阅读说明：本技术 Eb病毒balf4蛋白的抗原表位 (Epitope of EB virus BALF4 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BALF4蛋白的抗原表位。本发明提供了EB病毒BALF4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 BALF4 protein of EB virus. The invention provides an epitope of EB virus BALF4 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 BALF4 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 BALF4 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 BALF4 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 BALF4 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.

BALF4 is an EB virus lytic phase protein, and has been reported to be related to virus immunogenicity (PMID: 19244320, 16549597), virus infection host ability (PMID: 12409611), virus replication in a host (PMID: 8627735) and tumor-related Jak-STAT and Phosphonotdylinosol signaling pathway activation (PMID: 28415594). Therefore, the detection of the expression of the BALF4 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 BALF4 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 BALF4 antibody available for clinical detection and scientific research, and therefore, the expression detection of BALF4 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 BALF4 protein of epstein-barr virus, wherein the epitope prepared antibody can be used for specifically detecting expression of BALF4, 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 BALF4 protein provided by the invention is shown as SEQ ID NO: 1 is shown.

The invention obtains a conserved sequence of EB virus BALF4 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, namely amino acids 245-400 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 BALF4 protein in the tumor tissue is shown as SEQ ID NO: 4, respectively. Wherein the coding sequence of SEQ ID NO: 1, the total length of the nucleic acid molecules of the epitope shown in the specification is 156 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 BALF4 protein detection reagent.

The invention also claims a specific antibody of EB virus BALF4 protein, which is prepared by immunizing animals with the antigen epitope or the antigen 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 antigen epitope or the antigen protein. After immune antiserum is subjected to antigen affinity purification by using pGEX-4T-AB1-BALF4 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 BALF4 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 BALF4 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 BALF4 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 a conservative analysis of BALF4_ SNV;

FIG. 2 shows the alignment of the BALF4 protein sequence with the B95-8 reference sequence for 31 EB virus positive NK/T cell lymphoma patients, wherein BALF4_ temp is the reference sequence and BALF4_ SNV represents the actual sequencing data of 31 EBV positive NKT cell lymphoma samples we completed;

FIG. 3 shows the BALF4_ SNV transmembrane structural analysis;

FIG. 4 shows BALF4_ SNV post-translational modifications;

FIG. 5 shows BALF4_ SNV conserved domain;

FIG. 6 shows BALF4_ SNV tertiary structure analysis;

FIG. 7 shows secondary structure prediction of BALF4_ SNV;

figure 8 shows epitope prediction of BALF4_ SNV;

FIG. 9 shows validation of antigen expression plasmids;

FIG. 10 shows the fusion protein expressed by the constructed recombinant host before (left) and after (right) purification;

FIG. 11 shows the results of western blot detection of the antigen of the antibodies (E11787, E11788);

FIG. 12 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 BALF4 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 to enhance signal transduction and termination and polyadenylation. 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

Firstly, the invention analyzes and detects EB virus sequences in WGS data of 31 cases of patients with NK/T cell lymphoma with EB virus positive, extracts sequence information of BALF4, identifies single nucleotide variation site and amino acid variation site, compares with B95-8 reference sequence, identifies variation amino acid, determines virus conserved sequence (nucleic acid sequence is shown as SEQ ID NO: 4, protein sequence is shown as SEQ ID NO: 3),

2. comparing the conservation of the base sequence and the amino acid sequence of the BALF4 gene between different EB virus strains (figure 1), the result shows that the similarity between the BALF4 sequence of the different specific EB virus strains of the analyzed gene and the reference sequence is up to 99% in Human gamma herpesvims 4, and considering that the viral 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 BALF4, 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: FIG. 3

(2) Post-translational modification: FIG. 4

(3) Conserved domain: FIG. 5

(4) And (3) predicting a tertiary structure: FIG. 6

(4) And (3) secondary structure prediction: FIG. 7

Third, protein antigenicity analysis

(1) Epitope prediction (FIG. 8)

(2) Antigen region selection

245-400aa (SEQ ID NO: 1): the segment does not contain mutant amino acid sites, has better immunogenicity, stable secondary structure and easy expression, is suggested to construct pGEX4T-AB1 vectors, and adopts an escherichia coli prokaryotic system to express proteins to prepare antigens.

Fourth, antibody synthesis and efficacy verification

(1) Preparation of antigen expression plasmid

BALF4(245-400aa) PCR product was electrophoretically identified as correct in size, successfully cloned into pGEX-4T-AB1 vector, and sequenced as correct (FIG. 9).

