阅读说明：本技术 Eb病毒balf3蛋白的抗原表位 (Epitope of EB virus BALF3 protein ) 是由 赵维莅 熊杰 于 2020-03-20 设计创作，主要内容包括：本发明涉及分子生物学与免疫学技术领域,尤其涉及EB病毒BALF3蛋白的抗原表位。本发明提供了EB病毒BALF3蛋白的抗原表位,其如SEQ ID NO：1所示,并提供了编码该表位的核酸分子,还提供了其抗原蛋白、特异性抗体及其应用。本发明所述的抗原表位具有良好的保守性、敏感性及特异性,所制得的抗体效价较高,能够应用于临床诊断及分子机制的研究。(The invention relates to the technical field of molecular biology and immunology, in particular to an epitope of BALF3 protein of EB virus. The invention provides an epitope of EB virus BALF3 protein, which is shown as SEQ ID NO: 1, and provides a nucleic acid molecule for coding the epitope, and also provides an antigenic protein, a specific antibody and application thereof. The epitope has good conservation, sensitivity and specificity, and the prepared antibody has high titer and can be applied to clinical diagnosis and the research of molecular mechanism.)

An epitope of EB virus BALF3 protein, which is shown as SEQ ID NO: 1 is shown.

2. A nucleic acid molecule encoding the epitope of claim 1.

3. A nucleic acid vector comprising the nucleic acid molecule of claim 2.

4. A recombinant host comprising the nucleic acid vector of claim 3.

5. Expressing the antigenic protein obtained by the recombinant host of claim 4.

An antibody specific to EB virus BALF3 protein, which is produced by immunizing an animal with the antigenic epitope of claim 1 or the antigenic protein of claim 5.

7. Use of the epitope according to claim 1, the antigenic protein according to claim 5 or the specific antibody according to claim 6 in the preparation of reagents for detecting EB virus BALF3 protein.

8. An agent comprising the antigenic epitope of claim 1, the antigenic protein of claim 5 and/or the specific antibody of claim 6.

9. Use of the antigenic protein of claim 5 in the preparation of a therapeutic antibody and/or vaccine for the prevention and treatment of diseases caused by epstein-barr virus.

10. A therapeutic antibody and/or vaccine prepared from the antigenic protein of claim 5.

Technical Field

The invention relates to the technical field of molecular biology and immunology, in particular to an epitope of BALF3 protein of EB virus.

Background

Epstein-Barr virus (EBV) is a member of the genus lymphotropic virus of the family Herpesviridae, and its genome is DNA. EB virus has the biological property of specifically infecting human and certain primate B cells in vitro and in vivo. Humans are hosts for infection by the EB virus, and are transmitted mainly through saliva. Asymptomatic infection is frequently occurred in children, more than 90% of children 3-5 years old are infected with EB virus, and more than 90% of adults have virus antibodies. EB virus is a pathogen of infectious mononucleosis, and in addition, EB virus has close correlation with occurrence of nasopharyngeal carcinoma and lymphoma of children, and is listed as one of human tumor viruses which are possibly cancerogenic. The EB virus antibodies tested at present mainly comprise Capsid Antigen (CA), Early Antigen (EA) and nuclear antigen (EBNA) aiming at the virus.

BALF3 is a protein in EB virus lytic phase, and has been reported in literature to promote virus self-maturation (PMID: 24554665), participate in immune escape of virus in host human body (PMID: 30133498), and induce host cell genome instability, genetic abnormality and malignant transformation (PMID: 29108278, 25261366). Therefore, the detection of the expression of the BALF3 protein has great significance for researching the pathogenic mechanism and the like. However, the EB virus genome has high variability, and although the sequence of BALF3 is registered in NCBI database, it is still difficult to determine antigen structural domain with high conservation and high specificity, so that there is no BALF3 antibody available for clinical detection and scientific research, and therefore, the expression detection of BALF3 in tumor tissues and the research on pathogenic mechanism thereof are greatly limited.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide an epitope of BALF3 protein of epstein-barr virus, wherein the epitope prepared antibody can be used for specifically detecting expression of BALF3, can meet experimental requirements including immunoblotting, immunohistochemistry, co-immunoprecipitation, chromatin co-precipitation, etc., and is helpful for development of relevant clinical tests and scientific studies to clarify relevance of epstein-barr virus and tumor pathogenesis and molecular mechanisms thereof.

