CN111729078B

CN111729078B - Chicken infectious anemia virus gene engineering vaccine

Info

Publication number: CN111729078B
Application number: CN202010720531.3A
Authority: CN
Inventors: 曹文龙; 孔迪; 滕小锘
Original assignee: Suzhou Shinuo Biotechnology Co ltd
Current assignee: Suzhou Womei Biology Co ltd
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2020-11-20
Anticipated expiration: 2040-07-24
Also published as: CN111729078A

Abstract

The invention discloses a genetic engineering vaccine for chicken infectious anemia virus, which comprises a fusion protein with 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, pathogenicity to poultry such as chicken and the like is avoided, strong humoral immunity can be generated in the poultry, the immunized poultry can resist strong toxicity and attack, and the vaccine can be prepared through large-scale serum-free suspension culture of a bioreactor, so that the vaccine production cost is greatly reduced.

Description

Chicken infectious anemia virus gene engineering vaccine

Technical Field

The invention relates to a genetic engineering vaccine, in particular to a genetic engineering vaccine for chicken infectious anemia virus, a preparation method and application thereof, and belongs to the technical field of animal immunity drugs.

Background

Infectious Anemia of Chicken (CIA) is an immunosuppressive Infectious disease caused by Infectious Anemia Virus of Chicken (CIAV). Chickens are the main natural hosts of the CIA, and chickens of all ages are easy to infect, wherein chick embryos and chicks within 14 days of age have the highest susceptibility and the highest incidence. After CIAV infection, the characteristic pathological changes are anemia and pale visible mucosa, the clinical symptoms of the sick chicken mainly include mental depression, emaciation, beast and anus, bluish purple local skin, prolonged blood coagulation time and the like, and the pathological changes mainly include atrophy of whole bone marrow, aplastic anemia, atrophy of whole lymphoid tissues and the like. The sick chicken are killed in a large amount in 5-6 days after the sick chicken are attacked, the morbidity is generally 20-60%, the mortality can reach 100% in high cases, the mortality is generally 5-10%, and the mortality can reach more than 60% in serious cases. CIAV can be transmitted in chicken flocks in a vertical or horizontal transmission mode to cause immunosuppression, so that the chicken flocks are susceptible to other viruses, bacteria and fungi and are infected secondarily; can also interfere with the immune effect of other poultry disease vaccines, resulting in immune failure. Since Yuasa equals 1979, the virus was first reported in Germany, France and other countries, and was first isolated from Vat et al in 1992. At present, CIA spreads and has a popular trend in China, and causes great economic loss to the poultry industry.

The CIAV belongs to the circovirus of the circovirus family, is a single-chain circular DNA virus, is spherical or hexagonal when observed under an electron microscope, has no envelope on the surface, is in regular 20-face symmetry, and has an average particle size of 24-26 nm. The total length of the CIAV genome sequence is 2298 or 2319bp, which is related to whether 4 or 5 21bp repeated sequences exist on the genome. The genome is mainly composed of three Open Reading Frames (ORFs), a promoter region and a signal region of poly adenosine. The three ORFs encode the Cap gene encoding capsid protein VPl, phosphatase protein VP2, and apoptin VP3 protein (Meehan B M, Todd D, Creelan J L, et al. Characterization of viral DNAs from cells with a fed with a chip and an agent: sequence analysis of the closed functional form and transfection capabilities of the closed gene fragments [ J ]. Archives of Virology, 1992,124(3-4): 301-319), respectively). The VP1 protein is the main capsid protein and immunity protein of CIAV, and the VP1 protein is rich in arginine, is tightly combined with genome DNA, has protection function on DNA, and can stimulate the organism to produce antibody. The VP2 protein belongs to a non-structural protein, is a helper protein, and has the main function of assisting the assembly of viruses into mature virus particles. In baculovirus expression systems, VP1 and VP2 were synthesized simultaneously, which induced the body to produce neutralizing antibodies against CIAV and acted as a protective immune response, while simple mixing of the separately expressed products did not stimulate the body to produce neutralizing antibodies (Yamaguchi S. Identification of a genetic determination of genetic in chip and a virus [ J ]. Journal of General Virology, 2001,82(5):1233 + 1238.). The VP3 protein, commonly known as apoptin, plays an important role in the life cycle of the virus, and VP3 alone causes apoptosis.

There have been some reports in the literature of CIAV genetically engineered vaccines. For example, CN110028558A provides a method for preparing subunit vaccine by using the co-expression of the truncated CIAV VP1 and VP2 proteins, because the N terminal of VP1 has a large amount of R and has a large positive charge, the VP1 is easily degraded by alkaline protease, so that the expression level of VP1 is low, and the truncated VP1 completely removes the R-rich part at the N terminal and can not be assembled into VLP, even if the VP2 is co-expressed, the expression level and the assembly efficiency of VP1 protein are low. For another example, CN109136198A provides a method for preparing live vector vaccine of chicken pox virus expressing CIAV VP1 and VP2 genes, but compared with baculovirus, poxvirus vector safety is low, and repeated vaccination of poxvirus vector vaccine may generate anti-vector immunity, resulting in reduction of immune response; the inactivated vaccine has an undesirable immune effect because CIAV can cause immunosuppression; the attenuated live vaccine has residual toxicity and the possibility of strong toxicity.

Disclosure of Invention

The invention mainly aims to provide a genetic engineering vaccine for chicken infectious anemia 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 an amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence which is 95% identical to the full-length amino acid sequence of the SEQ ID NO. 2.

The embodiment of the invention also provides a coding gene of the fusion protein, which comprises a nucleic acid molecule with a sequence shown in SEQ ID NO. 1 or a nucleic acid molecule with the same nucleotide sequence of the SEQ ID NO. 1 of more than 95 percent.

The embodiment of the invention also provides a recombinant virus vector containing the coding gene.

The embodiment of the invention also provides a recombinant baculovirus containing the coding gene.

The embodiment of the invention also provides a host cell containing the coding gene, mainly an insect cell.

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:

constructing a recombinant baculovirus expression vector comprising the encoding gene of the fusion protein;

introducing the recombinant baculovirus expression vector into an insect cell and culturing the insect cell under conditions that allow expression of the protein, followed by isolation and recovery of the fusion protein from the cell culture of the insect cell.

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

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

transforming the recombinant shuttle vector into competent cells to obtain a recombinant baculovirus vector;

insect cells are transfected with the recombinant baculovirus vector and cultured, after which the fusion protein is isolated and recovered from the cell culture of the insect cells.

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

The embodiments also provide use of the fusion protein or the immune composition in the manufacture of a medicament for inducing an immune response against an infectious anemia virus antigen in a subject animal.

