectively: 0.06nM, 0.12nM, 3.12nM, 0.25nM. Affinity constants (Kd) of 20-IgG with EV71, CA16, CA10 and CA6 were: 0.6nM, 1.2nM, 3.12nM, 2.5nM. The affinity of 20-IgM to the corresponding virus was significantly higher than that of 20-IgG (10-fold). See fig. 6.

4) Identification of antibody neutralizing epitopes:

a) Identification of antibody-recognizing virus epitopes: after SDS-PAGE electrophoresis was performed with EV71, CA16, CA10 and CA6 whole viruses, western Blot analysis was performed by transferring PVDF membrane. The proteins after electrophoresis were transferred to PVDF membrane (purchased from Millipore) using a semi-dry transfer device (BIO-RAD) with a transfer current of 100mA and a transfer time of 45 minutes according to the instructions of the transfer apparatus. Immediately after the completion of the transfer, the PVDF membrane after the completion of the transfer was placed in 5% (W/V) of skimmed milk powder TBS [20mM Tris-HCl,500mM NaCl (pH 7.5) ] prepared in advance, slowly shaken on a shaker, and blocked at room temperature for 60 minutes. The purified monoclonal antibody was diluted 1:1000 with TBST wash solution [20mM Tris-HCl,500mM NaCl (pH 7.5) 0.01% Tween-20 (V/V) ] and added to the PVDF membrane after completion of blocking for overnight incubation. An anti-dilution solution is discarded. 5ml of TBST membrane washing solution was added. Shaking on a side shaking table was repeated 5 times. Incubation was then performed with HRP-labeled goat anti-human IgG as secondary antibody (1:10000 dilution with TBST) for 1h at room temperature. Discarding the secondary anti-dilution solution, repeating TBST film washing for 5 times, adding ECL (purchased from Millipore) reagent, tabletting in a darkroom, performing film washing or machine exposure through a developer fixing solution, and detecting the identification effect of the monoclonal antibody. The results show that: 20-IgM specifically recognized VP1 capsid proteins of EV71, CA16, CA10 and CA6 (FIGS. 7A-D).

Identification of antibody recognition amino acid sites: the identification results show that antibodies specifically recognize VP1 proteins of EV71, CA16, CA10 and CA6, and polypeptide sequences of VP1 are synthesized for further determining key amino acid sequences recognized by the antibodies: each peptide fragment consisted of 15 amino acids, as in table 2, synthesized by gill biochemistry (shanghai) limited. Identification by ELISA method: the ELISA plate is coated with different peptide fragments for identification, and the results show that: 20-IgM specifically recognizes the peptide fragment of VP1 protein (Phe Gly Glu His Lys Gln Glu Lys Asp. Leu Glu Tyr Gly Ala Cys, FGEHKQEKDLEYGAC). See fig. 7E-7G. And further confirming the binding specificity of 20-IgM and FGEHKQEKDLEYGAC peptide fragments by adopting an antigen peptide inhibition experiment: after incubation of antigen peptide fragments (15-For, 15-Back) with 20-IgM at 37℃for 1 hour, a trace neutralization experiment was added, and the results showed that: peptide No. 15, duan Te, affects the neutralizing potency of 20-IgM, see FIGS. 7H-7I. The above results confirm that: 20-IgM specifically recognizes VP1 capsid protein FGEHKQEKDLEYGAC peptide fragments. 20-IgG showed the same result.

TABLE 2 antigenic peptide amino acid sequences

1	Gly Asp Arg Val Ala Asp Val Ile Glu Ser Ser Ile Gly Asp Ser
		2	Val Ser Arg Ala Leu Thr His Ala Leu Pro Ala Pro Thr Gly Gln
3	Asn Thr Gln Val Ser Ser His Arg Leu Asp Thr Gly Lys Val Pro
		4	Ala Leu Gln Ala Ala Glu Ile Gly Ala Ser Ser Asn Ala Ser Asp
5	Glu Ser Met Ile Glu Thr Arg Cys Val Leu Asn Ser His Ser Thr
		6	Ala Glu Thr Thr Leu Asp Ser Phe Phe Ser Arg Ala Gly Leu Val
7	Gly Glu Ile Asp Leu Pro Leu Glu Gly Thr Thr Asn Pro Asn Gly
		8	Tyr Ala Asn Trp Asp Ile Asp Ile Thr Gly Tyr Ala Gln Met Arg
9	Arg Lys Val Glu Leu Phe Thr Tyr Met Arg Phe Asp Ala Glu
		10	Thr Phe Val Ala Cys Thr Pro Thr Gly Glu Val Val Pro Gln Leu
11	Leu Gln Tyr Met Phe Val Pro Pro Gly Ala Pro Lys Pro Asp Ser
		12	Arg Glu Ser Leu Ala Trp Gln Thr Ala Thr Asn Pro Ser Val Phe
13	Val Lys Leu Ser Asp Pro Pro Ala Gln Val Ser Val Pro Phe Met
		14	Ser Pro Ala Ser Ala Tyr Gln Trp Phe Tyr Asp Gly Tyr Pro Thr
15	Phe Gly Glu His Lys Gln Glu Lys Asp.Leu Glu Tyr Gly Ala Cys
		16	.Ser Asn Asn Met Met Gly Thr Phe Ser Val Arg Thr Val Gly Thr
17	Ser Lys Ser Lys Tyr Pro Leu Val Val Arg Ile Tyr Met Arg Met
		18	Lys His Val Arg Ala Trp Ile Pro Arg Pro Met Arg Asn Gln Asn
19	Tyr Leu Phe Lys Ala Asn Pro Asn Tyr Ala Gly Asn Ser Ile Lys
		20	Pro Thr Gly Ala Ser Arg Thr Ala Ile Thr Thr Leu
15For	Asp Gly Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp.Leu
		15Back	Gln Glu Lys Asp.Leu Glu Tyr Gly Ala Cys Ser Asn Asn Met Met

