CN113527441A

CN113527441A - Adenovirus antigen polypeptide and application thereof

Info

Publication number: CN113527441A
Application number: CN202110732142.7A
Authority: CN
Inventors: 田新贵; 刘振卫; 周志超; 李潇; 刘文宽; 周荣
Original assignee: First Affiliated Hospital of Guangzhou Medical University
Current assignee: First Affiliated Hospital of Guangzhou Medical University
Priority date: 2021-06-29
Filing date: 2021-06-29
Publication date: 2021-10-22

Abstract

The invention relates to the field of immunology, in particular to an adenovirus antigen polypeptide and application thereof. The antigen polypeptide provided by the invention is simple to prepare, can be expressed by using the simplest escherichia coli expression system, and the expressed knob polypeptide has a trimer structure, high expression quantity, strong immunogenicity, convenient production and purification and low cost; the knob polypeptide has strong immunogenicity, and can induce a neutralizing antibody with high titer; most importantly, neutralizing antibodies induced by HAdV-40L-Knob or HAdV-41L-Knob have cross-neutralization effects on HAdV-40 and HAdV-41, and the neutralizing titers are similar, so that the vaccine can be applied as a broad-spectrum human intestinal adenovirus vaccine.

Description

Adenovirus antigen polypeptide and application thereof

Technical Field

The invention relates to the field of immunology, in particular to an adenovirus antigen polypeptide and application thereof.

Background

Human adenovirus of the intestinal tract (HAdV) was first isolated from Human adenoid tissue in 1953 and was named adenovirus. It has affinity for lymph glands and can remain latent in lymph glands for a long time. They cause a variety of diseases including upper respiratory tract infections, conjunctivitis (eye infections), bladder infections, pneumonia, encephalitis and gastroenteritis. They are also associated with heart disease and obesity.

The HAdV comprises two types of HAdV-40 and HAdV-41, is a second important pathogen causing viral diarrhea in children, and no specific medicine or vaccine exists at present, and clinical support and symptomatic treatment are mainly used. The recombinant protein vaccine has low cost and convenient preparation, and a plurality of recombinant protein vaccines are clinically used at present, but no vaccine aiming at human intestinal adenovirus exists.

The adenovirus capsid mainly comprises ciliin, penton base and hexon, and the hexon is generally considered as the main neutralizing antigen of adenovirus in the prior art research, while the ciliin is a receptor binding protein for adsorbing cells, is a minor neutralizing antigen, has poor capability of inducing neutralizing antibody, and the main structural domain for binding with the receptor is positioned at the fiber hair protein head (knob). Adenovirus comprises 7 groups of 52 serotypes, and has no cross-neutralization among different serotypes, including between HAdV-40 and HAdV-41. The applicant of the invention has found and proved that an escherichia coli expression system respectively expresses 3, 7, 11 and 55 adenovirus knob polypeptides of human in a recombination manner, an immune mouse can induce high-titer neutralizing antibodies, and some types of knob antibodies have certain cross-neutralization effect and can be used as multivalent adenovirus vaccine candidates.

Complete HAdV-40 and HAdV-41 virion immunity can only induce type specific neutralizing antibody without type cross protection; recombination is easy to occur among different types of adenovirus gene groups, and a novel adenovirus with stronger toxicity can be generated; the human intestinal adenovirus cells are difficult to culture and have low yield; therefore, it is difficult to develop a conventional inactivated or attenuated live vaccine for human enteroadenovirus. Therefore, the problem to be solved is how to develop a vaccine which can prevent HAdV-40 and HAdV-41 simultaneously and has high immunogenicity and low cost.

Disclosure of Invention

The present inventors have unexpectedly found that, when the genomes of two adenoviruses, HAdV-40 and HAdV-41, are studied, the long fiber proteins of HAdV-40 and HAdV-41 have very high similarity, while the short fiber proteins have large difference, and it is presumed that the long fiber proteins may be receptor-binding proteins, and the cilium portion (L-Knob) of the long fiber proteins may be a receptor-binding domain, and may serve as a candidate for subunit vaccines. The invention is thus developed after testing. Specifically, in order to achieve the above object of the present invention, the following technical solutions are adopted:

the first aspect of the present invention relates to an antigenic polypeptide which is SEQ ID NO: 1 and/or SEQ ID NO: 2.

