CN111533811B

CN111533811B - Novel genetically engineered vaccine of avian encephalomyelitis virus

Info

Publication number: CN111533811B
Application number: CN202010394941.3A
Authority: CN
Inventors: 曹文龙; 孔迪; 滕小锘; 易小萍; 张大鹤
Original assignee: Suzhou Shinuo Biotechnology Co ltd
Current assignee: Suzhou Womei Biology Co ltd
Priority date: 2020-05-11
Filing date: 2020-05-11
Publication date: 2020-12-11
Anticipated expiration: 2040-05-11
Also published as: CN111533811A

Abstract

The invention discloses a novel genetic engineering vaccine of avian encephalomyelitis virus, which comprises a coding gene sequence shown as SEQ ID NO: 1. The antigenicity, immunogenicity and functions of the vaccine are similar to those of natural protein, the expression level is high, the immunogenicity is strong, strong humoral immunity can be generated in poultry bodies, the vaccine is not pathogenic to the poultry, the immunized poultry can resist strong toxicity and attack, and the vaccine can be prepared by large-scale serum-free suspension culture of a bioreactor, so that the production cost of the vaccine is greatly reduced.

Description

Novel genetically engineered vaccine of avian encephalomyelitis virus

Technical Field

The invention relates to a genetic engineering vaccine, in particular to a novel genetic engineering vaccine of avian encephalomyelitis virus, a preparation method and application thereof, belonging to the technical field of animal immunity drugs.

Background

Avian Encephalomyelitis (AE) is an infectious disease mainly affecting chicks and laying hens caused by Avian Encephalomyelitis Virus (AEV), and is characterized by non-suppurative Encephalomyelitis, mainly manifested by neurological symptoms such as dyskinesia, head and neck tremor, progressive paralysis and the like, so the infectious disease is also called epidemic tremor. The clinical symptoms are closely related to the day age of the chickens, the infection of the chickens with the age below four weeks can show typical neurological symptoms, the morbidity is 100% when the chicks are serious, and the mortality can reach 95%; the symptoms of the chickens with the age of more than four weeks are not obvious or have no symptoms. The disease is horizontally spread among chicken flocks through the digestive tract and can be vertically transferred through eggs, so that the culling rate of chicks is high, the laying rate of adult poultry is reduced, and the healthy development of the chicken industry is seriously influenced. The disease is reported for the first time in 1980 in China and is distributed worldwide.

AEV belongs to the family of picornaviridae, is a single-stranded positive-strand RNA virus with a genome length of 7055bp, has no envelope, has an average diameter of about 26.1 +/-0.4 nm, and has icosahedral symmetry and quintuple symmetry with 32-42 capsomeres. AEV was originally classified in enterovirus, however, the virus was later reclassified and, on the basis of limited sequence data, classified in a new genus, the hepadnavirus genus, which contains only human hepatitis virus. Recent data on its 5' -UTR structure and function show that AEV contains an IRES of type IV and arbovirus, similar to but different from hepatitis a virus. Thus, a new genus, the genus Tremavirus ((U.S. N.James MacLachlan Edward J.Dubovi. veterinary virology (fourth edition) [ M ] Kongxiong, Liu Sheng Wang translation. Beijing: Chinese agricultural Press 2015: 450.) was proposed. AEV genomic RNA possesses only one Open Reading Frame (ORF), encoding a polyprotein that is hydrolyzed by the cascade cleavage of viral proteases to produce the mature protein. As with other picornaviruses, the genomic P1 region of AEV encodes 4 structural proteins (VP4-VP3-VP2-VP1), the P2 and P3 regions encode 7 non-structural proteins (2A-2C and 3A-3D) (Marvil P, Knowles N J, et al. Avian encyclopy-infection virus a picornavirus and is most closed related to P-infection A virus [ J ]. J Gen Virus, 1999: 80: 653-. The VP0 protein has the main function of protecting AEV from being damaged by RNase in the external environment together with proteins VP1 and VP3, and is an antigen component constituting the virus. The VP1 protein is the main host protective immunity protein, encoding the main antigenic site and most type-specific neutralizing epitopes (M UIR P, KUNNRER U, K ORN K, ethyl. molecular typing of antigenic sequences: current states and future requirements [ J ]. Clin Microbiol Rev, 1998, 11 (1): 202-). 227.). VP1 has good antigenicity and immunogenicity, and can effectively stimulate the body to generate humoral immune response after the virus infects the body. The VP3 protein is an apoptosis-inducing factor (LIU J, W EI T, KAWANG J. Avian en center apoptosis virus a major structure protein VP3[ J ]. Viro-log, 2002, 300 (1): 39-49.), and it is involved in the induction of anti-viral neutralizing antibodies, and plays an important role in viral pathogenicity. VP2, VP3, VP4 are all involved in the assembly of viral particles and affect the immunogenicity of the viral particles. 2A is a protease that is required for early cleavage of the AEV polyprotein and for shut-down of host protein synthesis.

There is no effective drug treatment for this disease at present, and the prevention relies mainly on vaccination with vaccines. The whole virus inactivated vaccine applied in China is complicated in antigen production and preparation process, high in cost and high in virus dispersing risk, and the attenuated vaccine also has the disease condition of vaccination.

Disclosure of Invention

The invention mainly aims to provide a novel genetic engineering vaccine for avian encephalomyelitis virus, a preparation method and application thereof, so as to overcome the defects in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a fusion protein, which comprises SEQ ID NO:2 or an amino acid sequence corresponding to SEQ ID NO:2, and an amino acid sequence which is 95% or more identical to the full-length amino acid sequence of the polypeptide.

The embodiment of the invention also provides a coding gene of the fusion protein, which comprises a sequence shown as SEQ ID NO:1 or a nucleic acid molecule substantially identical to SEQ ID NO:1, or a nucleic acid molecule having a nucleotide sequence that is 95% or more identical to the nucleotide sequence of 1.

The embodiment of the invention also provides a recombinant gene vector, a recombinant baculovirus or an insect cell containing the encoding gene.

The embodiment of the invention also provides an immune composition, which comprises: the fusion protein; and, a pharmaceutically acceptable carrier.

The embodiment of the invention also provides a method for preparing the fusion protein, which comprises the following steps:

s1, cloning the encoding gene of the fusion protein to a baculovirus expression system transfer vector to obtain a recombinant expression vector;

s2, transforming the recombinant expression vector into a cell DH10Bac containing a baculovirus shuttle vector Bacmid to obtain a recombinant baculovirus gene;

s3, transfecting the recombinant baculovirus genome plasmid into an insect cell to obtain a recombinant baculovirus;

s4, inoculating the recombinant baculovirus into insect cells to obtain the fusion protein.

The embodiment of the invention also provides application of the fusion protein or the immune composition in preparing a detection reagent for the avian encephalomyelitis virus.

The embodiments also provide the use of the fusion protein or the immunological composition in the manufacture of a medicament for inducing an immune response against an avian encephalomyelitis virus antigen in a test animal.

The embodiment of the invention also provides the application of the fusion protein or the immune composition in the production of a medicament for preventing the animal from being infected by the avian encephalomyelitis virus.

The embodiment of the invention also provides application of the fusion protein or the immune composition in preparing a novel gene engineering vaccine of the avian encephalomyelitis virus.

Accordingly, the embodiment of the invention provides an avian encephalomyelitis virus genetic engineering subunit vaccine which comprises any one of the immune compositions. Further, the vaccine may further comprise a pharmaceutically acceptable carrier.

The embodiment of the invention also provides application of a recombinant gene vector containing the fusion protein coding gene, recombinant baculovirus or insect cells in producing a reagent for detecting the infection of animals by the avian encephalomyelitis virus.

The embodiment of the invention also provides application of a recombinant gene vector containing the fusion protein coding gene, recombinant baculovirus or insect cells in producing a medicament for inducing immune response against avian encephalomyelitis virus antigen in a test animal.

The embodiment of the invention also provides application of a recombinant gene vector containing the fusion protein coding gene, recombinant baculovirus or insect cells in producing a medicament for preventing animals from being infected by the avian encephalomyelitis virus.

The embodiment of the invention also provides a preparation method of the novel avian encephalomyelitis virus genetic engineering vaccine, which can comprise the following steps:

1) preparing a nucleic acid molecule for encoding the avian encephalomyelitis virus VP0M +2A + VP1+2A + VP3 fusion protein;

2) constructing a recombinant vector, and cloning the nucleic acid molecule in the step 1) into a shuttle vector to obtain a recombinant shuttle vector containing a target gene;

3) transforming the recombinant shuttle vector into DH10Bac bacteria, selecting recombinant bacteria, extracting a genome to transfect insect cells such as Sf9 cells and the like to obtain recombinant baculovirus;

4) cultivating the insect cell, and enabling the insect cell to be automatically assembled into VLP in the cell, and further performing recombinant expression to generate AEV-VLP;

5) adding the recombinant AEV-VLP to an adjuvant to obtain the vaccine.

Compared with the prior art, the embodiment of the invention uses insect cells to express a fusion protein (AEVVLP) specially designed for AEV, wherein the fusion protein is expressed by adopting molecules of VP0M (optimized VP0) +2A (optimized AEV 2A protein) + VP1+2A + VP3 and a promoter, and then three structural proteins of VP0/1/3 are formed under the self-digestion of AEV 2A protease, and the three structural proteins can form VLP. Furthermore, mutation of two sites (10 th site and 37 th site) is carried out on the AEV 2A protein, so that efficient enzyme digestion of the AEV 2A protein can be realized, and the enzyme digestion efficiency is greatly improved. Meanwhile, point mutation is carried out on one of VP0 proteins and a key amino acid site for VLP assembly, so that the acid resistance of AEV VLP is improved, and the AEV VLP is more suitable for the environment of intracellular acidity of insect cells. The avian encephalomyelitis virus genetic engineering vaccine prepared by the method of the embodiment of the invention has antigenicity, immunogenicity and functions similar to those of natural protein, has higher expression level and strong immunogenicity, has no pathogenicity to poultry, can be prepared by large-scale serum-free suspension culture of a bioreactor, and greatly reduces the production cost of the vaccine.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a gel electrophoresis image of a PCR product of a target gene in example 1 of the present invention.