b) Identification of key amino acid sites for antibody recognition: the identification result showed that the antibody specifically recognized peptide 15, and thus peptide 15 was subjected to amino acid site mutation, which was synthesized by Jier Biochemical (Shanghai) Co., ltd (Table 3). ELISA method is adopted for identification, and the ELISA plate is coated with the point mutation peptide fragment. The results show that: the specific recognition amino acid sites of 20-IgM are: DLEYG (219-223). The results are shown in FIG. 7J. Further this study shows by alignment of amino acid sequence homology of VP1 capsid proteins of EV71, CA16, CA10 and CA 6: the 20-IgM binding site was DLEYG (219-223) at EV71 capsid protein VP1 (FIG. 7K); CA16 is DLDYG (219-223) (FIG. 7L); CA10 is NTTYG (218-222) (FIG. 7N); CA6 is NLQYG (214-218) (FIG. 7M). 20-IgG showed the same result.

TABLE 3 antigenic peptide amino acid sequences

5) Neutralization potency identification: micro neutralization experiments. 50ul 2000TCID50/ml of virus (EV 71/CA16/CA10/CA6, 4 experiments, each group EV71, CA16, CA10, CA 6) was added to each well in 96-well plates and diluted in a gradient with 50ul (dilution ratio of 2) ¹ ～2 ⁸ 8 concentrations of antibody were incubated at 37℃for 1h, RD cells (15000) were added, 37℃and 5% CO ₂ Culturing for 3 days, and measuring cell viability by MTT method. At the same time, cytopathic effects were observed daily. The results show that: the 1nM 20-IgM antibody can inhibit EV71/CA16/CA10/CA6 infected cells developed lesions. In contrast, 1nM 20-IgG did not completely inhibit cytopathy. See fig. 8.

6) MTT method: after 3 days of RD cell culture, 20ul of thiazole blue (MTT) solution (5 mg/ml) was added to each well, incubation was continued for 4h, and the culture was terminated, taking care to discard the culture supernatant in the wells. 150ul DMSO was added to each well, and after 10 minutes of shaking, the absorbance values of each well were measured on an ELISA monitor at 490nm wavelength. Growth curves were plotted on Graphpad and half-effective inhibitory concentrations of antibodies were calculated (half maximal inhibitory concentration, IC 50). The results show that: EV 71-infected group, 20-IgM with IC50 of 0.11nM and IC80 of 0.35nM; the IC50 of 20-IgG was 1.31nM and the IC80 4.3nM. CA16 infected group, 20-IgM IC50 of 0.45nM and IC80 of 1.03nM; the IC50 of 20-IgG was 6.47nM and the IC80 was 14.14nM. CA10 infected group, 20-IgM IC50 of 1.47nM and IC80 of 3.33nM; the IC50 of 20-IgG was 10.85nM and the IC80 was 22.64nM; CA6 infected group, 20-IgM IC50 of 0.55nM and IC80 of 1.39nM; the IC50 of 20-IgG was 4.97nM and the IC80 11.37nM. See fig. 9.

7) Neutralization potency of antibodies against clinical isolates identification of: a trace neutralization experiment identifies the binding efficacy of the antibody; ELISA identified the binding efficacy of the antibodies. The results show that: 20-IgM has higher neutralizing effect and binding effect on the separated 5 EV71 strain and 5 CA16 clinical virus strain. Correspondingly, 20-IgG also has higher neutralizing and binding efficacy for both isolated 5 EV71 and 5 CA16 clinical strains, but both binding and neutralizing efficacy are lower than 20-IgM. See table 4.

Table 4 neutralizing efficacy of antibodies against clinical isolates 8) animal protection efficacy identification:

i. protective effects of antibodies in animals were evaluated using 1 day old rats as an infection model. Day 1 aged milk mice infection model: injection of cranial cavity 30ul 10 ⁷ TCID50/ml virus (EV 71 or CA 16) infected 1 day old rats (15 per group) and the survival rate of the mice as well as the weight change and clinical symptoms of the mice were recorded daily. The results show that: feel of the senseThe infected mice have clinical symptoms such as weight loss, quadriplegia and the like until death, and the suckling mice die basically all 10 days after infection. See arrows in fig. 10A.

Antibody protection effect evaluation: after 1 day of virus infection of the mice, the mice were given daily record of survival rates and weight changes and clinical symptoms by intraperitoneal injection of antibody (5 mg/kg). The results show that: clinical symptoms of mice in the 20-IgM treated group were reduced and weight gain was restored towards the control group (PBS) (fig. 10C and 10E); survival rate statistics show that: the protection rate of the EV71/CA16 infected mice by 5mg/kg of 20-IgM reached 100% (FIGS. 10B and 10D).

