CN109985235A - Infectious bronchitis of chicken genetic engineering subunit vaccine - Google Patents

Abstract

This application provides a kind of immune composition and subunit vaccines, include: the avian infectious bronchitis virus S1 albumen and S2 albumen being separately encoded using the nucleic acid molecules of SEQ ID NO:1 or with the nucleic acid molecules of the identical nucleic acid molecules of 95% or more nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:2 or with the identical nucleic acid molecules of 95% or more nucleotide sequence of SEQ ID NO:2, heterodimeric structure is capable of forming between S1 albumen and S2 albumen, it is similar with virus surface structure, the two albumen use eukaryotic expression, protein glycosylation is abundant, antigen protein immunogenicity is good, and expression quantity is very high, reach 2-3g/L, recombinant cell can suspend culture on a large scale, greatly reduce the complicated journey of vaccine preparation Degree, reduces production cost.

Description

Infectious bronchitis of chicken genetic engineering subunit vaccine

Technical field

This application involves animal immune technical field of pharmaceuticals, in particular to a kind of infectious bronchitis of chicken gene Engineering subunit vaccine.

Background technique

Infective bronchitis (Avian Infectious Bronchitis, IB) is by infectious bronchitis The acute of a breeder caused by malicious (Avian Infectious Bronchitis Virus, IBV), high degree in contact are communicable Respiratory diseases and genito-urinary disorders, using tracheae rale, cough, sneezing as main feature.Various age in days genders and kind Chicken have neurological susceptibility, but with chick more easy infection below 6 week old.There is no the Chickens Infected infectiousness branch gas of maternal antibody Cause permanent fallopian tubal to damage after the scorching virus of pipe, loses egg laying performance to sexal maturity, or even can be due to respiratory tract or kidney Infection and it is dead.Egg production is caused to decline after laying hen group's infection infectious bronchitis virus, egg type is whole, lopsided and matter Amount is low, and rate of fertilization reduces, and infective bronchitis also reduces the weight gain of chicken and the price of deed.Although the use of vaccine is to biography The prevalence of metachromia bronchitis plays certain prevention and control action, but since infectious bronchitis virus serotype is many It is more, have lesser intersecting protective even without intersecting protective between different serotypes strain.Therefore, infective bronchitis Serious economic loss still is caused to aviculture in vaccinated flock and nonimmune chicken mass-sending life and propagation at present.

IBV belongs to Nido virales (Nidovirales), coronaviridae (Coronaviridae), coronavirus genus (Coronavirus) member of third group in.Viral genome is made of the positive chain RNA of sub-thread non-segmented negative, is about 27.6kb. Containing 4 kinds of structural proteins, i.e., fine prominent (Spike, S) glycoprotein, nucleocapsid (Nucleocapsid, N) albumen, film (Membrane, M) glycoprotein and membranelle (Small Envelope, E) glycoprotein.

Existing patent is almost inactivated virus vaccine or live vaccine, such as disclosed in CN103007272A IBV vaccine is live vaccine, produces totivirus by suspension cell culture, needs serum to exist simultaneously and dissipates malicious risk, and antigen table It is low up to measuring；Currently available vaccines, existing vaccines is produced with chicken embryo simultaneously, and chicken embryo is difficult to handle, and easily causes pollution.CN104353070A is disclosed A kind of genetic engineering subunit vaccine of avian infectious bronchitis virus and preparation method thereof uses chicken bronchitis disease Malicious H120 strain S1 gene and H3N2 influenza virus HA2 gene fusion expression, utilize insect baculovirus expression system system It is standby, but there are larger differences with zooblast for insect cell glycosylation.

The present invention is therefore.

Summary of the invention

The application is intended to provide a kind of immune composition, to solve the problems of the prior art.

To achieve the goals above, according to the one aspect of the application, a kind of immune composition is provided, includes:

It is identical using the nucleic acid molecules of SEQ ID NO:1 or with 95% or more the nucleotide sequence of SEQ ID NO:1 Nucleic acid molecule encoding avian infectious bronchitis virus recombination S1 albumen and

The nucleic acid molecules of SEQ ID NO:2 or the identical nucleic acid of 95% or more nucleotide sequence with SEQ ID NO:2 The avian infectious bronchitis virus that molecule does not encode recombinates S2 albumen.

The further technical solution of the present invention is: the avian infectious bronchitis virus recombinant protein includes SEQ ID The amino acid sequence of NO:3 or the identical amino acid sequence of 95% or more full length amino acid sequence with SEQ ID NO:3；With And SEQ ID NO:4 amino acid sequence or the identical amino of 95% or more full length amino acid sequence with SEQ ID NO:4 Acid sequence.

Another object of the present invention is to provide the immune compositions described in one kind for producing in animal subject Purposes of the induction for the medicament of the immune response of avian infectious bronchitis virus antigen.

Another object of the present invention is to provide immune composition described in one kind for produce it is avian infectious for preventing The purposes of the medicament of bronchitis virus infection.

Another object of the present invention is to provide a kind of nucleic acid molecules composition, it can be used for encoding avian infectious branch gas The scorching viral recombinant protein of pipe, the sequential nucleotide sequence comprising SEQ ID NO:1 or the nucleotide sequence with SEQ ID NO:1 95% or more identical sequential nucleotide sequence；And SEQ ID NO:2 sequential nucleotide sequence or with SEQ ID NO:2 The identical sequential nucleotide sequence of 95% or more nucleotide sequence.

The embodiment of the invention also provides a kind of recombinant eukaryon expression vector pCI-S1-GS；The carrier includes for compiling Code avian infectious bronchitis virus recombinant protein SEQ ID NO:1 sequential nucleotide sequence or with SEQ ID NO:1 The identical sequential nucleotide sequence of 95% or more nucleotide sequence.

The embodiment of the invention also provides a kind of recombinant eukaryon expression vector pCI-S1-S2-GS, the carrier includes to be used for Encode avian infectious bronchitis virus recombinant protein SEQ ID NO:2 sequential nucleotide sequence or with SEQ ID NO: The 2 identical sequential nucleotide sequence of 95% or more nucleotide sequence.

Further, the recombinant eukaryon expression vector is mainly by under the CMV promoter of the pCI of expression vector It is inserted into the gene and is formed.

Another object of the present invention is to provide nucleic acid molecules described in one kind for produce for being lured in animal subject Purposes of the guide pin to the medicament of the immune response of avian infectious bronchitis virus antigen.

Another object of the present invention is to provide nucleic acid molecules described in one kind for produce passed by chicken for preventing animal The purposes of the medicament of metachromia bronchitis virus infection.

Another object of the present invention is to provide a kind of protein composition, selected from the group being made up of:

The amino acid sequence of SEQ ID NO:3 is identical as 95% or more the full length amino acid sequence of SEQ ID NO:3 Amino acid sequence；

And SEQ ID NO:4 amino acid sequence or 95% or more full length amino acid sequence with SEQ ID NO:4 Identical amino acid sequence.

The embodiment of the invention also provides the monoclonals for the high expression target gene for importing the recombinant eukaryon expression vector Host's mammalian cell.The CHO cell line can be as DG44, DXB11, CHO-K1, CHO-S cell strain, it is preferred that CHO-S Cell strain.

The embodiment of the invention also provides the immune composition, the gene or the recombinant mammalian cells to exist Prepare the application in chicken infectious bronchitis virogene engineering subunit vaccine.

The embodiment of the invention also provides chicken infectious bronchitis virogene engineering subunit vaccines, and it includes described Immune composition.Further, the vaccine includes the immune composition of pharmaceutical effective amount.

Further, the vaccine also includes excipient and/or adjuvant, and adjuvant can be including white oil (M52), tristearin Sour aluminium, department this, any one or two or more combinations in tween.

It is an object of that present invention to provide the IBV genetic engineering subunit vaccines that a kind of good immune effect, technique are safer.For Reaching object above, the present invention co-expresses IBV S1 albumen and S2 albumen using Chinese hamster ovary cell CHO-S cell, and And it is similar with virus surface structure to be capable of forming heterodimeric structure between S1 albumen and S2 albumen, and neutralization can be excited anti- Body.

The invention discloses a kind of recombination avian infectious bronchitis virus albumen of expressing cho cell and subunit's epidemic diseases The preparation method and application of seedling, and prove that the vaccine can generate stronger humoral immunity in chicken body, the chicken after being immunized can Resist it is virulent attack poison, belong to animal vaccine and veterinary biologics technical field, the present invention also provides one kind can large-scale industry The preparation method for the avian infectious bronchitis virus recombinant subunit vaccine that metaplasia produces: clone includes S1 protein coding gene first And the carrier for expression of eukaryon of truncated S2 gene expression frame；Then transfection CHO cell, by selection, screening obtains suspending steady The Chinese hamster ovary celI strain of fixed efficiently coexpression S1-S2 albumen；By the cell strain of fermented and cultured high efficient expression S1-S2 albumen, after purification Obtain recombination S1-S2 albumen；Finally recombination S1-S2 albumen and adjuvant are mixed well to obtain recombinant expression subunit vaccine.