A second aspect of the invention relates to an isolated nucleic acid expressing the antigenic polypeptide of claim 1.

Alternatively, a nucleic acid as described above which is SEQ ID NO: 3 and/or SEQ ID NO: 4.

A third aspect of the invention relates to a vector comprising a nucleic acid as described above.

A fourth aspect of the invention relates to a host cell comprising a nucleic acid as described above, or transformed with a vector as described above.

Alternatively, the host cell as described above, which is E.coli.

A fifth aspect of the invention relates to a method of producing an antigenic polypeptide as described above, comprising:

culturing a host cell as described above under suitable culture conditions; and

recovering the antigenic polypeptide so produced from the culture medium or from the cultured host cells.

A sixth aspect of the invention relates to a vaccine comprising an antigenic polypeptide as described above, or a nucleic acid as claimed in claim 2 or 3, or a vector as claimed in claim 4.

Optionally, the vaccine as described above, further comprising an adjuvant.

A seventh aspect of the invention relates to a kit comprising a vaccine as described above.

An eighth aspect of the invention relates to the use of an antigenic polypeptide as described above, or a nucleic acid as described above, or a vector as described above, or a vaccine as described above, in the manufacture of a medicament for the prevention of infection by an adenovirus of the type HAdV-40 and/or HAdV-41.

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

the antigen polypeptide provided by the invention is simple to prepare, can be expressed by using the simplest escherichia coli expression system, and the expressed knob polypeptide has a trimer structure, high expression quantity, strong immunogenicity, convenient production and purification and low cost; the knob polypeptide has strong immunogenicity, and can induce a neutralizing antibody with high titer; most importantly, neutralizing antibodies induced by HAdV-40L-Knob or HAdV-41L-Knob have cross-neutralization effects on HAdV-40 and HAdV-41, and the neutralizing titers are similar, so that the vaccine can be applied as a broad-spectrum human intestinal adenovirus vaccine.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an SDS-PAGE of purified products of adenovirus cilia protein knob according to one embodiment of the present invention; r is a boiled sample, and NR is an unboiled sample;

FIG. 2 shows the results of a cell micro-neutralization experiment according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention relates to an antigenic polypeptide which is SEQ ID NO: 1 and/or SEQ ID NO: 2.

When containing both SEQ ID NOs: 1 and SEQ ID NO: 2, the two can be used in the form of a mixture or a fusion protein.

The component of the invention also comprises a nucleotide sequence similar to SEQ ID NO: 1 and SEQ ID NO: 2, such as a polypeptide substantially similar to the polypeptide set forth in SEQ ID NO: 1 and SEQ ID NO: 2, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity, preferably with the respective function being retained.

A "substantially similar" amino acid sequence may also be a sequence comprising only SEQ ID NO: 1 and SEQ ID NO: 2, conservative amino acid substitution of the polypeptide shown in the figure. For example, a given amino acid sequence shares at least 85%, more preferably at least 90% and even more preferably at least 95% identity with a reference sequence. In addition, in general, only sequences describing or encoding proteins in which only conservative substitutions are made in conserved regions are substantially similar. Preferably, the substantially similar sequences also retain the unique activity of the polypeptide. Substitutions which are generally regarded as conservative substitutions are substitutions for one another in the aliphatic amino acids Ala, Val, Leu and Ile, for the hydroxyl residues Ser and Thr, for the acidic residues Asp and Glu, for the amide residues Asn and Gln, for the basic residues Lys and Arg and for the aromatic residues Phe, Tyr.