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> institute of medical biology at the national academy of medical science

<120> broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibody and application

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 123

<212> PRT

<213> Artificial sequence ()

<400> 1

Ile Pro Gly Ser Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro

1 5 10 15

Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr

20 25 30

Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu

35 40 45

Trp Ile Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln

50 55 60

Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr

65 70 75 80

Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Gly Gly Asn Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser

115 120

<210> 2

<211> 98

<212> PRT

<213> Artificial sequence ()

<400> 2

Glu Phe Asp His Cys Ser Gln Ser Ser Gln Ser Leu Leu Asn Ser Arg

1 5 10 15

Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser

20 25 30

Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro

35 40 45

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

50 55 60

Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln Ser

65 70 75 80

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

85 90 95

Gln Leu

<210> 3

<211> 359

<212> DNA

<213> Artificial sequence ()

<400> 3

caggttccaa gctgcagcag tctggacctg agctggtgaa gcctggggct tcagtgaaga 60

tatcctgcaa gacttctgga tacacattca ctgaatacac catgcactgg gtgaagcaga 120

gccatggaaa gagccttgag tggattggag gtattaatcc taacaatggt ggtactagct 180

acaaccagaa gttcaagggc aaggccacat tgactgtaga caagtcctcc agcacagcct 240

acatggagct ccgcagcctg acatctgagg attctgcagt ctattactgt gcaagaggtg 300

gtaactacta ctatgctatg gactactggg gtcaaggaac ctcagtcacc gtctcctcg 359

<210> 4

<211> 282

<212> DNA

<213> Artificial sequence ()

<400> 4

gatcattgtt ctcaatccag tcagagtctg ctcaacagta gaacccgaaa gaactacttg 60

gcttggtacc agcagaaacc agggcagtct cctaaactgc tgatctactg ggcatccact 120

agggaatctg gggtccctga tcgcttcaca ggcagtggat ctgggacaga tttcactctc 180

accatcagca gtgtgcaggc tgaagacctg gcagtttatt actgcaagca atcttataat 240

cttcggacgt tcggtggagg caccaagctg gaaataaaac gt 282

<210> 5

<211> 1362

<212> DNA

<213> Artificial sequence ()

<400> 5

ggatctgcat ccgctccaac cctgttcccc cttgtgagct gcgaaaattc tccatccgac 60

acaagcagcg tcgcggtcgg ctgcctggct caagactttc tccccgacag catcactttc 120

agttggaagt acaagaacaa cagtgacatt tctagtacaa gaggatttcc gagtgttctg 180

agaggcggga agtacgctgc tacatcccag gtgctgctgc cgtcaaaaga cgttatgcag 240

ggtaccgatg agcacgtagt gtgtaaggtg caacacccaa acggcaacaa agagaagaat 300

gtccccctgc cagtgattgc tgaactgccc cctaaagtct ccgtctttgt accccccaga 360

gacggattct tcggcaaccc acgaaagtcc aagctcatct gccaggccac gggattctct 420

cctcgccaga tccaggtgtc ttggttgagg gaggggaaac aggttggctc tggcgttacc 480

accgatcagg ttcaggccga ggccaaagag tccgggccaa ctacctataa ggttacaagt 540

accctgacta taaaagaaag tgactggctg tctcaatcca tgtttacatg cagagtcgat 600

catcgaggcc tgacgttcca acagaatgcc agttccatgt gcgtgccaga tcaagatacc 660

gctataagag tgttcgcaat ccctccttca ttcgcttcca tattccttac taagagcacc 720

aagctgacat gtcttgtgac tgatttgacc acttacgaca gcgttacaat cagctggacc 780

cgccagaatg gcgaagctgt gaaaacccac acaaacatct ccgagtctca ccccaacgct 840

acatttagcg ccgtcggcga ggcatccatc tgcgaggacg attggaacag tggggagcgg 900

ttcacgtgca ctgttacgca tacagacctg ccgtctcctc tcaaacaaac tatcagccga 960

cctaaaggag tggcactgca ccggccagac gtttacctgc tgccccctgc ccgggaacag 1020

ctgaacctgc gggaatccgc cacaattaca tgcctggtga ctggatttag cccagccgac 1080

gtgttcgtgc agtggatgca gagggggcag cccctctccc cggaaaaata cgtaacatcc 1140

gctcccatgc ctgagcctca ggcccctgga cggtatttcg cccatagtat tctgactgtg 1200

tctgaagagg aatggaatac aggcgaaaca tatacttgcg tggttgctca tgaggccctg 1260

cctaaccgag tcacagagag aacagtggat aaatctacag gcaagccgac cctctataat 1320

gtgtctctcg tgatgtcaga tacggctggc acttgctact ga 1362

<210> 6

<211> 323

<212> DNA

<213> Artificial sequence ()

<400> 6

gtacggtggc ggcgccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg 60

gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt 120

ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca 180

gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga 240

aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga 300

gcttcaacag gggagagtgt tag 323

<210> 7

<211> 8285

<212> DNA

<213> Artificial sequence ()

<400> 7

cctgcagggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga 60

aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca 120

ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca 180

ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc 240

ccactagtgg agccgagagt aattcataca aaaggaggga tcgccttcgc aaggggagag 300

cccagggacc gtccctaaat tctcacagac ccaaatccct gtagccgccc cacgacagcg 360

cgaggagcat gcgctcaggg ctgagcgcgg ggagagcaga gcacacaagc tcatagaccc 420

tggtcgtggg ggggaggacc ggggagctgg cgcggggcaa actgggaaag cggtgtcgtg 480

tgctggctcc gccctcttcc cgagggtggg ggagaacggt atataagtgc ggcagtcgcc 540

ttggacgttc tttttcgcaa cgggtttgcc gtcagaacgc aggtgagggg cgggtgtggc 600

ttccgcgggc cgccgagctg gaggtcctgc tccgagcggg ccgggccccg ctgtcgtcgg 660

cggggattag ctgcgagcat tcccgcttcg agttgcgggc ggcgcgggag gcagagtgcg 720

aggcctagcg gcaaccccgt agcctcgcct cgtgtccggc ttgaggccta gcgtggtgtc 780

cgcgccgccg ccgcgtgcta ctccggccgc actctggtct tttttttttt tgttgttgtt 840

gccctgctgc cttcgattgc cgttcagcaa taggggctaa caaagggagg gtgcggggct 900

tgctcgcccg gagcccggag aggtcatggt tggggaggaa tggagggaca ggagtggcgg 960

ctggggcccg cccgccttcg gagcacatgt ccgacgccac ctggatgggg cgaggcctgg 1020

ggtttttccc gaagcaacca ggctggggtt agcgtgccga ggccatgtgg ccccagcacc 1080

cggcacgatc tggcttggcg gcgccgcgtt gccctgcctc cctaactagg gtgaggccat 1140

cccgtccggc accagttgcg tgcgtggaaa gatggccgct cccgggccct gttgcaagga 1200

gctcaaaatg gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc cacacaaagg 1260