FIG. 2 is a gel electrophoresis image of the colony PCR product in example 1 of the present invention.

FIG. 3 is a schematic diagram of the transfer vector pF-VPOM-2A-VP1-2A-VP3 in example 1 of the present invention.

FIG. 4 is an SDS-PAGE gel electrophoresis chart in example 4 of the present invention.

FIG. 5 is a Western Blot identification chart in example 5 of the present invention.

FIG. 6 is a photograph of indirect immunofluorescence in example 6 of the present invention.

FIG. 7 is an SDS-PAGE gel electrophoresis chart in example 7 of the present invention.

FIG. 8 is an electron micrograph of a virus-like particle in example 8 of the present invention.

FIG. 9 is a photograph after ultracentrifugation of a sample in example 8 of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

One aspect of the embodiments of the present invention provides a fusion protein comprising SEQ ID NO:2 or an amino acid sequence corresponding to SEQ ID NO:2, and an amino acid sequence which is 95% or more identical to the full-length amino acid sequence of the polypeptide.

Furthermore, the fusion protein is assembled by a plurality of recombinant expressed structural proteins (AEVVP0, VP1, VP3 and the like), can rapidly induce humoral immunity and cellular immunity, and can provide complete protection for poultry with only small dose when being used as an AEV VLP vaccine.

In another aspect of the embodiments of the present invention, there is provided a gene encoding the fusion protein, which includes a sequence shown in SEQ ID NO:1 or a nucleic acid molecule substantially identical to SEQ ID NO:1, or a nucleic acid molecule having a nucleotide sequence that is 95% or more identical to the nucleotide sequence of 1.

In another aspect of the embodiments of the present invention, there is provided a recombinant gene vector comprising a gene encoding the fusion protein.

In another aspect of the embodiments of the present invention, there is provided a recombinant baculovirus, which includes the gene encoding the fusion protein.

In another aspect of the embodiments of the present invention, there is provided an insect cell comprising a gene encoding the fusion protein.

Further, the insect cell may be formed by transfection with a recombinant baculovirus genome plasmid containing the gene encoding the fusion protein.

In another aspect of the embodiments of the present invention, there is also provided an immunization composition comprising: the fusion protein; and, a pharmaceutically acceptable carrier. Further, the pharmaceutically acceptable carrier includes any one or a combination of two or more of white oil, aluminum stearate, span and tween, and is not limited thereto.

Another aspect of the embodiments of the present invention also provides a method of preparing the fusion protein, which includes:

Further, the baculovirus expression system transfer vector includes but is not limited to any one of pFastBac1, pVL1393, pFastBac dual, preferably pFastBac 1.

Further, the insect cells include but are not limited to Sf9, High Five or Sf21 cells, preferably Sf9 cells.

In the above examples of the present invention, the nucleic acid molecule encoding VP0M +2A + VP1+2A + VP3, a promoter, is used to express the fusion protein, and then three structural proteins of VP0, VP1 and VP3 are formed by self-digestion with AEV 2A protease, and these three structural proteins can constitute VLP (virus-like particle), and the positions of the three structural proteins can be exchanged.

In the above embodiments of the invention, the 2A protein may be FMDV 2A, AEV 2A or the like, most preferably AEV 2A.

In the above examples of the invention, VP0M refers to the optimized VP0 gene.

In the above embodiment of the invention, mutation is also performed on AEV 2A protein (the tenth E and the 37V are mutated into S, specifically shown in SEQ ID NO:2 and SEQ ID NO: 8), so that efficient enzyme digestion of 2A protein is realized, and enzyme digestion efficiency is greatly improved.

In the above embodiment of the present invention, a mutation (F, see SEQ ID NO: 2) is also made in one of the amino acid sites of VP0 protein that are critical for VLP assembly, which improves the acid resistance of the AEVVLP, especially in the environment of intracellular acidity of insect cells.

In the above embodiment of the present invention, by using a baculovirus insect cell expression system and performing expression using insect cells such as suspension culture Sf9 cells, the expression level is high and the protein immunogenicity is good.

The other aspect of the embodiment of the invention also provides application of the fusion protein or the immune composition in preparing a detection reagent for the avian encephalomyelitis virus.

Another aspect of the embodiments of the present invention also provides a use of the fusion protein or the immunological composition for the manufacture of a medicament for inducing an immune response against an avian encephalomyelitis virus antigen in a test animal.

In another aspect of the embodiments of the present invention, there is also provided a use of the fusion protein or the immunological composition in the manufacture of a medicament for preventing an infection of an animal with avian encephalomyelitis virus.

The other aspect of the embodiment of the invention also provides the application of the fusion protein or the immune composition in preparing a novel genetic engineering vaccine of the avian encephalomyelitis virus.

Accordingly, another aspect of the embodiments of the present invention provides an avian encephalomyelitis virus genetically engineered subunit vaccine comprising any one of the immunological compositions described above. Further, the vaccine may further comprise a pharmaceutically acceptable carrier.

In another aspect of the embodiment of the invention, the application of the recombinant gene vector containing the fusion protein coding gene, the recombinant baculovirus or the insect cell in producing the reagent for detecting the infection of the animal by the avian encephalomyelitis virus is also provided.

In another aspect of the embodiments of the present invention, there is also provided a use of a recombinant gene vector, a recombinant baculovirus or an insect cell comprising the gene encoding the fusion protein for the production of a medicament for inducing an immune response against an avian encephalomyelitis virus antigen in a test animal.

In another aspect of the embodiment of the invention, the application of the recombinant gene vector containing the fusion protein coding gene, the recombinant baculovirus or the insect cell in the production of a medicament for preventing the animal from being infected by the avian encephalomyelitis virus is also provided.

Accordingly, another aspect of the embodiments of the present invention also relates to a method of inducing an immune response against an avian encephalomyelitis virus antigen, said method comprising administering said avian encephalomyelitis virus genetically engineered subunit vaccine to a subject avian animal.

Accordingly, another aspect of the embodiments of the present invention also relates to a method of protecting a test animal from an avian encephalomyelitis virus infection, said method comprising administering said avian encephalomyelitis virus genetically engineered subunit vaccine to said test avian animal.

Yet another aspect of embodiments of the present invention provides a vaccine suitable for use in generating an immune response against avian encephalomyelitis virus in a test avian animal, the vaccine comprising: fusion proteins of the invention and adjuvant molecules.

Further, the adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof; and in some embodiments, can be IL-12, IL-15, IL-28 or RANTES.

Further, the adjuvant may preferably be related adjuvants produced by Suzhou Shino biotechnology, Inc. to improve the effect of the vaccine.

Furthermore, the AEV VLP vaccine provided by the above embodiment of the present invention can generate strong humoral immunity in the poultry, and the immunized poultry can resist strong toxicity.

1. And (4) defining.

The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

To the extent that numerical ranges are recited herein, each intervening number is specifically contemplated to be within the same precision. For example, for the range of 6-9, the numbers 7and 8 are encompassed in addition to 6 and 9, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly encompassed.

An "adjuvant" as used herein means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen encoded by the encoding nucleic acid sequence described below.

"antibody" as used herein means an antibody of the type IgG, IgM, IgA, IgD or IgE, or a fragment, fragment or derivative thereof, including Fab, F (ab') 2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody may be an antibody isolated from a serum sample of an animal, a polyclonal antibody, an affinity purified antibody, or a mixture thereof that exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

"coding sequence" or "coding nucleic acid" as used in the present specification means a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signals capable of directing expression in the cells of the subject or animal to which the nucleic acid is administered.

"complement" or "complementary" as used herein means that a nucleic acid can refer to Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of the nucleic acid molecule.

As used herein, "consensus" or "consensus sequence" means a polypeptide sequence based on analysis of a cohort of multiple subtypes of a particular avian encephalomyelitis virus antigen. Nucleic acid sequences encoding the consensus polypeptide sequence may be prepared. Vaccines comprising proteins comprising consensus sequences and/or nucleic acid molecules encoding these proteins can be used to induce broad immunity against multiple subtypes or serotypes of a particular avian encephalomyelitis virus antigen.

"electroporation", "electro-permeabilization" or "electrokinetic enhancement" ("EP") as used interchangeably herein means the use of transmembrane electric field pulses to induce microscopic pathways (pores) in a biological membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions and water to flow from one side of the cell membrane to the other.

"fragment" with respect to nucleic acid sequences as used herein means a nucleic acid sequence or a portion thereof that encodes a polypeptide that is capable of eliciting an immune response in an animal that is cross-reactive with the full-length wild-type strain avian encephalomyelitis virus antigen. The fragment may be a DNA fragment selected from at least one of various nucleotide sequences encoding protein fragments described below.

By "fragment" or "immunogenic fragment" with respect to a polypeptide sequence is meant a polypeptide capable of eliciting an immune response in an animal that is cross-reactive with the full-length wild-type strain avian encephalomyelitis virus antigen. A fragment of a protein may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the protein. In some embodiments, a fragment of a protein may comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more of the protein, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more.

