CN111574621A

CN111574621A - Monoclonal antibody for neutralizing EB virus and application thereof

Info

Publication number: CN111574621A
Application number: CN202010363338.9A
Authority: CN
Inventors: 曾木圣; 张林琦; 朱倩莹; 梁清泰; 左亚男
Original assignee: Tsinghua University; Sun Yat Sen University Cancer Center
Current assignee: Tsinghua University; Sun Yat Sen University Cancer Center
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2020-08-25
Anticipated expiration: 2040-04-30
Also published as: CN111574621B

Abstract

The invention discloses an antibody of EB virus for the first time, which consists of a light chain and a heavy chain, wherein the heavy chain variable region of the heavy chain is provided with 3 complementary regions CDR1, CDR2 and CDR3, the amino acid sequences of the complementary regions are respectively shown as SEQ ID No. 1-3, and the light chain variable region of the light chain is provided with 3 complementary regions CDR1 ', CDR2 ' and CDR3 ', the amino acid sequences of the complementary regions are respectively shown as SEQ ID No.4, GNN and SEQ ID No. 5. The antibody can block EBV from infecting B cells, IC50 is 203ng/ml, and the antibody has no neutralization effect on Ebola virus infection, which indicates that the antibody has high capacity of specifically neutralizing EBV infected B cells.

Description

Monoclonal antibody for neutralizing EB virus and application thereof

Technical Field

The invention relates to the technical field of antibodies, and particularly relates to a monoclonal antibody S54 for neutralizing EB virus and application thereof.

Background

EB virus was successfully cultured and established by Epstein and Barr from Burkitt lymphoma cells as early as 1964. EBV belongs to the gamma subtype of herpes virus and is the first human oncogenic virus to be discovered. EBV is very common among people and is reported to be carried by more than about 95% of the adults worldwide. In children and adolescents, many EBV infections cause infectious mononucleosis. Latent EBV infection is associated with a variety of lymphoid and epithelial neoplasia in humans, such as Hodgkin's lymphoma, Burkitt's lymphoma, and NK/T cell lymphoma, among others, and epithelial neoplasia including nasopharyngeal carcinoma and about 10% gastric carcinoma, among others. For the organ transplantation patients and the immune suppression patients such as AIDS patients, the probability of suffering EBV-related tumors is greatly increased. Statistically, there are approximately 200000 new EBV-associated tumor cases worldwide each year, and EBV has been formally listed in the 14 th edition carcinogen directory by the U.S. NIH in 2016.

At present, no effective vaccine exists for EB virus, and a specific treatment means is lacked for diseases caused by EBV infection. Most of the treatments for infectious mononucleosis use antiviral drugs such as acyclovir, which can relieve symptoms to some extent, but cannot eliminate EB virus in B lymphocytes and laryngeal epithelium. Treatment of EBV-associated tumors is mainly chemotherapy and radiotherapy, but is less effective in patients with relapses or metastases.

The monoclonal antibody can be produced in large scale, has high affinity and high specificity when being combined with antigen, and greatly reduces adverse reaction in clinical application. Antibody molecules can also be engineered to increase their antiviral efficacy. Antibodies, with their specificity and flexibility of use, are promising tools for the treatment of infectious diseases. However, no human monoclonal antibody against EBV envelope glycoprotein has yet been marketed. Therefore, the development of human monoclonal antibodies against EBV will provide more effective prevention and treatment means for diseases associated with EBV infection.

Disclosure of Invention

In the first aspect of the present invention, EB Virus (Epstein-Barr Virus, EBV) membrane protein gp350(glycoprotein 350) is used as bait to screen a library of human non-immune single chain variable antibody fragments (scFvs) displayed on the surface of yeast, thereby obtaining a monoclonal antibody capable of specifically binding to gp350 protein. Through EB virus infected cell model, monoclonal antibody with neutralizing activity is obtained through screening.

In a second aspect, the invention provides a nucleotide sequence encoding an antibody according to the first aspect of the invention.

It is an object of the third aspect of the invention to provide a transgenic cell line comprising a nucleotide sequence according to the second aspect of the invention.

In a fourth aspect of the present invention, there is provided a medicament for treating diseases associated with epstein barr virus, wherein the active ingredient of the medicament is the antibody according to the first aspect of the present invention.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided an antibody for neutralizing epstein-barr virus, comprising a light chain and a heavy chain, wherein the heavy chain has 3 complementary regions of CDR1, CDR2 and CDR3 in its heavy chain variable region, wherein the amino acid sequence of CDR1 is GYTFTTYY (SEQ ID No.1), the amino acid sequence of CDR2 is inp vggsa (SEQ ID No.2), and the amino acid sequence of CDR3 is ARVGPSRYSTSSPY (SEQ ID No. 3); the light chain has 3 complementary regions CDR1 ', CDR 2' and CDR3 'in the light chain variable region, wherein the amino acid sequence of CDR 1' is SSNIGSNT (SEQ ID NO.4), the amino acid sequence of CDR2 'is GNN, and the amino acid sequence of CDR 3' is SSYTSSSSLV (SEQ ID NO. 5).

The antibody according to the first aspect of the present invention, wherein the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID No. 11.

The antibody according to the first aspect of the present invention, wherein the amino acid sequence of the variable region of the light chain of the antibody is shown as SEQ ID No. 13.

According to the antibody of the first aspect of the present invention, the amino acid sequence of the heavy chain of the antibody is shown as SEQ ID No. 15.

The antibody according to the first aspect of the present invention, wherein the amino acid sequence of the light chain of the antibody is represented by SEQ id No. 17.

In a second aspect of the invention, there is provided a nucleotide sequence encoding an antibody according to the first aspect of the invention.

The nucleotide sequences encoding the 3 complementary regions CDR1, CDR2 and CDR3 in the variable region of the heavy chain according to the nucleotide sequence of the second aspect of the present invention are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

According to the nucleotide sequence of the second aspect of the present invention, the nucleotide sequence encoding the 3 complementary regions CDR1 ' in the light chain variable region is AGCTCCAACATCGGAAGTAATACT (SEQ ID NO.9), and the nucleotide sequence of the CDR2 ' is GGTAATAAT, CDR3 ' and is AGCTCATATACAAGCAGCAGCAGTCTGGTC (SEQ ID NO. 10).