The embodiment of the invention also provides application of the fusion protein or the immune composition in the production of a medicament for preventing the infection of animals with the chicken infectious anemia virus.

The embodiment of the invention also provides application of the fusion protein or the immune composition in preparing genetic engineering vaccines of chicken infectious anemia viruses.

Accordingly, the embodiment of the invention provides a chicken infectious anemia virus genetic engineering 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 the recombinant virus vector, the recombinant baculovirus or the insect cell containing the fusion protein coding gene in producing a reagent for detecting that an animal is infected by the chicken infectious anemia virus.

The embodiment of the invention also provides application of the recombinant viral vector, the recombinant baculovirus or the insect cell containing the fusion protein coding gene in producing a medicament for inducing an immune response against the chicken infectious anemia virus antigen in a test animal.

The embodiment of the invention also provides the application of the recombinant virus vector, the recombinant baculovirus or the insect cell containing the fusion protein coding gene in the production of a medicament for preventing the animal from being infected by the chicken infectious anemia virus.

Compared with the prior art, the embodiment of the invention forms the fusion protein by connecting and assembling the mutated chicken infectious anemia virus VP1 protein and the mutated chicken infectious anemia virus VP2 protein, obviously improves the expression quantity and the assembly efficiency of the VP1 protein, is safe and efficient, can provide a good immune effect only by a small amount of antigen, has antigenicity, immunogenicity and functions similar to those of natural protein, has higher expression level and strong immunogenicity, has no pathogenicity to poultry and the like, 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 chart of the PCR amplification product of the Fu gene in example 1, wherein the band of interest appears at the 1.7kbp position.

FIG. 2 is a gel electrophoresis chart of the colony PCR amplification product in example 1, wherein the band of interest appears at the 1.7kbp position.

FIG. 3 is a schematic diagram showing the structure of the transfer vector pF-Fu containing the desired gene in example 1.

FIG. 4 is a SDS-PAGE detection profile of the cell culture obtained in example 3.

FIG. 5 is a Western Blot detection pattern of the product after SDS-PAGE in example 4.

FIG. 6 is an indirect immunofluorescence assay profile of example 6.

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 the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence that is 95% or more identical to the full-length amino acid sequence of SEQ ID NO. 2.

The fusion protein provided by the embodiment of the invention is formed by mutated CIAV-VP1 protein and Linker + mutated CIAV-VP2 protein, wherein the stability of VP1 is increased without causing structural change of VP1 protein by mutating the largely positively charged R at positions 7, 28 and 42 of the amino acid sequence of VP1 protein to be A with neutral charge, and the mutated VP1 and the mutated VP2 protein are linked in the middle by using a Linker, the Linker has a sequence of GSSSSGPPGSSSSG, PP in the sequence can provide a flexible peptide connection, promote the mutual contact and interaction of VP2 and VP1, promote the assembly of VP1 into VLP, the mutant VP2 protein is truncated mutant VP2, and 66-138 amino acid fragments of the mutant VP2 are selected after a large number of experiments, and two D at positions 68 and 70 are mutated into two R, so that the stability and the assembly efficiency of the VP1 protein are further maintained.

Another aspect of the embodiment of the invention also provides a coding gene of the fusion protein, which comprises a nucleic acid molecule with a sequence shown in SEQ ID NO. 1 or a nucleic acid molecule with the same nucleotide sequence of the SEQ ID NO. 1 of more than 95 percent.

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:

constructing a recombinant baculovirus expression vector, wherein the recombinant baculovirus expression vector comprises a coding gene of the fusion protein;

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 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.

In another aspect of the embodiment of the invention, the application of the fusion protein or the immune composition in preparing a detection reagent for chicken infectious anemia virus is also provided.

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

Another aspect of the embodiments of the present invention also provides a use of the fusion protein or the immune composition in the manufacture of a medicament for preventing infection of an animal with chicken infectious anemia virus.

The other aspect of the embodiment of the invention also provides the application of the fusion protein or the immune composition in preparing the genetic engineering vaccine of the chicken infectious anemia virus.

Accordingly, another aspect of the embodiments of the present invention provides a chicken infectious anemia virus genetically engineered vaccine comprising any one of the immunization 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, the recombinant baculovirus or the insect cell containing the fusion protein coding gene in producing the reagent for detecting the infection of the animal with the chicken infectious anemia 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 infectious anemia virus antigen of chicken in a subject animal.

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 in the manufacture of a medicament for preventing an animal from being infected with chicken infectious anemia virus.

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

Accordingly, another aspect of the embodiments of the present invention also relates to a method of protecting a subject animal from infection by chicken infectious anemia virus, said method comprising administering said chicken infectious anemia virus genetically engineered vaccine to said subject avian animal.

Yet another aspect of the embodiments of the present invention provides a vaccine suitable for use in generating an immune response against chicken infectious anemia virus in a subject 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.

In some embodiments, the method for preparing the chicken infectious anemia virus genetic engineering vaccine specifically comprises the following steps:

1) preparing a nucleic acid molecule for encoding chicken infectious anemia virus fusion protein (which can be defined as CIAV-Fu);

2) constructing a recombinant vector, and cloning the nucleic acid molecule obtained 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 Sf9 cells, and obtaining recombinant baculovirus;

4) incubating the Sf9 cells, and automatically assembling the Sf9 cells into virus-like particles (VLPs) in the cells, and then performing recombinant expression to generate fusion proteins (defined as CIAV-VLPs);

5) adding the CIAV-VLP into an adjuvant to obtain the vaccine.

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.

An "adjuvant" as described in the present specification means any molecule added to the vaccine described in the present specification 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.

By "coding sequence" or "coding nucleic acid" as used herein is meant 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.

"consensus" or "consensus sequence" as used herein means a polypeptide sequence based on analysis of a cohort of subtypes of a particular chicken infectious anemia 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 chicken infectious anemia virus antigen.

"fragment" with respect to nucleic acid sequences as described 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 chicken infectious anemia virus antigen. The fragment may be a DNA fragment selected from at least one of various nucleotide sequences encoding protein fragments described below.

In the present specification, 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 chicken infectious anemia 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.

The term "genetic construct" as used herein 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 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 low stringency conditions. The term "substantially homologous" as used herein with respect to a single-stranded nucleic acid sequence means that the probe can hybridize to the single-stranded nucleic acid template sequence under low stringency conditions (i.e., is the complement of the single-stranded nucleic acid template sequence).

In the present specification, "identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences 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.

An "immune response" as described herein 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 a chicken infectious anemia virus consensus antigen. The immune response may be in the form of a cellular response or a humoral response or both.

A "nucleic acid" or "oligonucleotide" or "polynucleotide" as described herein 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.