aagagggcct ggtccctcac cggctgctgc ttcctgtgac cccgtggtcc tatcggccgc 1320

aatagtcacc tcgggctttt gagcacggct agtcgcggcg gggggagggg atgtaatggc 1380

gttggagttt gttcacattt ggtgggtgga gactagtcag gccagcctgg cgctggaagt 1440

catttttgga atttgtcccc ttgagttttg agcggagcta attctcgggc ttcttagcgg 1500

ttcaaaggta tcttttaaac ccttttttag gtgttgtgaa aaccaccgct aattcaaagc 1560

aaccggtatg gactggacct ggaggatcct cttcttggtg gcagcagcca caggagccca 1620

ctccctcgag tgcgcacgct ccaaccctgt tcccccttgt gagctgcgaa aattctccat 1680

ccgacacaag cagcgtcgcg gtcggctgcc tggctcaaga ctttctcccc gacagcatca 1740

ctttcagttg gaagtacaag aacaacagtg acatttctag tacaagagga tttccgagtg 1800

ttctgagagg cgggaagtac gctgctacat cccaggtgct gctgccgtca aaagacgtta 1860

tgcagggtac cgatgagcac gtagtgtgta aggtgcaaca cccaaacggc aacaaagaga 1920

agaatgtccc cctgccagtg attgctgaac tgccccctaa agtctccgtc tttgtacccc 1980

ccagagacgg attcttcggc aacccacgaa agtccaagct catctgccag gccacgggat 2040

tctctcctcg ccagatccag gtgtcttggt tgagggaggg gaaacaggtt ggctctggcg 2100

ttaccaccga tcaggttcag gccgaggcca aagagtccgg gccaactacc tataaggtta 2160

caagtaccct gactataaaa gaaagtgact ggctgtctca atccatgttt acatgcagag 2220

tcgatcatcg aggcctgacg ttccaacaga atgccagttc catgtgcgtg ccagatcaag 2280

ataccgctat aagagtgttc gcaatccctc cttcattcgc ttccatattc cttactaaga 2340

gcaccaagct gacatgtctt gtgactgatt tgaccactta cgacagcgtt acaatcagct 2400

ggacccgcca gaatggcgaa gctgtgaaaa cccacacaaa catctccgag tctcacccca 2460

acgctacatt tagcgccgtc ggcgaggcat ccatctgcga ggacgattgg aacagtgggg 2520

agcggttcac gtgcactgtt acgcatacag acctgccgtc tcctctcaaa caaactatca 2580

gccgacctaa aggagtggca ctgcaccggc cagacgttta cctgctgccc cctgcccggg 2640

aacagctgaa cctgcgggaa tccgccacaa ttacatgcct ggtgactgga tttagcccag 2700

ccgacgtgtt cgtgcagtgg atgcagaggg ggcagcccct ctccccggaa aaatacgtaa 2760

catccgctcc catgcctgag cctcaggccc ctggacggta tttcgcccat agtattctga 2820

ctgtgtctga agaggaatgg aatacaggcg aaacatatac ttgcgtggtt gctcatgagg 2880

ccctgcctaa ccgagtcaca gagagaacag tggataaatc tacaggcaag ccgaccctct 2940

ataatgtgtc tctcgtgatg tcagatacgg ctggcacttg ctactgatgt acagctagct 3000

ggccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa 3060

aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 3120

gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg 3180

tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggaa atgttaatta 3240

actagccatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 3300

agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 3360

aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 3420

ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 3480

gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 3540

aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 3600

aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 3660

gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 3720

aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 3780

aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 3840

cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 3900

cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 3960

tgctcacatg ttcttaatta acctgcaggc gttacataac ttacggtaaa tggcccgcct 4020

ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 4080

acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 4140

ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 4200

aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 4260

tacatctacg tattagtcat cgctattacc atgatgatgc ggttttggca gtacatcaat 4320

gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 4380

gggagtttgt tttgactagt ggagccgaga gtaattcata caaaaggagg gatcgccttc 4440

gcaaggggag agcccaggga ccgtccctaa attctcacag acccaaatcc ctgtagccgc 4500

cccacgacag cgcgaggagc atgcgcccag ggctgagcgc gggtagatca gagcacacaa 4560

gctcacagtc cccggcggtg gggggagggg cgcgctgagc gggggccagg gagctggcgc 4620

ggggcaaact gggaaagtgg tgtcgtgtgc tggctccgcc ctcttcccga gggtggggga 4680

gaacggtata taagtgcggt agtcgccttg gacgttcttt ttcgcaacgg gtttgccgtc 4740

agaacgcagg tgagtggcgg gtgtggcttc cgcgggcccc ggagctggag ccctgctctg 4800

agcgggccgg gctgatatgc gagtgtcgtc cgcagggttt agctgtgagc attcccactt 4860

cgagtggcgg gcggtgcggg ggtgagagtg cgaggcctag cggcaacccc gtagcctcgc 4920

ctcgtgtccg gcttgaggcc tagcgtggtg tccgccgccg cgtgccactc cggccgcact 4980

atgcgttttt tgtccttgct gccctcgatt gccttccagc agcatgggct aacaaaggga 5040

gggtgtgggg ctcactctta aggagcccat gaagcttacg ttggatagga atggaagggc 5100

aggaggggcg actggggccc gcccgccttc ggagcacatg tccgacgcca cctggatggg 5160

gcgaggcctg tggctttccg aagcaatcgg gcgtgagttt agcctacctg ggccatgtgg 5220

ccctagcact gggcacggtc tggcctggcg gtgccgcgtt cccttgcctc ccaacaaggg 5280

tgaggccgtc ccgcccggca ccagttgctt gcgcggaaag atggccgctc ccggggccct 5340

gttgcaagga gctcaaaatg gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc 5400