The term "genetic construct" as used in this specification refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence comprises an initiation signal and a termination signal operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. The term "expression form" as used herein refers to a genetic construct containing the necessary regulatory elements operably linked to a coding sequence encoding a protein such that the coding sequence will be expressed when present in the cells of the individual.

The term "homology" as used in the present specification refers to the degree of complementarity. There may be partial homology or complete homology (i.e., identity). Partial complementary sequences that at least partially inhibit hybridization of a fully complementary sequence to a target nucleic acid are referred to using the functional term "substantially homologous". The term "substantially homologous" as used herein when used with respect to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, means that the probe can hybridize to a strand of the double-stranded nucleic acid sequence under conditions of low stringency. The term "substantially homologous" as used herein with respect to a single-stranded nucleic acid sequence means that the probe can hybridize to a single-stranded nucleic acid template sequence (i.e., is the complement of the single-stranded nucleic acid template sequence) under low stringency conditions.

In the case of two or more nucleic acid or polypeptide sequences, "identical" or "identity" as used herein means that the sequences have a specified percentage of identical residues in a specified region. The percentage may be calculated by: optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions of the identical residue in the two sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions within the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. Where two sequences are of different lengths or the alignment produces one or more staggered ends and the specified regions of comparison include only a single sequence, the residues of the single sequence are included in the denominator of the calculation rather than in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

As used herein, "immune response" means the activation of the immune system of a host (e.g., the immune system of an animal) in response to the introduction of an antigen, such as an avian encephalomyelitis virus consensus antigen. The immune response may be in the form of a cellular response or a humoral response or both.

As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" means at least two nucleotides covalently linked together. The description of single strands also defines the sequence of the complementary strand. Thus, nucleic acids also encompass the complementary strand of the single strand described. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, nucleic acids also encompass substantially the same nucleic acids and their complements. Single strands provide probes that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acids also encompass probes that hybridize under stringent hybridization conditions.

The nucleic acid may be single-stranded or double-stranded or may contain portions of both double-stranded or single-stranded sequences. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherein the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, as well as a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acid may be obtained by chemical synthesis methods or by recombinant methods.

The expression of the gene is carried out under the control of a promoter which is spatially linked thereto. Under its control, the promoter may be positioned 5 '(upstream) or 3' (downstream) of the gene. The distance between the promoter and the gene may be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, this change in distance can be adjusted without loss of promoter function. By "promoter" is meant a molecule of synthetic or natural origin that is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. The promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or alter spatial and/or temporal expression thereof. A promoter may also contain distal enhancer or repressor elements, which can be located as much as several thousand pairs of base pairs from the start of transcription. Promoters may be obtained from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may regulate expression of a gene component either substantially or differentially with respect to the cell, tissue or organ in which expression occurs or with respect to the developmental stage at which expression occurs or in response to an external stimulus such as a physiological stress, pathogen, metal ion or inducer. Representative examples of promoters include the phage T7 promoter, the phage T3 promoter, the SP6 promoter, the lactose operon-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter, and the CMVIE promoter.

"Signal peptide" and "leader sequence" refer to amino acid sequences that can be attached to the amino terminus of an avian encephalomyelitis virus protein as described herein. The signal peptide/leader sequence is generally indicative of the location of the protein. The signal peptide/leader sequence used in the present specification preferably promotes secretion of the protein from the cell in which it is produced. The signal peptide/leader sequence is often cleaved from the remainder of the protein, which is often referred to as the mature protein after secretion from the cell. The signal peptide/leader sequence is linked to the N-terminus of the protein.

By "stringent hybridization conditions" is meant conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10 ℃ lower than the thermodynamic melting point (Tm) of the particular sequence at a defined ionic strength pH. The Tm can be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (at Tm, 50% of the probes are occupied at equilibrium because the target sequence is present in excess). Stringent conditions may be those in which the salt concentration is less than about 1.0M sodium ion, such as about 0.01-1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30 ℃ for short probes (e.g., about 10-50 nucleotides) and at least about 60 ℃ for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For a selected or specific hybridization, the positive signal can be at least 2 to 10 times the background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5 XSSC and 1% SDS, incubated at 42 ℃ or 5 XSSC, 1% SDS, incubated at 65 ℃ washed with 0.2 XSSC and 0.1% SDS at 65 ℃.

"substantially complementary" as used herein means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or that two sequences hybridize under stringent hybridization conditions.

"substantially identical" as used herein means that the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 80%, 90%, 95%, 100%, 180%, 270%, 360, 450, 540 or more nucleotides or amino acid regions at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical, or, in the case of nucleic acids, if the first and second sequences are substantially complementary, so are the first and second sequences, within 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 540 or more nucleotide or amino acid regions.

"subtype" or "serotype": as used interchangeably herein and with respect to avian encephalomyelitis virus, is meant a genetic variant of avian encephalomyelitis virus such that one subtype is recognized by the immune system and separated from a different subtype.

"variant" as used herein with respect to a nucleic acid means (i) a portion or fragment of a reference nucleotide sequence; (ii) a complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to a reference nucleic acid or a complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

"variants" in the case of peptides or polypeptides differ in amino acid sequence by insertion, deletion or conservative substitution of amino acids, but retain at least one biological activity. A variant also means a protein having substantially the same amino acid sequence as a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., the replacement of an amino acid with a different amino acid of similar characteristics (e.g., hydrophilicity, extent and distribution of charged regions) are believed in the art to typically involve minor changes. As understood in the art, these minor changes may be identified in part by considering the hydropathic index of amino acids. Ketjet (Kyte), et al, journal of molecular biology (j.mol.biol.) 157: 105-132(1982). The hydropathic index of the amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indices can be substituted and still retain protein function. In one aspect, amino acids with a hydropathic index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the case of peptides allows the calculation of the maximum local average hydrophilicity of the peptide, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. As is understood in the art, substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity (e.g., immunogenicity). The substitution may be with amino acids having hydrophilicity values within soil 2 of each other. Both the hydropathic index and the hydropathic value of an amino acid are affected by the specific side chain of the amino acid. Consistent with the observations, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of these amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

"vector" as used herein means a nucleic acid sequence containing an origin of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector, and is preferably a DNA vector.

2. Vaccine

The vaccines of the present invention can be designed to control the extent or magnitude of an immune response in a subject animal against one or more avian encephalomyelitis virus serotypes. The vaccine may comprise elements or agents that inhibit its integration into the chromosome. The vaccine can be an RNA encoding an avian encephalomyelitis virus structural protein. An RNA vaccine can be introduced into the cells. The vaccines of the present invention may comprise avian encephalomyelitis virus structural proteins. Avian encephalomyelitis virus structural proteins are targets for immune-mediated viral clearance by inducing 1) a Cytotoxic T Lymphocyte (CTL) response, 2) a T helper cell response, and/or 3) a B cell response, or preferably all of the above-mentioned responses, to achieve cross-presentation.

The antigens may comprise protein epitopes that make them particularly effective as immunogens against which an immune response against avian encephalomyelitis virus can be induced. The avian encephalomyelitis virus antigen can include a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof.

Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences of the present specification. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 96% homology to the nucleic acid coding sequences of the present specification. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that have 97% homology to the nucleic acid coding sequences of the present specification. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that have 98% homology to the nucleic acid coding sequences of the present specification. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins having 99% homology to the nucleic acid coding sequences of the present specification. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to a coding sequence of a protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader.

Some embodiments relate to SEQ ID NO: 1. A fragment may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence of SEQ ID NO: 1. the fragment can be similar to SEQ ID NO:1 is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical. The fragment can be similar to SEQ ID NO:1 is at least 80%, at least 85%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, a fragment comprises a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, a fragment does not contain a coding sequence that encodes a leader sequence. In some embodiments, the fragment does not contain a coding leader sequence, such as, for example, a coding sequence of an IgE leader.

Some embodiments relate to a polypeptide that differs from SEQ ID NO:2 homologous protein. Some embodiments relate to a polypeptide as set forth in SEQ ID NO:2, or an immunogenic protein having 95% homology to the protein sequence set forth in claim 2. Some embodiments relate to a polypeptide as set forth in SEQ ID NO:2, or an immunogenic protein having 96% homology to the protein sequence described in claim 2. Some embodiments relate to a polypeptide as set forth in SEQ ID NO:2, or an immunogenic protein having 97% homology to the protein sequence described in 2. Some embodiments relate to a polypeptide as set forth in SEQ ID NO:2, or an immunogenic protein having 98% homology to the protein sequence described in 2. Some embodiments relate to a polypeptide as set forth in SEQ ID NO:2, or an immunogenic protein having 99% homology to the protein sequence described in claim 2.

Some embodiments relate to a polypeptide that differs from SEQ ID NO:2 the same protein. Some embodiments relate to a polypeptide having a sequence as set forth in SEQ ID NO:2, 80%, 85%, 91%, 92%, 93%, 95, 97%, 98%, or 99% identical over the entire amino acid sequence length of the full length consensus amino acid sequence.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader. A fragment of a protein may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the protein. The nucleic acid sequence of SEQ ID NO: 2. An immunogenic fragment may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence of SEQ ID NO: 2. in some embodiments, the fragment comprises a leader sequence, such as, for example, an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader.

Amino acid sequences similar to SEQ ID NO:2, or an immunogenic fragment of a protein homologous thereto. The immunogenic fragment may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a polypeptide that is identical to SEQ ID NO: 295% homologous protein. Some embodiments relate to immunogenic fragments having 96% homology to the immunogenic fragments of the protein sequences of the present specification. Some embodiments relate to immunogenic fragments that are 97% homologous to immunogenic fragments of the protein sequences of the present specification. Some embodiments relate to immunogenic fragments that are 98% homologous to immunogenic fragments of the present specification protein sequences. Some embodiments relate to immunogenic fragments that are 99% homologous to immunogenic fragments of the present specification protein sequences. In some embodiments, the fragment comprises a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader.