In the present specification, a 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.

In the present specification, the expression of a gene is carried out under the control of a promoter 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.

In the present specification, "signal peptide" and "leader sequence" refer to amino acid sequences that can be linked to the amino terminus of the chicken infectious anemia virus protein described in the present specification. 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.

In the present specification, "stringent hybridization conditions" means 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 ℃.

In the present specification, "subtype" or "serotype": as used interchangeably herein and with respect to chicken infectious anemia virus, means a genetic variant of chicken infectious anemia virus such that one subtype is recognized by the immune system and is 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.

In the present specification, "variants" in the context of a peptide or polypeptide 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. Kate (Kyte), et al, 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). Substitutions may be made with amino acids having hydrophilicity values within ± 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 comprising 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.

The baculovirus expression system referred to herein is a gene expression system comprising a Baculovirus Expression Vector (BEV) and a host insect cell thereof. Baculovirus is a large DNA virus that infects invertebrates, the main host being insects and partial arthropods. Since neither humans nor other vertebrates host baculoviruses, the Baculovirus Expression Vector System (BEVS) developed by baculoviruses is very safe. The baculovirus expression vector refers to a recombinant baculovirus (recombinant baculovirus) carrying a foreign gene. Once the baculovirus expression vector infects host insect cells, the strong promoter in the baculovirus expression vector can be used to control the expression of the foreign gene and produce a large amount of recombinant protein.

In the present specification, post-translational modifications (post-translational modifications) of recombinant proteins obtained from baculovirus expression vector systems, for example, are similar to those produced in mammalian cells, and thus recombinant proteins produced using baculovirus expression vector systems, whether antigenically, immunogenically, and biologically, are substantially similar to, or even nearly identical to, the original proteins.

In one example of the present specification, the coding gene sequence of the fusion protein is constructed in a baculovirus vector, and the 5' end of the coding gene sequence is more selectively linked to the gene sequence of the signal peptide of baculovirus membrane glycoprotein (membrane protein) gp 64. Thus expressing the recombinant fusion antigen protein with the signal peptide of gp64, the signal peptide of gp64 can be cleaved off during intracellular delivery.

The vaccines of the present invention can be designed to control the extent or intensity of an immune response in a subject animal against one or more serotypes of chicken infectious anemia virus. The vaccine may comprise elements or agents that inhibit its integration into the chromosome. The vaccine may be RNA encoding structural proteins of chicken infectious anemia virus. An RNA vaccine can be introduced into the cells. The vaccine of the present invention may comprise chicken infectious anemia virus structural proteins. Chicken infectious anemia 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 the chicken infectious anemia virus can be induced. The chicken infectious anemia 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 a fragment of 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 SEQ ID NO. 1.

Some embodiments relate to proteins homologous to SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having 95%, 96%, 97%, 98% or 99% homology to the protein sequence as set forth in SEQ ID NO 2.

Some embodiments relate to the same protein as SEQ ID NO 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 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 as set forth in SEQ ID No. 2.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader. 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.

Immunogenic fragments of proteins having amino acid sequences homologous to the immunogenic fragment of SEQ ID NO. 2 can be provided. The immunogenic fragment can 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 protein that is 295% homologous to SEQ ID NO. Some embodiments relate to immunogenic fragments having 96%, 97%, 98%, 99% homology to the immunogenic fragments of the protein sequences of the present specification. Some embodiments relate to immunogenic fragments having homology to immunogenic fragments of the protein sequences of the present specification. 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.

In the present specification, immunogenic fragments of proteins having the same amino acid sequence as the immunogenic fragment of SEQ ID NO. 2 can be provided. 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.

In the present specification, the vaccine may comprise a nucleic acid construct or plasmid encoding said chicken infectious anemia virus fusion protein. The present specification provides genetic constructs that may comprise nucleic acid sequences encoding the chicken infectious anemia 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.

In the present specification, the genetic construct may also be part of the genome of a recombinant viral vector, including a recombinant baculovirus vector and the like. The genetic construct may be part of the genetic material in a recombinant microbial vector in a live attenuated microorganism or in a cell.

In the present description, 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.

In the present specification, a 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.

In the present specification, 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.

In the present specification, 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.

In the present specification, the vector may further comprise a polyadenylation signal, which may be downstream of the chicken infectious anemia 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).

In the present specification, the vector may also comprise an enhancer upstream of the consensus chicken infectious anemia virus core protein coding sequence or the consensus chicken infectious anemia 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.

In the present specification, 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).

In the present specification, the sequence of the chicken infectious anemia virus fusion protein CIAV-Fu of the present invention 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, SV40 polyA transfection termination signal, HSV tk polyA transfection termination signal or OpIE polyA termination signal, and other transcription termination signals are also possible. Wherein the insect cell line may be selected from but not limited to Sf9, High Five, S2 or Sf21 cells, preferably Sf 9. The animal of the invention mainly refers to poultry, in particular to chicken.

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 chicken infectious anemia virus fusion protein. The encoding gene of the chicken infectious anemia virus fusion protein can use P10 promoter, PH promoter, prawn beta-actin gene promoter, OpIE promoter and the like, and the corresponding transcription termination signal can be SV40 polyA transfection termination signal, HSV tk polyA transfection termination signal, OpIE polyA termination signal and the like. Furthermore, the shuttle plasmid and the baculovirus genome plasmid can be constructed to transfect insect cells together, recombinant baculovirus can be obtained through screening, and the recombinant baculovirus can be inoculated to Sf9 cells to express the chicken infectious anemia virus fusion protein.

Specifically, the designed codon-optimized fusion protein coding gene can be cloned to the enzyme cutting sites of BamH I and Pst I of pFastBac1 plasmid vector to construct recombinant plasmidGroup shuttleAnd (3) a carrier. And then co-transfecting the Sf9 cells with the constructed recombinant shuttle vector and the baculovirus genome plasmid to obtain the recombinant baculovirus.

The codon optimization referred to herein is to perform codon optimization according to the preference of the recombinant baculovirus expression system to each amino acid codon without changing the original amino acid sequence, so that the recombinant protein can be correctly translated and modified to exert immune protection effect. The reason for this is that, although Escherichia coli is currently the most common protein expression system and has the advantages of a rapid growth cycle, high yield, and low cost, it cannot be subjected to post-translational modification such as accurate saccharification, and thus has a high probability of forming insoluble inclusion bodies. In contrast, recombinant baculovirus expression systems produce large quantities of recombinant proteins and allow for post-translational modifications such as correct glycation. In one embodiment, the recombinant fusion antigen protein thus expressed has the correct glycosyl and folding structure.