The "substantially similar" amino acid sequence may also be SEQ ID NO: 1 and SEQ ID NO: 2. The term "derivative" refers to a chemically modified protein or polypeptide that has been chemically modified by natural processes (such as processing and other post-translational modifications) as well as by chemical modification techniques, such as by the addition of one or more polyethylene glycol molecules, sugars, phosphates, and/or other such molecules, wherein the one or more molecules are not naturally attached to the wild-type protein. Derivatives include salts. Such chemical modifications are described in detail in basic texts and in more detailed monographs, as well as in a large number of research documents, and they are well known to those skilled in the art. It is understood that the same type of modification may be present to the same or different degrees at several sites in a given protein or polypeptide. In addition, a given protein or polypeptide may contain many types of modifications. Modifications can occur anywhere in a protein or polypeptide, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamic acid, methylation, gamma-carboxylation, glycosylation, GPI-anchoring, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation and ADP-ribosylation, selenization, sulfation, transfer RNA-mediated addition of amino acids to proteins (such as arginylation), and ubiquitination. They may also be bound to vitamins such as biotin, folic acid or vitamin B12. See, e.g., Proteins-Structure And Molecular Properties,2nd Ed., T.E.Creighton, W.H.Freeman And Company, New York (1993) And world, F., "Postrelational Protein Modifications: perspectra and Prospectra, "pgs.1-12 in Posttranslation equivalent Modification Of Proteins, B.C. Johnson, Ed., Academic Press, New York (1983); seifter et al, meth. enzymol.182: 626 + 646(1990) and Rattan et al, "Protein Synthesis: posttranslation Modifications and Aging, "Ann.N.Y.Acad.Sci.663: 48-621992). The term "derivative" includes chemical modifications that result in a protein or polypeptide becoming branched or cyclic, with or without branching. Cyclic, branched and branched circular proteins or polypeptides can be naturally processed post-translationally and can also be made entirely by synthetic methods. In some embodiments, the compound may be covalently linked to a carrier protein, such as serum albumin or other plasma proteins.

In some embodiments, the fusion polypeptide further comprises one or more linker peptides.

In some embodiments, the number of amino acids of the linker peptide is 1 to 30; there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

In some embodiments, the amino acids of the linker peptide are nonsense polypeptides that do not have additional functions (e.g., protein localization, cleavage sites, etc.) other than linking.

In some embodiments, the linker peptide is a flexible linker peptide;

in some embodiments, the amino acid sequence of the linking peptide is selected from one or more of Gly, Ser, Pro, Ala, and Glu.

In some embodiments, the amino acid sequence of the linker peptide is selected from (GGGGS) n, (GGGS) n, (GGS) n, (GS) n, or (G) n, wherein n represents the number of repeats selected from 1, 2, 3, 4, 5, or 6.

The linker peptide is generally flexible and can reduce steric hindrance between the fusion protein and the protein of interest, thereby facilitating proper folding of the protein.

In further embodiments, the linker peptide is a rigid linker peptide; i.e. a relatively inflexible peptide linker. Rigid linker peptides do not require a complete lack of flexibility, but are less flexible than flexible linker peptides such as glycine-rich peptide linkers. Due to its relative lack of flexibility, the rigid linker peptide reduces the movement of two protein domains (in the present case a stabilizer protein and a thermostable reverse transcriptase) linked together by the rigid linker peptide. A linker peptide providing an ordered chain (e.g., an alpha helical structure) can provide a rigid linker peptide. For example, arginine, leucine, glutamic acid, glutamine and methionine all show a tendency to have a relatively high helical structure. However, non-helical linkers containing many proline residues may also exhibit significant rigidity. Examples of rigid linking peptides include polylysine and poly-DL-alanine polylysine. Further description of rigid peptide linkers is provided by Wriggers et al, Biopolymers, 80, pages 736-46 (2005). In addition, rigid linker peptides are described in the linker database described by George et al, Protein Engineering, 15, pp 871-79 (2003). Preferably, the rigid linking peptide is also a non-cleavable linker peptide, i.e. a non-cleavable rigid linking peptide.

The invention also relates to an isolated nucleic acid expressing an antigenic polypeptide as described above.

The nucleic acid may be RNA or DNA.