Method provided by the invention can harvest destination protein from cells and supernatant, and yield is up to 2-3g/L, no It only shortens the protein purification time and simplifies production of vaccine step, be greatly reduced production of vaccine cost.

After adopting the above scheme, the present invention has the advantages that following prominent and effect compared with prior art:

Immune composition of the invention is not related to totivirus, has natural safety, very by the expression quantity of the method Height is secreted into supernatant, is easy to purify, and the expression of zooblast eukaryotic expression is glycosylation modified perfect, closest to original point Son, suspend culture, is easy to be mass produced.

The present invention uses the important antigen protein S1-S2 albumen of expressing cho cell IBV, uses eukaryotic expression, protein glycosylation Sufficiently, antigen protein immunogenicity is good, and expression quantity is very high, reaches 2-3g/L, and recombinant cell can suspend training on a large scale It supports, greatly reduces the complexity of vaccine preparation, reduce production cost.Subunit vaccine of the invention can give birth on a large scale It produces, is recombinated for amount, Quality Control is easy；Highly-safe, immunogenicity is good；Stablize between batch；Production cost is low.

Present invention uses the S1 protein sequence of optimization, S1 albumen and S2 albumen, two eggs are co-expressed in the same carrier The white heterodimer that is capable of forming is similar with virus surface structure, and using suspending, culture Chinese hamster ovary celI is expressed, and expression quantity is very Height, protein immunogenic are good.

Detailed description of the invention

The accompanying drawings constituting a part of this application is used to provide further understanding of the present application, and the application's shows Meaning property embodiment and its explanation are not constituted an undue limitation on the present application for explaining the application.In the accompanying drawings:

Fig. 1 is that PCR product after the amplification of S1 gene PCR is carried out gel electrophoresis result, it can be seen that the item of a 1.6kbp Band；Wherein 1 is IBV-S1 gene；2 be negative control；M is molecular weight marker；

Fig. 2 be multiple S1 genetic transformation bacterium colony sample PCR amplification after PCR product carry out gel electrophoresis as a result, 1.6kbp band occurs being positive sample.Wherein 1~3,5~9 be the production after the bacterium colony sample P CR amplification of S1 genetic transformation Object, 4 be non-positive sample；M is molecular weight marker；

Fig. 3 is the result that PCR product after IBV-S2 gene expression cassette PCR amplification is carried out to gel electrophoresis, it can be seen that one The band of a 3.4kbp；Wherein 1~7 is IBV-S2 gene expression cassette；M is molecular weight marker；

Fig. 4 be after the conversion of IBV-S2 gene expression cassette after colony PCR amplification PCR product carry out gel electrophoresis as a result, 3.4kbp band occurs being positive sample.Wherein 1 is the production after bacterium colony sample P CR amplification after the conversion of IBV-S2 gene expression cassette Object, 2 be negative control；M is molecular weight marker；

Fig. 5 is the cell culture supernatant progress PAGE gel electrophoresis harvested in embodiment 3 as a result, in molecular weight Nearby there are two purpose bands in about 100kDa and 50kDa, and wherein 100kDa or so is IBV-S1 albumen；50kDa is IBV-S2 Albumen.Wherein 1 cell culture supernatant to be harvested in embodiment 3；2 be negative control；M is molecular weight marker；

Fig. 6 is that the recombinaant CHO cell culture supernatant of single expression S1 albumen in embodiment 4 carries out PAGE gel electricity The result of swimming；Wherein 1 be single expression S1 albumen recombinaant CHO cell culture supernatant；2 be negative control；M is molecular weight mark Note；

Fig. 7 is that the recombinaant CHO cell culture supernatant of single expression S2 albumen in embodiment 4 carries out PAGE gel electricity The result of swimming；Wherein 1 be single expression S2 albumen recombinaant CHO cell culture supernatant；2 be negative control；M is molecular weight mark Note；

Fig. 8 is recombinant C HO Supernatant samples Western Blot testing result in embodiment 3, and there are two cell bands；Wherein 1 is recombination CHO Supernatant samples；2 be negative control；M is molecular weight marker；

Fig. 9 is that the recombinaant CHO cell culture supernatant Western Blot of single expression S1 albumen in embodiment 5 detects knot Fruit；Wherein 1 be single expression S1 albumen recombinaant CHO cell culture supernatant；2 be negative control；M is molecular weight marker；

Figure 10 is the recombinaant CHO cell culture supernatant Western Blot detection of single expression S2 albumen in embodiment 5 As a result；Wherein 1 be single expression S2 albumen recombinaant CHO cell culture supernatant；2 be negative control；M is molecular weight marker；

Figure 11 is recombination CHO (pCI-S1-S2-GS) cell expression product co-immunoprecipitation testing result, and 1 is negative right According to；2 be recombination CHO (pCI-S1-S2-GS) cell expression product；It can be seen that the combination by S1 protein monoclonal antibody produces Raw to precipitate, in precipitated product other than antibody band, there is also S1 and S2 protein bands, illustrate the S1 of CHO expression, S2 albumen Form a kind of compound.

Figure 12 is after protein purification as a result, 1,6,7 being wherein positive sample, 2,3,4,5 be negative sample, and 8 be feminine gender Control, M is molecular weight marker；

Figure 13 is to attack the comparing result of negative control group and the dissection of vaccine group chicken kidney after poison after using vaccine of the present invention, Left side is negative control group；Right side is vaccine group, it can be seen that the kidney of vaccine group chicken is normal, and negative control group chicken kidney goes out Now apparent congested, lesion spot (as shown by arrows).

Figure 14 is the carrier for expression of eukaryon pCI-S1-S2-GS map of building.

Specific embodiment

It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field The identical meanings of understanding.

It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singular Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.

The present invention provides a kind of immune compositions, include:

It is identical using the nucleic acid molecules of SEQ ID NO:1 or with 95% or more the nucleotide sequence of SEQ ID NO:1 The nucleic acid molecules or identical with 95% or more the nucleotide sequence of SEQ ID NO:2 of nucleic acid molecules and SEQ ID NO:2 The avian infectious bronchitis virus recombinant protein that nucleic acid molecules are separately encoded.

The present invention also relates to a kind of inductions for the method for the immune response of IBV antigen, and the method includes moving to tested Object applies vaccine of the invention.

The present invention also relates to a kind of method for protecting animal subject to infect from IBV, the method includes to described tested Animal applies vaccine of the invention.

The invention also includes the vaccines for being suitable for inducing the immune response for IBV.Vaccine of the invention can be for comprising upper State the plasmid of nucleic acid molecules.Nucleic acid molecules can be incorporated into that in virion.Vaccine can also include adjuvant molecules.Adjuvant can be IL- 12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNF α, TNF β, GM-CSF, epidermal growth The factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33 or combinations thereof；And It in some embodiments, can be IL-12, IL-15, IL-28 or RANTES.

Provided herein is one group of protein compositions selected from the group being made up of: the albumen comprising SEQ ID NO:3；? 95% identical albumen in the whole length of the amino acid sequence of SEQ ID NO:3；And the segment of SEQ ID NO:4；With SEQ The identical albumen of segment 95% of ID NO:4.

The present invention also provides a kind of protein compositions for constituting heterodimer: a kind of albumen selected from the following: (a) SEQ ID NO:3；(b) the 95% identical albumen on the entire length amino acid sequence of the full length sequence as described in SEQ ID NO:3；(c) The immunogenic fragments of 20 or more the amino acid comprising SEQ ID NO:3 of SEQ ID NO:3；(d) in SEQ ID The immunogenicity comprising 20 or more amino acid of 95% identical albumen in the whole length of the amino acid sequence of NO:3 Segment；A kind of and albumen selected from the following: (a) SEQ ID NO:4；(b) the full length sequence as described in SEQ ID NO:4 95% identical albumen on entire length amino acid sequence；(c) 20 comprising SEQ ID NO:4 of SEQ ID NO:4 or more The immunogenic fragments of multiple amino acid；(d) the 95% identical egg in the whole length of the amino acid sequence of SEQ ID NO:4 The white immunogenic fragments comprising 20 or more amino acid.

The present invention also provides the nucleic acid molecules of the sequence comprising encoding one or more protein groups.In some implementations In scheme, the nucleic acid molecules include the sequence selected from the group being made up of: SEQ ID NO:1；In the core of SEQ ID NO:1 95% identical nucleic acid sequence in the whole length of nucleotide sequence；The segment of SEQ ID NO:1；With the segment of SEQ ID NO:1 95% identical nucleotide sequence；In other embodiments, the nucleic acid molecules include selected from the group being made up of Sequence: SEQ ID NO:2；The 95% identical nucleic acid sequence in the whole length of the nucleotide sequence of SEQ ID NO:2；SEQ The segment of ID NO:2；Nucleotide sequence identical with the segment 95% of SEQ ID NO:2.