When in use, the recombinant fusion protein can be prepared into a subunit vaccine composition with a pharmaceutically acceptable carrier, and an effective amount of the subunit vaccine composition is inoculated to poultry such as chickens. In one embodiment, the pharmaceutically acceptable carrier comprises an adjuvant and/or an immunopotentiator, wherein the adjuvant is not limited in kind, and specific examples thereof may include, but are not limited to, an alumina gel adjuvant, an oily adjuvant (e.g., Freund's complete adjuvant, Freund's incomplete adjuvant, etc.), or any combination thereof.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of disease; a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts.

The fusion protein is expressed by using the baculovirus and the Sf9 cell, the antigenicity, the immunogenicity and the 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 such as chicken.

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, 1987 and 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 transfer vector pF-CIAV-Fu

CIAV-Fu gene amplification and purification CIAV-Fu gene (SEQ ID NO: 1) after codon optimization is synthesized by Shanghai Sangni biological science and technology limited and cloned to pUC17 vector to obtain pUC-Fu plasmid vector. PCR amplification was performed using pUC-Fu plasmid as template and CIAV-Fu-F, CIAV-Fu-R as upstream and downstream primers (the sequence of CIAV-Fu-F, CIAV-Fu-R is shown in SEQ ID NO:3, 4), and the amplification system is shown in Table 1.

TABLE 1 CIAV-Fu 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 1.7kbp, and the target gene was successfully amplified and recovered and purified using a gel recovery and purification kit.

2. Enzyme digestion and purification the pFastBac1 plasmid and the PCR amplification product of the CIAV-Fu gene were digested simultaneously at 37 ℃ for 3 hours using BamHI and Hind III, and the specific 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 Fu gene fragment by using a gel recovery and purification kit respectively.

TABLE 2 CIAV-Fu Gene restriction system

TABLE 3 pFastBac1 plasmid digestion reaction System

3. Ligation the digested pFastBac1 plasmid and the digested product of CIAV-Fu gene were ligated overnight using T4 DNA ligase in a 16 ℃ water bath, the ligation system is shown in Table 4.

TABLE 4 CIAV-Fu Gene and pFastBac1 plasmid ligation System

4. Mu.l of the ligation product was added to 100. mu.l of DH 5. alpha. competent cells, mixed well, heat-shocked at 42 ℃ for 90 seconds, ice-bathed for 2 minutes, added to 900. mu.l of LB medium without Amp, and incubated at 37 ℃ for 1 hour. 1.0 mL of the cell suspension was concentrated by centrifugation to 100. mu.l, and the concentrated solution was applied to LB solid medium containing Amp and cultured at 37 ℃ for 16 hours.

5. Colony PCR and sequencing identification single colonies on the selected plate are respectively inoculated into an LB liquid culture medium, cultured for 2 hours at 37 ℃, and colony PCR is carried out by taking a bacterial liquid as a template and CIAV-Fu-F and CIAV-Fu-R as primers. The size of the gene of interest was confirmed by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 2, a sample showing a band of approximately 1.7kbp was positive. And (4) sending the bacterial liquid with positive colony PCR identification to a sequencing company for sequencing, and selecting the bacterial liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector pF-CIAV-Fu containing the target gene is shown in FIG. 3.

Example 2 recombinant baculovirus genome Bac-CIAV-Fu construction

DH10Bac transformation mu.l pF-CIAV-Fu plasmid from example 1 was added to 100. mu.l DH10Bac competent cells and mixed, ice-bathed for 30min, water-bathed heat shock at 42 ℃ for 90 sec, ice-bathed for 2 min, 900. mu.l LB liquid medium without Amp was added, 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, then 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 a strain, and extracting a plasmid to obtain a recombinant plasmid Bacmid-CIAV-Fu.

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-CIAV-Fu plasmid from example 2 was diluted with 100. mu.l of transfection culture T1 medium, and the diluted transfection reagent and the recombinant Bacmid-Fu plasmid were mixed and gently blown down to prepare a transfection mixture. And adding the transfection mixture after the cells adhere to the wall, incubating for 5 hours at 27 ℃, removing the supernatant, adding 2mLSF-SFM fresh culture medium, and culturing for 4-5 days at 27 ℃ to obtain the supernatant. Obtaining recombinant baculovirus rBac-CIAV-Fu, detecting virus titer of the harvested P1 generation recombinant baculovirus by using an MTT relative efficacy method, wherein the titer of rBac-CIAV-Fu P1 virus is 3.4 multiplied by 10⁷pfu/mL, and amplifying the recombinant baculovirus rBac-CIAV-Fu to be used as seed virus for standby.

Cell lines expressing the proteins shown in Table 5 were also constructed according to the above example:

TABLE 5

Example 4 SDS-PAGE detection

The cell cultures and groups harvested in example 3 were subjected to SDS-PAGE detection 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 expression level of the mutated CIAV-Fu protein was higher than that of the remaining four groups.

Example 5 Western Blot assay

The product of example 4 after SDS-PAGE electrophoresis was transferred to an NC (nitrocellulose) membrane, blocked with 5% skim milk for 2 hours, incubated with chicken-derived anti-CIAV positive serum for 2 hours, rinsed, incubated with 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 has wider and brighter bands for correctly expressing CIAV-Fu in Sf9 cells, which indicates that the CIAV-Fu has high expression level and good immunogenicity.

Example 6 Indirect immunofluorescence assay

Sf9 cell suspension transfected by rBac-CIAV-Fu is added into a 96-well cell culture plate, and the suspension is 100 mul/well (the cell concentration is 2.5 multiplied by 10)⁵～4.0×10⁵piece/mL), 4 wells are inoculated, the mixture is kept still at 27 ℃ for 15 minutes, Sf9 cells are attached to the bottom wall of the culture plate, and then 10 mul of virus seeds diluted by 10 times are added into 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-CIAV 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 inoculated with the empty baculovirus Sf9 cells, whereas the recombinant baculovirus Sf9 cells could be observedFluorescence was observed, indicating that the antigen of interest was correctly expressed in Sf9 cells and that the recombinant baculovirus was correctly constructed.

Example 7 purification of CIAV-Fu and Electron microscopy

1. Sucrose density gradient centrifugation was performed on the recombinant baculovirus rBac-CIAV-Fu and the four groups of cell cultures in example 3 by gradient centrifugation, specifically as follows: freeze thawing cell culture for three times, centrifuging at 12000r/min for 30min, collecting supernatant, filtering with 0.22 μm filter membrane, removing impurities, and concentrating with ultrafiltration tube with molecular weight cutoff of 500 kDa by 10 times. 10mL of 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.