Amino acid sequences similar to SEQ ID NO:2, an immunogenic fragment of the same protein. The immunogenic fragment may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence set forth in SEQ ID NO:2, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical over the entire length of the amino acid sequence recited in claim 2. In some embodiments, the fragment comprises a leader sequence, such as, for example, an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader.

3. Vaccine constructs and plasmids

The vaccine may comprise a nucleic acid construct or plasmid encoding said avian encephalomyelitis virus fusion protein. The present specification provides genetic constructs that may comprise nucleic acid sequences encoding the avian encephalomyelitis virus fusion proteins disclosed herein. The genetic construct may be present as a functional extrachromosomal molecule. The genetic construct may be a linear minichromosome comprising a centromere, telomere or plasmid or cosmid.

The genetic construct may also be part of the genome of a recombinant viral vector, including recombinant adenovirus, recombinant adeno-associated virus, and recombinant vaccinia. The genetic construct may be part of the genetic material in a recombinant microbial vector in a live attenuated microorganism or in a cell.

The genetic construct may comprise regulatory elements for gene expression of the coding sequence of the nucleic acid. The regulatory element may be a promoter, enhancer, start codon, stop codon or polyadenylation signal.

The nucleic acid sequence may constitute a genetic construct which may be a vector. The vector is capable of expressing an antigen in cells of an animal in an amount effective to elicit an immune response in the animal. The vector may be recombinant. The vector may comprise a heterologous nucleic acid encoding an antigen. The vector may be a plasmid. The vector may be suitable for transfecting cells with nucleic acid encoding an antigen, the transformed host cells being cultured and maintained under conditions in which expression of the antigen occurs.

The coding sequence can be optimized for stability and high levels of expression. In some cases, the codons are selected to reduce the formation of RNA secondary structures, such as those due to intramolecular bonds.

The vector may comprise a heterologous nucleic acid encoding an antigen, and may further comprise a start codon that may be upstream of the antigen encoding sequence and a stop codon that may be downstream of the antigen encoding sequence. The initiation codon and the stop codon can be in frame with the antigen coding sequence. The vector further comprises a promoter operably linked to the antigen coding sequence. The promoter operably linked to the antigen-encoding sequence may be a promoter from simian virus 40(SV40), mouse mammary virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) promoter such as the Bovine Immunodeficiency Virus (BIV) Long Terminal Repeat (LTR) promoter, Moloney (Moloney) virus promoter, Avian Leukemia Virus (ALV) promoter, Cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr Virus (EBV) promoter, or Rous Sarcoma Virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human heme, human muscle creatine or human metallothionein. The promoter may also be a tissue-specific promoter, such as a natural or synthetic muscle or skin-specific promoter.

The vector may further comprise a polyadenylation signal, which may be downstream of the avian encephalomyelitis virus core protein coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β -globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from the pCEP4 vector (Invitrogen, San Diego, CA).

The vector may also comprise an enhancer upstream of the consensus avian encephalomyelitis virus core protein coding sequence or the consensus avian encephalomyelitis virus surface antigen protein coding sequence. The enhancer is necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as one from CMV, HA, RSV or EBV.

The vector may also comprise an animal origin of replication, in order to maintain the vector extrachromosomally and to produce multiple copies of the vector in the cell. The vector may be pVAX1, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may contain the replication origin of epstein-barr virus and the nuclear antigen EBNA-1 coding region, which may produce high copy episomal replication without integration. The vector may be a pVAX1 or a pVAX1 variant, such as a variant plasmid as described in the present specification, with a variation. The variant pVax1 plasmid is a 2998 base pair variant of the backbone vector plasmid pVax1(Invitrogen, Carlsbad CA). The CMV promoter is located at base 137-724. The T7 promoter/initiation site was located at base 664-683. The multiple cloning site is located at bases 696-811. The bovine GH polyadenylation signal is at base 829-1053. The Kanamycin (Kanamycin) resistance gene is at base 1226-containing 2020. The pUC origin is at base 2320-2993.

The vector may be pSE420(Invitrogen, San Diego, Calif), which can be used to produce proteins in e. The vector may be pYES2(Invitrogen, San Diego, Calif.) which can be used to produce proteins in a Saccharomyces cerevisiae strain of yeast (Saccharomyces cerevisiae strain). The vector may also have a MAXBACTM complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used to produce proteins in insect cells. The vector may also be pcDNA I or pcDNA3(Invitrogen, san diego, Calif.) which can be used to produce proteins in animal cells such as the Sf9 cell line. The vector may be an expression vector or system for producing a protein by conventional techniques and readily available starting materials, including Sambrook et al, Molecular Cloning and Laboratory Manual, 2 nd edition, Cold spring Harbor (1989).

The protein sequence of the avian encephalomyelitis virus fusion protein can be an original sequence, an increased sequence and a truncated sequence.

The baculovirus expression system transfer vector in the recombinant baculovirus vector of the present invention includes, but is not limited to, pFastBac1, pVL1393, pFastBac dual, etc., and pFastBac1 is preferably used. The promoter can be selected from, but not limited to, P10 promoter, PH promoter, prawn beta-actin gene promoter, OpIE promoter, and other baculovirus promoters. Wherein the transcription termination signal may be selected from any one of, but not limited to, the SV40polyA transfection termination signal, the HSV tk polyA transfection termination signal, or the OpIE poly A termination signal, and may be other transcription termination signals. The insect cell line can be selected from but not limited to Sf9, High Five, S2 or Sf21 cells, and Sf9 is preferably adopted. The animal of the invention mainly refers to poultry.

The principle of the invention lies in that a recombinant baculovirus shuttle plasmid is constructed, and the plasmid contains an expression gene for expressing the avian encephalomyelitis virus fusion protein. The gene encoding the avian encephalomyelitis virus fusion protein may use P10 promoter, PH promoter, prawn beta-actin gene promoter, OpIE promoter, etc., and the corresponding transcription termination signal may be SV40polyA transfection termination signal, HSV tk polyA transfection termination signal, OpIE polyA termination signal, etc. Furthermore, the shuttle plasmid can be constructed to transform DH10Bac bacteria to obtain recombinant bacmid (baculoviral plasmid), the obtained recombinant bacmid transfects Sf9 cells to obtain recombinant Baculovirus, and the recombinant Baculovirus is inoculated with Sf9 cells to simultaneously express the avian encephalomyelitis virus fusion protein.

Specifically, the designed codon-optimized fusion protein coding gene can be cloned to the enzyme cutting sites of BamH I and Hind III of pFastBac1 plasmid vector to construct a recombinant shuttle vector. And then transforming the constructed recombinant shuttle vector into DH10Bac bacteria, selecting recombinant bacteria, extracting a genome to transfect Sf9 cells, and obtaining the recombinant baculovirus.

The fusion protein is expressed by using baculovirus and Sf9 cells, the antigenicity, immunogenicity and function of the obtained fusion protein are similar to those of natural protein, the expression level is higher, the immunogenicity is strong, and the fusion protein is not pathogenic to poultry.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The reagents and starting materials used in the following examples are commercially available, and the test methods in which specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the respective manufacturers. Further, unless otherwise indicated, the assays, detection methods, and preparations disclosed herein are performed using molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and techniques conventional in the art. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 construction and characterization of the transfer vector pF-VP0M-2A-VP1-2A-VP3

VP0M-2A-VP1-2A-VP3 gene amplification and purification VP0M-2A-VP1-2A-VP3 gene (SEQ ID NO: 1) after codon optimization is synthesized by Nanjing Kingsri Biotech Co., Ltd and cloned to pUC17 vector to obtain pUC-VP0M-2A-VP1-2A-VP3 plasmid vector. The pUC-VP0M-2A-VP1-2A-VP3 plasmid is used as a template, VP0M-2A-VP1-2A-VP3-F, VP0M-2A-VP1-2A-VP3-R is used as an upstream and downstream primer for PCR amplification (the gene sequences of VP0M-2A-VP1-2A-VP3-F, VP0M-2A-VP1-2A-VP3-R are respectively shown in SEQ ID NO.3 and SEQ ID NO. 4), and the amplification system is shown in Table 1.

TABLE 1 VP0M-2A-VP1-2A-VP3 Gene amplification System

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 45 seconds, annealing at 54 ℃ for 45 seconds, extension at 72 ℃ for 1 minute, 35 cycles; extension at 72 ℃ for 10 min.

The PCR product was subjected to gel electrophoresis to verify the size of the target gene, and as shown in FIG. 1, a band of interest appeared at a position of 2.6kbp, and the target gene was successfully amplified and recovered and purified using a gel recovery and purification kit.

2. Enzyme digestion and purification the PCR amplification product of pFastBac1 plasmid and VP0M-2A-VP1-2A-VP3 gene was subjected to double enzyme digestion at BamH I and Hind III37 ℃ for 3 hours, and the specific enzyme digestion reaction systems are shown in tables 2 and 3.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBac1 plasmid and the VP0M-2A-VP1-2A-VP3 gene fragment by using a gel recovery and purification kit respectively.

TABLE 2 enzyme digestion reaction system of VP0M-2A-VP1-2A-VP3 gene

TABLE 3 pFastBac1 plasmid digestion reaction System

3. Ligation the double-digested pFastBac1 plasmid and the product of the VP0M-2A-VP1-2A-VP3 gene digestion were ligated using T4 DNA ligase in a 16 ℃ water bath overnight. The specific ligation reaction system is shown in Table 4.