The nucleotide sequence according to the second aspect of the present invention, the nucleotide sequence encoding the heavy chain variable region of the antibody is shown in SEQ ID NO. 12.

The nucleotide sequence of the second aspect of the invention, the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO. 14.

The nucleotide sequence according to the second aspect of the invention, the nucleotide sequence encoding the heavy chain of the antibody is shown in SEQ ID No. 16.

The nucleotide sequence encoding the light chain of the antibody according to the second aspect of the invention is shown in SEQ ID No. 18.

In a third aspect of the invention there is provided a transgenic cell line comprising a nucleotide sequence according to the second aspect of the invention.

The invention has the beneficial effects that:

the invention discloses an antibody for neutralizing EB virus for the first time, which separates yeast monoclonal antibody of specific binding antigen from a yeast surface human non-immune single chain variable region antibody fragment (scFvs) library, and then clones the monoclonal antibody gene, researches the structure and the function and screens. Not only avoids the long process required by antigen immunity, hybridoma fusion and monoclonal antibody screening, but also greatly reduces the side effect caused by animal source antibody.

The invention adopts the method, the gp350 is utilized to screen the human non-immune single chain variable region antibody fragment library displayed on the surface of the yeast, the yeast monoclonal antibody which can be specifically combined with the antigen is obtained, the antibody variable region gene is obtained, then the antibody variable region gene is cloned to a eukaryotic cell expression vector containing a corresponding constant region and is transfected to 293T cells, and the monoclonal antibody which is specifically combined with the gp350 protein can be obtained. Then, a monoclonal antibody with the anti-EBV neutralizing activity is obtained by screening through an EB virus infected cell model.

The antibody for neutralizing the EB virus provided by the invention can block EBV from infecting B cells, IC50 is 203ng/ml, and the antibody has no neutralizing effect on Ebola virus infection, thereby indicating that the antibody has high capacity of specifically neutralizing EBV from infecting B cells. ,

drawings

FIG. 1 neutralizing effect of the S54 antibody on EBV-infected Raji cells in example 3.

FIG. 2 neutralizing effect of the S54 antibody in example 3 on Ebola pseudovirus infected Huh7 cells.

Detailed Description

(1) And (3) preparing the EBV gp350 recombinant protein.

The gene segment coding the EBV gp350 protein is expressed and purified in a mammalian cell expression system. The recombinant antigen protein C end is provided with 6 XHis-avi tag, and affinity purification of gp350 protein is carried out by using a nickel column.

(2) And (3) screening to obtain the yeast monoclonal specifically combined with the gp350 protein by using a yeast surface display scFv screening technology.

The antibody heavy chain and light chain variable regions respectively amplified from normal human bodies are connected together by a G4S linker to construct a yeast display carrier, namely a successful yeast surface display scFv library is constructed (the yeast surface display scFv library used in the experiment is from Michael J. Gp350 labeled with biotin is incubated with the yeast surface display scFv library, and positive scFv yeast clones which react with gp350 are obtained by flow sorting. Extracting scFv expression vector in yeast clone, then respectively utilizing heavy chain and light chain specific primer to make nested PCR, sequencing so as to obtain antibody gene.

(3) And (5) expression and purification of the monoclonal antibody.

Performing overlapping PCR on the upstream and CMV fragments, the downstream and the constant region of human IgG1 and the ployA fragment of the antibody heavy chain variable region obtained by screening to obtain a fragment capable of expressing a complete heavy chain; and (3) performing overlapping PCR on the upstream and CMV fragments, the downstream and constant region of the light chain kappa/lambda and the ploy A fragment of the antibody light chain variable region obtained by screening to obtain a fragment capable of expressing the complete light chain. These two fragments were then constructed into the PMD18T (Takara) vector. The expression of the antibody was carried out by co-transfecting 293T cells with PMD18T plasmid carrying full-length sequences of the heavy and light chains of the antibody, and the purification of the antibody was carried out using protein A beads.

(4) And (5) detecting the neutralizing activity of the antibody.

Detection of EBV neutralizing antibodies was performed using a virus-infected cell model. Diluting the expressed and purified full-length antibody according to a certain multiple ratio, and incubating with EB virus; spreading the EBV corresponding susceptible cells into the co-incubation system, and culturing the cells for 48-72 hours; detecting the proportion of GFP positive cells by using a flow cytometer for infected cells; IC50 for the monoclonal antibody was calculated using Prism 5 software.

The invention will be further illustrated with reference to specific examples. The examples are given solely for the purposes of illustration and understanding and are not intended to limit the scope of the invention.

EXAMPLE 1 preparation of EBV gp350 recombinant protein

The gp350 protein plays an important role in the process of EBV B cells. Thus, the inventors' research team selected gp350 as a bait protein to screen yeast surface display scFv libraries in an attempt to obtain neutralizing antibodies that block EBV infection.

Then, the genome of the EBV-M81 strain is selected as a template, and the sequence of gp350 is amplified.

gp350 DNA sequence:

ATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCTGCCTCGGAATGGAGGCAGCCTTGCTTGTGTGTCAGTACACCATCCAGAGCCTGATCCATCTCACGGGTGAAGATCCTGGTTTTTTCAATGTTGAGATTCCGGAATTCCCATTTTACCCCACATGCAATGTTTGTACGGCAGATGTCAATGTAACTATCAATTTCGATGTCGGGGGCAAAAAGCATCAACTTGATCTTGACTTTGGCCAGCTGACACCCCATACGAAGGCTGTCTACCAACCTCGAGGTGCATTTGGTGGCTCAGAAAATGCCACCAATCTCTTTCTACTGGAGCTCCTTGGTGCAGGAGAATTGGCTCTAACTATGCGGTCTAAGAAGCTTCCAATTAACGTCACCACCGGAGAGGAGCAACAAGTAAGCCTGGAATCTGTAGATGTCTACTTTCAAGATGTGTTTGGAACCATGTGGTGCCACCATGCAGAAATGCAAAACCCCGTGTACCTGATACCAGAAACAGTGCCATACATAAAGTGGGATAACTGTAATTCTACCAATATAACGGCAGTAGTGAGGGCACAGGGGCTGGATGTCACGTTACCCTTAAGTTTGCCAACGTCAGCTCAAGACTCGAATTTCAGCGTAAAAACACAAATGCTCGGTAATGAGATAGATATTGAGTGTATTATGGAGGATGGCGAAATTTCACAAGTTCTGCCCGGAGACAACAAATTTAACATCACCTGCAGTGGATACGAGAGCCATGTTCCCAGCGGCGGAATTCTCACATCAACGAGTCCCGTGGCCACCCCAATACCTGGTACAGGGTATGCATACAGCCTGCGTCTGACACCACGTCCAGTGTCACGATTTCTTGGCAATAACAGTATCCTGTACGTGTTTTACTCTGGGAATGGACCGAAGGCGAGCGGGGGAGATTACTGCATTCAGTCCAACATTGTGTTCTCTGATGAGATTCCAGCTTCACAGGACATGCCGACAAACACCACAGACATCACATATGTGGGTGACAATGCTACCTATTCAGTGCCAATGGTCACTTCTGAGGACGCAAACTCGCCAAATGTTACAGTGACTGCCTTTTGGGCCTGGCCAAACAACACTGAAACTGACTTTAAGTGCAAATGGACTCTCACCTCGGGGACACCTTCGGGTTGTGAAAATATTTCTGGTGCATTTGCGAGCAATCGGACATTTGACATTACTGTCTCGGGTCTTGGCACGGCCCCCAAAACACTCATTATCACACGAACGGCTACCAATGCCACCACAACAACCCACAAGGTTATATTCTCCAAGGCACCCCATCATCACCATCACCACGGTCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAA(SEQ ID NO.19)。

construction of eukaryotic expression vectors

The gp350 gene fragment was ligated into the mammalian cell expression vector pcDNA3.1+, as follows:

1) amplification of gp350 protein Gene

The signal peptide of gp350 protein itself was replaced with CD5 Signal Peptide (SP), and KOZAK sequence was added in front of the signal peptide to enhance the expression of the protein. The signal peptide will be cleaved off during expression and secretion of the protein in mammalian cells. Since the transmembrane region of the protein cannot be secreted, the transmembrane region of gp350 protein is not included in the expressed protein gene. Finally, 6 XHis-avi tag is added at the end of the protein for subsequent purification. Therefore, the recombinant protein is constructed as SP-gp 350-his-avi.

Using EBV-M81-BAC DNA as a template, gp350 was amplified with the following primers:

gp350 forward primer:

TAGTCCAGTGTGGTGGAATTCGCCACCATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCTGCCTCGGAATGGAGGCAGCCTTGCTTG(SEQ ID NO.20)。

gp350 reverse primer:

GCCCTCTAGACTCGAGCGGCCGCTTATTCGTGCCATTCGATTTTCTGAGCCTCGAAGATGTCGTTCAGACCGTGGTGATGGTGATGATGGGGTGCCTTGGAGAATATAAC(SEQ ID NO.21)。

a50. mu.l PCR reaction was selected:

and analyzing the amplified target fragment by agarose gel electrophoresis, cutting a band with correct molecular weight under an ultraviolet lamp, and recovering a PCR product according to the operation of a kit instruction.

2) And (3) enzyme digestion and connection of the target fragment and the vector:

the vector used eukaryotic expression plasmid pcDNA3.1 +. Both the fragment of interest and the vector were digested with EcoRI and NotI using a 50. mu.l reaction:

the enzyme digestion is carried out at 37 ℃ for 3-5 hr. And (3) recovering the fragment by using a DNA purification kit, and performing gel recovery after the gel is run by enzyme digestion of the vector.

The ligation reaction was carried out using a 20. mu.l system and was carried out at 37 ℃ for 30 min:

3) conversion of ligation products

Adding the ligation product to freshly thawed DH5 α competent cells; standing on ice for 30 min; heat shock at 42 deg.C for 90s, and placing on ice for 5 min; adding 200 μ l LB culture medium, and resuscitating at 30 deg.C for 40 min; the culture broth was pipetted and spread on ampicillin-resistant LB plates and cultured overnight at 37 ℃.

4) Screening for Positive clones

Selecting single colony, inoculating into 5ml culture medium containing ampicillin, and culturing for about 12 hr; extracting plasmids by using a plasmid miniextraction kit; carrying out enzyme digestion identification to obtain positive clones; and (5) sequencing and verifying to obtain a completely correct recombinant plasmid. The plasmids were extracted in large quantities.

Expression and extraction of recombinant proteins

293F cells were cultured to 1L and transfected with PEI transfection reagent at cell densities of 1.5X 10^ 6/ml. Adding 1mg of recombinant plasmid and 5ml of 1mg/ml PEI into 100ml of DMEM culture medium, shaking uniformly, standing at room temperature for 20min, and adding into cells. Collecting cell supernatant after culturing for 5 days, centrifuging at 4000rpm for 30min, discarding cell precipitate, and collecting supernatant containing target protein.

gp350 recombinant protein purification

1) Affinity chromatography

The gp350 recombinant protein C-terminal has 6 XHis-avi tag, and can be affinity purified by using nickel column.

Adding nickel column beads into the supernatant after high-speed centrifugation, and incubating overnight at 4 ℃; centrifuging at low speed to remove supernatant; rinsing the beads with a total of 100mL of loading buffer containing 20mM imidazole; the protein of interest was eluted with 5 column volumes of elution buffer containing 250mM imidazole.

2) Gel filtration chromatography

Concentrating the protein eluted from the nickel column to a volume of less than 1ml by using a 30kD concentration tube; samples were purified using superdex200 column.

The amino acid sequence finally obtained:

MPMGSLQPLATLYLLGMLVASCLGMEAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGAFGGSENATNLFLLELLGAGELALTMRSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNCNSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTQMLGNEIDIECIMEDGEISQVLPGDNKFNITCSGYESHVPSGGILTSTSPVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKASGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWAWPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTTHKVIFSKAPHHHHHHGLNDIFEAQKIEWHE(SEQ ID NO.22)。

biotin labeling of gp350 recombinant proteins

The gp350 protein was biotinylated using the reagent EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo). To gp350 at a concentration of 2mg/ml, 10mM biotin reagent was added and the reaction was carried out at room temperature for 30 min. Finally, the reaction system was dialyzed with PBS.