2. On Sf9 cells which have just grown in a monolayer, recombinant baculovirus rBac-CIAV-Fu is inoculated with a multiplicity of infection of 3-5pfu/cell under electron microscope observation, and diseased cells are collected and ultrasonically lysed 72 hours later. 8000g at 4 ℃ for 30min, and then concentrated to 1/10 in bulk with sucrose, the supernatant contained the expressed VLP particles. The supernatant was subjected to ultracentrifugation at 27000rpm/min for 3 hours, the pellet was resuspended in 2ml of PBS and blown repeatedly with a 1ml syringe, then the virus suspension was subjected to ultracentrifugation at 27000rpm/min for 16 hours with a 20-60% sucrose density gradient, and the uppermost protein band was collected and observed by electron microscopy. Baculovirus was observed under an electron microscope and typical CIAV-like virus-like particles were formed. 3. The purified CIAV-Fu and each group of 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 VLP strips in the ultracentrifuge tube is observed, and the CIAV-Fu is obviously deeper, which shows that the modified CIAV-Fu can be automatically assembled, and the method has the advantages of high assembly efficiency, better stability and higher expression quantity.

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

In 1000mL shake flaskCulturing Sf9 insect cells for 3-4 days until the concentration reaches 3-5 × 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, the CIAV-Fu is inoculated, 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 harvested by centrifugation or hollow fiber filtration, and the CIAV-VLP vaccine stock solution is stored at 2-8 ℃.

The prepared vaccine antigen has CIAV-VLP content detected by Elisa method. The operation mode is as follows: diluting the chicken anti-CIAV multi-antiserum to a proper concentration by using a coating buffer solution, wherein each well is 100 mu l, the temperature is kept overnight at 4 ℃, PBST is washed for three times, and 1% BSA is blocked for 1 h. Antigen standard substances (CIAV-VLP obtained by sucrose density gradient centrifugation and purification) with different concentrations and samples to be detected are added and diluted in a gradient manner, incubated for 1 hour at 37 ℃ and washed for three times by PBST. Add detection antibody per well: CIAV-VLP protein monoclonal antibody (a monoclonal antibody recognizing the structure of CIAV virus-like particles), incubated at 37 ℃ for 1 hour, and washed three times with PBST. 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 CIAV-VLP in the sample to be detected through a standard curve.

CIAV-VLPs were prepared on a large scale according to example 8, and the average content of CIAV-VLPs in the vaccine stock solution was about 142 ug/mL as determined by Elisa as follows.

Protein titers of the expressed recombinant CIAV-VLPs and the four panels of example 3 were determined using the agar-agar method. Punching plum blossom holes on an agarose gel plate, adding CIAV 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 72 h, 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 CIAV-VLP protein agar expansion titer is 1:512, the group 1 protein agar expansion titer is 1:128, the group 2 protein agar expansion titer is 1:64, the group 3 protein agar expansion titer is 1:32, and the group 4 protein agar expansion titer is 1: 32.

Example 9 vaccine preparation

The vaccine stock solution expressed in example 8 was taken, diluted with physiological saline so that the concentration of the antigen protein reached 10 μ g/mL, and then the vaccine stock solution was mixed with an oil adjuvant in the following ratio of 2: 3 is prepared into oil emulsion vaccine. Specifically, 1429 g of white oil, 70.2 g of span, 8.43 g of aluminum stearate and 53.3 g of tween are added into 1L of vaccine stock solution. Then crushing and emulsifying by an emulsifying crusher to prepare the oil emulsion adjuvant inactivated vaccine. The cell culture fluid of the control group 4 in example 3 was prepared into an oil emulsion adjuvant inactivated vaccine according to the same method.

Example 10 immunization experiment

30 SPF chickens of 21 days old are taken and randomly divided into 3 groups of 10, namely a CIAV-VLP immune group, a group 4 immune group and a control group. The CIAV-VLP immunization group and the group 4 immunization group were fed separately by injecting the vaccine prepared in example 9 subcutaneously into the neck of the chicken, 0.2 ml/chicken, and injecting the same volume of white oil into the control group for control. Blood is collected 21 days after immunization, serum is separated, and the fowl anemia antibody detection kit of IDXEE is used for determining antibody titer, so that the result shows that the average value of S/N of the CIAV-VLP immune group is 0.0531, the average value of S/N of the control group 4 immune group is 0.2083, and the average value of S/N of the control group is 0.9561. The CIAV-VLP has stronger immunogenicity, higher protein expression and higher assembly efficiency. See table 6 for specific results.

TABLE 6 serological test results for vaccines

Note: positive if the S/N value is less than or equal to 0.6

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> chicken infectious anemia virus gene engineering vaccine