Some aspects of the present invention provide the method for immune response of the induction for IBV, the described method comprises the following steps: to Individual application IBV antigen and/or combination thereof object.

The other aspect of the present invention provides the method for protecting individuals from IBV infection.It the described method comprises the following steps: to The nucleic acid molecules or composition comprising such nucleic acid sequence of the individual application prevention effective dose；Wherein the nucleic acid sequence exists It is expressed in the cell of the individual, and for the protein induced protective immunological reaction by the nucleic acid sequence encoding.

Some aspects of the invention provide a kind of method for inducing the immune response for IBV antigen, the method includes Nucleic acid molecules of the invention are applied to animal subject.

Some aspects of the invention provide a kind of method for protecting animal subject to infect from IBV, the method includes to The animal subject applies nucleic acid molecules of the invention.

To the vaccine of the invention for the chicken application immune effective dose for needing to defend virus infection.It can be very by routine test It is easily determined or easily titrates the immune effective dose or immunogenicity amount of chicken to be inoculated with.Effective quantity is obtained to vaccine The dosage of enough immune responses, to protect the chicken for being exposed to IBV.Preferably, chicken is protected to such degree, wherein virus Property disease a kind of to all bad physiological signs or effect reduced, improved or prevent completely significantly.

The vaccine can be applied with single dose or with repeated doses.Dosage range can be for example from about 1 microgram to about 1,000 microgram contains IBV DNA (concentration of the immune active ingredient depending on vaccine), preferably 100 to 200 microgram chicken IBV DNA, but the dosage for the adverse reaction for being enough to cause virus infection should not be contained.It is known in the state of the art to be used to measure or titrate The active antigens of suitable dose find the side of minimum effective dose according to the weight of chicken, the concentration of antigen and other typical factors Method.

On the other hand, the present invention provides a kind of a kind of protein composition for constituting heterodimer: albumen selected from the following: (a)SEQ ID NO:3；(b) the 98% identical albumen in the whole length of the amino acid sequence of SEQ ID NO:3；(c)SEQ The immunogenic fragments comprising 20 or more amino acid of ID NO:3；And (d) in the amino acid sequence of SEQ ID NO:3 The immunogenic fragments comprising 20 or more amino acid of 98% identical albumen in the whole length of column.And selected from Under another albumen: (a) SEQ ID NO:4；(b) 98% phase in the whole length of the amino acid sequence of SEQ ID NO:4 Same albumen；(c) immunogenic fragments comprising 20 or more amino acid of SEQ ID NO:4；And (d) in SEQ ID The immunogenicity comprising 20 or more amino acid of 98% identical albumen in the whole length of the amino acid sequence of NO:4 Segment

Some aspects of the invention provide a kind of epidemic disease suitable for generating the immune response for IBV animal subject Seedling, the vaccine includes: nucleic acid molecules composition of the invention and adjuvant molecules.The adjuvant can for 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 combinations thereof；And in some implementations It can be IL-12, IL-15, IL-28 or RANTES in scheme.

Vaccine of the invention may include by the protein composition of the nucleic acid molecule encoding.Vaccine of the invention can be wrapped also Nucleic acid molecules composition described in including and the protein composition by the nucleic acid molecule encoding.

1. defining

The term as used herein is not intended to limit merely for the sake of the purpose for describing specific embodiment.Such as illustrating Used in book and the claim, in addition to the other clear stipulaties of context, singular "one", "an" and " institute State " it include plural form.

For numberical range cited herein, clearly cover in the number for having each insertion between identical precision Word.For example, also covering number 7 and 8 other than 6 and 9 for the range of 6-9, and for the range of 6.0-7.0, clearly cover Number 6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9 and 7.0.

" adjuvant " means to be added in DNA plasmid vaccine as described herein and enhance by described below as used herein DNA plasmid and nucleic acid sequence encoding encoded antigen immunogenicity any molecule.

As used herein " antibody " mean type IgG, IgM, IgA, IgD or IgE antibody or segment, its segment or Derivative, including Fab, F (ab') 2, Fd and single-chain antibody, double-chain antibody, bispecific antibody, bifunctional antibody and its spread out Biology.The antibody can be the antibody isolated from the serum sample of animal, polyclonal antibody, affinity purification antibody or its Mixture, to required epitope or derived from it, sequence shows enough binding specificities to the mixture.

" coded sequence " or " code nucleic acid " means the nucleic acid of the nucleotide sequence comprising coding albumen as used herein (RNA or DNA molecular).The coded sequence may further include the initial signal for being operably coupled to controlling element and end Stop signal, the controlling element include the promoter and more that expression can be instructed in the individual of administration of nucleic acid or the cell of animal Polyadenylation signal.

" complement " or " complementation " means that nucleic acid can refer to the nucleotide in nucleotide or nucleic acid molecules as used herein Watson-Crick (for example, A-T/U and C-G) or Hoogsteen base pairing between analog.

" shared " or " consensus sequence " means multiple Asias based on the queue for analyzing specific IBV antigen as used herein The polypeptide sequence of type.The nucleic acid sequence for encoding shared polypeptide sequence can be prepared.Induction needle can be used to by wrapping protein-contg vaccine Extensive immune, consensus sequence of the vaccine comprising encoding these albumen of a variety of hypotypes or serotype to specific IBV antigen And/or nucleic acid molecules.

As " electroporation " used interchangeably herein, " electricity-permeabilization " or " electronic enhancing " (" EP ") mean using across Film electric field pulse induces the microcosmic approach (hole) in biomembrane；Their presence allows biomolecule such as plasmid, few core Thuja acid, siRNA, drug, ion and water flow to the other side from the side of cell membrane.

As used herein relative to " segment " of nucleic acid sequence mean coding can with overall length wild-type strain IBV The nucleic acid sequence or part of it of the polypeptide triggered an immune response in the animal of antigenic cross-reaction.The segment, which can be, to be selected from Encode at least one DNA fragmentation of the various nucleotide sequences of protein fragments described below.

For polypeptide sequence, " segment " or " immunogenic fragments " mean can with overall length wild-type strain IBV The polypeptide triggered an immune response in the animal of antigenic cross-reaction.The segment of albumen can wrap protein-contg 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%.In some embodiments, the segment of albumen can wrap protein-contg at least 20 amino acid or more, at least 30 ammonia Base acid or more, at least 40 amino acid or more, at least 50 amino acid or more, at least 60 amino acid or more, extremely Few 70 amino acid or more, at least 80 amino acid or more, at least 90 amino acid or more, at least 100 amino acid Or more, at least 110 amino acid or more, at least 120 amino acid or more, at least 130 amino acid or more, at least 140 amino acid or more, at least 150 amino acid or more, at least 160 amino acid or more, at least 170 amino acid Or more, at least 180 amino acid or more, at least 190 amino acid or more, at least 200 amino acid or more, at least 210 amino acid or more, at least 220 amino acid or more, at least 230 amino acid or more or at least 240 amino Acid or more.

Term " genetic constructs " as used herein refers to DNA or RNA points of the nucleotide sequence comprising coding albumen Son.The coded sequence includes the initial signal and termination signal for being operably coupled to controlling element, the controlling element packet Include the promoter and polyadenylation signal that expression can be instructed in the cell of the individual of administration of nucleic acid molecule.Such as this paper institute Term " expression-form " refers to that gene construct, the gene construct contain the volume for being operably coupled to coding albumen The necessary controlling element of code sequence, so that coded sequence will express when in the cell for being present in the individual.

Term " homology " as used herein refers to complementary degree.Homoeology or complete homology may be present (that is, identity).At least partly inhibiting fully-complementary sequence to hybridize in the partial complementarity sequence of target nucleic acids is using function art Language " substantially homologous " refers to.It is when the double-strandednucleic acid sequence about such as cDNA or genomic clone in use, as used herein Term " substantially homologous " refer to that probe can hybridize under the conditions of property low strict in the chain of the double-strandednucleic acid sequence.When about single-stranded Nucleic acid sequence is in use, term " substantially homologous " as used herein refers to that probe can hybridize under the conditions of property low strict in list Chain sequence of template of nucleic acid (that is, being the complementary series of single stranded nucleic acid template sequence).

In the case where two or more nucleic acid or polypeptide sequence, " identical " or " identity " as used herein means Sequence has the prescribed percentage of identical residue in specified region.The percentage can be calculated by following: most preferably Compare two sequences, specified region compare two sequences, the quantity for the position for determining residue identical in the two sequences with Generate the quantity of matching position, the total quantity with the quantity of matching position divided by the position in specified region, and by result The percentage of sequence identity is generated multiplied by 100.There is different length in two sequences or compare generation one or more In the case that a staggered end and the specified region compared only include unique sequence, the residue of unique sequence is included in meter In the denominator of calculation rather than in molecule.When comparison dna and RNA, thymidine (T) and uracil (U) are considered With.Identity can be executed manually or by computer sequence algorithm such as BLAST or BLAST 2.0 is used.