In some embodiments, the nucleic acid is codon optimized.

In some embodiments, the nucleic acid is SEQ ID NO: 3 and/or SEQ ID NO: 4.

When containing both SEQ ID NOs: 3 and SEQ ID NO: 4, the two can be expressed as a fusion.

According to an aspect of the invention, it also relates to a vector comprising a nucleic acid as described above.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the present invention.

In some embodiments, the vector of the present invention may further comprise a gene used for screening (e.g., an antibiotic resistance gene), a nucleic acid for producing a fluorescent protein, or the like. The fluorescent protein can be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein.

The green fluorescent protein can adopt common GFP, and can also adopt modified GFP genes, such as enhanced GFP gene EGFP and the like; the blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP and the like; the yellow fluorescent protein can be selected from EYFP, Ypct, PhiYFP and the like; the orange fluorescent protein can be selected from mKO, mOrange, mBanana and the like; the red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKate and the like.

The invention also relates to a host cell containing a nucleic acid as described above, or transformed with a vector as described above.

The term "host cell" refers to a cell which can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. The host cell is preferably a prokaryotic cell, more preferably E.coli.

The present invention also relates to a method for producing an antigenic polypeptide as described above, comprising:

culturing a host cell as described above under suitable culture conditions; and

The invention also relates to a vaccine comprising an antigenic polypeptide as described above, or a nucleic acid as described above, or a vector as described above.

The vaccine may be in the form of a solution or a dry powder, preferably a lyophilized powder.

In some embodiments, the vaccine further comprises an adjuvant.

As used herein, an "adjuvant" is a substance that is capable of favoring or amplifying an immunological cascade of events, ultimately leading to a better immune response, i.e., an integrated physical response against an antigen. Adjuvants are generally not required for the immune response to occur, but facilitate or amplify the response.

Adjuvants suitable for use in the vaccine of the present invention include adjuvants that enhance the immune response against the above-described antigenic peptides, preferably those capable of activating the activity of Dendritic Cells (DCs) or T cells (particularly CTL cells) to facilitate the generation of an immune response, and such adjuvants are well known in the art.

The adjuvant may be at least one of aluminum salt, liposome, incomplete Freund's adjuvant, complete Freund's adjuvant, alum adjuvant, MF59, monophosphoryl lipid A, flagellin, CpG-ODN, and Poly (I: C). Preferred adjuvants are those suitable for use in humans. In some embodiments, the aluminum salt is selected from the group consisting of aluminum phosphate, potassium aluminum phosphate, and aluminum hydroxide. Other well known adjuvants include hydrocarbon oils, polymers, saponins and/or adjuvants composed of gelled particles of sodium acrylate in water, for example, montanide m PET GEL ATM (Seppic, Paris France). A low molecular weight copolymer adjuvant may form crosslinks in solution to become a high molecular weight gel, such as POLYGENTM (mvplabases, Omaha). When added, the amount of adjuvant in the vaccine is typically between about 1% and 20% (v/v). In particular embodiments, the amount of adjuvant is between about 2% and 10% (v/v). In a more specific embodiment, the amount of adjuvant is between about 3% and 6% (v/v).

In some embodiments, the vaccine is a water-in-oil emulsion having an aqueous phase and an oil phase.

In some embodiments, the vaccine is an oil-in-water emulsion having an aqueous phase and an oil phase.

Vaccines are typically formulated for parenteral administration. Typical immunizations are achieved by Subcutaneous (SC) or Intramuscular (IM) injection, but the invention also contemplates vaccinations by the nasal route, or oral, Intravenous (IV), Intraperitoneal (IP), or Intradermal (ID) injection. Alternatively, the vaccine may also be administered via a skin patch, in a delayed release implant, scarification or topical application. Administration can also be via drinking water and/or food of the recipient animal/human.