Example 2 screening of Yeast libraries

The yeast library screening technology is utilized to obtain the yeast positive clone specifically combined with the gp350 protein.

(1) Materials and apparatus

The yeast surface display human non-immune scFv library is an antibody heavy chain and light chain variable region respectively amplified from lymphocytes of spleen and lymph node of 58 normal persons, and 3G is passed in the middle₄S linker links two genes together to construct a yeast display carrier, namely a successful yeast surface display scFv library is constructed, and the library capacity of the library is 10⁹(the Yeast surface display scFv library used in this experiment was from Michael J. laboratory), Omega Yeast Plasmid Mini kit, Amp + plate, LB medium, streptavidin-microbeads (milenyi biotech), anti-biotin-microbeads (milenyi biotech), streptavidin-APC (ebioscience), anti-biotin-PE (ebioscience), YPD medium, SDCAA plate, SGCAA medium, Yeast air EBY100, E.Colistancins DH5 α, pNL6 Plasmid, flow sorter (Aril II, BD).

(2) Method of producing a composite material

1) Induced yeast cellsAfter OD measurement, 10 is taken¹⁰The cells were centrifuged, washed 1-2 times with PBS, resuspended in 10ml PBS and 100nM gp350-biotin protein was added. Incubate at 4 ℃ for 5-10 minutes, wash twice with PBS, and then mix as 1: at a dilution of 2500 streptavidin-microbeads were added and incubated for 10 min at 4 ℃. PBS was washed twice, and 40ml PBS was resuspended and then magnetic bead sorted. And (3) after the sorted yeast cells are amplified in an SD-CAA culture medium and induced by SG-CAA, carrying out second round of magnetic bead sorting, and replacing the secondary antibody with anti-biotin-microbeads. And directly taking the yeast positive cells obtained by the second round of sorting, adding 100nM gp350 protein again, incubating for 30 minutes at 4 ℃, washing twice with PBS, adding streptavidin-APC, incubating for 30 minutes at 4 ℃, and washing twice with PBS for flow sorting. And (3) after amplification and SG-CAA induction in the sorted yeast cell SD-CAA culture medium, carrying out second alternate sorting, replacing a secondary antibody with streptavidin-APC and anti-biotin-PE, observing whether yeast enrichment aiming at the streptavidin-APC exists or not, and obtaining the human scFv yeast positive clone combined with gp 350.

2) And (3) coating the scFv yeast mixed solution obtained by the third round of flow sorting on a SDCAA plate, selecting 60 monoclonal yeast colonies, amplifying by using an SDCAA liquid culture medium and inducing by using an SGCAA culture medium, and then verifying the binding activity of the monoclonal yeast colonies and the antigen by flow detection.

EXAMPLE 3 sequencing and expression purification of monoclonal antibodies

(1) The human scFv yeast positive clone which is combined with gp350 and obtained by screening in the example 2 is extracted from the plasmid in the yeast and sequenced to obtain the antibody gene.

(2) And (5) expression and purification of the monoclonal antibody. Performing overlapping PCR on the upstream and CMV fragments, the downstream and the constant region of human IgG1 and the ploy A fragment of the antibody heavy chain variable region obtained by screening to obtain a fragment capable of expressing a complete heavy chain; and (3) performing overlapping PCR on the upstream and CMV fragments, the downstream and constant region of the light chain kappa/lambda and the ploy A fragment of the antibody light chain variable region obtained by screening to obtain a fragment capable of expressing the complete light chain. These two fragments were then constructed into the PMD18T (Takara) vector. The expression of the antibody was carried out by co-transfecting 293T cells with the PMD18T plasmid containing the full-length sequences of the heavy and light chains of the antibody amplified from the same B cell, and purifying the antibody with protein A beads.

Example 4 detection of neutralizing Activity of antibodies in EBV Virus-infected cell model

Preparation of EBV Virus

(1) EBV-GFP infected Akata cells were cultured to a density of 2 x 10^6/ml, in serum-free 1640 medium, goat anti-human IgG was added to a final concentration of 8ul/ml, and cultured at 37 ℃ in a 5% CO2 environment for 6 h.

(2) After 6h, the medium of the cells was changed to 1640 medium containing 5% FBS, and the cells were cultured at 37 ℃ in an atmosphere of 5% CO2 for another 3 days.

(3) Collecting cell supernatant after 3 days, filtering the supernatant with 0.45um filter membrane, and centrifuging at 4 deg.C for 2h at 20000rpm with ultra-high speed centrifuge.

(4) The virus pellet was resuspended in serum-free 1640 medium.

2. Detection of neutralizing Activity of monoclonal antibodies

Diluting the monoclonal antibody according to a certain fold ratio, and incubating the diluted monoclonal antibody and the EBV for 3 hours at 4 ℃;

adding the incubated virus liquid into Raji cells, and culturing the cells for 48-72 hours;

preparing Raji cell suspension, and detecting the percentage of GFP positive cells by using a flow cytometer; IC50 of the antibody was calculated using Prism 5 software.

3. Neutralization test results

Through virus neutralization capacity verification, 1 strain antibody S54 for B cells with neutralizing EBV infection is obtained, the basic information is shown in Table 1, and the results of the neutralization experiment are shown in FIGS. 1 and 2.

TABLE 1S 54 basic information on antibody sequences

The sequence information for the variable regions of the S54 antibody, including antibody family distribution, antibody germline conservation and CDR3 amino acid sequence, are listed in table 1.

As can be seen from the results of the neutralization experiments shown in FIGS. 1 and 2, S54 blocked EBV infection of B cells, IC50 was 203ng/ml, and the negative control antibody 2G4 did not block EBV infection of B cells. The S54 antibody has high capability of neutralizing EBV infected B cells.

The full-length heavy chain of S54 has 470 amino acids, and the sequence is shown as follows:

wherein the underlined portion is the amino acid sequence of the heavy chain variable region; the amino acid sequences of the 3 complementary regions CDR1, CDR2 and CDR3 in the heavy chain variable region are underlined and in bold.