It " is immunoreacted " introducing for meaning that antigen is shared in response to antigen such as IBV, the siberian crabapple of host as used herein The activation of system (such as immune system of animal).The immune response can be the shape of cell effect or humoral response or both Formula.

" nucleic acid " or " oligonucleotides " or " polynucleotides " mean at least two be covalently joined together as used herein A nucleotide.Single-stranded description also defines the sequence of complementary strand.Therefore, nucleic acid also covers described single-stranded complementation Chain.Many variants of nucleic acid can be used for purpose identical with given nucleic acid.Therefore, nucleic acid also covers substantially the same Nucleic acid and its complement.The probe that single-stranded offer can hybridize under stringent hybridization conditions with target sequence.Therefore, nucleic acid is also Cover the probe hybridized under stringent hybridization conditions.

Nucleic acid can be single-stranded or double-strand or can be containing the part of both double-strand or single stranded sequence.The core Acid can be both DNA, genome and cDNA, RNA or heterozygote, wherein the nucleic acid can containing deoxyribonucleotide and The combination of ribonucleotide, and it is yellow including uracil, adenine, thymidine, cytimidine, the fast quinoline of bird, inosine, xanthine time The combination of the base of purine, iso-cytosine and isoguanine.Nucleic acid can by chemical synthesis process or pass through recombination side Method obtains.

It " is operably connected " as used herein and means that the expression of gene is the promoter being spatially attached thereto The lower progress of control.At the control, promoter can be positioned in the upstream 5'(of gene) or the downstream 3'().The starting Son and the distance between gene can about with the promoter and its base controlled in the promoter therefrom gene of derivation The distance between cause is identical.As it is known in the art, the variation of this distance can be the case where not losing promoter function Under be adjusted.

" promoter " means that synthesis or natural source molecule, the molecule can be assigned, be activated as used herein Or the expression of the nucleic acid in enhancing cell.Promoter may include one or more specific transcription regulating nucleotide sequences further to increase Strongly expressed and/or change the expression in its space and/or the expression of time.Promoter comprising Distal enhancer or can also check member Part, they can be located at the almost thousands of pairs of base-pairs since the starting point of transcription.Promoter can from include virus, Bacterium, fungi, plant, insect and animal source in obtain.Promoter can relative to wherein express cell, group It knits or organ or relative to the stage of development expressed or in response to outside stimulus such as physiological stress, pathogen, metal ion Or inducer and basically or the distinctively expression of controlling gene component.The representative example of promoter includes phage t7 starting Son, bacteriophage T3 promoter, SP6 promoter, lactose operon-promoter, tac promoter, SV40 late promoter, SV40 are early Phase promoter, RSV-LTR promoter, CMVIE promoter, SV40 early promoter or SV40 late promoter and CMVIE Promoter.

" signal peptide " and " leader sequence " is used interchangeably herein and refer to and can be connected IBV as described herein The amino acid sequence of the amino terminal of albumen.Signal peptide/leader sequence is indicated generally at the position of albumen.Signal used herein Peptide/leader sequence preferably facilitates albumen and secretes from the cell for generating it.Signal peptide/leader sequence is usually from the residue of albumen Part cracks, and the albumen from cell after secreting through being commonly referred to as maturation protein.Signal peptide/leader sequence is connected to the egg White N-terminal.

" stringent hybridization conditions " mean such condition as used herein, i.e. such as answering in nucleic acid under the described conditions The first nucleic acid sequence (for example, probe) will hybridize with second nucleotide sequence (for example, target) in miscellaneous mixture.Stringent item Part is sequence dependent and will be different in different environment.Stringent condition can at the ionic strength pH of restriction To be selected as about 5-10 DEG C lower than the thermal melting point of particular sequence (Tm).The Tm can be such temperature and (limit Ionic strength, under pH and nucleic acid concentration), the probe and target sequence with the 50% of target-complementary are balancing at said temperatures Hybridized (because target sequence is present in excess, at Tm, 50% probe is occupied in the state of the equilibrium) under state.Stringent item Part can be those conditions, i.e., wherein salinity is less than about the sodium ion of 1.0M, the about 0.01-1.0M such as at pH 7.0 to 8.3 Na ion concentration (or other salt), and temperature for short probe (for example, about 10-50 nucleotide) be at least about 30 DEG C It and is at least about 60 DEG C for long probe (for example, greater than about 50 nucleotide).Stringent condition can also be gone by addition Stabilizer such as formamide is realized.For selection or specific hybridization, positive signal can be at least the 2 to 10 of background hybridization Times.Illustrative stringent hybridization conditions include the following: 50% formamide, 5x SSC and 1%SDS, it is incubated at 42 DEG C, or Person 5x SSC, 1%SDS, it is incubated at 65 DEG C, washed at 65 DEG C with 0.2x SSC and 0.1%SDS.

" be substantially complementary " as used herein mean First ray 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, in the region of 450,540 or more nucleotide or amino acid with the complement at least 60% of the second sequence, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% are identical, or mean two sequences stringent miscellaneous Hybridized under the conditions of friendship.

As used herein " substantially the same " mean First ray and the second sequence 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, be at least 60% in 180,270,360,450,540 or more nucleotide or amino acid region, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% are identical, or for nucleic acid, if First ray and the second sequence The complement of column is substantially complementary, then First ray and the second sequence are also identical in this way.

" hypotype " or " serotype ": as be used interchangeably herein and about IBV, it is intended that the genetic mutation of IBV so that One hypotype is identified and separated from different hypotypes by immune system.

" variant " used for nucleic acid means a part or segment of (i) reference nucleotide sequence herein；(ii) join Examine the complement of nucleotide sequence or part thereof；(iii) nucleic acid substantially the same with reference nucleic acid or its complement；Or (iv) nucleic acid hybridized under strict conditions with reference nucleic acid, its complement or the sequence substantially the same with its.

" variant " for peptide or polypeptide is by the insertion of amino acid, missing or conservative replaces in amino acid sequence Upper difference, but retain at least one bioactivity.Variant, which is still meant that, has the amino acid sequence substantially the same with reference protein The albumen of column, the reference protein have the amino acid sequence for retaining at least one bioactivity.The conservative replaces of amino acid, Amino acid is replaced with the different aminoacids of similar characteristic (for example, hydrophily, the degree of charging zone and distribution), in ability It is considered being usually directed to minor change in domain.As understood in the art, these minor changes can be partially by considering amino The hydrophilic and hydrophobic index of acid identifies.Kate (Kyte) etc., J. Mol. BioL (J.Mol.Biol.) 157:105-132 (1982).The hydrophilic and hydrophobic index of the amino acid is based on the considerations of its hydrophobicity and charge.It is known in the art that similar Hydrophilic and hydrophobic index amino acid can be substituted and still retain protein function.In one aspect, hydrophilic and hydrophobic index is ± 2 amino acid is substituted.The hydrophily of amino acid, which can be utilized to disclose, can generate taking for the albumen for retaining biological function Generation.Consider that the hydrophily of amino acid allows to calculate the peptide maximum local average hydrophilicity in the case of peptide, this is It is reported and antigenicity and the good associated useful measurement of immunogenicity.As this field is understood, there is similar hydropathic The substitution of the amino acid of value can produce the peptide for retaining bioactivity (such as immunogenicity).Can use has each other in ± 2 The amino acid of hydrophilicity value is replaced.The hydrophilic and hydrophobic index and hydrophilicity value of amino acid are both by the spy of the amino acid Determine side chain influence.Consistent with the observation to be, the amino acid substitution compatible with biological function is understood to depend on these ammonia The opposite similitude of base acid, and especially those amino acid side chain, such as by hydrophobicity, hydrophily, charge, size and Other characteristics are revealed.

" carrier " means the nucleic acid sequence containing replication orgin as used herein.Carrier can be viral vectors, phagocytosis Body, bacterial artificial chromosome or yeast artificial chromosome.Carrier can be DNA or RNA carrier.Carrier can be self-replacation The outer carrier of chromosome, and preferably DNA plasmid.

2. vaccine

The present invention relates to a kind of infectious bronchitis of chicken genetic engineering subunit vaccines.Infectious bronchitis of chicken gene Engineering subunit vaccine (IBV) may include the nucleic acid for encoding IBV antigen.Vaccine of the invention can be designed to control animal subject In for one or more IBV serotypes immune response degree or intensity.

Vaccine of the invention may include IBV S1 albumen and/or S2 albumen.IBV recombinant protein is by inducing 1) cell toxicant Property T lymphocyte (CTL) react, 2) t helper cell reaction and/or 3) B cell reaction, or it is preferably all above-mentioned anti- It answers, intersects submission to reach come the target of the immune-mediated virus sweep carried out.

The antigen may include the protein epitope for making them particularly effectively be used as immunogene, can be directed to the immunogene Anti- IBV is induced to be immunoreacted.IBV antigen may include overall length translation product, its variant, its segment or combinations thereof.