The vaccines are administered in a manner compatible with the dosage formulation, and in amounts such as a therapeutically effective amount and an immunogenically effective amount. The amount administered will depend on the subject being treated, the ability of the subject's immune system to synthesize antibodies, and the degree of protection desired. The exact amount of active ingredient to be administered will depend on the judgment of the practitioner, and will vary from individual to individual. Suitable regimens for initial administration and booster vaccination may also vary, but are typically 1 injection or otherwise administered after a certain interval of time (weeks or months) after the first administration.

Vaccines may also deliver antigenic peptides, nucleic acids or vectors to a subject via a delivery system. Methods for non-viral delivery of nucleic acids include lipofection, nuclear transfection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipids nucleic acid conjugates, naked DNA, artificial viral particles and agent-enhanced uptake of DNA.

The methods of viral delivery of nucleic acids can be administered directly to the subject or can be used to treat cells (particularly immune cells) in vitro, and the treated cells can optionally be administered to the subject.

The vaccine may also comprise a pharmaceutically acceptable carrier.

A "pharmaceutically acceptable carrier" is intended to aid in the stabilization and administration of the vaccine, while being harmless and well tolerated by the target. Such carriers may be, for example, sterile water or sterile physiological saline solution. In a more complex form, the carrier may for example be a buffer, which may contain further additives, such as stabilisers or preservatives. Aqueous or aqueous solutions saline solutions and aqueous solutions of sugars (e.g., dextrose and/or glycerol) may be employed as carriers, particularly for injectable solutions. Furthermore, the carrier may be and/or comprise a hydrocolloid and/or a polymer solution, e.g. to thicken the vaccine.

In some embodiments, the vaccine further comprises an inactivated virus and/or an inactivated bacterium (e.g., a bacterin) and/or an antigen of a bacterin.

The invention also relates to a kit comprising an antigenic peptide as described above, or a nucleic acid as described above, or a vector as described above, or a vaccine as described above.

The kit may further comprise a container for vaccination with the vaccine. The inoculation container is preferably a medical syringe.

The term "kit" refers to any article of manufacture (e.g., a package or container) that includes at least one device. The kit may further comprise instructions for use, supplemental reagents, and/or components for use in the methods described herein or steps thereof.

The invention also relates to the use of an antigenic polypeptide as described above, or a nucleic acid as described above, or a vector as described above, or a vaccine as described above, for the preparation of a medicament for the prevention of an adenovirus infection of type HAdV-40 and/or HAdV-41.

The invention further provides a method of protecting a subject against infection by an adenovirus of type HAdV-40 and/or HAdV-41, comprising administering to the subject an effective amount of an antigenic peptide, nucleic acid, vector or vaccine according to the invention.

In some embodiments, the subject is an animal, preferably a mammal, such as a cow, pig, dog, cat, sheep, rat, mouse, rabbit, horse. The subject is further preferably a primate, more preferably a human.

The term "effective amount" as used herein refers to an amount of a component to which the term corresponds that achieves treatment, prevention, alleviation and/or amelioration of a disease or disorder described herein in a subject.

Factors affecting the preferred dosage regimen can include, for example, the species or breed of the subject (e.g., the species or breed of the subject), age, weight, diet, activity, lung size, and condition; the route of administration; efficacy, safety and immune duration profiles of the particular vaccine used; whether a delivery system is used; and whether the vaccine is administered as part of a medicament and/or vaccine combination. Thus, the dosage actually employed may vary for a particular animal, and thus may deviate from the typical dosages described above. Determination of such dosage adjustments is generally within the skill of those in the art of vaccine development using conventional methods.

Embodiments of the present invention will be described in detail with reference to examples.

In this example, the L-Knob genes of HAdV-40 and HAdV-41 were cloned, and after gene codon optimization according to different expression systems, the genes were cloned into different expression vectors, including prokaryotic expression vector pQE30, BL21(DE3) was transformed for expression, bacteria were collected, lysozyme was lysed, and purification was performed with a nickel column; insect cell-baculovirus expression system pFastBac vector, expressed in insect cell sf 9; pcDNA3.1 vector, expressed in mammalian cells CHO. The purified protein is presented as a homotrimer structure, and the immune mouse after purification can induce high titer neutralizing antibody, and can be used as an adenovirus vaccine candidate; finally, it was found that anti-HAdV-40L-Knob serum can neutralize HAdV-40 and HAdV-41 at similar high titers; HAdV-41L-Knob serum can also neutralize HAdV-40 and HAdV-41 with similar high potency, and HAdV-40L-Knob and HAdV-41L-Knob can be used as HAdV-40 and HAdV-41 bivalent intestinal adenovirus vaccine candidates.