TABLE 4 VP0M-2A-VP1-2A-VP3 Gene and pFastBac1 plasmid ligation System

4. Transformation mu.l of the ligation product was added to 100. mu.l of DH 5. alpha. competent cells and mixed well, ice-cooled for 30 minutes, heat-shocked in a water bath at 42 ℃ for 90 seconds, ice-cooled for 2 minutes again, added to 900. mu.l of LB liquid medium without Amp, and cultured at 37 ℃ for 1 hour. 1.0mL of the cell suspension was concentrated to 100. mu.l and applied to LB solid medium containing Amp, and cultured at 37 ℃ for 16 hours.

5. Colony PCR and sequencing identification Single colonies on the picked plates are respectively inoculated into LB liquid culture medium, cultured for 2 hours at 37 ℃, colony PCR identification is carried out by taking bacterial liquid as a template and VP0M-2A-VP1-2A-VP3-F and VP0M-2A-VP1-2A-VP3-R as primers, PCR products are subjected to gel electrophoresis to verify the size of target genes, and as shown in figure 2, a sample with a 2.6kbp band is a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector pF-VP0M-2A-VP1-2A-VP3 containing the target gene is shown in FIG. 3.

Example 2 construction of recombinant baculovirus genome Bac-VP0M-2A-VP1-2A-VP3

DH10Bac transformation mu.l pF-VP0M-2A-VP1-2A-VP3 plasmid from example 1 was added to 100. mu.l DH10Bac competent cells and mixed well, ice-cooled for 30 min, water-bath heat shock at 42 ℃ for 90 sec, ice-cooled for 2 min, added to 900. mu.l LB liquid medium without Amp, and cultured at 37 ℃ for 5 h. After 100. mu.l of the diluted bacterial solution was diluted 81 times, 100. mu.l of the diluted bacterial solution was applied to LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37 ℃ for 48 hours.

2. Selecting a single colony, selecting a large white colony by using an inoculating needle, streaking on an LB solid culture medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, culturing for 48 hours at 37 ℃, selecting a single colony, inoculating an LB liquid culture medium containing gentamicin, kanamycin and tetracycline, culturing, preserving strains, and extracting plasmids to obtain the recombinant plasmid Bacmid-VP0M-2A-VP1-2A-VP 3.

Example 3 recombinant baculovirus transfection

Six well plates were seeded 0.8X 10 per well⁶The confluency of Sf9 cells is 50-70%. The following complexes were prepared for each well: diluting 4. mu.l of Cellffectin transfection reagent with 100. mu.l of transfection medium T1, and shaking briefly with vortex; mu.g of the recombinant Bacmid-VP0M-2A-VP1-2A-VP3 plasmid from example 2 was diluted with 100. mu.l of transfection culture T1 medium, and the diluted transfection reagent and plasmid were mixed and gently blown down to prepare a transfection mixture. Adding the transfection complex after the cells adhere to the wall, incubating for 5 hours at 27 ℃, removing the supernatant, adding 2mLSF-SFM fresh culture medium, and culturing at 27 DEGAnd culturing for 4-5 days and harvesting the supernatant. Obtaining recombinant baculovirus rBac-VPOM-2A-VP1-2A-VP3, detecting virus titer of the harvested P1 generation recombinant baculovirus by using an MTT relative efficacy method, wherein the titer of rBac-VP0M-2A-VP1-2A-VP 3P 1 viruses is 3.4 multiplied by 10⁷pfu/mL, and amplifying recombinant baculovirus rBac-VP1M-2A-VP1-2A-VP3 as seed virus for later use.

Three control groups of recombinant baculoviruses were additionally constructed as described in the above example (table 5):

TABLE 5

Example 4 SDS-PAGE detection

The cell culture harvested in example 3 and each control group were subjected to SDS-PAGE detection while using Sf9 cells infected with empty baculovirus as a negative control. The specific operation is as follows: mu.l of the harvested cell culture was taken, 10. mu.l of 5 × loading buffer was added, the mixture was centrifuged in a boiling water bath for 5 minutes at 12000r/min for 1 minute, the supernatant was subjected to SDS-PAGE gel (12% strength gel) electrophoresis, and the gel was stained and decolored after electrophoresis to observe the band. As shown in FIG. 4, the rBac-VP0M-2A-VP1-2A-VP3 component showed the desired bands at molecular weights around 27kDa and 31kDa, respectively, the control group 1 showed the desired bands at molecular weights around 27kDa, 31kDa and 94kDa, respectively, the control group 2 showed the desired bands at molecular weights around 27kDa and 31kDa, the control group 3 showed the desired band at molecular weight around 31kDa, and the negative control showed no band at the corresponding position.

Example 5 Western Blot identification

The product after SDS-PAGE electrophoresis in example 4 was transferred to an NC (nitrocellulose) membrane, blocked with 5% skim milk for 2 hours, incubated with chicken-derived anti-AEV positive serum for 2 hours, rinsed, incubated with a secondary goat anti-chicken polyclonal antibody labeled with HRP for 2 hours, rinsed, and then added dropwise with an enhanced chemiluminescent fluorescent substrate and photographed using a chemiluminescent imager. As shown in FIG. 5, the target protein was correctly expressed in Sf9 cells, and the cleavage efficiency of the fusion protein was greatly improved after the mutation of 2A protein.

Example 6 Indirect immunofluorescence assay

A suspension of Sf9 cells transfected with rBac-VP0M-2A-VP1-2A-VP3 was added to a 96-well cell culture plate at 100. mu.l/well (cell concentration 2.5X 10)⁵～4.0×10⁵one/mL), 4 wells were seeded, left at 27 ℃ for 15 minutes, Sf9 cells were attached to the bottom wall of the plate, and 10. mu.l of a 10-fold diluted seed was added to each well. Meanwhile, a blank cell control is set. After inoculation, the cells are placed in a constant temperature incubator at 27 ℃ for culture for 72-96 hours, the culture solution is discarded, and cold methanol/acetone (1: 1) is used for fixation. Firstly reacting with chicken source anti-AEV multi-antiserum, then reacting with FITC labeled goat anti-chicken IgG, and observing the result by an inverted fluorescence microscope. As shown in FIG. 6, no fluorescence could be observed when the cells inoculated with the empty baculovirus Sf9, while fluorescence could be observed when the cells inoculated with the recombinant baculovirus Sf9 indicated that the target antigen was correctly expressed in Sf9 cells and the recombinant baculovirus was correctly constructed.

Example 7 AEVVLP purification and Electron microscopy

1. And (3) respectively freezing and thawing the recombinant baculovirus rBac-VP0M-2A-VP1-2A-VP3 and cell cultures of three control groups for three times by sucrose density gradient centrifugation, centrifuging for 30 minutes at 12000r/min, taking supernatant, filtering by a 0.22 mu m filter membrane, removing impurities, and concentrating by 10 times by using an ultrafiltration tube with the molecular weight cutoff of 500 kDa. 10ml of a 40% sucrose solution was added to each centrifuge tube, then 2.0ml of the ultrafiltration concentrated sample was added, ultracentrifugation was performed at 29000r/min for 2 hours, the supernatant was discarded, and the pellet was resuspended in 2.0ml PBS. Then the suspension is centrifuged by sucrose with gradient concentration of 50 percent, 60 percent, 70 percent and 80 percent respectively, ultracentrifuged at 29000r/min for 2 hours, and then strips positioned at the junction of 60 percent to 70 percent concentration are collected.

And performing protein electrophoresis detection by taking a strip at a 60-70% concentration boundary to perform SDS-PAGE detection. The electrophoretogram is shown in FIG. 7, and AEV-VLP, control 1 and control 2 all showed bands at molecular weights 27kDa and 31kDa, indicating that three proteins, VP0, VP3 and VP1, purified by sucrose gradient centrifugation, could form VLPs, and VP1 alone could not form VLPs. The AEV-VLP sample bands were significantly larger than control 2 and control 3, indicating that it was easier to assemble into VLPs.

2. And (3) respectively dripping the small amount of purified and concentrated AEV-VLP sample and the three control group samples on a copper net with a carbon film for electron microscope observation, naturally drying, dripping 2% sodium phosphotungstate solution for negative dyeing, and carrying out electron microscope observation after the negative dyeing. The virus-like particles with the same size and morphology as AEV virus particles are observed under an electron microscope, and the diameter of VLP is about 20-25nm, and the specific result is shown in figure 8.

3. The purified AEV-VLP and control 2 samples were diluted to the same concentration with PBS, treated at 37 ℃ for 12h, and ultracentrifuged at 29000r/min for 2 hours. The depth of the VLP band in the ultracentrifuge tube was observed. As shown in fig. 9, AEV-VLPs were significantly higher, indicating that the engineered VLPs were more stable and more tolerant to high temperatures.

Example 8 bioreactor serum-free suspension culture of insect cells and quantification of expression of AEV-VLP and agar expansion titer determination

Aseptically culturing Sf9 insect cells in 1000ml shake flask for 3-4 days until the concentration reaches 3-5X 10⁶cell/mL, when the activity is more than 95%, inoculating the cells into a 5L bioreactor, wherein the inoculation concentration is 3-8 × 10⁵cell/mL. When the cell concentration reaches 3-55X 10⁶At cell/mL, cells were seeded into a 50L bioreactor until the cells grew to a concentration of 3-55X 10⁶cell/mL, inoculating into 500L bioreactor until cell concentration reaches 2-85 × 10⁶When cell/mL, AEV-VLP is inoculated, and the culture conditions of the reactor are that the pH value is 6.0-6.5, the temperature is 25-27 ℃, the dissolved oxygen is 30-80 percent, and the stirring speed is 100-. In view of the optimum conditions for cell culture, it is preferable to set pH6.2, the temperature at the stage of cell culture at 27 ℃, the dissolved oxygen at 50%, and the stirring speed at 100-180 rpm. Culturing for 5-9 days after infection, adding one-thousandth final concentration BEI, acting at 37 deg.C for 48 hr, adding two-thousandth final concentration Na₂S₂O₃The inactivation is terminated. Cell culture supernatant is obtained by centrifugation or hollow fiber filtration, and the vaccine stock solution is stored at 2-8 ℃.