What some embodiments were related to encoding immunogenic albumen has 95% homology with this paper nucleic acid coding sequence Nucleic acid molecules.What some embodiments were related to encoding immunogenic albumen has 96% homology with this paper nucleic acid coding sequence Nucleic acid molecules.What some embodiments were related to encoding immunogenic albumen has 97% homology with this paper nucleic acid coding sequence Nucleic acid molecules.What some embodiments were related to encoding immunogenic albumen has 98% homology with this paper nucleic acid coding sequence Nucleic acid molecules.What some embodiments were related to encoding immunogenic albumen has 99% homology with this paper nucleic acid coding sequence Nucleic acid molecules.In some embodiments, have and homologous disclosed herein of the coded sequence of albumen disclosed herein The nucleic acid molecules of coded sequence are connected to the volume for encoding homologous protein sequence disclosed herein comprising coding IgE leader sequence The sequence of 5 ' ends of code sequence.

In some embodiments, the nucleic acid sequence is free of the coded sequence of encoding leader sequence.In some embodiment party In case, coded sequence of the nucleic acid sequence without coding IgE lead.

Some embodiments are related to the segment of SEQ ID NO:1 or 2.Segment can 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% SEQ ID NO:1 or 2.Segment can be with the segment of SEQ ID NO:1 or 2 at least 95%, at least 96%, at least 97%, at least 98% or at least 99% are identical.Segment can be with the segment of SEQ ID NO:1 or 2 extremely Few 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% are identical.In some embodiments, segment includes encoding leader sequence Sequence, such as immunoglobulin leader object, such as IgE lead.In some embodiments, segment is without encoding leader sequence Coded sequence.In some embodiments, segment is free of encoding leader sequence, the such as coded sequence of IgE lead.

Some embodiments are related to the albumen homologous with SEQ ID NO:3.Some embodiments are related to and such as SEQ ID Protein sequence described in NO:3 has the immunogenic protein of 95% homology.Some embodiments are related to and such as SEQ ID Protein sequence described in NO:3 has the immunogenic protein of 96% homology.Some embodiments are related to and such as SEQ ID Protein sequence described in NO:3 has the immunogenic protein of 97% homology.Some embodiments are related to and such as SEQ ID Protein sequence described in NO:3 has the immunogenic protein of 98% homology.Some embodiments are related to and such as SEQ ID Protein sequence described in NO:3 has the immunogenic protein of 99% homology.Some embodiments are related to and SEQ ID NO: 4 homologous albumen.Some embodiments are related to having exempting from for 95% homology with the protein sequence as described in SEQ ID NO:4 Epidemic disease immunogenic peptide.Some embodiments are related to having exempting from for 96% homology with the protein sequence as described in SEQ ID NO:4 Epidemic disease immunogenic peptide.Some embodiments are related to having exempting from for 97% homology with the protein sequence as described in SEQ ID NO:4 Epidemic disease immunogenic peptide.Some embodiments are related to having exempting from for 98% homology with the protein sequence as described in SEQ ID NO:4 Epidemic disease immunogenic peptide.Some embodiments are related to having exempting from for 99% homology with the protein sequence as described in SEQ ID NO:4 Epidemic disease immunogenic peptide.

Some embodiments are related to albumen identical with SEQ ID NO:3 or 4.Some embodiments are related to having such as 80% identical amino on the entire length amino acid sequence of overall length consensus amino acid sequences described in SEQ ID NO:3 or 4 The immunogenic protein of acid sequence.Some embodiments, which are related to having, shares ammonia in the overall length as described in SEQ ID NO:3 or 4 The immunogenic protein of 85% identical amino acid sequence on the entire length amino acid sequence of base acid sequence.Some embodiments It is related to having on the entire length amino acid sequence of the overall length consensus amino acid sequences as described in SEQ ID NO:3 or 4 The immunogenic protein of 90% identical amino acid sequence.Some embodiments are related to having the institute in such as SEQ ID NO:3 or 4 The immunogenicity egg of 91% identical amino acid sequence on the entire length amino acid sequence for the overall length consensus amino acid sequences stated It is white.Some embodiments are related to the entire amino in the overall length consensus amino acid sequences as described in SEQ ID NO:3 or 4 The immunogenic protein of 92% identical amino acid sequence in acid sequence length.Some embodiments are related to having in such as SEQ ID 93% identical amino acid sequence on the entire length amino acid sequence of overall length consensus amino acid sequences described in NO:3 or 4 Immunogenic protein.Some embodiments are related to having in the overall length consensus amino acid sequences as described in SEQ ID NO:3 or 4 Entire length amino acid sequence on 94% identical amino acid sequence immunogenic protein.Some embodiments are related to having 95% is identical on the entire length amino acid sequence of the overall length consensus amino acid sequences as described in SEQ ID NO:3 or 4 The immunogenic protein of amino acid sequence.Some embodiments are related to having total in the overall length as described in SEQ ID NO:3 or 4 There is the immunogenic protein of 96% identical amino acid sequence on the entire length amino acid sequence of amino acid sequence.Some implementations Scheme is related to the entire length amino acid sequence in the overall length consensus amino acid sequences as described in SEQ ID NO:3 or 4 The immunogenic protein of upper 97% identical amino acid sequence.Some embodiments are related to having in such as SEQ ID NO:3 or 4 The immunogenicity of 98% identical amino acid sequence on the entire length amino acid sequence of the overall length consensus amino acid sequences Albumen.Some embodiments are related to the entire ammonia in the overall length consensus amino acid sequences as described in SEQ ID NO:3 or 4 The immunogenic protein of 99% identical amino acid sequence in base acid sequence length.

In some embodiments, albumen is free of leader sequence.In some embodiments, albumen is free of IgE lead. The segment of albumen may include 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% albumen.It can The immunogenic fragments of SEQ ID NO:3 or 4 are provided.Immunogenic fragments may include 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% SEQ ID NO:3 or 4.In some embodiments, segment includes leading sequence Column, such as immunoglobulin leader object, such as IgE lead.In some embodiments, segment is free of leader sequence.One In a little embodiments, segment is free of leader sequence, such as IgE lead.

It can provide the immunogenic fragments of the amino acid sequence albumen homologous with the immunogenic fragments of SEQ ID NO:4. The immunogenic fragments may include 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% and SEQ ID NO:495% homologous albumen.Some embodiments are related to same with 96% with the immunogenic fragments of this paper protein sequence The immunogenic fragments of source property.Some embodiments are related to having 97% homology with the immunogenic fragments of this paper protein sequence Immunogenic fragments.Some embodiments are related to having exempting from for 98% homology with the immunogenic fragments of this paper protein sequence Epidemic disease immunogenic fragment.Some embodiments are related to the immunogene for having 99% homology with the immunogenic fragments of this paper protein sequence Property segment.In some embodiments, segment includes leader sequence, such as immunoglobulin leader sequence, as IgE is leading Object.In some embodiments, segment is free of leader sequence.In some embodiments, segment is free of leader sequence, such as IgE lead.

It can provide the immunogenic fragments of amino acid sequence albumen identical with the immunogenic fragments of SEQ ID NO:4. The immunogenic fragments may include 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% in SEQ 80% in the whole length of amino acid sequence described in ID NO:4,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical albumen.In some embodiments, segment includes leader sequence, such as immune ball Protein leader object, such as IgE lead.In some embodiments, segment is free of leader sequence.In some embodiments, piece Duan Buhan leader sequence, such as IgE lead.

D. vaccine constructs and plasmid

Vaccine may include the combined nucleic acid construct or plasmid for encoding IBV S1-S2 albumen.Provided herein is may include The genetic constructs of the nucleic acid sequence of IBV antigen disclosed herein are encoded, the core antigen includes protein sequence and albumen The sequence of sequence homology, the segment of protein sequence and the sequence homologous with the segment of protein sequence.In addition, provided herein is can wrap The IBV surface antigen disclosed herein containing coding is (including protein sequence and the homologous sequence of protein sequence, the segment of protein sequence And the homologous sequence with the segment of protein sequence) nucleic acid sequence genetic constructs.The genetic constructs can be used as Molecule outside functional genomics and exist.The genetic constructs can be including centromere, telomere or plasmid or clay Linear minichromosome.

The genetic constructs can also be a part of the genome of recombinant viral vector, the recombinant viral vector packet Include recombined adhenovirus, recombinant adeno-associated virus and recombinant vaccinia.Genetic constructs can be the viable microbial in attenuation Or the part of the inhereditary material in the recombinant microorganism carrier living in cell.

Genetic constructs may include the controlling element of the gene expression of the coded sequence for nucleic acid.Controlling element can be with It is promoter, enhancer, initiation codon, terminator codon or polyadenylation signal.

Nucleic acid sequence can be the genetic constructs of carrier.The carrier can be exempted from effectively causing in animal The amount of epidemic disease reaction expresses antigen in the cell of animal.Institute+state carrier can be recombinant.It is anti-that the carrier may include coding Former heterologous nucleic acids.The carrier can be plasmid.The carrier can be adapted for transfecting cell with the nucleic acid of coding for antigens, The host cell of the conversion is cultivated and is maintained under conditions of antigen presentation wherein occurs.