The epitope of the EB virus BALF3 protein provided by the invention is shown as SEQ ID NO: 1 is shown.

The invention obtains a conserved sequence of EB virus BALF3 protein from 31 EBV-infected tumor tissues, wherein the sequence is shown as SEQ ID NO: 3, finally determining that the epitope is SEQ ID NO: 3, i.e. amino acids 510-700 of the amino acid sequence shown in SEQ ID NO: 1. The segment does not contain mutant amino acid sites, has better immunogenicity, stable secondary structure and easy expression.

The invention also provides a nucleic acid encoding SEQ ID NO: 1, or a nucleic acid molecule comprising an epitope of an antigen represented by formula (I).

The nucleotide sequence of the DNA molecule for coding BALF3 protein in the tumor tissue is shown as SEQ ID NO: 4, respectively. Wherein the coding sequence of SEQ ID NO: 1, the total number of 573 bp. In order to ensure that the epitope can be smoothly expressed in a prokaryotic system, the nucleic acid molecule for coding the epitope is subjected to codon optimization, and the optimized sequence is shown as SEQ ID NO: 2, respectively.

The invention also provides a nucleic acid vector comprising a nucleic acid sequence encoding SEQ ID NO: 1, or a nucleic acid molecule comprising an epitope of an antigen represented by formula (I).

In the nucleic acid vector of the present invention, the nucleic acid sequence encoding SEQ ID NO: 1 is shown as SEQ ID NO: 2, respectively. The skeleton vector of the vector is pGEX4T-AB 1.

The invention also provides a recombinant host comprising the nucleic acid vector of the invention.

The nucleic acid vector can express SEQ ID NO: 1, which host is referred to herein as a recombinant host. The host cell is selected from escherichia coli, yeast, insect cells or mammalian cells. In order to achieve more efficient expression of epitopes, prokaryotic expression systems are employed. In an embodiment of the invention, the host cell of the recombinant host is escherichia coli. Coli Rosetta is specifically adopted.

The recombinant host is induced, expressed, separated and purified to obtain the expressed antigen protein. Therefore, the invention also provides the antigen protein obtained by expressing the recombinant host of the invention.

The antigen protein provided by the invention can stimulate a receptor to generate an antibody, and the antibody can be used for preparing an EB virus BALF3 protein detection reagent.

The invention also claims a specific antibody of EB virus BALF3 protein, which is prepared by immunizing animals with the antigen epitope or the fusion protein. Preferably, the specific antibody may be a monoclonal antibody or a polyclonal antibody. In the embodiment of the invention, the antibody is prepared by immunizing rabbits with the epitope or the fusion protein. After immune antiserum is subjected to antigen affinity purification by using pGEX-4T-AB1-BALF3 protein, and concentrated antibody is obtained.

The antigen epitope, the antigen protein or the specific antibody provided by the invention can be applied to preparation of an EB virus BALF3 protein detection reagent.

The antigen epitope, the antigen protein or the specific antibody provided by the invention can be applied to an immunological detection method of EB virus BALF3 protein, such as: enzyme-linked immunosorbent assay, immunoblotting, immunohistochemistry, co-immunoprecipitation, chromatin co-precipitation, and the like. The detection may comprise direct or indirect methods.

The invention also provides an agent comprising an epitope according to the invention, an antigenic protein according to the invention and/or a specific antibody according to the invention.

The reagent provided by the invention can be applied to immunological detection, and comprises a buffer solution, a secondary antibody and a solid support required by detection besides the antigen epitope, the antigen protein and/or the specific antibody.

The antigen protein provided by the invention can stimulate a receptor to generate an antibody, and the antibody has good specificity and can also be used for protecting the receptor against infection of EB virus.