Example 1 expression of recombinant human adenovirus knob polypeptide

The entire genome sequence GenBank number of the HAdV-40 virus strain (Dugan strain) described in the examples of the present specification: NC-001454.1; whole genome sequence GenBank No. of HAdV-41 virus strain (Tak strain): DQ 315364.2. The knob gene in the examples of the present specification is identical to those of these strains, and the amino acid sequence is free from mutation; these strains were also used in neutralization experiments and the like in the present examples.

Carrying out codon optimization on Knob genes (L-Knob) of long fibers of HAdV-40 and HAdV-41 to adapt to escherichia coli expression, cloning the genes into a prokaryotic expression vector pQE30 after gene synthesis, transforming BL21(DE3) for low-temperature induced expression, collecting bacteria, resuspending PBS buffer solution (1mM EDTA, 10% glycerol, 1% Triton X100 and 10mM imidazole), lysing the bacteria by lysozyme, taking supernatant after high-speed centrifugation, purifying by using a nickel column, washing and eluting by using gradient imidazole, and centrifuging by using a 10kDa ultrafiltration tube to remove the imidazole; the purified protein exhibits a homotrimeric structure.

Amino acid sequence

HAdV-40L-Knob:

GSIAVSPTTTTPTTLWTTADPSPNATFYESLDAKVWLVLVKCNGMVNGTISIKAQKGTLLKPTASFISFVMYFYSDGTWRKNYPVFDNEGILANSATWGYRQGQSANTNVSNAVEFMPSSKRYPNEKGSEVQNMALTYTFLQGDPNMAISFQSIYNHAIEGYSLKFTWRVRNNERFDIPCCSFSYVTEQ(SEQ ID NO：1)

The gene base sequence after the codon optimization of the escherichia coli is as follows:

GGTAGCATCGCCGTGAGTCCAACCACGACCACCCCTACGACACTGTGGACGACCGCTGATCCTAGCCCGAATGCCACGTTTTATGAATCATTAGATGCTAAAGTTTGGTTAGTGTTAGTTAAATGTAATGGCATGGTTAATGGTACCATCTCTATCAAAGCCCAGAAAGGCACACTGCTGAAACCGACAGCGAGCTTTATTAGCTTTGTGATGTATTTTTATAGTGATGGTACCTGGCGTAAAAATTATCCGGTGTTTGATAATGAAGGTATTCTGGCCAATAGCGCAACCTGGGGCTATCGTCAGGGTCAGAGTGCCAATACAAATGTGTCTAATGCGGTGGAATTTATGCCGTCAAGTAAACGCTATCCGAATGAAAAAGGCTCAGAAGTTCAGAATATGGCCTTAACGTATACATTTTTGCAAGGCGATCCTAATATGGCAATCTCATTTCAGAGCATCTATAATCATGCGATCGAAGGCTATAGTCTGAAATTTACGTGGCGCGTTCGCAATAATGAACGCTTTGATATTCCTTGTTGCAGCTTTTCTTATGTGACGGAACAG(SEQ ID NO：3)

HAdV-41L-Knob：

GSITVSPTTTTPTTLWTTADPSPNATFYESLDAKVWLVLVKCNGMVNGTISIKAQKGILLRPTASFISFVMYFYSDGTWRKNYPVFDNEGILANSATWGYRQGQSANTNVSNAVEFMPSSKRYPNQKGSEVQNMALTYTFLQGDPNMAISFQSIYNHALEGYSLKFTWRVRNNERFDIPCCSFSYVTEQ(SEQ ID NO：2)