The content of AEV-VLP in the prepared vaccine antigen was determined using Elisa method. The operation mode is as follows: chicken anti-AEV polyclonal antiserum was diluted with coating buffer to appropriate concentration, 100. mu.l per well, overnight at 4 ℃, PBST washedThree times, 1% BSA blocking for 1 h. Antigen standards (AEV-VLP obtained by sucrose density gradient centrifugation purification) with different concentrations and gradient dilution of samples to be detected are added, incubated for 1 hour at 37 ℃, and washed three times by PBST. Add detection antibody per well: the AEV-VLP protein monoclonal antibody (a monoclonal antibody recognizing the structure of AEV virus-like particles) was incubated at 37 ℃ for 1 hour and PBST washed three times. A secondary antibody, i.e., HRP-labeled goat anti-chicken IgG, was added to each well, incubated at 37 ℃ for 1 hour, and washed three times with PBST. TMB development for 10 min, 2M H₂SO₄The reaction was terminated. Reading by a microplate reader, and calculating the amount of AEV-VLP in the sample to be detected through a standard curve.

AEV-VLPs were prepared on a large scale according to example 8, and the results of Elisa test were as follows, with the average content of AEV-VLPs in the vaccine stock being about 176 mg/L.

Protein titers of the expressed recombinant AEV-VLPs and three control groups were determined using the agar-agar method. Punching plum blossom holes on an agarose gel plate, adding AEV agar detection standard serum in the middle of the plum blossom holes, and adding expression antigens diluted by 2 to the power of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9and 10 on the periphery of the plum blossom holes respectively. After incubation in an inverted position for 72h, the line of precipitation was observed. The maximum dilution at which a precipitate line appears is its agar titer. The agar titer detection results are as follows: the AEV-VLP protein agar-agar titer is 1: 64, the control group 1 protein agar titer is 1: 32, the control group 2 protein agar titer is 1: 32, and the control group 3 protein agar titer is 1: 4.

EXAMPLE 9 preparation of the vaccine

The recombinant protein stock solution expressed in the example 8 is taken and diluted by using normal saline, vaccine stock solutions with different concentrations are prepared according to requirements, and then the diluted vaccine stock solution and an oil adjuvant are prepared into an oil emulsion vaccine according to the proportion of 2: 3. Specifically, 1429g of white oil, 70.2g of span, 8.43g of aluminum stearate and 53.3g of tween are added into 1L of mixed vaccine stock solution. Then crushing and emulsifying by an emulsifying crusher to prepare an oil emulsion adjuvant inactivated vaccine, and storing at 4 ℃ after quality inspection is qualified.

Example 10 immunopotency assay

AEV-VLP vaccines were prepared at concentrations of 100. mu.g/mL, 50. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL and 5. mu.g/mL, respectively, according to example 9, and the same concentration of AEV-VP1 vaccine was prepared from the cell culture of control 3 in example 3 according to the method of example 9, and stored at 4 ℃ until use.

110 SPF chickens of 21-35 days old are randomly divided into three groups, namely 50 AEV-VLP immune groups, 50 control 3 immune groups and 10 negative control groups. The AEV-VLP immune group and the control group 3 immune group are divided into 5 groups, and 10 vaccines prepared in different concentrations are injected into the neck part subcutaneously or intramuscularly respectively; the negative control group was injected with physiological saline in the same manner, and after 21 days, all chickens were individually bled, and serum was separated and antibody was detected using IDEXX kit. After blood collection, 0.03mL (containing 1000 EID) of AEV LC-95 strain virus solution is inoculated in each group of chicken brains₅₀) And observing for 21 days, and counting the protection rate. The specific results are shown in Table 6.

TABLE 6 neutralizing index of antibody after chicken immunization and protection rate after challenge

It is to be understood that the above-described embodiments are part of the present invention, and not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Sequence listing