cacacaaagg aagagggcct tgcccctcgc cggccgctgc ttcctgtgac cccgtggtct 5460

atcggccgca tagtcacctc gggcttctct tgagcaccgc tcgtcgcggc ggggggaggg 5520

gatctaatgg cgttggagtt tgttcacatt tggtgggtgg agactagtca ggccagcctg 5580

gcgctggaag tcattcttgg aatttgcccc tttgagtttg gagcgaggct aattctcaag 5640

cctcttagcg gttcaaaggt attttctaaa cccgtttcca ggtgttgtga aagccaccgc 5700

taattcaaag caatccggaa tgttgccatc acaactcatt gggtttctgc tgctctgggt 5760

tccagctagc cgcggtgaat tcgtttaaac gtacggtggc ggcgccatct gtcttcatct 5820

tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc ctgctgaata 5880

acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc caatcgggta 5940

actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc ctcagcagca 6000

ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc gaagtcaccc 6060

atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt tagggatccc 6120

gtacgcctag gagcaggttt ccccaatgac acaaaacgtg caacttgaaa ctccgcctgg 6180

tctttccagg tctagagggg taacactttg tactgcgttt ggctccacgc tcgatccact 6240

ggcgagtgtt agtaacagca ctgttgcttc gtagcggagc atgacggccg tgggaactcc 6300

tccttggtaa caaggaccca cggggccaaa agccacgccc acacgggccc gtcatgtgtg 6360

caaccccagc acggcgactt tactgcgaaa cccactttaa agtgacattg aaactggtac 6420

ccacacactg gtgacaggct aaggatgccc ttcaggtacc ccgaggtaac acgcgacact 6480

cgggatctga gaaggggact ggggcttcta taaaagcgct cggtttaaaa agcttctatg 6540

cctgaatagg tgaccggagg tcggcacctt tcctttgcaa ttactgaccc tatgaataca 6600

ctgactgttt gacaattaat catcggcata gtatatcggc atagtataat acgactcact 6660

ataggagggc caccatgaag aaacctgaac tgacagcaac ttctgttgag aagtttctca 6720

ttgaaaaatt tgattctgtt tctgatctca tgcagctgtc tgaaggtgaa gaaagcagag 6780

ccttttcttt tgatgttgga ggaagaggtt atgttctgag ggtcaattct tgtgctgatg 6840

gtttttacaa agacagatat gtttacagac actttgcctc tgctgctctg ccaattccag 6900

aagttctgga cattggagaa ttttctgaat ctctcaccta ctgcatcagc agaagagcac 6960

aaggagtcac tctccaggat ctccctgaaa ctgagctgcc agctgttctg caacctgttg 7020

ctgaagcaat ggatgccatt gcagcagctg atctgagcca aacctctgga tttggtcctt 7080

ttggtcccca aggcattggt cagtacacca cttggaggga tttcatttgt gccattgctg 7140

atcctcatgt ctatcactgg cagactgtga tggatgacac agtttctgct tctgttgctc 7200

aggcactgga tgaactcatg ctgtgggcag aagattgtcc tgaagtcaga cacctggtcc 7260

atgctgattt tggaagcaac aatgttctga cagacaatgg cagaatcact gcagtcattg 7320

actggtctga agccatgttt ggagattctc aatatgaggt tgccaacatt tttttttgga 7380

gaccttggct ggcttgcatg gaacaacaaa caagatattt tgaaagaaga cacccagaac 7440

tggctggttc ccccagactg agagcctaca tgctcagaat tggcctggac caactgtatc 7500

aatctctggt tgatggaaac tttgatgatg ctgcttgggc acaaggaaga tgtgatgcca 7560

ttgtgaggtc tggtgctgga actgttggaa gaactcaaat tgcaagaagg tctgctgctg 7620

tttggactga tggatgtgtt gaagttctgg ctgactctgg aaacaggaga ccctccacaa 7680

gacccagagc caaggaatga atattagcta gattatccct aatacctgcc accccactct 7740

taatcagtgg tggaagaacg gtctcagaac tgtttgtttc aattggccat ttaagtttag 7800

tagtaaaaga ctggttaatg ataacaatgc atcgtaaaac cttcagaagg aaaggagaat 7860

gttttgtgga ccactttggt tttctttttt gcgtgtggca gttttaagtt attagttttt 7920

aaaatcagta ctttttaatg gaaacaactt gaccaaaaat ttgtcacaga attttgagac 7980

ccattaaaaa agttaaatga gaaacctgtg tgttcctttg gtcaacaccg agacatttag 8040

gtgaaagaca tctaattctg gttttacgaa tctggaaact tcttgaaaat gtaattcttg 8100

agttaacact tctgggtgga gaatagggtt gttttccccc cacataattg gaaggggaag 8160

gaatatcatt taaagctatg ggagggttgc tttgattaca acactggaga gaaatgcagc 8220

atgttgctga ttgcctgtca ctaaaacagg ccaaaaactg agtccttggg ttgcatagaa 8280

agctg 8285

<210> 8

<211> 7930

<212> DNA

<213> Artificial sequence ()