GGTAGTATCACGGTGTCACCGACCACAACGACACCTACCACACTGTGGACGACAGCAGATCCGTCTCCGAATGCGACGTTTTATGAATCATTAGATGCTAAAGTTTGGTTAGTGCTGGTTAAATGTAATGGCATGGTTAATGGTACGATCTCAATCAAAGCCCAGAAAGGCATCTTACTGCGTCCTACCGCTTCATTTATTAGCTTTGTGATGTATTTTTATAGCGATGGTACGTGGCGTAAAAATTATCCGGTGTTTGATAATGAAGGCATTCTGGCCAATTCAGCAACCTGGGGCTATCGCCAGGGTCAGAGCGCCAATACAAATGTGAGCAATGCCGTGGAATTTATGCCGAGTTCTAAACGCTATCCTAATCAGAAAGGCTCTGAAGTGCAGAATATGGCCTTAACATATACATTTTTGCAGGGTGATCCGAATATGGCGATTAGCTTTCAGTCTATCTATAATCATGCACTGGAAGGTTATAGTCTGAAATTTACGTGGCGCGTTCGCAATAATGAACGCTTTGATATTCCTTGTTGCTCATTTAGTTATGTGACGGAACAG(SEQ ID NO：4)

example 2: recombinant protein immunogenicity assays

To analyze the immunogenicity of recombinant proteins, mice were immunized 3 times with the recombinant proteins emulsified with aluminum phosphate adjuvant (1: 1), each mouse was immunized 40ug protein each time, and serum was collected 7 days after the last immunization for ELISA detection and cell micro-neutralization. The recombinant knob immunized mice can induce high-titer antibody responses, and the serum neutralizing antibody titer is greater than 4608. The results in Table 1 and FIG. 2 show that L-Knob antisera to HAdV-40 and HAdV-41 can cross-neutralize HAdV-40 and HAdV-41.

TABLE 1 cell microneutralization experiments to detect serum neutralizing antibody titers

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

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 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. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Guangzhou medical university affiliated first hospital (Guangzhou respiratory center)

<120> adenovirus antigen polypeptide and application thereof

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

1 5 10 15

Thr Thr Ala Asp Pro Ser Pro Asn Ala Thr Phe Tyr Glu Ser Leu Asp

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Ala Lys Val Trp Leu Val Leu Val Lys Cys Asn Gly Met Val Asn Gly

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

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Ser Phe Ile Ser Phe Val Met Tyr Phe Tyr Ser Asp Gly Thr Trp Arg

65 70 75 80

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

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Thr Trp Gly Tyr Arg Gln Gly Gln Ser Ala Asn Thr Asn Val Ser Asn

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

115 120 125

Ser Glu Val Gln Asn Met Ala Leu Thr Tyr Thr Phe Leu Gln Gly Asp

130 135 140

Pro Asn Met Ala Ile Ser Phe Gln Ser Ile Tyr Asn His Ala Ile Glu

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Gly Tyr Ser Leu Lys Phe Thr Trp Arg Val Arg Asn Asn Glu Arg Phe

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Asp Ile Pro Cys Cys Ser Phe Ser Tyr Val Thr Glu Gln

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<210> 2

<211> 189

<212> PRT

<213> artificial sequence

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Gly Ser Ile Thr Val Ser Pro Thr Thr Thr Thr Pro Thr Thr Leu Trp