The antigen protein of the invention is applied to the preparation of therapeutic antibodies and/or vaccines for preventing and treating diseases caused by EB virus.

A therapeutic antibody and/or vaccine prepared from the antigenic protein of the invention.

The therapeutic antibodies provided by the invention include antibodies produced by receptors stimulated by the antigenic proteins described herein. The composition can also comprise pharmaceutically acceptable auxiliary materials, and the preparation form of the composition comprises an injection or an oral preparation according to the requirement of practical application.

The invention also provides a vaccine comprising the antigenic protein of the invention and an adjuvant.

Preferably, the adjuvant is aluminum hydroxide adjuvant, Freund's complete adjuvant or Freund's incomplete adjuvant.

The vaccine also comprises a stabilizer, a pH regulator, a preservative and the like. Modulators of the immune response may also be included.

The diseases caused by EB virus include pharyngitis, fever and lymphadenopathy caused by EB virus infection, as well as infectious mononucleosis, nasopharyngeal carcinoma and children's lymphoma.

The invention provides an epitope of EB virus BALF3 protein, which is shown as SEQ ID NO: 1, and provides a nucleic acid molecule for coding the epitope, and also provides an antigenic protein, a specific antibody and application thereof. The epitope has good specificity, and the prepared antibody has high titer and can be applied to clinical diagnosis and the research of molecular mechanism.

Drawings

FIG. 1 shows the alignment of the BALF3 protein sequence with the B95-8 reference sequence for 31 EB virus positive NK/T cell lymphoma patients, wherein BALF3_ temp is the reference sequence and BALF3_ SNV represents the actual sequencing data of 31 EBV positive NKT cell lymphoma samples we completed;

FIG. 2 shows the conserved domain of BALF3_ SNV;

FIG. 3 shows secondary structure prediction of BALF3_ SNV;

figure 4 shows epitope prediction of BALF3_ SNV;

FIG. 5 shows validation of antigen expression plasmids;

FIG. 6 shows the fusion protein expressed by the constructed recombinant host before (left) and after (right) purification;

FIG. 7 shows two antibody strains obtained by immunization for antiserum ELISA detection;

FIG. 8 shows the results of western blot detection of the antigen of the antibodies (E11797, E11798);

FIG. 9 shows the results of antibody-specific detection (i.e., negative for the sample containing no viral protein).

Detailed Description

The invention provides the epitope of EB virus BALF3 protein, and the technical personnel can use the content for reference and appropriately improve the technological parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The "epitope" (epitope) in the present invention is also called an antigenic determinant or epitope, and refers to a specific chemical group in an antigenic molecule that determines the specificity of an antigen. The epitope of the present invention is a polypeptide fragment comprising 191 amino acid residues, and thus, in the present invention, the epitope, the polypeptide are used alternately.

In the present invention, the "nucleic acid molecule" refers to a biological macromolecular compound formed by polymerizing a plurality of nucleotides, wherein the nucleotides can be ribonucleic acid or deoxyribonucleic acid and modifications thereof, including double-stranded or single-stranded DNA, cDNA, RNA, mRNA, and the like, and can be circular or linear, or can be a part of a circular vector or a fragment in a genome.

In the present invention, the "nucleic acid vector" refers to a recombinant DNA molecule comprising a desired coding sequence and suitable nucleic acid sequences necessary for the expression of the operably linked coding gene in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotic cells include promoters, optionally including operator sequences, ribosome binding sites and possibly other sequences. Prokaryotic cells are known to utilize promoters, enhancers and termination and polyadenylation signals. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In the present specification, "plasmid" and "vector" may sometimes be used interchangeably, since plasmids are the most commonly used form of vector at present. However, the present invention is intended to include such other forms of expression vectors which serve equivalent functions, which are or will become known in the art, including, but not limited to: plasmids, phage particles, viral vectors and/or simply potential genomic inserts.

In the present invention, a "host cell" is generally a prokaryotic or eukaryotic host containing a nucleic acid vector and/or a gene of interest. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are competent to replicate the vector encoding the protein or to express the desired protein.