<110> Suzhou Shino Biotechnology Ltd

<120> novel gene engineering vaccine of avian encephalomyelitis virus

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2637

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

ggatccatgt ctaagctgtt ctccaccgtg ggtagaacag ttgacgaagt gttgtccgtg 60

ctgaacgacg aagataccga atcttacgca ggtccagaca gaacagcagt tgttggagga 120

ggtttcctga ctacagtgga tcagtcttcc gtgtctacag ctactatggg ttctctgcaa 180

gacgtgcaat accgtacagc agtggatatc ccaggttccc gcgtgactca gggcgaacgt 240

ttcttcttga tcgaccaacg cgagtggaac tctactcaaa gcgagtggca attgctgggt 300

aagatcgaca tcgtgaagga gttgctggac caatcatacg ctgttgacgg tttgctgaag 360

taccactcct acgctagatt cggtttggac gtgatcgtgc agatcaaccc tacttctttc 420

caggctggag gtttgattgc agctttggtg ccttacgacc aagtggatat cgagtctatc 480

gcatttatga ccacctattg ccacggcaag ctgaattgca acatcaacaa cgtggtgcgc 540

atgaaggtgc cttacatcta ctccagaggt tgctacaacc tgcgtaactc agcctactcc 600

atttggatgc tggtgatcag agtttggagc agactgcaat tgggttccgg tacttctacc 660

cagatcacta tcaccactct ggctagattc gtggacttgg aactgcacgg tttgtctcct 720

ttggtggctc aacatcacca tcaccatcac agcagccatt ttagctttga tgaaattagc 780

gaagcgcagt gcagcaaatg caaaattgat ctgggcgata ttgtgagctg cagcggcgaa 840

aaagcgaaac attttggcag ctatgtgggc gatggcgtgg tgcatgtgga tccggaaatg 900

atgcgtaacg agttccgtct gtcctcttct tccaacatcg tgaacctggc caactacgaa 960

gacgctaggg ctaaagtttc tctcgctctg ggtcaagaag gtttctcccg cgactcctca 1020

tctacaggag gaggtatgct gtaccacttc agtcagtgga cttccatccc ttgtctggct 1080

ttcatcttca ccttcccagg tacagtgggt cctggtactc gtatttggtc tactaccgtc 1140

gaccctttct cttgcaactt gagagctttc tccaccgttc accctactaa cctgtcttcc 1200

atcgcaggta tgttctgctt ctggagagga gatatcgtgt tcgagttcca ggtgttccgt 1260

actaagtacc actccggtag actgatgttc gtgtacgttc caggagacga aaacaccaag 1320

atctccactc tgaccgctac tcaggcttcc tcaggtctga cagcagtgtt cgacatcaac 1380

ggagtgaact caactctggt gttccgttgc cctttcatct cagacactcc ttacagggtg 1440

aaccctacta ctcacaagtc tccttggcct tacgctacag gtaagctggt ttgctacgtg 1500

tacaaccgtt tgaacgctcc agcttctgtg tctccttccg tgtctatcaa cgtgtacaag 1560

tcagccgtgg acttggaatt gtacgctcca gtctacggag tgtctcctac taacacttcc 1620

gtgttcgctc aacatcacca tcaccatcac agcagccatt ttagctttga tgaaattagc 1680

gaagcgcagt gcagcaaatg caaaattgat ctgggcgata ttgtgagctg cagcggcgaa 1740

aaagcgaaac attttggcag ctatgtgggc gatggcgtgg tgcatgtgga tccggaaggt 1800

aaaggagacg aaggaggttt ctcctcagtt ccagaagtgg aacaacacgt ggtggaagac 1860

aaggaacctc aaggtccttt gcacgttact cctttcggag cagttaaggc tatggaggac 1920

cctcaactgg ctagaaagac tccaggtact ttcccagaat tggctccagg taagcctaga 1980

catacagtgg accacatgga cctgtacaag ttcatgggta gggctcacta cctctggggt 2040

cataagttca ccaagaccga catgcagtac accttccaga tccctctgtc tcctatcaag 2100

gagggtttcg tgacaggtac tttgcgttgg ttcctgtcac tgttccaact gtacaggggt 2160

tctttggaca tcaccatgac cttcgctggt aagactaacg tggacggtat cgtgtacttc 2220

gtaccagaag gagtggctat cgaaaccgaa cgtaaggaac agactcctct gctgactctg 2280

aactacaaga cctcagtggg agctatccgt ttcaacacag gtcagactac caacgtgcag 2340

ttcagaatcc ccttctacac tcctctggaa cacatcgcta ctcactccaa gaacgctatg 2400

gactcagtgt tgggagctat cactacccag atcaccaact actccgctca agacgaatac 2460

ctgcaggtga cctactacat ctccttcaac gaggactccc aattctcagt gcctagagct 2520

gttccagtgg tgtcttcttt caccgatacc tcctccaaga ccgtgatgaa cacttactgg 2580

ctggacgacg acgaattggt tgaagaacat caccatcacc atcactaatg aaagctt 2637

<210> 2

<211> 855

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 2

Met Ser Lys Leu Phe Ser Thr Val Gly Arg Thr Val Asp Glu Val Leu

1 5 10 15

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

20 25 30

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

35 40 45

Val Ser Thr Ala Thr Met Gly Ser Leu Gln Asp Val Gln Tyr Arg Thr

50 55 60

Ala Val Asp Ile Pro Gly Ser Arg Val Thr Gln Gly Glu Arg Phe Phe

65 70 75 80

Leu Ile Asp Gln Arg Glu Trp Asn Ser Thr Gln Ser Glu Trp Gln Leu

85 90 95

Leu Gly Lys Ile Asp Ile Val Lys Glu Leu Leu Asp Gln Ser Tyr Ala

100 105 110

Val Asp Gly Leu Leu Lys Tyr His Ser Tyr Ala Arg Phe Gly Leu Asp

115 120 125

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

130 135 140

Ala Ala Leu Val Pro Tyr Asp Gln Val Asp Ile Glu Ser Ile Ala Phe

145 150 155 160

Met Thr Thr Tyr Cys His Gly Lys Leu Asn Cys Asn Ile Asn Asn Val

165 170 175

Val Arg Met Lys Val Pro Tyr Ile Tyr Ser Arg Gly Cys Tyr Asn Leu

180 185 190

Arg Asn Ser Ala Tyr Ser Ile Trp Met Leu Val Ile Arg Val Trp Ser

195 200 205

Arg Leu Gln Leu Gly Ser Gly Thr Ser Thr Gln Ile Thr Ile Thr Thr

210 215 220

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

225 230 235 240

Ala Gln Ser Ser His Phe Ser Phe Asp Glu Ile Ser Glu Ala Gln Cys

245 250 255

Ser Lys Cys Lys Ile Asp Leu Gly Asp Ile Val Ser Cys Ser Gly Glu

260 265 270

Lys Ala Lys His Phe Gly Ser Tyr Val Gly Asp Gly Val Val His Val

275 280 285

Asp Pro Glu Met Met Arg Asn Glu Phe Arg Leu Ser Ser Ser Ser Asn

290 295 300

Ile Val Asn Leu Ala Asn Tyr Glu Asp Ala Arg Ala Lys Val Ser Leu

305 310 315 320

Ala Leu Gly Gln Glu Gly Phe Ser Arg Asp Ser Ser Ser Thr Gly Gly

325 330 335

Gly Met Leu Tyr His Phe Ser Gln Trp Thr Ser Ile Pro Cys Leu Ala

340 345 350

Phe Ile Phe Thr Phe Pro Gly Thr Val Gly Pro Gly Thr Arg Ile Trp

355 360 365

Ser Thr Thr Val Asp Pro Phe Ser Cys Asn Leu Arg Ala Phe Ser Thr

370 375 380

Val His Pro Thr Asn Leu Ser Ser Ile Ala Gly Met Phe Cys Phe Trp

385 390 395 400

Arg Gly Asp Ile Val Phe Glu Phe Gln Val Phe Arg Thr Lys Tyr His

405 410 415

Ser Gly Arg Leu Met Phe Val Tyr Val Pro Gly Asp Glu Asn Thr Lys

420 425 430

Ile Ser Thr Leu Thr Ala Thr Gln Ala Ser Ser Gly Leu Thr Ala Val

435 440 445

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

450 455 460

Ile Ser Asp Thr Pro Tyr Arg Val Asn Pro Thr Thr His Lys Ser Pro

465 470 475 480

Trp Pro Tyr Ala Thr Gly Lys Leu Val Cys Tyr Val Tyr Asn Arg Leu

485 490 495

Asn Ala Pro Ala Ser Val Ser Pro Ser Val Ser Ile Asn Val Tyr Lys

500 505 510

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

515 520 525

Thr Asn Thr Ser Val Phe Ala Gln Ser Ser His Phe Ser Phe Asp Glu

530 535 540

Ile Ser Glu Ala Gln Cys Ser Lys Cys Lys Ile Asp Leu Gly Asp Ile

545 550 555 560

Val Ser Cys Ser Gly Glu Lys Ala Lys His Phe Gly Ser Tyr Val Gly

565 570 575

Asp Gly Val Val His Val Asp Pro Glu Gly Lys Gly Asp Glu Gly Gly

580 585 590

Phe Ser Ser Val Pro Glu Val Glu Gln His Val Val Glu Asp Lys Glu

595 600 605

Pro Gln Gly Pro Leu His Val Thr Pro Phe Gly Ala Val Lys Ala Met

610 615 620

Glu Asp Pro Gln Leu Ala Arg Lys Thr Pro Gly Thr Phe Pro Glu Leu

625 630 635 640

Ala Pro Gly Lys Pro Arg His Thr Val Asp His Met Asp Leu Tyr Lys

645 650 655

Phe Met Gly Arg Ala His Tyr Leu Trp Gly His Lys Phe Thr Lys Thr

660 665 670

Asp Met Gln Tyr Thr Phe Gln Ile Pro Leu Ser Pro Ile Lys Glu Gly

675 680 685

Phe Val Thr Gly Thr Leu Arg Trp Phe Leu Ser Leu Phe Gln Leu Tyr

690 695 700

Arg Gly Ser Leu Asp Ile Thr Met Thr Phe Ala Gly Lys Thr Asn Val

705 710 715 720

Asp Gly Ile Val Tyr Phe Val Pro Glu Gly Val Ala Ile Glu Thr Glu

725 730 735

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

740 745 750

Gly Ala Ile Arg Phe Asn Thr Gly Gln Thr Thr Asn Val Gln Phe Arg

755 760 765

Ile Pro Phe Tyr Thr Pro Leu Glu His Ile Ala Thr His Ser Lys Asn

770 775 780

Ala Met Asp Ser Val Leu Gly Ala Ile Thr Thr Gln Ile Thr Asn Tyr

785 790 795 800

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

805 810 815

Glu Asp Ser Gln Phe Ser Val Pro Arg Ala Val Pro Val Val Ser Ser

820 825 830

Phe Thr Asp Thr Ser Ser Lys Thr Val Met Asn Thr Tyr Trp Leu Asp

835 840 845

Asp Asp Glu Leu Val Glu Glu

850 855

<210> 3

<211> 35

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

ataggatcca tgtctaagct gttctccacc gtggg 35

<210> 4

<211> 38

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ataaagcttt cattagtgat ggtgatggtg atgttctt 38