1 5 10 15

Thr Thr Ala Asp Pro Ser Pro Asn Ala Thr Phe Tyr Glu Ser Leu Asp

20 25 30

Ala Lys Val Trp Leu Val Leu Val Lys Cys Asn Gly Met Val Asn Gly

35 40 45

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

50 55 60

Ser Phe Ile Ser Phe Val Met Tyr Phe Tyr Ser Asp Gly Thr Trp Arg

65 70 75 80

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

85 90 95

Thr Trp Gly Tyr Arg Gln Gly Gln Ser Ala Asn Thr Asn Val Ser Asn

100 105 110

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

115 120 125

Ser Glu Val Gln Asn Met Ala Leu Thr Tyr Thr Phe Leu Gln Gly Asp

130 135 140

Pro Asn Met Ala Ile Ser Phe Gln Ser Ile Tyr Asn His Ala Leu Glu

145 150 155 160

Gly Tyr Ser Leu Lys Phe Thr Trp Arg Val Arg Asn Asn Glu Arg Phe

165 170 175

Asp Ile Pro Cys Cys Ser Phe Ser Tyr Val Thr Glu Gln

180 185

<210> 3

<211> 567

<212> DNA

<213> artificial sequence

<400> 3

ggtagcatcg ccgtgagtcc aaccacgacc acccctacga cactgtggac gaccgctgat 60

cctagcccga atgccacgtt ttatgaatca ttagatgcta aagtttggtt agtgttagtt 120

aaatgtaatg gcatggttaa tggtaccatc tctatcaaag cccagaaagg cacactgctg 180

aaaccgacag cgagctttat tagctttgtg atgtattttt atagtgatgg tacctggcgt 240

aaaaattatc cggtgtttga taatgaaggt attctggcca atagcgcaac ctggggctat 300

cgtcagggtc agagtgccaa tacaaatgtg tctaatgcgg tggaatttat gccgtcaagt 360

aaacgctatc cgaatgaaaa aggctcagaa gttcagaata tggccttaac gtatacattt 420

ttgcaaggcg atcctaatat ggcaatctca tttcagagca tctataatca tgcgatcgaa 480

ggctatagtc tgaaatttac gtggcgcgtt cgcaataatg aacgctttga tattccttgt 540

tgcagctttt cttatgtgac ggaacag 567

<210> 4

<211> 567

<212> DNA

<213> artificial sequence

<400> 4

ggtagtatca cggtgtcacc gaccacaacg acacctacca cactgtggac gacagcagat 60

ccgtctccga atgcgacgtt ttatgaatca ttagatgcta aagtttggtt agtgctggtt 120

aaatgtaatg gcatggttaa tggtacgatc tcaatcaaag cccagaaagg catcttactg 180

cgtcctaccg cttcatttat tagctttgtg atgtattttt atagcgatgg tacgtggcgt 240

aaaaattatc cggtgtttga taatgaaggc attctggcca attcagcaac ctggggctat 300

cgccagggtc agagcgccaa tacaaatgtg agcaatgccg tggaatttat gccgagttct 360

aaacgctatc ctaatcagaa aggctctgaa gtgcagaata tggccttaac atatacattt 420

ttgcagggtg atccgaatat ggcgattagc tttcagtcta tctataatca tgcactggaa 480

ggttatagtc tgaaatttac gtggcgcgtt cgcaataatg aacgctttga tattccttgt 540

tgctcattta gttatgtgac ggaacag 567

Claims

1. An antigenic polypeptide which is SEQ ID NO: 1 and/or SEQ ID NO: 2.

2. An isolated nucleic acid expressing the antigenic polypeptide of claim 1.

3. The nucleic acid of claim 2 which is SEQ ID NO: 3 and/or SEQ ID NO: 4.

4. A vector comprising the nucleic acid of claim 2 or 3.

5. A host cell comprising the nucleic acid of claim 2 or 3, or transformed with the vector of claim 4.

6. The host cell of claim 5, which is E.coli.

7. A method of producing the antigenic polypeptide of claim 1, comprising:

culturing the host cell of claim 5 or 6 under suitable culture conditions; and

8. A vaccine comprising the antigenic polypeptide of claim 1, or the nucleic acid of claim 2 or 3, or the vector of claim 4.

9. The vaccine of claim 8, further comprising an adjuvant.

10. A kit comprising the vaccine of claim 8 or 9.

11. Use of an antigenic polypeptide as defined in claim 1, or a nucleic acid as defined in claim 2 or 3, or a vector as defined in claim 4, or a vaccine as defined in claim 8 or 9, for the manufacture of a medicament for the prevention of infection by an adenovirus of type HAdV-40 and/or HAdV-41.