In the present invention, the therapeutic antibody is an antibody that can exert a therapeutic effect by a mechanism such as neutralization, tracing or targeting, competitive inhibition/antagonism, cytotoxic effect of antibody-dependent cell streets and complement-dependent cytolysis, or antigen simulation by an internal imaging effect.

In the present invention, the immunological detection is a technology for qualitatively, quantitatively or locally studying intracellular antigens (polypeptides and proteins) or antibodies by using the basic immunological principle of antigen-antibody reaction, i.e. the principle of antigen-antibody specific binding, and developing a color-developing agent (fluorescein, enzyme, metal ion, isotope) for labeling antibodies through a chemical reaction, and such technologies include, but are not limited to, enzyme-linked immunoassay, immunofluorescence, radioimmunoassay, immunoblotting, immunohistochemistry, co-immunoprecipitation, chromatin co-precipitation, and the like.

In the present invention, the solid support refers to any support to which the epitope, specific antibody and/or antigenic protein described herein can be attached, including but not limited to nitrocellulose membrane, polyvinylidene fluoride (PVDF) membrane, iPDMS chip, microwell plate, microparticle, microcarrier, gel, etc.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:

examples

First, the EBV virus fragments in 31 cases of EBV-infected tumor tissues were detected by whole genome sequencing. Selecting one from more than 120 strains of viruses in NCBI library as a reference nucleic acid sequence, determining virus conserved sequence (the nucleic acid sequence is shown as SEQ ID NO: 4, the protein sequence is shown as SEQ ID NO: 3) by sequence comparison,

2. comparing the conservation of the base sequence and the amino acid sequence of the BALF3 gene between different EB virus strains (figure 1), the result shows that in Human gamma heresvirus 4, the similarity between the BALF3 sequence of the different EB virus strains with the specificity of the gene and the reference sequence is up to 99 percent, and considering that the virus genome has higher variability, the application screens the BALF3 antigen segment based on the relatively conserved whole exon sequence. The antibody designed and prepared on the basis of the segment sequence has reliable detection efficiency, and can avoid false negative results caused by different subtypes of the infected virus strains.

3. Comparison of the conservation between the EB virus strain sequence and the above reference sequence in clinical samples (FIG. 2): the onset of NK/T cell lymphoma is closely related to EB virus infection. By analyzing and detecting EB virus sequences in WGS data of 31 EB virus positive NK/T cell lymphoma patients, extracting sequence information of BALF3, identifying single nucleotide variation sites and amino acid variation sites, comparing the single nucleotide variation sites and the amino acid variation sites with a B95-8 reference sequence, and identifying changed amino acids.

Second, protein expressibility analysis

(1) Analysis of transmembrane structure: non-transmembrane structure

(2) Post-translational modification: temporarily do not have

(3) Conserved domains (FIG. 2)

(4) Secondary structure prediction (FIG. 3)

Third, protein antigenicity analysis

(1) Epitope prediction (FIG. 4)

(2) Antigen region selection

510-700aa (SEQ ID NO: 1): the segment does not contain mutant amino acid sites, has better immunogenicity, stable secondary structure and easy expression, constructs a pGEX4T-AB1 vector, and adopts an escherichia coli prokaryotic system to express protein to prepare antigen.

Fourth, antibody synthesis and efficacy verification

(1) Preparation of antigen expression plasmid

BALF3(510-700aa) PCR product was electrophoretically identified as correct in size (FIG. 5), successfully cloned into pGEX-4T-AB1 vector, and sequenced for correct identification.

(2) Antigenic protein expression

Transformation of recombinant plasmid into expression strain e.coli Rosetta, expression induction conditions: culturing until OD600nm 0.5-0.6, adding 0.8mM IPTG, and inducing at 37 deg.C for 4 hr

Results identification (fig. 6):

pGEX-4T-AB1-BALF3(510-700aa) was expressed in inclusion bodies.

2. The protein concentration after the inclusion body purification is 0.6mg/mL, and the purity reaches the immune requirement.