Coded sequence can be optimized to be used in the stability and high level of expression.In some cases, codon is selected To reduce the formation of RNA secondary structure, the secondary structure such as formed due to intramolecular bond.

The present invention is used to encode the nucleotide sequence of the important antigen protein S1 albumen of IBV, preferably original series, increase with And truncated sequence；The present invention is used to encode the nucleotide sequence of IBV antigen protein S2 albumen, preferably original series, increases And truncated sequence；Using carrier pSV2-GS, pCI-GS, pcDNA4-GS, pCI-GS is preferably used.CHO cell line can be with It is for DG44, DXB11, CHO-K1, CHO-S cell strain, preferred CHO-S.

The building of 1 recombinant eukaryon expression vector pCI-S1-GS of embodiment

1. the S1 gene of codon optimization comes from Nanjing Genscript Biotechnology Co., Ltd., by S1 gene cloning to pUC- On 57 carriers, pUC-S1 plasmid vector is constructed.S1 gene order after optimization is as shown in SEQ ID NO.1.

Using pUC-S1 as template, S1-F, S1-R carry out PCR amplification (S1-F, S1-R as primer for 2.S1 gene magnification Gene order as shown in SEQ ID NO.6,7), amplification system is shown in Table 1.Reaction condition are as follows: 94 DEG C initial denaturation 5 minutes；95℃ Denaturation 45 seconds, 60 DEG C renaturation 45 seconds, 72 DEG C extend 2 minutes, 30 circulation；72 DEG C extend 10 minutes, 4 DEG C of preservations.

1 S1 gene magnification system of table

PCR product is subjected to gel electrophoresis and identifies target gene size, as shown in Figure 1, item occurs in the position in 1.6kbp Band, target gene expand successfully, carry out recovery purifying with gel purification kit.

3. S1 gene PCR product of the digestion by pCI-GS plasmid and after purification uses Xho I, Kpn I enzyme, 37 DEG C of digestions respectively 3 hours, reaction system was shown in Table 2, table 3.It is separately recovered, is carried out with gel purification kit pure after digestion products gel electrophoresis Change.

2 S1 gene endonuclease reaction system of table

3 pCI-GS plasmid enzyme restriction reaction system of table

4. connection connects the pCI-GS plasmid of digestion and S1 gene digestion products using the 16 DEG C of water-baths of T4DNA ligase It connects, overnight, linked system is shown in Table 4.

4 S1 gene of table and pCI-GS plasmid linked system

5. conversion take 10 μ l connection products be added 100 μ l DH5 α competent cell, mix, 42 DEG C heat shock 90 seconds, ice The LB culture medium that 900 μ l are free of Amp is added in bath 2 minutes, and 37 DEG C are cultivated 1 hour.1.0ml bacterium solution centrifugal concentrating is taken to apply at 100 μ l It is distributed on the LB solid medium containing Amp, 37 DEG C are cultivated 16 hours.

6. the single colonie on bacterium colony PCR and sequencing identification picking plate is inoculated with LB liquid medium, 37 DEG C of cultures 2 respectively Hour, using bacterium solution as template, S1-F and S1-R are as primer progress bacterium colony PCR.PCR product is subjected to gel electrophoresis and verifies mesh Gene size, as shown in Fig. 2, occur 1.6kbp band sample be positive sample.The positive bacterium solution of bacterium colony PCR identification is sent Sequencing company sequencing, selection are sequenced correct bacterium solution and are saved.Obtain carrier for expression of eukaryon pCI-S1-GS.

The building of 2 recombinant eukaryon expression vector pCI-S1-S2-GS of embodiment.

1. the S2 gene expression frame (sequence is as shown in SEQ ID NO:5) of codon optimization comes from Nanjing Jin Sirui biology section Skill Co., Ltd (expression cassette include CMV promoter, truncated S2 gene (sequence is as shown in SEQ ID NO:2) and SV40polyA transcription stop signals, and be cloned on pUC-57 carrier, construct pUC-S2 plasmid vector.

2.S2 gene expression frame is expanded using pUC-S2 as template, and S2-F, S2-R are as the primer (gene of S2-F, S2-R Sequence is as shown in SEQ ID NO.8,9) PCR amplification is carried out, amplification system is shown in Table 5.Reaction condition are as follows: 94 DEG C initial denaturation 5 minutes； 95 DEG C be denaturalized 45 seconds, 60 DEG C renaturation 45 seconds, 72 DEG C extend 4 minutes, 30 circulation；72 DEG C extend 10 minutes, 4 DEG C of preservations.

5 S2 gene expression frame amplification system of table

It is as shown in Figure 3 that PCR product is subjected to gel electrophoresis identification target gene size, it can be seen that the item of a 3.4kbp Band, target gene expand successfully, carry out recovery purifying with gel purification kit.

3. S2 gene expression frame PCR product of the digestion by pCI-S1-GS plasmid and after purification uses Hpa I, Cla I respectively 37 DEG C digestion 3 hours, reaction system is shown in Table 6, table 7.It is separately recovered after digestion products gel electrophoresis, with gel purification reagent Box is purified.

6 S2 gene expression frame endonuclease reaction system of table

7 pCI-S1-GS plasmid enzyme restriction reaction system of table

4. the pCI-S1-GS plasmid of digestion and S2 gene expression frame digestion products are used T4 DNA ligase by connection Overnight, linked system is shown in Table 8 for 16 DEG C of water-bath connections.

4 S2 gene expression frame of table and pCI-S1-GS plasmid linked system

5. conversion take 10 μ l connection products be added 100 μ l DH5 α competent cell, mix, 42 DEG C heat shock 90 seconds, ice The LB culture medium that 900 μ l are free of Amp is added in bath 2 minutes, and 37 DEG C are cultivated 1 hour.1.0ml bacterium solution centrifugal concentrating is taken to apply at 100 μ l It is distributed on the LB solid medium containing Amp, 37 DEG C are cultivated 16 hours.

6. the single colonie on bacterium colony PCR and sequencing identification picking plate is inoculated with LB liquid medium respectively, 37 DEG C of cultures 2 are small When, using bacterium solution as template, S2-F and S2-R are as primer progress bacterium colony PCR.PCR product is subjected to gel electrophoresis verifying purpose Gene size, as shown in figure 4, the sample for 3.4kbp band occur is positive sample.Send the positive bacterium solution of bacterium colony PCR identification to survey The sequencing of sequence company, selection are sequenced correct bacterium solution and are saved.Obtain carrier for expression of eukaryon pCI-S1-S2-GS.It builds Vector map is as shown in figure 14.

Embodiment 3: recombinant C HO-S cell construction screening

1. cell transfecting

1.1 prepare the Chinese hamster ovary celI of cell logarithmic growth phase, and sampling counts, with 1 × 10⁶The cell density of cells/ml Continue to pass on, maintain seed, remaining cell centrifugation after 1000rpm is centrifuged 4 minutes, abandons supernatant, with the fresh CHO- of 20ml or so WM culture medium is resuspended, and is centrifuged again, and 1000rpm is centrifuged 4 minutes, abandons after supernatant and counting is resuspended with a small amount of culture medium, finally will be thin Born of the same parents' density is adjusted to 1.43 × 10⁷cells/ml。

PCI-S1-S2-GS plasmid vector 5ug in 1.2 plasmids and mixing with cells Example 2 is added into EP pipe, adds Add 0.7ml cell, after mixing, stands 15 minutes.

1.3 electrotransformation 280V20ms shock by electricity 2 pulses, and after the completion of electric shock, cell is transferred into shaking flask at once, is suspended It cultivates, observes cell state after 48h, change liquid culture, cell densities is waited to grow into 0.6 × 10⁶When cells/ml, 50uM is added MSX pressurization screening.

2. monoclonal screens

2.1 use the Chinese hamster ovary celI Serum-free and protein-free medium CHO-WM for being purchased from Suzhou City Wo Mei Bioisystech Co., Ltd Cell culture medium+50uM MSX suspension cell again counts.

To 5/mL, the cell for taking 200ul to mix is added in 96 orifice plates 2.2 bed board diluting cells, is placed into 37 DEG C, 5%CO₂4-6h is incubated in cell incubator.Record the hole of individual cells.

2.3 when the hole length of individual cells in 96 orifice plates is got up, discard culture medium, PBS is washed once, 100ul0.25% Trypsin-EDTA, room temperature digest 2min or so, and 2mLCHO-WM culture medium (MSX containing 10%FBS+50uM) is added and terminates digestion Reaction, and dispelled cell with pipettor.Cell is transferred to 12 orifice plates, when 12 orifice plates cover with, takes supernatant, Elisa detection Whether clone is the positive, and the positive colony of high efficient expression continues to expand culture, freeze.

3. cell shake flask fermentation

The configuration of 3.1 secondary culture bases: it uses CHO-WM culture medium addition 50uM MSX as secondary culture base, is placed in 37 37 DEG C are preheated in DEG C water-bath.