The coding gene of the full-length heavy chain of S54 has 2499 bases in total, and the sequence is shown as follows:

wherein the underlined portion is the nucleotide sequence of the heavy chain variable region; the nucleotide sequences of the 3 complementary regions CDR1, CDR2 and CDR3 in the heavy chain variable region are underlined and in bold; underlined and italicized parts are the start, stop codons.

The full-length light chain of S54 has 234 amino acids, and the sequence is shown as follows:

wherein the underlined portion is the amino acid sequence of the light chain variable region; the amino acid sequences of the 3 complementary regions CDR1, CDR2 and CDR3 in the light chain variable region are underlined and in bold.

The coding gene of the full-length light chain of S54 has total 1741 bases, and the sequence is shown as follows:

wherein the underlined portion is the nucleotide sequence of the light chain variable region; the nucleotide sequences of the 3 complementary regions CDR1 ', CDR2 ' and CDR3 ' in the light chain variable region are underlined and in bold; underlined and italicized parts are the start, stop codons.

The heavy chain variable region of the heavy chain of S54 has 3 complementary regions CDR1, CDR2 and CDR3, and CDR1, CDR2 and CDR3 in the heavy chain variable region are the 45 th to 52 th amino acid residues, the 70 th to 77 th amino acid residues and the 116 th and 129 th amino acid residues from the N terminal of SEQ ID NO.15 of the sequence table (corresponding to the bold partial sequence of SEQ ID NO. 15); the light chain has 3 complementary regions CDR1 ', CDR 2' and CDR3 'in the light chain variable region, and CDR 1', CDR2 'and CDR 3' in the light chain variable region are respectively the 46 th to 51 th amino acid residues, the 69 th to 71 th amino acid residues and the 108 th and 116 th amino acid residues from the N terminal of the sequence table SEQ ID NO.17 (corresponding to the bold partial sequence of the SEQ ID NO. 17).

The amino acid sequence of CDR1 is: GYTFTTYs (SEQ ID NO. 1);

the amino acid sequence of CDR2 is: INPSVGSA (SEQ ID NO. 2);

the amino acid sequence of CDR3 is: ARVGPSRYSTSSPY (SEQ ID NO. 3);

the amino acid sequence of CDR 1' is: SSNIGSNT (SEQ ID NO. 4);

the amino acid sequence of CDR 2' is: GNN;

the amino acid sequence of CDR 3' is: SSYTSSSSLV (SEQ ID NO. 5).

The nucleotide sequences encoding CDR1, CDR2, CDR3, CDR1 ', CDR2 ' and CDR3 ' are shown below:

the nucleotide sequence of CDR1 is: GGATACACCTTCACCACATATTAT (SEQ ID NO. 6);

the nucleotide sequence of CDR2 is: ATCAACCCCAGCGTTGGTAGCGCA (SEQ ID NO. 7);

the nucleotide sequence of CDR3 is:

GCGCGAGTAGGTCCGTCAAGATATAGCACTTCGTCACCCTAC(SEQ ID NO.8)；

the nucleotide sequence of CDR 1' is: AGCTCCAACATCGGAAGTAATACT (SEQ ID NO. 9);

the nucleotide sequence of CDR 2' is: GGTAATAAT, respectively;

the nucleotide sequence of CDR 3' is: AGCTCATATACAAGCAGCAGCAGTCTGGTC (SEQ ID NO. 10).

The amino acid sequence of the heavy chain variable region of S54 consists of amino acid residues from the 20 th to 140 th positions from the N-terminus of SEQ ID No.15 of the sequence listing, as shown below (corresponding to the underlined sequence in SEQ ID No. 15):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYLHWMRQAPGEGLEWVGLINPSVGSASSAQKFKGRVTMTSDTSTSIAYLEVNSLTSEDTAVYYCARVGPSRYSTSSPYWGQGTLVTVSS(SEQ ID NO.11)。

the nucleotide sequence is shown below (corresponding to the underlined sequence in SEQ ID NO. 16):

CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCACATATTATTTACACTGGATGCGTCAGGCCCCTGGAGAAGGGCTTGAGTGGGTGGGGCTAATCAACCCCAGCGTTGGTAGCGCAAGCTCCGCACAGAAGTTCAAGGGCAGAGTCACGATGACCAGCGACACGTCCACGAGCATTGCCTATTTGGAGGTAAACAGCCTGACATCTGAAGACACGGCCGTATATTATTGTGCGCGAGTAGGTCCGTCAAGATATAGCACTTCGTCACCCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA(SEQ IDNO.12)。

the light chain variable region of S54 consists of amino acid residues 20 to 128 from the N-terminus of SEQ ID No.17 of the sequence listing, as shown below (corresponding to the underlined sequence in SEQ ID No. 17):

QLVLTQSPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYGNNQRPSGVPDRFSGSKSGTSASLAISGLQSDDEADYYCSSYTSSSSLVFGGGTKLTVL(SEQ ID NO.13)。

the nucleotide sequence is shown below (corresponding to the underlined sequence in SEQ ID NO. 18):

CAGCTTGTGCTGACTCAGTCACCCTCAGCGTCTGGGACCCCCGGACAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTAAACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGACGATGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCAGTCTGGTCTTCGGCGGAGGGACCAAGCTCACCGTCCTA(SEQ ID NO.14)。

example 5 detection of neutralizing Activity of antibodies in model of Ebola pseudovirus infected cells

1. Preparation of Ebola (Ebola-Zaire) pseudovirus

(1) 293T cells were co-transfected with eukaryotic expression plasmids (pcDNA3.1+, available from Invitrogen) for full-length membrane proteins of Ebola and the backbone plasmid pNL 4-3R-E-luciferase;

(2) after 48 hours, cell supernatants were collected and assayed for viral titer using an ELISA kit for quantitative detection of p 24.

2. Detection of neutralizing Activity of monoclonal antibodies

(4) Diluting the monoclonal antibody according to a certain multiple ratio, and incubating the diluted monoclonal antibody and the Ebola pseudovirus at 37 ℃ for 1 h;

(5) adding the incubated virus liquid into Huh7 cells, and culturing the cells for 48-72 hours;

(6) lysing cells Huh 7; luciferase activity in the lysates was detected and IC50 of the antibodies was calculated using Prism 5 software.