(3) Rabbit immunization

The flow is shown in table 1:

TABLE 1 immunization protocol

Number of immunizations	Immune cycle	Immunization dose	Immunologic adjuvant	Immunizing animal conditions
					First immunization	1 day	0.3mg	Complete Freund's adjuvant	Good effect
Second immunization	12 days	0.15mg	Incomplete Freund's adjuvant	Good effect
					Third immunization	26 days	0.15mg	Incomplete Freund's adjuvant	Good effect
The fourth immunization	40 days	0.15mg	Incomplete Freund's adjuvant	Good effect
					Blood sampling for immunized animals	For 52 days			Normal blood sampling

(4) The antiserum ELISA examination is carried out by setting a blank control group without adding antibody or serum, a negative control group with adding antibody but without adding serum, and an experimental group with adding serum and antibody with different concentrations. The two antibodies obtained by the construction are respectively marked as BALF3-E11787 and BALF3-E11788, and the detection results are shown in FIG. 7. The results show that both antibodies show good titers.

(5) Antiserum purification

The pGEX-4T-AB1-BALF3 protein for affinity purification is detected to have the concentration of 0.5mg/ml, has little difference with the concentration and the purity after bacteria breaking and purification, and can be used for antigen affinity purification.

The antiserum was purified by antigen affinity using pGEX-4T-AB1-BALF3 protein to obtain concentrated antibodies: e11797 concentration of 2.65mg/ml, E11798 concentration of 2.78mg/ml

(6) Antigen western blot detection

The purified antibody is used for detecting the antigen, and the result is shown in figure 8, and the result shows that:

the sizes of antigen bands detected by E11797 and E11798 antibodies are about 49 KD;

② the 500pg antigen can be detected by diluting the E11797 and E11798 antibodies 1: 1000;

and the concentrations of E11797 and E11798 antibodies are normal.

The results show that the epitope designed and obtained by the invention can specifically detect BALF3 protein, and the antibody has good titer.

(7) Specificity verification

The human cell line lysate samples were tested negative with the above antibodies (FIG. 9), as shown in the figure, BALF3 theoretical size 74,764Da, and the red boxes should be the target protein blot. B95-8 is a cell strain capable of secreting EBV, endogenously expressed BALF3 can be detected (positive control), and BALF3 is not detected by SNK6 cell strain without EBV components (negative control).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

<110> Renjin Hospital affiliated to Shanghai university of transportation medical school

<120> epitope of BALF3 protein of EB virus

<130> MP1914455

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 191

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Pro Glu Val Pro Arg Pro Ala Gly Ala Arg Glu Pro Gly Pro Ser Gly

1 5 10 15

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

20 25 30

Ala Thr Glu Gly Leu Ala Lys Leu Gln Ser Cys Leu Ala Gln Gln Ser

35 40 45

Glu Thr Leu Thr Glu Thr Leu Cys Leu Arg Val Trp Gly Asp Val Val

50 55 60

Tyr Trp Glu Leu Ala Arg Met Arg Asn His Phe Leu Tyr Arg Arg Ala

65 70 75 80

Phe Val Ser Gly Pro Trp Glu Asp Arg Arg Ala Gly Glu Gly Ala Ala

85 90 95

Phe Glu Asn Ser Lys Tyr Ile Lys Thr His Leu Phe Thr Gln Thr Leu

100 105 110

Ser Ser Glu His Leu His Ala Leu Thr His Ser Leu Tyr Thr Phe Ile

115 120 125

Thr Gly Pro Leu Ala Glu Glu Ser Gly Leu Phe Pro Pro Pro Ser Asn

130 135 140

Val Ala Leu Ala Arg Cys Cys Asp Ala Ala Gly Thr Leu Pro His Gln

145 150 155 160

Lys Ala Phe Leu Thr Ser Leu Ile Trp Pro Gly Ile Glu Pro Ser Asp

165 170 175

Trp Ile Glu Thr Ser Phe Asn Ser Phe Tyr Ser Val Pro Gly Gly

180 185 190

<210> 2

<211> 573

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ccggaggtgc cccggccggc tggggcacga gagcccggcc cgtccggcgc cctctcggac 60