(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

Analysis of results (fig. 10):

pGEX-4T-AB1-BALF4(245-400aa) was expressed in inclusion bodies.

2. The concentration of the inclusion body protein is 8mg/mL, the purity meets the requirement of immunity, and the immunity is transferred.

(3) Rabbit immunization

The process is as follows:

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-BALF4 protein for affinity purification is detected to have the concentration of 6mg/ml, has little difference with the concentration and 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-BALF4 protein to obtain concentrated antibodies: the concentration of E11443 is 2.93 mg/ml; the concentration of E11444 was 1.96 mg/ml.

(6) Antigen western blot detection

The purified antibody is used for detecting the antigen, and the result is shown in figure 11, and the result shows that:

and (4) analyzing results:

1. the sizes of the antigen bands detected by the E11443 and E11444 antibodies are about 45 KDa;

2. e11443, E11444 antibody 1: 500pg antigen was detectable at 1000 dilution;

3. e11443, E11444 antibody concentrations were 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

When the antibody is used for detecting a sample without virus protein, the detection result is negative (figure 12), and the figure shows that: BALF4 has a theoretical size of 95,639Da and should be a mark of the protein of interest as shown in the red box. Description of the drawings: b95-8 is a cell strain capable of secreting EBV, endogenously expressed BALF4 (positive control) can be detected, SNK6 is a cell strain without EBV components, and BALF4 (negative control) is not detected.

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 BALF4 protein of EB virus

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caaacctttg actcgaccat cgccacagaa acagggaagt caatacattt tgtgactgac 1440

gagggcacct ctagcttcgt gaccaacaca accgtgggca tagagctccc ggacgccttc 1500

aagtgcatcg aagagcaggt gaacaagacc atgcatgaga agtacgaggc cgtccaggat 1560

cgttacacga agggccagga agccattaca tattttataa cgagcggagg attgttatta 1620

gcttggctac ctctgacccc gcgctcgttg gccaccgtca agaacctgac ggagcttacc 1680

actccgactt cctcaccccc cagcagtcca tcgcccccag ccccatccgc ggcccgcggg 1740

agcacccccg ccgccgttct gaggcgtcgg aggcgggatg cggggaacgc caccacaccg 1800

gtgcccccca cggcccccgg gaagtccctg ggcaccctca acaatcccgc caccgtccag 1860

atccaatttg cctacgactc cctgcgccgc cagatcaacc gcatgctggg agaccttgcg 1920

cgggcctggt gcctggagca gaagaggcag aacatggtgc tgagagaact aaccaagatt 1980

aatccaacca ccgtcatgtc cagcatctac ggtaaggcgg tggcggccaa gcgcctgggg 2040

gatgtcatct cagtctccca gtgcgtgccc gttaaccagg ccaccgtcac cctgcgcaag 2100

agcatgaggg tccctggctc cgagaccatg tgctactcgc gccccctggt gtccttcagc 2160

tttatcaacg acaccaagac ctacgaggga cagctgggca ccgacaacga gatcttcctc 2220

acaaaaaaga tgacggaggt gtgccaggcg accagccagt actacttcca gtccggcaac 2280

gagatccacg tctacaacga ctaccaccac tttaaaacca tcgagctgga cggcattgcc 2340

accctgcaga ccttcatctc actaaacacc tccctcatcg agaacattga ctttgcctcc 2400

ctggagctgt actcacggga cgaacagcgt gcctccaacg tctttgacct ggagggcatc 2460

ttccgggagt acaacttcca ggcgcaaaac atcgccggcc tgcggaagga tttggacaat 2520

gcagtgtcaa acggaagaaa tcaattcgtg gacggcctgg gggaacttat ggacagtctg 2580

ggtagcgtgg gtcagtccat caccaaccta gtcagcacgg tggggggttt gtttagcagc 2640

ctggtctctg gtttcatctc cttcttcaaa aaccccttcg gcggcatgct cattctggtc 2700

ctggtggcgg gggtggtgat cctggttatt tccctcacga ggcgcacgcg ccagatgtcg 2760

cagcagccgg tgcagatgct ctaccccggg atcgacgagc tcgctcagca acatgcctct 2820

ggtgagggtc caggcattaa tcccattagt aagacagaat tacaagccat catgttagcg 2880

ctgcatgagc aaaaccagga gcaaaagaga gcagctcaga gggcggccgg accctcagtg 2940

gccagcagag cattgcaggc agccagggac cgttttccag gcctacgcag aagacgctat 3000

cacgatccag agaccgccgc cgcactgctt ggggaggcag agactgagtt ttaa 3054