3. Neutralization test results

Through virus neutralization capacity verification, the antibody S54 has no neutralization capacity on the Ebola pseudovirus, and the antibody 2G4 serves as a positive control for the neutralization of the Ebola pseudovirus. The results of the neutralization experiments are shown in FIG. 2.

It can be seen from FIG. 2 that S54 could not block the infection of Huh7 cells by Ebola pseudovirus, while 2G4 antibody as a positive control could block the infection of Ebola pseudovirus with high efficiency, and IC50 was 330 ng/ml. Taken together, it was suggested that S54 is a specific antibody with high neutralizing activity against EBV.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

SEQUENCE LISTING

<110> center for tumor prevention and treatment of Zhongshan university (Zhongshan university affiliated tumor Hospital, Zhongshan university tumor research)

Institute)

<120> monoclonal antibody neutralizing EB virus and application thereof

<130>

<160>22

<170>PatentIn version 3.5

<210>1

<211>8

<212>PRT

<213> Artificial sequence

<400>1

Gly Tyr Thr Phe Thr Thr Tyr Tyr

1 5

<210>2

<211>8

<212>PRT

<213> Artificial sequence

<400>2

Ile Asn Pro Ser Val Gly Ser Ala

1 5

<210>3

<211>14

<212>PRT

<213> Artificial sequence

<400>3

Ala Arg Val Gly Pro Ser Arg Tyr Ser Thr Ser Ser Pro Tyr

1 5 10

<210>4

<211>8

<212>PRT

<213> Artificial sequence

<400>4

Ser Ser Asn Ile Gly Ser Asn Thr

1 5

<210>5

<211>10

<212>PRT

<213> Artificial sequence

<400>5

Ser Ser Tyr Thr Ser Ser Ser Ser Leu Val

1 5 10

<210>6

<211>24

<212>DNA

<213> Artificial sequence

<400>6

ggatacacct tcaccacata ttat 24

<210>7

<211>24

<212>DNA

<213> Artificial sequence

<400>7

atcaacccca gcgttggtag cgca 24

<210>8

<211>42

<212>DNA

<213> Artificial sequence

<400>8

gcgcgagtag gtccgtcaag atatagcact tcgtcaccct ac 42

<210>9

<211>24

<212>DNA

<213> Artificial sequence

<400>9

agctccaaca tcggaagtaa tact 24

<210>10

<211>30

<212>DNA

<213> Artificial sequence

<400>10

agctcatata caagcagcag cagtctggtc 30

<210>11

<211>121

<212>PRT

<213> Artificial sequence

<400>11

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

1 5 10 15

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

20 25 30

Tyr Leu His Trp Met Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val

35 40 45

Gly Leu Ile Asn Pro Ser Val Gly Ser Ala Ser Ser Ala Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Ser Asp Thr Ser Thr Ser Ile Ala Tyr

65 70 75 80

Leu Glu Val Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Pro Ser Arg Tyr Ser Thr Ser Ser Pro Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>12

<211>363

<212>DNA

<213> Artificial sequence

<400>12

caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc acatattatt tacactggat gcgtcaggcc 120

cctggagaag ggcttgagtg ggtggggcta atcaacccca gcgttggtag cgcaagctcc 180

gcacagaagt tcaagggcag agtcacgatg accagcgaca cgtccacgag cattgcctat 240

ttggaggtaa acagcctgac atctgaagac acggccgtat attattgtgc gcgagtaggt 300

ccgtcaagat atagcacttc gtcaccctac tggggccagg gaaccctggt caccgtctcc 360

tca 363

<210>13

<211>109

<212>PRT

<213> Artificial sequence

<400>13

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser

85 90 95

Ser Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210>14

<211>327

<212>DNA

<213> Artificial sequence

<400>14

cagcttgtgc tgactcagtc accctcagcg tctgggaccc ccggacagag ggtcaccatc 60

tcttgttctg gaagcagctc caacatcgga agtaatactg taaactggta ccagcaactc 120

ccaggaacgg cccccaaact cctcatctat ggtaataatc agcggccctc aggggtccct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgacgatg aggctgatta ttactgcagc tcatatacaa gcagcagcag tctggtcttc 300

ggcggaggga ccaagctcac cgtccta 327

<210>15

<211>470

<212>PRT

<213> Artificial sequence

<400>15

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

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

35 40 45

Thr Thr Tyr Tyr Leu His Trp Met Arg Gln Ala Pro Gly Glu Gly Leu

50 55 60

Glu Trp Val Gly Leu Ile Asn Pro Ser Val Gly Ser Ala Ser Ser Ala

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Met Thr Ser Asp Thr Ser Thr Ser

85 90 95

Ile Ala Tyr Leu Glu Val Asn Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Val Gly Pro Ser Arg Tyr Ser Thr Ser Ser Pro