gcgctcaagc gcaaggagca gtacctgcgc caggtggcca ccgagggtct ggccaagctg 120

cagtcctgcc tggcgcaaca gagcgagacc ctgaccgaga ccctgtgcct gcgcgtctgg 180

ggggacgtgg tctactggga gctggcccgc atgcgcaacc acttcctcta cagacgggcc 240

ttcgtctcgg gtccctggga ggacaggcgc gccggcgagg gtgccgcctt tgagaattcc 300

aagtacatca aaacacacct gtttacccag accctgagct cggagcacct gcacgcgctg 360

acgcacagcc tgtacacctt catcacgggg cccctggcgg aggagagcgg gctctttccc 420

ccacccagca acgtggccct ggctcgctgc tgtgacgccg caggtacgct gccccaccag 480

aaggcattcc tgacttccct gatatggcca ggcatcgagc cgagcgactg gatagagacc 540

tccttcaaca gcttctacag cgtacccggg ggc 573

<210> 3

<211> 789

<212> PRT

<213> EB virus (Epstein-Barr virus)

<400> 3

Gly Arg Arg Gly Val Leu Ile Gly Pro Leu Leu Arg Pro Gly Gly Gln

1 5 10 15

Arg Pro Arg Asn Pro Gly Asp His Cys Leu Gln Arg Asp Arg Val Asp

20 25 30

Gly Gly Gly Arg Ser Gly Leu Ser Gly Gly Ser Val Gly Trp Ala Arg

35 40 45

Gly Ser Pro Gly Gln Leu Tyr Gln Arg Gly Arg Ala Gly Gln His Arg

50 55 60

Val Ala Gly Leu Gly Arg Ala Ala Ala Gly Trp Arg Arg Ala Gly Leu

65 70 75 80

Arg Gly Gln Thr Gln Ala Pro Ser Gly His Arg Ser Pro Arg Thr Arg

85 90 95

Gly Leu Glu Thr Pro Gly Ala Ala Met Ser Gly Leu Leu Ala Ala Ala

100 105 110

Tyr Ser Gln Val Tyr Ala Leu Ala Val Glu Leu Ser Val Cys Thr Arg

115 120 125

Leu Asp Pro Arg Ser Leu Asp Val Ala Ala Val Val Arg Asn Ala Gly

130 135 140

Leu Leu Ala Glu Leu Glu Ala Ile Leu Leu Pro Arg Leu Arg Arg Gln

145 150 155 160

Asn Asp Arg Ala Cys Ser Ala Leu Ser Leu Glu Leu Val His Leu Leu

165 170 175

Glu Asn Ser Arg Glu Ala Ser Ala Ala Leu Leu Ala Pro Gly Arg Lys

180 185 190

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

195 200 205

Glu Phe Tyr Gly Gly His Lys Val Asp Val Ser Leu Cys Leu Ile Asn

210 215 220

Asp Ile Glu Ile Leu Met Lys Arg Ile Asn Ser Val Phe Tyr Cys Met

225 230 235 240

Ser His Thr Met Gly Leu Glu Ser Leu Glu Arg Ala Leu Asp Leu Leu

245 250 255

Gly Arg Phe Arg Gly Val Ser Pro Ile Pro Asp Pro Arg Leu Tyr Ile

260 265 270

Thr Ser Val Pro Cys Trp Arg Cys Val Gly Glu Leu Met Val Leu Pro

275 280 285

Asn His Gly Asn Pro Ser Thr Ala Glu Gly Thr His Val Ser Cys Asn

290 295 300

His Leu Ala Val Pro Val Asn Pro Glu Pro Val Ser Gly Leu Phe Glu

305 310 315 320

Asn Glu Val Arg Gln Ala Gly Leu Gly His Leu Leu Glu Ala Glu Glu