<400> 8

cctgcagggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga 60

aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca 120

ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca 180

ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc 240

ccactagtgg agccgagagt aattcataca aaaggaggga tcgccttcgc aaggggagag 300

cccagggacc gtccctaaat tctcacagac ccaaatccct gtagccgccc cacgacagcg 360

cgaggagcat gcgctcaggg ctgagcgcgg ggagagcaga gcacacaagc tcatagaccc 420

tggtcgtggg ggggaggacc ggggagctgg cgcggggcaa actgggaaag cggtgtcgtg 480

tgctggctcc gccctcttcc cgagggtggg ggagaacggt atataagtgc ggcagtcgcc 540

ttggacgttc tttttcgcaa cgggtttgcc gtcagaacgc aggtgagggg cgggtgtggc 600

ttccgcgggc cgccgagctg gaggtcctgc tccgagcggg ccgggccccg ctgtcgtcgg 660

cggggattag ctgcgagcat tcccgcttcg agttgcgggc ggcgcgggag gcagagtgcg 720

aggcctagcg gcaaccccgt agcctcgcct cgtgtccggc ttgaggccta gcgtggtgtc 780

cgcgccgccg ccgcgtgcta ctccggccgc actctggtct tttttttttt tgttgttgtt 840

gccctgctgc cttcgattgc cgttcagcaa taggggctaa caaagggagg gtgcggggct 900

tgctcgcccg gagcccggag aggtcatggt tggggaggaa tggagggaca ggagtggcgg 960

ctggggcccg cccgccttcg gagcacatgt ccgacgccac ctggatgggg cgaggcctgg 1020

ggtttttccc gaagcaacca ggctggggtt agcgtgccga ggccatgtgg ccccagcacc 1080

cggcacgatc tggcttggcg gcgccgcgtt gccctgcctc cctaactagg gtgaggccat 1140

cccgtccggc accagttgcg tgcgtggaaa gatggccgct cccgggccct gttgcaagga 1200

gctcaaaatg gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc cacacaaagg 1260