115 120 125

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

130 135 140

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

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

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

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

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

260 265 270

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

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

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

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

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

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210>16

<211>2499

<212>DNA

<213> Artificial sequence

<400>16

agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 60

ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 120

tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 180

atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 240

ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 300

gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 360

ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 420

tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 480

aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 540

tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct 600

gttttgacct ccatagaaga caccgggacc gatccagcct ccgcggccgg gaacggtgca 660

ttggaacgcg gattccccgt gccaagagtg acgtaagtac cgcctataga gtctataggc 720

ccaccccctt ggcttcgtta gaacgcggct acaattaata cataacctta tgtatcatac 780

acatacgatt taggtgacac tatagaataa catccacttt gcctttctct ccacaggtgt 840

ccactcccag gtccaactgc acctcggttc tatcgattga attccaccat gggatggtca 900

tgtatcatcc tttttctagt agcaactgca accggtgtac attcccaggt ccagcttgtg 960

cagtctgggg ctgaggtgaa gaagcctggg gcctcagtga aggtttcctg caaggcatct 1020

ggatacacct tcaccacata ttatttacac tggatgcgtc aggcccctgg agaagggctt 1080

gagtgggtgg ggctaatcaa ccccagcgtt ggtagcgcaa gctccgcaca gaagttcaag 1140

ggcagagtca cgatgaccag cgacacgtcc acgagcattg cctatttgga ggtaaacagc 1200

ctgacatctg aagacacggc cgtatattat tgtgcgcgag taggtccgtc aagatatagc 1260

acttcgtcac cctactgggg ccagggaacc ctggtcaccg tctcctcagc gtcgaccaag 1320

ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 1380

ctgggctgcc tggtcaagga ctacttcccc gaacctgtga cggtctcgtg gaactcaggc 1440

gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 1500

ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 1560

gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 1620

aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 1680

ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 1740

gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 1800

gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 1860

gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 1920

aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1980

cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 2040

caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 2100

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 2160

ggctccttct tcctctatag caagctcacc gtggacaaga gcaggtggca gcaggggaac 2220

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 2280

tccctgtccc cgggtaaatg agtgcgacgg ccggcaagcc cccgctcccc gggctctcgc 2340

ggtcgtacga ggaaagcttg gccgccatgg cccaacttgt ttattgcagc ttataatggt 2400

tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 2460

agttgtggtt tgtccaaact catcaatgta tcttatcat 2499

<210>17

<211>234

<212>PRT

<213> Artificial sequence

<400>17

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 510 15

Ser Leu Ser Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr

20 25 30

Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile

35 40 45

Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro

50 55 60

Lys Leu Leu Ile Tyr Gly Asn Asn Gln Arg Pro Ser Gly Val Pro Asp

65 70 75 80

Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser

85 90 95

Gly Leu Gln Ser Asp Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr

100 105 110

Ser Ser Ser Ser Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

115 120 125

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

130 135 140

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

145 150 155 160

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

165 170175

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

180 185 190

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

195 200 205

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

210 215 220

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

225 230

<210>18

<211>1741

<212>DNA

<213> Artificial sequence

<400>18

agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 60

ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 120

tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 180

atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 240

ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 300

gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 360

ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 420

tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 480

aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 540

tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct 600

gttttgacct ccatagaaga caccgggacc gatccagcct ccgcggccgg gaacggtgca 660

ttggaacgcg gattccccgt gccaagagtg acgtaagtac cgcctataga gtctataggc 720

ccaccccctt ggcttcgtta gaacgcggct acaattaata cataacctta tgtatcatac 780

acatacgatt taggtgacac tatagaataa catccacttt gcctttctct ccacaggtgt 840

ccactcccag gtccaactgc acctcggttc tatcgattga attccaccat gggatggtca 900

tgtatcatcc tttttctagt agcaactgca accggttctc tctcccagct tgtgctgact 960

cagtcaccct cagcgtctgg gacccccgga cagagggtca ccatctcttg ttctggaagc 1020

agctccaaca tcggaagtaa tactgtaaac tggtaccagc aactcccagg aacggccccc 1080

aaactcctca tctatggtaa taatcagcgg ccctcagggg tccctgaccg attctctggc 1140

tccaagtctg gcacctcagc ctccctggcc atcagtgggc tccagtctga cgatgaggct 1200

gattattact gcagctcata tacaagcagc agcagtctgg tcttcggcgg agggaccaag 1260

ctcaccgtcc taggtcagcc caaggctgcc ccctcggtca ctctgttccc gccctcgagt 1320

gaggagcttc aagccaacaa ggccacactg gtgtgtctca taagtgactt ctacccggga 1380

gccgtgacag tggcctggaa ggcagatagc agccccgtca aggcgggagt ggagaccacc 1440

acaccctcca aacaaagcaa caacaagtac gcggccagca gctacctgag cctgacgcct 1500

gagcagtgga agtcccacag aagctacagc tgccaggtca cgcatgaagg gagcaccgtg 1560

gagaagacag tggcccctac agaatgttca tagaagcttg gccgccatgg cccaacttgt 1620

ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 1680

catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 1740

t 1741

<210>19

<211>1410

<212>DNA

<213> Artificial sequence

<400>19

atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60

tcctgcctcg gaatggaggc agccttgctt gtgtgtcagt acaccatcca gagcctgatc 120

catctcacgg gtgaagatcc tggttttttc aatgttgaga ttccggaatt cccattttac 180

cccacatgca atgtttgtac ggcagatgtc aatgtaacta tcaatttcga tgtcgggggc 240

aaaaagcatc aacttgatct tgactttggc cagctgacac cccatacgaa ggctgtctac 300

caacctcgag gtgcatttgg tggctcagaa aatgccacca atctctttct actggagctc 360

cttggtgcag gagaattggc tctaactatg cggtctaaga agcttccaat taacgtcacc 420

accggagagg agcaacaagt aagcctggaa tctgtagatg tctactttca agatgtgttt 480

ggaaccatgt ggtgccacca tgcagaaatg caaaaccccg tgtacctgat accagaaaca 540

gtgccataca taaagtggga taactgtaat tctaccaata taacggcagt agtgagggca 600

caggggctgg atgtcacgtt acccttaagt ttgccaacgt cagctcaaga ctcgaatttc 660

agcgtaaaaa cacaaatgct cggtaatgag atagatattg agtgtattat ggaggatggc 720

gaaatttcac aagttctgcc cggagacaac aaatttaaca tcacctgcag tggatacgag 780

agccatgttc ccagcggcgg aattctcaca tcaacgagtc ccgtggccac cccaatacct 840

ggtacagggt atgcatacag cctgcgtctg acaccacgtc cagtgtcacg atttcttggc 900

aataacagta tcctgtacgt gttttactct gggaatggac cgaaggcgag cgggggagat 960

tactgcattc agtccaacat tgtgttctct gatgagattc cagcttcaca ggacatgccg 1020

acaaacacca cagacatcac atatgtgggt gacaatgcta cctattcagt gccaatggtc 1080

acttctgagg acgcaaactc gccaaatgtt acagtgactg ccttttgggc ctggccaaac 1140

aacactgaaa ctgactttaa gtgcaaatgg actctcacct cggggacacc ttcgggttgt 1200

gaaaatattt ctggtgcatt tgcgagcaat cggacatttg acattactgt ctcgggtctt 1260

ggcacggccc ccaaaacact cattatcaca cgaacggcta ccaatgccac cacaacaacc 1320

cacaaggtta tattctccaa ggcaccccat catcaccatc accacggtct gaacgacatc 1380

ttcgaggctc agaaaatcga atggcacgaa 1410

<210>20

<211>118

<212>DNA

<213> Artificial sequence

<400>20

tagtccagtg tggtggaatt cgccaccatg cccatggggt ctctgcaacc gctggccacc 60

ttgtacctgc tggggatgct ggtcgcttcc tgcctcggaa tggaggcagc cttgcttg 118

<210>21

<211>110

<212>DNA

<213> Artificial sequence

<400>21

gccctctaga ctcgagcggc cgcttattcg tgccattcga ttttctgagc ctcgaagatg 60

tcgttcagac cgtggtgatg gtgatgatgg ggtgccttgg agaatataac 110

<210>22

<211>470

<212>PRT

<213> Artificial sequence

<400>22

Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly

1 5 10 15

Met Leu Val Ala Ser Cys Leu Gly Met Glu Ala Ala Leu Leu Val Cys

20 25 30

Gln Tyr Thr Ile Gln Ser Leu Ile His Leu Thr Gly Glu Asp Pro Gly

35 40 45

Phe Phe Asn Val Glu Ile Pro Glu Phe Pro Phe Tyr Pro Thr Cys Asn

50 55 60

Val Cys Thr Ala Asp Val Asn Val Thr Ile Asn Phe Asp Val Gly Gly

65 70 75 80

Lys Lys His Gln Leu Asp Leu Asp Phe Gly Gln Leu Thr Pro His Thr

85 90 95

Lys Ala Val Tyr Gln Pro Arg Gly Ala Phe Gly Gly Ser Glu Asn Ala

100 105 110

Thr Asn Leu Phe Leu Leu Glu Leu Leu Gly Ala Gly Glu Leu Ala Leu

115120 125

Thr Met Arg Ser Lys Lys Leu Pro Ile Asn Val Thr Thr Gly Glu Glu

130 135 140

Gln Gln Val Ser Leu Glu Ser Val Asp Val Tyr Phe Gln Asp Val Phe

145 150 155 160

Gly Thr Met Trp Cys His His Ala Glu Met Gln Asn Pro Val Tyr Leu

165 170 175

Ile Pro Glu Thr Val Pro Tyr Ile Lys Trp Asp Asn Cys Asn Ser Thr

180 185 190

Asn Ile Thr Ala Val Val Arg Ala Gln Gly Leu Asp Val Thr Leu Pro

195 200 205

Leu Ser Leu Pro Thr Ser Ala Gln Asp Ser Asn Phe Ser Val Lys Thr

210 215 220

Gln Met Leu Gly Asn Glu Ile Asp Ile Glu Cys Ile Met Glu Asp Gly

225 230 235 240

Glu Ile Ser Gln Val Leu Pro Gly Asp Asn Lys Phe Asn Ile Thr Cys

245 250 255

Ser Gly Tyr Glu Ser His Val Pro Ser Gly Gly Ile Leu Thr Ser Thr

260 265 270

Ser Pro Val Ala Thr Pro Ile Pro Gly Thr Gly Tyr Ala Tyr Ser Leu

275280 285

Arg Leu Thr Pro Arg Pro Val Ser Arg Phe Leu Gly Asn Asn Ser Ile

290 295 300

Leu Tyr Val Phe Tyr Ser Gly Asn Gly Pro Lys Ala Ser Gly Gly Asp

305 310 315 320

Tyr Cys Ile Gln Ser Asn Ile Val Phe Ser Asp Glu Ile Pro Ala Ser

325 330 335

Gln Asp Met Pro Thr Asn Thr Thr Asp Ile Thr Tyr Val Gly Asp Asn

340 345 350

Ala Thr Tyr Ser Val Pro Met Val Thr Ser Glu Asp Ala Asn Ser Pro

355 360 365

Asn Val Thr Val Thr Ala Phe Trp Ala Trp Pro Asn Asn Thr Glu Thr

370 375 380

Asp Phe Lys Cys Lys Trp Thr Leu Thr Ser Gly Thr Pro Ser Gly Cys

385 390 395 400

Glu Asn Ile Ser Gly Ala Phe Ala Ser Asn Arg Thr Phe Asp Ile Thr

405 410 415

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

420 425 430

Ala Thr Asn Ala Thr Thr Thr Thr His Lys Val Ile Phe Ser Lys Ala

435440 445

Pro His His His His His His Gly Leu Asn Asp Ile Phe Glu Ala Gln

450 455 460

Lys Ile Glu Trp His Glu

465 470

Claims

1. An antibody neutralizing EB virus consisting of a light chain and a heavy chain having 3 complementary regions CDR1, CDR2 and CDR3 in the heavy chain variable region,

wherein the amino acid sequence of the CDR1 is GYTFTTYY (SEQ ID NO.1),

the amino acid sequence of CDR2 is INPSVGSA (SEQ ID NO.2),

the amino acid sequence of CDR3 is ARVGPSRYSTSSPY (SEQ ID NO. 3);

the light chain has 3 complementary regions CDR1 ', CDR2 ' and CDR3 ' in the light chain variable region,

wherein the amino acid sequence of CDR 1' is SSNIGSNT (SEQ ID NO.4),

the amino acid sequence of CDR 2' is GNN,

the amino acid sequence of CDR 3' is SSYTSSSSLV (SEQ ID NO. 5).

2. The antibody of claim 1, wherein the heavy chain variable region of said antibody has the amino acid sequence set forth in SEQ ID No. 11.

3. The antibody of claim 1, wherein the light chain variable region of said antibody has the amino acid sequence set forth in SEQ ID No. 13.

4. The antibody of any one of claims 1 to 3, wherein the amino acid sequence of the heavy chain of said antibody is as set forth in SEQ ID No. 15.

5. The antibody of any one of claims 1 to 3, wherein the light chain of said antibody has the amino acid sequence set forth in SEQ ID No. 17.

6. A nucleotide sequence encoding the antibody of any one of claims 1 to 5.

7. The nucleotide sequence of claim 6, wherein the nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID No.14, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID No. 14.

8. The nucleotide sequence of claim 6 or 7, wherein the nucleotide sequence encoding the heavy chain of the antibody is represented by SEQ ID No.16 and the nucleotide sequence encoding the light chain of the antibody is represented by SEQ ID No. 18.

9. A transgenic cell line comprising a nucleotide sequence according to any one of claims 6 to 8.

10. A medicament for the treatment of epstein barr virus-related disease, wherein the active ingredient of said medicament is an antibody according to any one of claims 1 to 5.