<130> 20200629

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1674

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

atggcgcgcc gcgcgcgcgc gccgcgcggc cgcttttatg cgtttcgccg cggccgctgg 60

catcatctga aacgcctgcg cgcgcgctat aaatttcgcc atcgccgccg ccagcgctat 120

cgcgcgcgcg cgtttcgcaa agcgtttcat aacccgcgcc cgggcaccta tagcgtgcgc 180

ctgccgaacc cgcagagcac catgaccatt cgctttcagg gcgtgatttt tctgaccgaa 240

ggcctgattc tgccgaaaaa cagcaccgcg ggcggctatg cggatcatat gtatggcgcg 300

cgcgtggcga aaattagcgt gaacctgaaa gaatttctgc tggcgagcat gaacctgacc 360

tatgtgagca aactgggcgg cccgattgcg ggcgaactga ttgcggatgg cagcaaaagc 420

caggcggcgg aaaactggcc gaactgctgg ctgccgctgg ataacaacat gccgagcgcg 480

accccgagcg cgtggtgggg ctgggcgctg atgatgatgc agccgaccga tagctgccgc 540

ttttttaacc atccgaaaca gatggcgctg caggatatgg gccgcatgtt tggcggctgg 600

catctgtttc gccatattga aacccgcttt cagctgctgg cgaccaaaaa cgaaggcagc 660

tttagcccgg tggcgagcct gctgagccag ggcgaatatc tgacccgccg cgatgatgtg 720

aaatatagca gcgatcatca gaaccgctgg cgcaaaggcg aacagccgat gaccggcggc 780

attgcgtatg cgaccggcaa aatgcgcccg gatgaacagc agtatccggc gatgccgccg 840

gatccgccga ttattaccag caccaccgcg cagggcaccc aggtgcgctg catgaacagc 900

acccaggcgt ggtggagctg ggatacctat atgagctttg cgaccctgac cgcgctgggc 960

gcgcagtgga gctttccgcc gggccagcgc agcgtgagcc gccgcagctt taaccatcat 1020

aaagcgcgcg gcgcgggcga tccgaaaggc cagcgctggc ataccctggt gccgctgggc 1080

accgaaacca ttaccgatag ctatatgggc gcgccggcga gcgaaattga taccaacttt 1140

tttaccctgt atgtggcgca gggcaccaac aaaagccagc agtataaatt tggcaccgcg 1200

acctatgcgc tgaaagaacc ggtgatgaaa agcgatagct gggcggtggt gcgcgtgcag 1260

agcgtgtggc agctgggcaa ccgccagcgc ccgtatccgt gggatgtgaa ctgggcgaac 1320

agcaccatgt attggggcag ccagccgggc agcagcagca gcggcccgcc gggcagcagc 1380

agcagcggcc agcgcaaacc gaaatggtat cgctggaact ataaccatag cattgcggtg 1440

tggctgcgcg aatgcagccg cagccatgcg aaaatttgca actgcggcca gtttcgcaaa 1500

cattggtttc aggaatgcgc gggcctggaa gatcgcagca cccaggcgag cctggaagaa 1560

gcgattctgc gcccgctgcg cgtgcagggc aaacgcgcga aacgcaaact ggattatcat 1620

tatagccagc cgaccccgaa ccgcaaaaaa gtgtataaaa ccgtgcgcta atga 1674

<210> 2

<211> 556

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 2

Met Ala Arg Arg Ala Arg Ala Pro Arg Gly Arg Phe Tyr Ala Phe Arg

1 5 10 15

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

20 25 30

Arg His Arg Arg Arg Gln Arg Tyr Arg Ala Arg Ala Phe Arg Lys Ala

35 40 45

Phe His Asn Pro Arg Pro Gly Thr Tyr Ser Val Arg Leu Pro Asn Pro

50 55 60

Gln Ser Thr Met Thr Ile Arg Phe Gln Gly Val Ile Phe Leu Thr Glu

65 70 75 80

Gly Leu Ile Leu Pro Lys Asn Ser Thr Ala Gly Gly Tyr Ala Asp His

85 90 95

Met Tyr Gly Ala Arg Val Ala Lys Ile Ser Val Asn Leu Lys Glu Phe

100 105 110

Leu Leu Ala Ser Met Asn Leu Thr Tyr Val Ser Lys Leu Gly Gly Pro

115 120 125

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

130 135 140

Asn Trp Pro Asn Cys Trp Leu Pro Leu Asp Asn Asn Met Pro Ser Ala

145 150 155 160

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

165 170 175

Asp Ser Cys Arg Phe Phe Asn His Pro Lys Gln Met Ala Leu Gln Asp

180 185 190

Met Gly Arg Met Phe Gly Gly Trp His Leu Phe Arg His Ile Glu Thr

195 200 205

Arg Phe Gln Leu Leu Ala Thr Lys Asn Glu Gly Ser Phe Ser Pro Val

210 215 220

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

225 230 235 240

Lys Tyr Ser Ser Asp His Gln Asn Arg Trp Arg Lys Gly Glu Gln Pro

245 250 255

Met Thr Gly Gly Ile Ala Tyr Ala Thr Gly Lys Met Arg Pro Asp Glu

260 265 270

Gln Gln Tyr Pro Ala Met Pro Pro Asp Pro Pro Ile Ile Thr Ser Thr

275 280 285

Thr Ala Gln Gly Thr Gln Val Arg Cys Met Asn Ser Thr Gln Ala Trp

290 295 300

Trp Ser Trp Asp Thr Tyr Met Ser Phe Ala Thr Leu Thr Ala Leu Gly

305 310 315 320

Ala Gln Trp Ser Phe Pro Pro Gly Gln Arg Ser Val Ser Arg Arg Ser

325 330 335

Phe Asn His His Lys Ala Arg Gly Ala Gly Asp Pro Lys Gly Gln Arg

340 345 350

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

355 360 365

Met Gly Ala Pro Ala Ser Glu Ile Asp Thr Asn Phe Phe Thr Leu Tyr

370 375 380

Val Ala Gln Gly Thr Asn Lys Ser Gln Gln Tyr Lys Phe Gly Thr Ala

385 390 395 400

Thr Tyr Ala Leu Lys Glu Pro Val Met Lys Ser Asp Ser Trp Ala Val

405 410 415

Val Arg Val Gln Ser Val Trp Gln Leu Gly Asn Arg Gln Arg Pro Tyr

420 425 430

Pro Trp Asp Val Asn Trp Ala Asn Ser Thr Met Tyr Trp Gly Ser Gln

435 440 445

Pro Gly Ser Ser Ser Ser Gly Pro Pro Gly Ser Ser Ser Ser Gly Gln

450 455 460

Arg Lys Pro Lys Trp Tyr Arg Trp Asn Tyr Asn His Ser Ile Ala Val

465 470 475 480

Trp Leu Arg Glu Cys Ser Arg Ser His Ala Lys Ile Cys Asn Cys Gly

485 490 495

Gln Phe Arg Lys His Trp Phe Gln Glu Cys Ala Gly Leu Glu Asp Arg

500 505 510

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

515 520 525

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

530 535 540

Thr Pro Asn Arg Lys Lys Val Tyr Lys Thr Val Arg

545 550 555

<210> 3

<211> 37

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

atggcgcgcc gcgcgcgcgc gccgcgcggc cgctttt 37

<210> 4

<211> 38