3.2 from CO₂Constant-temperature table takes out shaking flask cell, is counted.

3.3 diluting cells are to 2.5-3.5 × 10⁵A cell/mL inoculation 30mL culture is based in a 125mL shaking flask.Carefully Born of the same parents' culture bottle is placed into 37 DEG C, 5%CO₂100rpm/min is incubated overnight in constant-temperature table.

3.4, every counting cell density and vigor for 24 hours, survey glucose, when sugar is lower than 2g/L, add glucose To 4g/L；1mL sample is taken daily, and supernatant is for detecting protein expression situation.

4 SDS-PAGE of embodiment detection

The cell culture supernatant harvested in embodiment 3 is subjected to SDS-PAGE detection, while being made using empty Chinese hamster ovary celI For negative control.Concrete operations are as follows: the cell culture for taking 40 μ l to harvest, and 5 × loadingbuffer of 10 μ l, boiling is added Water-bath 5 minutes, 12000r/min was centrifuged 1 minute, took supernatant to carry out PAGE gel (12% concentration gel) electrophoresis, after electrophoresis Take gel to be dyed, decolourize after observe purpose band.As shown in figure 5, occurring two near molecular weight about 100kDa and 50kDa Purpose band, wherein S1 albumen is 100kDa or so, and S2 albumen is 50kDa.The present embodiment also constructs single expression S1 and S2 The recombinaant CHO cell of albumen, two albumen difference electrophoresis are as shown in Figure 6 and Figure 7, illustrate that S1, S2 albumen are correctly expressed. Because S1 albumen has more glycosylation modified, molecular weight is high about compared with by the molecular weight of amino acid sequence theoretical calculation 40kDa, S2 albumen size and calculated value are in the same size, and level of glycosylation is lower, and negative control does not have item in corresponding position Band.

5 WesternBlot of embodiment detection

Product after SDS-PAGE electrophoresis in embodiment 4 is transferred on NC (nitrocellulose) film, with 5% skim milk Closing 2 hours, the anti-IBV polyvalent antibody of Ji Yuan are incubated for 2 hours, and the goat-anti chicken polyclonal antibody secondary antibody of rinsing, HRP label is incubated for 2 Hour, then rinsing is added dropwise enhanced chemical luminous fluorescent substrate, is taken pictures using chemiluminescence imaging instrument.As shown in figure 8, weight For group CHO Supernatant samples there are two cell band, Fig. 9 and Figure 10 are the S1 and S2 of single expression, WB detection figure, negative control respectively There is no purpose band, illustration purpose antigen protein is correctly expressed in recombinaant CHO cell.

The detection of 6 recombinant C HO (pCI-S1-S2-GS) cell expression product co-immunoprecipitation of embodiment

1. IBV-S1 protein monoclonal antibody 10ul is added in the supernatant 1mL for taking CHO to express, 4 DEG C are rocked overnight incubation.

2. taking 10ulproteinA sepharose 4B, washed 3 times with appropriate PBS buffer solution, each 3000rpm is centrifuged 3min；

3. pretreated 10ul protein A sepharose 4B is added to and expresses supernatant with the overnight CHO of antibody incubation Incubation 2-4h is slowly rocked for 4 DEG C in liquid, keeps antibody and proteinA sepharose 4B coupled；

4. after immune precipitation, being centrifuged 3min at 4 DEG C with 3000rpm speed, sepharose 4B being centrifuged to tube bottom；It will be upper It carefully sucks clearly, sepharose 4B is washed 3-4 times with 1ml PBS buffer solution；It is eventually adding the 2xSDS sample-loading buffer of 15ul, boiling water It boils 5 minutes；

5.SDS-PAGE testing result is as shown in figure 11.Precipitating, precipitating are generated by the combination of S1 protein monoclonal antibody In product other than antibody band, there is also S1 and S2 protein band, illustrate that the S1 of CHO expression, S2 albumen form a kind of multiple Close object.

7 protein purification of embodiment

Destination protein contains his label, purifies destination protein using affinity chromatography.

The regeneration and balance of 1.Ni column:

The NaOH back flush Ni column 10min of 1.1 0.5mol/L after flushing, is rinsed with ultrapure water forward direction.

The NiSO of 1.2 50mol/L₄Rinse 5 column volumes.After, it is rinsed with ultrapure water forward direction.

1.3 with containing 300mM imidazoles PBS (pH=7.4) buffer rinse 3 column volumes after continue to be rushed with PBS buffer solution Wash 5 column volumes of balance.

2.Ni purification process:

After PBS balances Ni column, the supernatant loading of culture is taken, 5 column volumes to baseline is washed with PBS after end of the sample and puts down Weighing apparatus.Then respectively using 30mM is contained, the PBS of 150mM, 300mM and 500mM imidazoles are eluted.As a result as shown in figure 12, Destination protein is eluted in the PBS solution of 150mM imidazole concentration, Simultaneous purification obtains S1, S2 albumen composition.

The preparation of 8 subunit vaccine of embodiment

The avian infectious bronchitis virus S1-S2 albumen purified in right amount is added to (volume ratio in ISA201VG adjuvant For 46:54), make final concentration of protein 100ug/mL, emulsify, quality inspection qualification is placed on 4 DEG C of preservations.

Immunization experiment: using 14-42 age in days SPF chicken 40, wherein 20 be negative control group, 20 experimental groups.Every chicken First each eye droppings inoculation 1 part of infectious bronchitis of chicken live vaccine (H120 plants), 21-28 days after inoculation, takes a blood sample respectively, separates blood Clearly, experimental group is respectively inoculated with this vaccine 0.5ml, and negative control group is inoculated with the vaccine of physiological saline configuration.21-28 after secondary inoculation Day, then take a blood sample respectively, separate serum.The measurement of HI antibody titer will be made respectively by serum twice.As a result as shown in the table.From result As can be seen that the geometrical mean of HI potency logarithm is greater than 4 before and after experimental group chicken immune recombination IBV inactivated vaccine, meet the requirements.

Test group	HI potency average value before immune	HI potency average value after immune	Difference
				Group	22.6	26.8	24.2
Negative control group	22.7	22.5	2-0.2

To immune group and negative control group attack respectively malicious IBV GD/2015 plants and to 0.1mL (viral level >= 10^6.4EID₅₀), isolated rearing, observation to 14d, clinical symptoms monitor after attacking poison, attack the appetite of daily monitoring test chicken after poison, essence Refreshing state, the clinical pictures such as breathing.20 chickens of control group attack malicious hinterland continued reveal expiratory dyspnea, issue rale, cough, beat The clinical manifestations such as sneeze, the death rate 50%, dissect die of illness the pathological changes such as the visible kidney of chicken, tracheae than more serious (such as Figure 13 Left side).The clinical manifestation of vaccine group chicken is normal (on the right side of Figure 13).

The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.

Sequence table

<110>Suzhou Shi Nuo Bioisystech Co., Ltd

<120>infectious bronchitis of chicken genetic engineering subunit vaccine

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1644

<212> DNA/RNA

<213>artificial sequence (artificial sequence)

<400> 1

ggtaccgccg ccaccatgga aacagataca ctcctcctct gggtgctgct cctctgggtg 60

ccaggatcta caggcgccct gtacgactcc agctcttacg tgtactatta ccagtccgcc 120

ttcaggccac ctaacggatg gcacctgcat ggcggagctt acgctgtggt gaatatctcc 180

agcgagtcta acaatgctgg ctcttcccca ggatgcatcg tgggcacaat ccacggcgga 240

agggtggtga acgctagctc tatcgccatg accgctccct ccagcggaat ggcttggtct 300

tccagccagt tctgcacagc tcactgtaat ttttccgaca ccacagtgtt cgtgacacat 360

tgctataagt acgatggctg tcccatcacc ggcatgctgc agaagaactt tctgagagtg 420

agcgccatga agaacggcca gctgttctac aatctgacag tgtccgtggc taagtatcct 480

acctttaaga gcttccagtg cgtgaacaac ctgacctccg tgtacctgaa cggcgacctg 540

gtgtatacat ctaatgagac cacagatgtg acctccgccg gcgtgtactt taaggctggc 600

ggcccaatca cctataaagt gatgagagag gtgaaggccc tggcttactt cgtgaatggc 660

acagcccagg acgtgatcct gtgcgatggc tcccccaggg gcctgctggc ttgtcagtac 720

aacaccggca acttctccga tggcttttat cctttcatca actcttccct ggtgaagcag 780

aagtttatcg tgtatcgaga gaactctgtg aataccacat tcaccctgca caacttcaca 840

tttcataatg agaccggcgc caaccctaat ccatccggcg tgcagaacat ccagacctac 900

cagacccaga cagctcagag cggctattac aacttcaact tctccttcct gtcctccttc 960

gtgtacaagg agtctaactt catgtacggc tcttatcacc cctcctgcaa cttcaggctg 1020

gagaccatca acaatggcct gtggttcaac tccctgagcg tgtctatcgc ctacggccct 1080

ctgcagggcg gctgtaagca gtccgtgttc tccggccggg ccacatgctg ttatgcttac 1140

agctatggcg gcccatctct gtgcaagggc gtgtactccg gagagctggc cctgaatttc 1200

gagtgtggcc tgctggtgta tgtgaccaag agcggcggct ccagaatcca gaccgctaca 1260

gagccacccg tgatcacacg gcataactac aacaatatca ccctgaacac atgcgtggac 1320

tacaatatct atggcaggac cggccagggc tttatcacca acgtgacaga ctccgccgtg 1380

agctacaatt atctggccga tgctggcctg gctatcctgg acacctccgg cagcatcgat 1440

atctttgtgg tgcagggcga gtacggcctg acatattaca aggtgaaccc ctgtgaggat 1500

gtgaatcagc agttcgtggt gtccggcggc aagctggtgg gcatcctgac atcccggaac 1560

gagaccggca gccagctgct ggagaaccag ttctatatca agatcaccaa tggccatcac 1620

catcaccatc actgatgact cgag 1644

<210> 2

<211> 1773

<212> DNA/RNA

<213>artificial sequence (artificial sequence)