325 330 335

Lys Ala Arg Pro Gly Gly Pro Glu Glu Gly Ala Val Pro Gly Pro Gly

340 345 350

Arg Pro Glu Ala Glu Gly Ala Thr Arg Ala Leu Asp Thr Tyr Asn Val

355 360 365

Phe Ser Thr Val Pro Pro Glu Val Ala Glu Leu Ser Glu Leu Leu Tyr

370 375 380

Trp Asn Ser Gly Gly His Ala Ile Gly Ala Thr Gly Gln Gly Glu Gly

385 390 395 400

Gly Gly His Ser Arg Leu Ser Ala Leu Phe Ala Arg Glu Arg Arg Leu

405 410 415

Ala Leu Val Arg Gly Ala Cys Glu Glu Ala Leu Ala Gly Ala Arg Leu

420 425 430

Thr His Leu Phe Asp Ala Val Ala Pro Gly Ala Thr Glu Arg Leu Phe

435 440 445

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

450 455 460

Ala Asp Cys Ala Ala Ala Phe Thr Ala His Pro Gln Tyr Arg Ala Ile

465 470 475 480

Leu Gln Lys Arg Asn Glu Leu Tyr Thr Arg Leu Asn Arg Ala Met Gln

485 490 495

Arg Leu Gly Arg Gly Glu Glu Glu Ala Ser Arg Glu Ser Pro Glu Val

500 505 510

Pro Arg Pro Ala Gly Ala Arg Glu Pro Gly Pro Ser Gly Ala Leu Ser

515 520 525

Asp Ala Leu Lys Arg Lys Glu Gln Tyr Leu Arg Gln Val Ala Thr Glu

530 535 540

Gly Leu Ala Lys Leu Gln Ser Cys Leu Ala Gln Gln Ser Glu Thr Leu

545 550 555 560

Thr Glu Thr Leu Cys Leu Arg Val Trp Gly Asp Val Val Tyr Trp Glu

565 570 575

Leu Ala Arg Met Arg Asn His Phe Leu Tyr Arg Arg Ala Phe Val Ser

580 585 590

Gly Pro Trp Glu Asp Arg Arg Ala Gly Glu Gly Ala Ala Phe Glu Asn

595 600 605

Ser Lys Tyr Ile Lys Thr His Leu Phe Thr Gln Thr Leu Ser Ser Glu

610 615 620

His Leu His Ala Leu Thr His Ser Leu Tyr Thr Phe Ile Thr Gly Pro

625 630 635 640

Leu Ala Glu Glu Ser Gly Leu Phe Pro Pro Pro Ser Asn Val Ala Leu

645 650 655

Ala Arg Cys Cys Asp Ala Ala Gly Thr Leu Pro His Gln Lys Ala Phe

660 665 670

Leu Thr Ser Leu Ile Trp Pro Gly Ile Glu Pro Ser Asp Trp Ile Glu

675 680 685

Thr Ser Phe Asn Ser Phe Tyr Ser Val Pro Gly Gly Ser Leu Ala Ser

690 695 700

Ser Gln Gln Ile Leu Cys Arg Ala Leu Arg Glu Ala Val Leu Thr Val

705 710 715 720

Ser Leu Tyr Asn Lys Thr Trp Gly Arg Ser Leu Ile Leu Arg Arg Ala

725 730 735

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

740 745 750

Thr Tyr Asp Ser Asp Arg Pro Leu Ile Leu Leu Tyr Lys Gly Arg Gly

755 760 765

Trp Val Phe Lys Asp Leu Tyr Ala Leu Leu Tyr Leu His Leu Gln Met

770 775 780

Arg Asp Asp Ser Ala

785

<210> 4

<211> 2367

<212> DNA

<213> EB virus (Epstein-Barr virus)