<210> 5

<211> 2637

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

ggatccatgt ctaagctgtt ctccaccgtg ggtagaacag ttgacgaagt gttgtccgtg 60

ctgaacgacg aagataccga atcttacgca ggtccagaca gaacagcagt tgttggagga 120

ggtttcctga ctacagtgga tcagtcttcc gtgtctacag ctactatggg ttctctgcaa 180

gacgtgcaat accgtacagc agtggatatc ccaggttccc gcgtgactca gggcgaacgt 240

ttcttcttga tcgaccaacg cgagtggaac tctactcaaa gcgagtggca attgctgggt 300

aagatcgaca tcgtgaagga gttgctggac caatcatacg ctgttgacgg tttgctgaag 360

taccactcct acgctagatt cggtttggac gtgatcgtgc agatcaaccc tacttctttc 420

caggctggag gtttgattgc agctttggtg ccttacgacc aagtggatat cgagtctatc 480

gcatttatga ccacctattg ccacggcaag ctgaattgca acatcaacaa cgtggtgcgc 540

atgaaggtgc cttacatcta ctccagaggt tgctacaacc tgcgtaactc agcctactcc 600

atttggatgc tggtgatcag agtttggagc agactgcaat tgggttccgg tacttctacc 660

cagatcacta tcaccactct ggctagattc gtggacttgg aactgcacgg tttgtctcct 720

ttggtggctc aacatcacca tcaccatcac agcagccatt ttagctttga tgaaattgaa 780

gaagcgcagt gcagcaaatg caaaattgat ctgggcgata ttgtgagctg cagcggcgaa 840

aaagcgaaac attttggcgt gtatgtgggc gatggcgtgg tgcatgtgga tccggaaatg 900

atgcgtaacg agttccgtct gtcctcttct tccaacatcg tgaacctggc caactacgaa 960

gacgctaggg ctaaagtttc tctcgctctg ggtcaagaag gtttctcccg cgactcctca 1020

tctacaggag gaggtatgct gtaccacttc agtcagtgga cttccatccc ttgtctggct 1080

ttcatcttca ccttcccagg tacagtgggt cctggtactc gtatttggtc tactaccgtc 1140

gaccctttct cttgcaactt gagagctttc tccaccgttc accctactaa cctgtcttcc 1200

atcgcaggta tgttctgctt ctggagagga gatatcgtgt tcgagttcca ggtgttccgt 1260

actaagtacc actccggtag actgatgttc gtgtacgttc caggagacga aaacaccaag 1320

atctccactc tgaccgctac tcaggcttcc tcaggtctga cagcagtgtt cgacatcaac 1380

ggagtgaact caactctggt gttccgttgc cctttcatct cagacactcc ttacagggtg 1440

aaccctacta ctcacaagtc tccttggcct tacgctacag gtaagctggt ttgctacgtg 1500

tacaaccgtt tgaacgctcc agcttctgtg tctccttccg tgtctatcaa cgtgtacaag 1560

tcagccgtgg acttggaatt gtacgctcca gtctacggag tgtctcctac taacacttcc 1620

gtgttcgctc aacatcacca tcaccatcac agcagccatt ttagctttga tgaaattgaa 1680

gaagcgcagt gcagcaaatg caaaattgat ctgggcgata ttgtgagctg cagcggcgaa 1740

aaagcgaaac attttggcgt gtatgtgggc gatggcgtgg tgcatgtgga tccggaaggt 1800

aaaggagacg aaggaggttt ctcctcagtt ccagaagtgg aacaacacgt ggtggaagac 1860

aaggaacctc aaggtccttt gcacgttact cctttcggag cagttaaggc tatggaggac 1920

cctcaactgg ctagaaagac tccaggtact ttcccagaat tggctccagg taagcctaga 1980

catacagtgg accacatgga cctgtacaag ttcatgggta gggctcacta cctctggggt 2040

cataagttca ccaagaccga catgcagtac accttccaga tccctctgtc tcctatcaag 2100

gagggtttcg tgacaggtac tttgcgttgg ttcctgtcac tgttccaact gtacaggggt 2160

tctttggaca tcaccatgac cttcgctggt aagactaacg tggacggtat cgtgtacttc 2220

gtaccagaag gagtggctat cgaaaccgaa cgtaaggaac agactcctct gctgactctg 2280

aactacaaga cctcagtggg agctatccgt ttcaacacag gtcagactac caacgtgcag 2340

ttcagaatcc ccttctacac tcctctggaa cacatcgcta ctcactccaa gaacgctatg 2400

gactcagtgt tgggagctat cactacccag atcaccaact actccgctca agacgaatac 2460

ctgcaggtga cctactacat ctccttcaac gaggactccc aattctcagt gcctagagct 2520

gttccagtgg tgtcttcttt caccgatacc tcctccaaga ccgtgatgaa cacttactgg 2580

ctggacgacg acgaattggt tgaagaacat caccatcacc atcactaatg aaagctt 2637

<210> 6

<211> 2619

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

ggatccatga gcaaactgtt tagcaccgtg ggccgcaccg tggatgaagt gctgagcgtg 60

ctgaacgatg aagataccga aagctatgcg ggcccggatc gcaccgcggt ggtgggcggc 120

ggctttctga ccaccgtgga tcagagcagc gtgagcaccg cgaccatggg cagcctgcag 180

gatgtgcagt atcgcaccgc ggtggatatt ccgggcagcc gcgtgaccca gggcgaacgc 240

ttttttctga ttgatcagcg cgaatggaac agcacccaga gcgaatggca gctgctgggc 300

aaaattgata ttgtgaaaga actgctggat cagagctatg cggtggatgg cctgctgaaa 360

tatcatagct atgcgcgctt tggcctggat gtgattgtgc agattaaccc gaccagcttt 420

caggcgggcg gcctgattgc ggcgctggtg ccgtatgatc aggtggatat tgaaagcatt 480

gcggcgatga ccacctattg ccatggcaaa ctgaactgca acattaacaa cgtggtgcgc 540

atgaaagtgc cgtatattta tagccgcggc tgctataacc tgcgcaacag cgcgtatagc 600

atttggatgc tggtgattcg cgtgtggagc cgcctgcagc tgggcagcgg caccagcacc 660

cagattacca ttaccaccct ggcgcgcttt gtggatctgg aactgcatgg cctgagcccg 720

ctggtggcgc agagcagcca ttttagcttt gatgaaatta gcgaagcgca gtgcagcaaa 780

tgcaaaattg atctgggcga tattgtgagc tgcagcggcg aaaaagcgaa acattttggc 840

agctatgtgg gcgatggcgt ggtgcatgtg gatccggaaa tgatgcgtaa cgagttccgt 900

ctgtcctctt cttccaacat cgtgaacctg gccaactacg aagacgctag ggctaaagtt 960

tctctcgctc tgggtcaaga aggtttctcc cgcgactcct catctacagg aggaggtatg 1020

ctgtaccact tcagtcagtg gacttccatc ccttgtctgg ctttcatctt caccttccca 1080

ggtacagtgg gtcctggtac tcgtatttgg tctactaccg tcgacccttt ctcttgcaac 1140

ttgagagctt tctccaccgt tcaccctact aacctgtctt ccatcgcagg tatgttctgc 1200

ttctggagag gagatatcgt gttcgagttc caggtgttcc gtactaagta ccactccggt 1260

agactgatgt tcgtgtacgt tccaggagac gaaaacacca agatctccac tctgaccgct 1320

actcaggctt cctcaggtct gacagcagtg ttcgacatca acggagtgaa ctcaactctg 1380

gtgttccgtt gccctttcat ctcagacact ccttacaggg tgaaccctac tactcacaag 1440

tctccttggc cttacgctac aggtaagctg gtttgctacg tgtacaaccg tttgaacgct 1500

ccagcttctg tgtctccttc cgtgtctatc aacgtgtaca agtcagccgt ggacttggaa 1560

ttgtacgctc cagtctacgg agtgtctcct actaacactt ccgtgttcgc tcaacatcac 1620

catcaccatc acagcagcca ttttagcttt gatgaaatta gcgaagcgca gtgcagcaaa 1680

tgcaaaattg atctgggcga tattgtgagc tgcagcggcg aaaaagcgaa acattttggc 1740

agctatgtgg gcgatggcgt ggtgcatgtg gatccggaag gtaaaggaga cgaaggaggt 1800

ttctcctcag ttccagaagt ggaacaacac gtggtggaag acaaggaacc tcaaggtcct 1860

ttgcacgtta ctcctttcgg agcagttaag gctatggagg accctcaact ggctagaaag 1920

actccaggta ctttcccaga attggctcca ggtaagccta gacatacagt ggaccacatg 1980

gacctgtaca agttcatggg tagggctcac tacctctggg gtcataagtt caccaagacc 2040

gacatgcagt acaccttcca gatccctctg tctcctatca aggagggttt cgtgacaggt 2100

actttgcgtt ggttcctgtc actgttccaa ctgtacaggg gttctttgga catcaccatg 2160

accttcgctg gtaagactaa cgtggacggt atcgtgtact tcgtaccaga aggagtggct 2220

atcgaaaccg aacgtaagga acagactcct ctgctgactc tgaactacaa gacctcagtg 2280

ggagctatcc gtttcaacac aggtcagact accaacgtgc agttcagaat ccccttctac 2340

actcctctgg aacacatcgc tactcactcc aagaacgcta tggactcagt gttgggagct 2400

atcactaccc agatcaccaa ctactccgct caagacgaat acctgcaggt gacctactac 2460

atctccttca acgaggactc ccaattctca gtgcctagag ctgttccagt ggtgtcttct 2520

ttcaccgata cctcctccaa gaccgtgatg aacacttact ggctggacga cgacgaattg 2580

gttgaagaac atcaccatca ccatcactaa tgaaagctt 2619

<210> 7

<211> 840

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

ggtaaaggag acgaaggagg tttctcctca gttccagaag tggaacaaca cgtggtggaa 60

gacaaggaac ctcaaggtcc tttgcacgtt actcctttcg gagcagttaa ggctatggag 120

gaccctcaac tggctagaaa gactccaggt actttcccag aattggctcc aggtaagcct 180

agacatacag tggaccacat ggacctgtac aagttcatgg gtagggctca ctacctctgg 240

ggtcataagt tcaccaagac cgacatgcag tacaccttcc agatccctct gtctcctatc 300

aaggagggtt tcgtgacagg tactttgcgt tggttcctgt cactgttcca actgtacagg 360

ggttctttgg acatcaccat gaccttcgct ggtaagacta acgtggacgg tatcgtgtac 420

ttcgtaccag aaggagtggc tatcgaaacc gaacgtaagg aacagactcc tctgctgact 480

ctgaactaca agacctcagt gggagctatc cgtttcaaca caggtcagac taccaacgtg 540

cagttcagaa tccccttcta cactcctctg gaacacatcg ctactcactc caagaacgct 600

atggactcag tgttgggagc tatcactacc cagatcacca actactccgc tcaagacgaa 660

tacctgcagg tgacctacta catctccttc aacgaggact cccaattctc agtgcctaga 720

gctgttccag tggtgtcttc tttcaccgat acctcctcca agaccgtgat gaacacttac 780

tggctggacg acgacgaatt ggttgaagaa catcaccatc accatcacta atgaaagctt 840

<210> 8

<211> 49

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

Ser Ser His Phe Ser Phe Asp Glu Ile Ser Glu Ala Gln Cys Ser Lys

1 5 10 15

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

20 25 30

Lys His Phe Gly Ser Tyr Val Gly Asp Gly Val Val His Val Asp Pro

35 40 45

Glu

Claims

1. The amino acid sequence of the fusion protein is shown as SEQ ID NO. 2.

2. The gene encoding the fusion protein of claim 1.

3. The coding gene of claim 2, wherein: the sequence of the coding gene is shown as SEQ ID NO. 1.

4. A recombinant gene vector comprising the coding gene of claim 2 or 3.

5. A recombinant baculovirus comprising the encoding gene of claim 2 or 3.

6. An insect cell comprising the gene of claim 2 or 3.

7. An immunological composition characterized by comprising: a fusion protein of claim 1; and, a pharmaceutically acceptable carrier.

8. The immunogenic composition of claim 7, wherein: the pharmaceutically acceptable carrier comprises one or more of white oil, aluminum stearate, span and Tween.

9. The method of producing a fusion protein according to claim 1, comprising:

10. The method of claim 9, wherein: the baculovirus expression system transfer vector comprises any one of pFastBac1, pVL1393 and pFastBac dual.

11. The method of claim 9, wherein: the insect cells include Sf9, High Five or Sf21 cells.

12. Use of the fusion protein of claim 1 or the immunological composition of claim 7 or 8 in the manufacture of a medicament for inducing an immune response against an avian encephalomyelitis virus antigen in a subject animal.

13. Use of the fusion protein of claim 1 or the immunogenic composition of claim 7 or 8 in the manufacture of a medicament for preventing infection of an animal by avian encephalomyelitis virus.

14. Use of the fusion protein according to claim 1 or the immunological composition according to claim 7 or 8 for the preparation of a novel genetically engineered vaccine of avian encephalomyelitis virus.