aagagggcct ggtccctcac cggctgctgc ttcctgtgac cccgtggtcc tatcggccgc 1320

aatagtcacc tcgggctttt gagcacggct agtcgcggcg gggggagggg atgtaatggc 1380

gttggagttt gttcacattt ggtgggtgga gactagtcag gccagcctgg cgctggaagt 1440

catttttgga atttgtcccc ttgagttttg agcggagcta attctcgggc ttcttagcgg 1500

ttcaaaggta tcttttaaac ccttttttag gtgttgtgaa aaccaccgct aattcaaagc 1560

aaccggtatg gactggacct ggaggatcct cttcttggtg gcagcagcca caggagccca 1620

ctccctcgag tgcgcacccg ctagcaccaa gggcccatcg gtcttccccc tggcaccctc 1680

ctccaagagc acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc 1740

cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc 1800

ggctgtccta cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag 1860

cagcttgggc acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt 1920

ggacaagaaa gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc 1980

acctgaactc ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct 2040

catgatctcc cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc 2100

tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc 2160

gcgggaggag cagtacaaca gcacgtaccg ggtggtcagc gtcctcaccg tcctgcacca 2220

ggactggctg aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc 2280

catcgagaaa accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct 2340

gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg 2400

cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta 2460

caagaccacg cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac 2520

cgtggacaag agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc 2580

tctgcacaac cactacacgc agaagagcct ctccctgtct ccgggtaaat gatgtacagc 2640

tagctggcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 2700

gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 2760

aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg 2820

ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggaaatgtt 2880

aattaactag ccatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 2940

cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 3000

ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 3060

actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta 3120

gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 3180

ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 3240

gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 3300

acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 3360

tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 3420

gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 3480

cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 3540

cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 3600

ccttttgctc acatgttctt aattaacctg caggcgttac ataacttacg gtaaatggcc 3660

cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 3720

tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 3780

cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 3840

acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 3900

ggcagtacat ctacgtatta gtcatcgcta ttaccatgat gatgcggttt tggcagtaca 3960

tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 4020

tcaatgggag tttgttttga ctagtggagc cgagagtaat tcatacaaaa ggagggatcg 4080

ccttcgcaag gggagagccc agggaccgtc cctaaattct cacagaccca aatccctgta 4140

gccgccccac gacagcgcga ggagcatgcg cccagggctg agcgcgggta gatcagagca 4200

cacaagctca cagtccccgg cggtgggggg aggggcgcgc tgagcggggg ccagggagct 4260

ggcgcggggc aaactgggaa agtggtgtcg tgtgctggct ccgccctctt cccgagggtg 4320

ggggagaacg gtatataagt gcggtagtcg ccttggacgt tctttttcgc aacgggtttg 4380

ccgtcagaac gcaggtgagt ggcgggtgtg gcttccgcgg gccccggagc tggagccctg 4440

ctctgagcgg gccgggctga tatgcgagtg tcgtccgcag ggtttagctg tgagcattcc 4500

cacttcgagt ggcgggcggt gcgggggtga gagtgcgagg cctagcggca accccgtagc 4560

ctcgcctcgt gtccggcttg aggcctagcg tggtgtccgc cgccgcgtgc cactccggcc 4620

gcactatgcg ttttttgtcc ttgctgccct cgattgcctt ccagcagcat gggctaacaa 4680

agggagggtg tggggctcac tcttaaggag cccatgaagc ttacgttgga taggaatgga 4740

agggcaggag gggcgactgg ggcccgcccg ccttcggagc acatgtccga cgccacctgg 4800

atggggcgag gcctgtggct ttccgaagca atcgggcgtg agtttagcct acctgggcca 4860

tgtggcccta gcactgggca cggtctggcc tggcggtgcc gcgttccctt gcctcccaac 4920

aagggtgagg ccgtcccgcc cggcaccagt tgcttgcgcg gaaagatggc cgctcccggg 4980

gccctgttgc aaggagctca aaatggagga cgcggcagcc cggtggagcg ggcgggtgag 5040

tcacccacac aaaggaagag ggccttgccc ctcgccggcc gctgcttcct gtgaccccgt 5100

ggtctatcgg ccgcatagtc acctcgggct tctcttgagc accgctcgtc gcggcggggg 5160

gaggggatct aatggcgttg gagtttgttc acatttggtg ggtggagact agtcaggcca 5220

gcctggcgct ggaagtcatt cttggaattt gcccctttga gtttggagcg aggctaattc 5280

tcaagcctct tagcggttca aaggtatttt ctaaacccgt ttccaggtgt tgtgaaagcc 5340

accgctaatt caaagcaatc cggaatgttg ccatcacaac tcattgggtt tctgctgctc 5400

tgggttccag ctagccgcgg tgaattcgtt taaacgtacg gtggcggcgc catctgtctt 5460

catcttcccg ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct 5520

gaataacttc tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc 5580

gggtaactcc caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag 5640

cagcaccctg acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt 5700

cacccatcag ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttaggg 5760

atcccgtacg cctaggagca ggtttcccca atgacacaaa acgtgcaact tgaaactccg 5820

cctggtcttt ccaggtctag aggggtaaca ctttgtactg cgtttggctc cacgctcgat 5880

ccactggcga gtgttagtaa cagcactgtt gcttcgtagc ggagcatgac ggccgtggga 5940

actcctcctt ggtaacaagg acccacgggg ccaaaagcca cgcccacacg ggcccgtcat 6000

gtgtgcaacc ccagcacggc gactttactg cgaaacccac tttaaagtga cattgaaact 6060

ggtacccaca cactggtgac aggctaagga tgcccttcag gtaccccgag gtaacacgcg 6120

acactcggga tctgagaagg ggactggggc ttctataaaa gcgctcggtt taaaaagctt 6180

ctatgcctga ataggtgacc ggaggtcggc acctttcctt tgcaattact gaccctatga 6240

atacactgac tgtttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac 6300

tcactatagg agggccacca tgaagaaacc tgaactgaca gcaacttctg ttgagaagtt 6360

tctcattgaa aaatttgatt ctgtttctga tctcatgcag ctgtctgaag gtgaagaaag 6420

cagagccttt tcttttgatg ttggaggaag aggttatgtt ctgagggtca attcttgtgc 6480

tgatggtttt tacaaagaca gatatgttta cagacacttt gcctctgctg ctctgccaat 6540

tccagaagtt ctggacattg gagaattttc tgaatctctc acctactgca tcagcagaag 6600

agcacaagga gtcactctcc aggatctccc tgaaactgag ctgccagctg ttctgcaacc 6660

tgttgctgaa gcaatggatg ccattgcagc agctgatctg agccaaacct ctggatttgg 6720

tccttttggt ccccaaggca ttggtcagta caccacttgg agggatttca tttgtgccat 6780

tgctgatcct catgtctatc actggcagac tgtgatggat gacacagttt ctgcttctgt 6840

tgctcaggca ctggatgaac tcatgctgtg ggcagaagat tgtcctgaag tcagacacct 6900

ggtccatgct gattttggaa gcaacaatgt tctgacagac aatggcagaa tcactgcagt 6960

cattgactgg tctgaagcca tgtttggaga ttctcaatat gaggttgcca acattttttt 7020

ttggagacct tggctggctt gcatggaaca acaaacaaga tattttgaaa gaagacaccc 7080

agaactggct ggttccccca gactgagagc ctacatgctc agaattggcc tggaccaact 7140

gtatcaatct ctggttgatg gaaactttga tgatgctgct tgggcacaag gaagatgtga 7200

tgccattgtg aggtctggtg ctggaactgt tggaagaact caaattgcaa gaaggtctgc 7260

tgctgtttgg actgatggat gtgttgaagt tctggctgac tctggaaaca ggagaccctc 7320

cacaagaccc agagccaagg aatgaatatt agctagatta tccctaatac ctgccacccc 7380

actcttaatc agtggtggaa gaacggtctc agaactgttt gtttcaattg gccatttaag 7440

tttagtagta aaagactggt taatgataac aatgcatcgt aaaaccttca gaaggaaagg 7500

agaatgtttt gtggaccact ttggttttct tttttgcgtg tggcagtttt aagttattag 7560

tttttaaaat cagtactttt taatggaaac aacttgacca aaaatttgtc acagaatttt 7620

gagacccatt aaaaaagtta aatgagaaac ctgtgtgttc ctttggtcaa caccgagaca 7680

tttaggtgaa agacatctaa ttctggtttt acgaatctgg aaacttcttg aaaatgtaat 7740

tcttgagtta acacttctgg gtggagaata gggttgtttt ccccccacat aattggaagg 7800

ggaaggaata tcatttaaag ctatgggagg gttgctttga ttacaacact ggagagaaat 7860

gcagcatgtt gctgattgcc tgtcactaaa acaggccaaa aactgagtcc ttgggttgca 7920

tagaaagctg 7930

Claims (10)