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

tcattagcgc acggttttat acactttttt gcggttcg 38

<210> 5

<211> 1674

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

atggcgcgcc gcgcgcgcgc gccgcgcggc cgcttttatg cgtttcgccg cggccgctgg 60

catcatctga aacgcctgcg cgcgcgctat aaatttcgcc atcgccgccg ccagcgctat 120

cgcgcgcgcg cgtttcgcaa agcgtttcat aacccgcgcc cgggcaccta tagcgtgcgc 180

ctgccgaacc cgcagagcac catgaccatt cgctttcagg gcgtgatttt tctgaccgaa 240

ggcctgattc tgccgaaaaa cagcaccgcg ggcggctatg cggatcatat gtatggcgcg 300

cgcgtggcga aaattagcgt gaacctgaaa gaatttctgc tggcgagcat gaacctgacc 360

tatgtgagca aactgggcgg cccgattgcg ggcgaactga ttgcggatgg cagcaaaagc 420

caggcggcgg aaaactggcc gaactgctgg ctgccgctgg ataacaacat gccgagcgcg 480

accccgagcg cgtggtgggg ctgggcgctg atgatgatgc agccgaccga tagctgccgc 540

ttttttaacc atccgaaaca gatggcgctg caggatatgg gccgcatgtt tggcggctgg 600

catctgtttc gccatattga aacccgcttt cagctgctgg cgaccaaaaa cgaaggcagc 660

tttagcccgg tggcgagcct gctgagccag ggcgaatatc tgacccgccg cgatgatgtg 720

aaatatagca gcgatcatca gaaccgctgg cgcaaaggcg aacagccgat gaccggcggc 780

attgcgtatg cgaccggcaa aatgcgcccg gatgaacagc agtatccggc gatgccgccg 840

gatccgccga ttattaccag caccaccgcg cagggcaccc aggtgcgctg catgaacagc 900

acccaggcgt ggtggagctg ggatacctat atgagctttg cgaccctgac cgcgctgggc 960

gcgcagtgga gctttccgcc gggccagcgc agcgtgagcc gccgcagctt taaccatcat 1020

aaagcgcgcg gcgcgggcga tccgaaaggc cagcgctggc ataccctggt gccgctgggc 1080

accgaaacca ttaccgatag ctatatgggc gcgccggcga gcgaaattga taccaacttt 1140

tttaccctgt atgtggcgca gggcaccaac aaaagccagc agtataaatt tggcaccgcg 1200

acctatgcgc tgaaagaacc ggtgatgaaa agcgatagct gggcggtggt gcgcgtgcag 1260

agcgtgtggc agctgggcaa ccgccagcgc ccgtatccgt gggatgtgaa ctgggcgaac 1320

agcaccatgt attggggcag ccagccgggc agcagcagca gcggcccgcc gggcagcagc 1380

agcagcggcc agcgcgatcc ggattggtat cgctggaact ataaccatag cattgcggtg 1440

tggctgcgcg aatgcagccg cagccatgcg aaaatttgca actgcggcca gtttcgcaaa 1500

cattggtttc aggaatgcgc gggcctggaa gatcgcagca cccaggcgag cctggaagaa 1560

gcgattctgc gcccgctgcg cgtgcagggc aaacgcgcga aacgcaaact ggattatcat 1620

tatagccagc cgaccccgaa ccgcaaaaaa gtgtataaaa ccgtgcgcta atga 1674

<210> 6

<211> 1674

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

atggcgcgcc gcgcgcgccg cccgcgcggc cgcttttatg cgtttcgccg cggccgctgg 60

catcatctga aacgcctgcg ccgccgctat aaatttcgcc atcgccgccg ccagcgctat 120

cgccgccgcg cgtttcgcaa agcgtttcat aacccgcgcc cgggcaccta tagcgtgcgc 180

ctgccgaacc cgcagagcac catgaccatt cgctttcagg gcgtgatttt tctgaccgaa 240

ggcctgattc tgccgaaaaa cagcaccgcg ggcggctatg cggatcatat gtatggcgcg 300

cgcgtggcga aaattagcgt gaacctgaaa gaatttctgc tggcgagcat gaacctgacc 360

tatgtgagca aactgggcgg cccgattgcg ggcgaactga ttgcggatgg cagcaaaagc 420

caggcggcgg aaaactggcc gaactgctgg ctgccgctgg ataacaacat gccgagcgcg 480

accccgagcg cgtggtgggg ctgggcgctg atgatgatgc agccgaccga tagctgccgc 540

ttttttaacc atccgaaaca gatggcgctg caggatatgg gccgcatgtt tggcggctgg 600

catctgtttc gccatattga aacccgcttt cagctgctgg cgaccaaaaa cgaaggcagc 660

tttagcccgg tggcgagcct gctgagccag ggcgaatatc tgacccgccg cgatgatgtg 720

aaatatagca gcgatcatca gaaccgctgg cgcaaaggcg aacagccgat gaccggcggc 780

attgcgtatg cgaccggcaa aatgcgcccg gatgaacagc agtatccggc gatgccgccg 840

gatccgccga ttattaccag caccaccgcg cagggcaccc aggtgcgctg catgaacagc 900

acccaggcgt ggtggagctg ggatacctat atgagctttg cgaccctgac cgcgctgggc 960

gcgcagtgga gctttccgcc gggccagcgc agcgtgagcc gccgcagctt taaccatcat 1020

aaagcgcgcg gcgcgggcga tccgaaaggc cagcgctggc ataccctggt gccgctgggc 1080

accgaaacca ttaccgatag ctatatgggc gcgccggcga gcgaaattga taccaacttt 1140

tttaccctgt atgtggcgca gggcaccaac aaaagccagc agtataaatt tggcaccgcg 1200

acctatgcgc tgaaagaacc ggtgatgaaa agcgatagct gggcggtggt gcgcgtgcag 1260

agcgtgtggc agctgggcaa ccgccagcgc ccgtatccgt gggatgtgaa ctgggcgaac 1320

agcaccatgt attggggcag ccagccgggc agcagcagca gcggcccgcc gggcagcagc 1380

agcagcggcc agcgcaaacc gaaatggtat cgctggaact ataaccatag cattgcggtg 1440

tggctgcgcg aatgcagccg cagccatgcg aaaatttgca actgcggcca gtttcgcaaa 1500

cattggtttc aggaatgcgc gggcctggaa gatcgcagca cccaggcgag cctggaagaa 1560

gcgattctgc gcccgctgcg cgtgcagggc aaacgcgcga aacgcaaact ggattatcat 1620

tatagccagc cgaccccgaa ccgcaaaaaa gtgtataaaa ccgtgcgcta atga 1674

<210> 7

<211> 1674

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

atggcgcgcc gcgcgcgccg cccgcgcggc cgcttttatg cgtttcgccg cggccgctgg 60

catcatctga aacgcctgcg ccgccgctat aaatttcgcc atcgccgccg ccagcgctat 120

cgccgccgcg cgtttcgcaa agcgtttcat aacccgcgcc cgggcaccta tagcgtgcgc 180

ctgccgaacc cgcagagcac catgaccatt cgctttcagg gcgtgatttt tctgaccgaa 240

ggcctgattc tgccgaaaaa cagcaccgcg ggcggctatg cggatcatat gtatggcgcg 300

cgcgtggcga aaattagcgt gaacctgaaa gaatttctgc tggcgagcat gaacctgacc 360

tatgtgagca aactgggcgg cccgattgcg ggcgaactga ttgcggatgg cagcaaaagc 420

caggcggcgg aaaactggcc gaactgctgg ctgccgctgg ataacaacat gccgagcgcg 480

accccgagcg cgtggtgggg ctgggcgctg atgatgatgc agccgaccga tagctgccgc 540

ttttttaacc atccgaaaca gatggcgctg caggatatgg gccgcatgtt tggcggctgg 600