<400> 4

ggacgacgcg gagtacttat tgggccgctt ctccgtcctg gcggacagcg tcctagaaac 60

cctggcgacc attgcctcca gcgggataga gtggacggcg gaggccgctc gggactttct 120

ggagggagtg tggggtgggc ccggggcagc ccaggacaac tttatcagcg tggccgagcc 180

ggtcagcacc gcgtcgcagg cctcggccgg gctgctgctg ggtggaggag ggcagggctc 240

cgggggcaga cgcaagcgcc gtctggccac cgttctcccc ggactcgagg tctagagacc 300

cctggggcgg cgatgtcggg gctgctggcg gcggcgtaca gccaggtgta cgccctggcg 360

gttgagctga gcgtgtgcac ccggctggac ccccggagtc tggacgtggc tgcggtggtg 420

cgcaacgccg gcctgctggc cgagctggag gccatcctcc ttccccgttt gagacggcag 480

aatgaccgtg catgcagcgc cctgtccctg gagctggtgc acctgctaga gaactcgaga 540

gaggcctctg ccgcgctgct cgcccctggt agaaagggta cccgggtccc gcctctccgt 600

accccctcag tcgcgtactc tgtggagttt tacggggggc ataaagtcga tgtaagtttg 660

tgcctaataa atgacataga gattttaatg aagagaatca atagcgtgtt ttattgcatg 720

tctcacacca tggggctgga gagcctggaa cgggccctgg atctgctggg ccgctttcgg 780

ggcgtaagtc ccatcccaga cccgcgcctc tacatcacct ctgtgccctg ctggcgctgt 840

gtgggcgagc tgatggttct gcccaaccac ggcaaccctt ccacggcaga ggggacccac 900

gtctcctgta accacctggc ggtgccggtg aatccggagc cggtctcggg actgtttgag 960

aatgaagtcc gccaggcggg gctcgggcac ctgttggagg ctgaggagaa ggcgaggccg 1020

ggcggcccag aggagggcgc ggtcccgggc ccggggcggc cggaggcaga gggggcgacc 1080

agagcgctgg acacctacaa cgtcttctcg acagtgcccc cggaggtggc ggagctctcg 1140

gagctcctct attggaactc tggcggccat gctatcggtg caacggggca gggggagggt 1200

ggcggccatt cccgcctctc tgccctgttt gcccgggagc gtcgcctggc cctggtgcgg 1260

ggggcctgcg aggaggcgct ggcgggggca aggctgactc acctgtttga cgccgtggct 1320

cccggggcca cggagcggct cttctgcggc ggggtctaca gctcctcggg cgacgcggtg 1380

gaggcgctga aggcggactg cgccgccgcc ttcacggcgc acccccagta ccgggccatc 1440

ctgcaaaaga ggaacgagct gtacacgcgg ctcaaccgag ccatgcagcg gttgggccga 1500

ggcgaggagg aggcgtcccg ggagagcccg gaggtgcccc ggccggctgg ggcacgagag 1560

cccggcccgt ccggcgccct ctcggacgcg ctcaagcgca aggagcagta cctgcgccag 1620

gtggccaccg agggtctggc caagctgcag tcctgcctgg cgcaacagag cgagaccctg 1680

accgagaccc tgtgcctgcg cgtctggggg gacgtggtct actgggagct ggcccgcatg 1740

cgcaaccact tcctctacag acgggccttc gtctcgggtc cctgggagga caggcgcgcc 1800

ggcgagggtg ccgcctttga gaattccaag tacatcaaaa cacacctgtt tacccagacc 1860

ctgagctcgg agcacctgca cgcgctgacg cacagcctgt acaccttcat cacggggccc 1920

ctggcggagg agagcgggct ctttccccca cccagcaacg tggccctggc tcgctgctgt 1980

gacgccgcag gtacgctgcc ccaccagaag gcattcctga cttccctgat atggccaggc 2040

atcgagccga gcgactggat agagacctcc ttcaacagct tctacagcgt acccgggggc 2100

tcactggcat ctagccagca aattctgtgc cgggcgctgc gcgaggccgt cctaaccgtg 2160

tccctctaca acaagacctg ggggcggtcc ttgatcctgc gccgggcgga cgcggtcagc 2220

cccggccagg ccctgccccc ggatgggctt tacctcacgt acgactctga ccgcccccta 2280

attcttctgt ataagggcag ggggtgggta tttaaggatc tatatgccct tctctacctg 2340

cacctccaaa tgagagatga ctcggcg 2367