1. The preparation method of the broad-spectrum neutralizing antibody hybridoma cell strain for resisting EV71, CA16, CA10 and CA6 is characterized by comprising the following steps:

step (1), preparation of immunogen: purified EV71 and CA16 viruses are used as immunogens;

step (2), animal immunization: four immunizations were performed at 2 week intervals on 6-8 week old Balb/c mice with the following procedure: after primary immunization, fully mixing Freund's complete adjuvant and an equal amount of immunogen for emulsification, performing subcutaneous multipoint injection on the back; the second immunization and the third immunization, the adjuvant is replaced by Freund's incomplete adjuvant, and the method and the metering are the same as the first immunization; the fourth time of intraperitoneal injection of immunogen without adjuvant is adopted, and the injection quantity is the same as that of the first immunization; after 3 days, spleen cells of immunized mice were taken for preparation of hybridoma cells;

step (3), preparation and screening of hybridoma cell strains: carrying out cell fusion on SP2/0 cells in the logarithmic growth phase and spleen cells of the immunized mice in the step (2) under the action of PEG4000, culturing the fused hybridoma cells by using a1 XHAT selective medium, detecting cell supernatants 10-14 days after cell fusion by an ELISA (enzyme-linked immunosorbent assay) test to screen hybridoma cell strains capable of specifically secreting broad-spectrum neutralizing antibodies against EV71, CA16, CA10 and CA 6.

2. The anti-EV 71, CA16, CA10 and anti-EV of claim 1A method for preparing a broad-spectrum neutralizing antibody hybridoma cell strain of CA6, which is characterized in that in the step (2), the first immunization injection amount is 50ul 10 ⁷ TCID50/ml EV71 and 50ul 10 ⁷ TCID50/ml CA16；

In the step (3), SP2/0 cells in the logarithmic growth phase and spleen cells of the immunized mice in the step (2) are subjected to cell fusion under the action of PEG4000, and the fused hybridoma cells are cultured by using a1 XHAT selective medium, and the specific method comprises the following steps:

selecting a Balb/c mouse with the age of 6-8 weeks, taking abdominal macrophages of the Balb/c mouse to prepare feeder cells; mixing SP2/0 cells in logarithmic growth phase with spleen cells of immunized mice in step (2) according to cell number ratio of 1:10, performing cell fusion with polyethylene glycol 4000, adding hybridoma obtained after cell fusion into culture plate containing feeder layer cells, using 1 XHAT selective medium, culturing at 37deg.C and 5% CO ₂ Culturing under the condition;

also included are the cloning of hybridoma cells, specifically: subcloning and culturing the hybridoma cell strain by limiting dilution method, continuously culturing for multiple times until the antibody in the cloning hole detects 100% positive, enlarging culturing and freezing storing.

3. A method for producing broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM-type monoclonal antibodies, characterized by using the broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 hybridoma cell strain produced by the method for producing broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 hybridoma cell strain according to any one of claims 1 to 2; the method comprises the following steps:

extracting RNA of broad-spectrum neutralizing antibody hybridoma cell strains resisting EV71, CA16, CA10 and CA6, and performing reverse transcription to obtain cDNA; then amplifying the antibody heavy chain variable region by nested PCR, and amplifying the antibody light chain variable region by nested PCR; then, connecting the variable regions of the antibody 20-IgM by using PVITRO 1-hygro-mcs-IgM/kappa connection vectors to obtain IgM type antibodies; meanwhile, PVITRO 1-hygro-mcsIgG/kappa linking vector is adopted to link the variable region of the antibody 20-IgM, so as to obtain an IgG antibody; then, the positive monoclonal antibody obtained by connection is transfected into CHO-S cells, and then purified, thus obtaining broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM type monoclonal antibodies and IgG type monoclonal antibodies; the DNA sequence of the PVITRO 1-hygro-mcs-IgM/kappa linking vector is shown as SEQ ID NO. 7; the DNA sequence of the PVITRO 1-hygro-mcsIgG/kappa linking vector is shown as SEQ ID NO. 8.

4. An anti-EV 71, CA16, CA10 and CA6 human-murine chimeric broad-spectrum neutralizing IgM-type monoclonal antibody prepared by the method for preparing a broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-murine chimeric IgM-type monoclonal antibody according to claim 3.

5. A broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-mouse chimeric IgM monoclonal antibody is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.1 in a sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2 in the sequence table.

6. A DNA encoding a broad spectrum neutralizing anti-EV 71, CA16, CA10, and CA6 human-murine chimeric IgM type monoclonal antibody according to claim 5, characterized by comprising a heavy chain variable region and a light chain variable region;

the coding DNA sequence of the heavy chain variable region is shown as SEQ ID NO. 3;

the coding DNA sequence of the light chain variable region is shown as SEQ ID NO. 4.

7. An expression vector comprising a heavy chain constant region DNA sequence and a light chain constant region DNA sequence for expressing the broad-spectrum neutralizing anti-EV 71, CA16, CA10, and CA6 human-murine chimeric IgM-type monoclonal antibody of claim 5;

the coding DNA sequence of the heavy chain constant region is shown as SEQ ID NO. 5;

the coding DNA sequence of the light chain constant region is shown in SEQ ID NO. 6.

8. A host cell comprising the expression vector of claim 7.

9. A pharmaceutical composition comprising a broad spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-murine chimeric IgM type monoclonal antibody according to claim 4 or 5 and a pharmaceutically acceptable carrier.

10. Use of a broad-spectrum neutralizing anti-EV 71, CA16, CA10 and CA6 human-murine chimeric IgM-type monoclonal antibody according to claim 4 or 5 for the preparation of a medicament for the treatment or prevention of hand-foot-and-mouth disease caused by EV71, CA16, CA10 and CA6 infections.

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