catctgtttc gccatattga aacccgcttt cagctgctgg cgaccaaaaa cgaaggcagc 660

tttagcccgg tggcgagcct gctgagccag ggcgaatatc tgacccgccg cgatgatgtg 720

aaatatagca gcgatcatca gaaccgctgg cgcaaaggcg aacagccgat gaccggcggc 780

attgcgtatg cgaccggcaa aatgcgcccg gatgaacagc agtatccggc gatgccgccg 840

gatccgccga ttattaccag caccaccgcg cagggcaccc aggtgcgctg catgaacagc 900

acccaggcgt ggtggagctg ggatacctat atgagctttg cgaccctgac cgcgctgggc 960

gcgcagtgga gctttccgcc gggccagcgc agcgtgagcc gccgcagctt taaccatcat 1020

aaagcgcgcg gcgcgggcga tccgaaaggc cagcgctggc ataccctggt gccgctgggc 1080

accgaaacca ttaccgatag ctatatgggc gcgccggcga gcgaaattga taccaacttt 1140

tttaccctgt atgtggcgca gggcaccaac aaaagccagc agtataaatt tggcaccgcg 1200

acctatgcgc tgaaagaacc ggtgatgaaa agcgatagct gggcggtggt gcgcgtgcag 1260

agcgtgtggc agctgggcaa ccgccagcgc ccgtatccgt gggatgtgaa ctgggcgaac 1320

agcaccatgt attggggcag ccagccgggc agcagcagca gcggcccgcc gggcagcagc 1380

agcagcggcc agcgcgatcc ggattggtat cgctggaact ataaccatag cattgcggtg 1440

tggctgcgcg aatgcagccg cagccatgcg aaaatttgca actgcggcca gtttcgcaaa 1500

cattggtttc aggaatgcgc gggcctggaa gatcgcagca cccaggcgag cctggaagaa 1560

gcgattctgc gcccgctgcg cgtgcagggc aaacgcgcga aacgcaaact ggattatcat 1620

tatagccagc cgaccccgaa ccgcaaaaaa gtgtataaaa ccgtgcgcta atga 1674

<210> 8

<211> 1632

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

atggcgcgcc gcgcgcgccg cccgcgcggc cgcttttatg cgtttcgccg cggccgctgg 60

catcatctga aacgcctgcg ccgccgctat aaatttcgcc atcgccgccg ccagcgctat 120

cgccgccgcg cgtttcgcaa agcgtttcat aacccgcgcc cgggcaccta tagcgtgcgc 180

ctgccgaacc cgcagagcac catgaccatt cgctttcagg gcgtgatttt tctgaccgaa 240

ggcctgattc tgccgaaaaa cagcaccgcg ggcggctatg cggatcatat gtatggcgcg 300

cgcgtggcga aaattagcgt gaacctgaaa gaatttctgc tggcgagcat gaacctgacc 360

tatgtgagca aactgggcgg cccgattgcg ggcgaactga ttgcggatgg cagcaaaagc 420

caggcggcgg aaaactggcc gaactgctgg ctgccgctgg ataacaacat gccgagcgcg 480

accccgagcg cgtggtgggg ctgggcgctg atgatgatgc agccgaccga tagctgccgc 540

ttttttaacc atccgaaaca gatggcgctg caggatatgg gccgcatgtt tggcggctgg 600

catctgtttc gccatattga aacccgcttt cagctgctgg cgaccaaaaa cgaaggcagc 660

tttagcccgg tggcgagcct gctgagccag ggcgaatatc tgacccgccg cgatgatgtg 720

aaatatagca gcgatcatca gaaccgctgg cgcaaaggcg aacagccgat gaccggcggc 780

attgcgtatg cgaccggcaa aatgcgcccg gatgaacagc agtatccggc gatgccgccg 840

gatccgccga ttattaccag caccaccgcg cagggcaccc aggtgcgctg catgaacagc 900

acccaggcgt ggtggagctg ggatacctat atgagctttg cgaccctgac cgcgctgggc 960

gcgcagtgga gctttccgcc gggccagcgc agcgtgagcc gccgcagctt taaccatcat 1020

aaagcgcgcg gcgcgggcga tccgaaaggc cagcgctggc ataccctggt gccgctgggc 1080

accgaaacca ttaccgatag ctatatgggc gcgccggcga gcgaaattga taccaacttt 1140

tttaccctgt atgtggcgca gggcaccaac aaaagccagc agtataaatt tggcaccgcg 1200

acctatgcgc tgaaagaacc ggtgatgaaa agcgatagct gggcggtggt gcgcgtgcag 1260

agcgtgtggc agctgggcaa ccgccagcgc ccgtatccgt gggatgtgaa ctgggcgaac 1320

agcaccatgt attggggcag ccagccgcag cgcgatccgg attggtatcg ctggaactat 1380

aaccatagca ttgcggtgtg gctgcgcgaa tgcagccgca gccatgcgaa aatttgcaac 1440

tgcggccagt ttcgcaaaca ttggtttcag gaatgcgcgg gcctggaaga tcgcagcacc 1500

caggcgagcc tggaagaagc gattctgcgc ccgctgcgcg tgcagggcaa acgcgcgaaa 1560

cgcaaactgg attatcatta tagccagccg accccgaacc gcaaaaaagt gtataaaacc 1620

gtgcgctaat ga 1632

<210> 9

<211> 285

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

cagcgcgatc cggattggta tcgctggaac tataaccata gcattgcggt gtggctgcgc 60

gaatgcagcc gcagccatgc gaaaatttgc aactgcggcc agtttcgcaa acattggttt 120

caggaatgcg cgggcctgga agatcgcagc acccaggcga gcctggaaga agcgattctg 180

cgcccgctgc gcgtgcaggg caaacgcgcg aaacgcaaac tggattatca ttatagccag 240

ccgaccccga accgcaaaaa agtgtataaa accgtgcgct aatga 285

Claims

1. The fusion protein has the sequence shown in 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 viral vector comprising the coding gene of claim 2.

5. A host cell comprising the gene encoding of claim 2.

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

7. The immunogenic composition of claim 6, wherein: the pharmaceutically acceptable carrier comprises one or a combination of more than two of white oil, aluminum stearate, span and Tween.

8. A method for producing a fusion protein, comprising:

constructing a recombinant baculovirus expression vector comprising a gene encoding the fusion protein of claim 2 or 3;

9. The method for producing the 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 6 or 7 in the manufacture of a medicament for inducing an immune response against an infectious anemia virus antigen of chicken in a subject animal.

13. Use of the fusion protein of claim 1 or the immunogenic composition of claim 6 or 7 in the manufacture of a medicament for preventing infection of an animal by chicken infectious anemia virus.

14. Use of the fusion protein of claim 1 or the immunological composition of claim 6 or 7 for the preparation of genetically engineered vaccines for chicken infectious anemia virus.