<400> 2

gttaacgccg ccaccatgga aacagataca ctcctcctct gggtgctgct cctctgggtg 60

ccaggatcta caggcagcat caccgagaac gtggccaatt gtccttacgt gtcttatggc 120

aagttctgca tcaagccaga cggctccatc gctaccatcg tgcccaagca gctggagcag 180

ttcgtggccc ctctgctgaa cgtgacagag aatgtgctga tcccaaacag ctttaatctg 240

accgtgacag acgagtacat ccagacacgg atggataagg tgcagatcaa ctgtctgcag 300

tacgtgtgcg gcaattctct ggactgtaga gatctgtttc agcagtacgg cccagtgtgc 360

gacaacatcc tgagcgtggt gaactccatc ggccagaagg aggatatgga gctgctgaac 420

ttctattcca gcaccaagcc cgccggcttc aacacacctt ttctgtccaa tgtgagcacc 480

ggcgagttta atatctccct gctgctgacc acaccatctt cccccaggag gaggtccttc 540

atcgaggacc tgctgtttac atctgtggag tccgtgggac tgccaaccga cgatgcttac 600

aagaactgta cagccggccc tctgggcttc ctgaaggatc tggcctgcgc tcgagagtat 660

aatggcctgc tggtgctgcc ccctatcatc accgctgaga tgcagaccct gtacacaagc 720

tctctggtgg ctagcatggc cttcggcgga atcaccgctg ctggagctat cccctttgcc 780

acacagctgc aggccaggat caaccacctg ggcatcaccc agagcctgct gctgaagaac 840

caggagaaga tcgccgcttc tttcaataag gctatcggcc gcatgcagga gggctttagg 900

tctacatccc tggccctgca gcagatccag gacgtggtga acaagcagtc cgctatcctg 960

accgagacaa tggccagcct gaacaagaat tttggcgcta tctccagcgt gatccaggag 1020

atctatcagc agctggacgc catccaggcc aatgctcagg tggatcgcct gatcaccggc 1080

aggctgtctt ccctgtccgt gctggcttcc gccaagcagg ctgagcatat ccgggtgagc 1140

cagcagagag agctggccac ccagaagatc aacgagtgcg tgaagagcca gtctatccgc 1200

tactctttct gcggcaatgg caggcacgtg ctgacaatcc ctcagaacgc tccaaatggc 1260

atcgtgttta tccatagctc ttataccccc gattctttcg tgaacgtgac agccatcgtg 1320

ggcttttgcg tgaagccagc caatgcttcc cagtacgcta tcgtgccagc taacggaagg 1380

ggcatcttca tccaagtgaa tggctcttac tatatcaccg ctcgggacat gtatatgcct 1440

agagctatca cagccggcga tatcgtgacc ctgacatctt gccaggccaa ctacgtgtcc 1500

gtgaataaga ccgtgatcac cacatttgtg gacaacgacg atttcgactt taatgatgag 1560

ctgtctaagt ggtggaacga taccaagcac gagctgcctg acttcgataa gtttaattac 1620

acagtgccaa tcctggacat cgattccgag atcgacagaa tccagggcgt gatccagggc 1680

ctgaacgaca gcctgatcga tctggagaag ctgtctatcc tgaagaccta catcaagtgg 1740

cctcaccacc accatcatca ctgatgaatc gat 1773

<210> 3

<211> 539

<212> PRT

<213>avian infectious bronchitis virus (Avian infectious bronchitis virus)

<400> 3

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Ala Leu Tyr Asp Ser Ser Ser Tyr Val Tyr Tyr Tyr

20 25 30

Gln Ser Ala Phe Arg Pro Pro Asn Gly Trp His Leu His Gly Gly Ala

35 40 45

Tyr Ala Val Val Asn Ile Ser Ser Glu Ser Asn Asn Ala Gly Ser Ser

50 55 60

Pro Gly Cys Ile Val Gly Thr Ile His Gly Gly Arg Val Val Asn Ala

65 70 75 80

Ser Ser Ile Ala Met Thr Ala Pro Ser Ser Gly Met Ala Trp Ser Ser

85 90 95

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

100 105 110

Val Thr His Cys Tyr Lys Tyr Asp Gly Cys Pro Ile Thr Gly Met Leu

115 120 125

Gln Lys Asn Phe Leu Arg Val Ser Ala Met Lys Asn Gly Gln Leu Phe

130 135 140

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

145 150 155 160

Gln Cys Val Asn Asn Leu Thr Ser Val Tyr Leu Asn Gly Asp Leu Val

165 170 175

Tyr Thr Ser Asn Glu Thr Thr Asp Val Thr Ser Ala Gly Val Tyr Phe

180 185 190

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

195 200 205

Leu Ala Tyr Phe Val Asn Gly Thr Ala Gln Asp Val Ile Leu Cys Asp

210 215 220

Gly Ser Pro Arg Gly Leu Leu Ala Cys Gln Tyr Asn Thr Gly Asn Phe

225 230 235 240

Ser Asp Gly Phe Tyr Pro Phe Ile Asn Ser Ser Leu Val Lys Gln Lys

245 250 255

Phe Ile Val Tyr Arg Glu Asn Ser Val Asn Thr Thr Phe Thr Leu His

260 265 270

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

275 280 285

Val Gln Asn Ile Gln Thr Tyr Gln Thr Gln Thr Ala Gln Ser Gly Tyr

290 295 300

Tyr Asn Phe Asn Phe Ser Phe Leu Ser Ser Phe Val Tyr Lys Glu Ser

305 310 315 320

Asn Phe Met Tyr Gly Ser Tyr His Pro Ser Cys Asn Phe Arg Leu Glu

325 330 335

Thr Ile Asn Asn Gly Leu Trp Phe Asn Ser Leu Ser Val Ser Ile Ala

340 345 350

Tyr Gly Pro Leu Gln Gly Gly Cys Lys Gln Ser Val Phe Ser Gly Arg

355 360 365

Ala Thr Cys Cys Tyr Ala Tyr Ser Tyr Gly Gly Pro Ser Leu Cys Lys

370 375 380

Gly Val Tyr Ser Gly Glu Leu Ala Leu Asn Phe Glu Cys Gly Leu Leu

385 390 395 400

Val Tyr Val Thr Lys Ser Gly Gly Ser Arg Ile Gln Thr Ala Thr Glu

405 410 415

Pro Pro Val Ile Thr Arg His Asn Tyr Asn Asn Ile Thr Leu Asn Thr

420 425 430

Cys Val Asp Tyr Asn Ile Tyr Gly Arg Thr Gly Gln Gly Phe Ile Thr

435 440 445

Asn Val Thr Asp Ser Ala Val Ser Tyr Asn Tyr Leu Ala Asp Ala Gly

450 455 460

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

465 470 475 480

Gly Glu Tyr Gly Leu Thr Tyr Tyr Lys Val Asn Pro Cys Glu Asp Val

485 490 495

Asn Gln Gln Phe Val Val Ser Gly Gly Lys Leu Val Gly Ile Leu Thr

500 505 510

Ser Arg Asn Glu Thr Gly Ser Gln Leu Leu Glu Asn Gln Phe Tyr Ile

515 520 525

Lys Ile Thr Asn Gly His His His His His His

530 535

<210> 4

<211> 582

<212> PRT

<213>avian infectious bronchitis virus (Avian infectious bronchitis virus)

<400> 4

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Ser Ile Thr Glu Asn Val Ala Asn Cys Pro Tyr Val

20 25 30

Ser Tyr Gly Lys Phe Cys Ile Lys Pro Asp Gly Ser Ile Ala Thr Ile

35 40 45

Val Pro Lys Gln Leu Glu Gln Phe Val Ala Pro Leu Leu Asn Val Thr

50 55 60

Glu Asn Val Leu Ile Pro Asn Ser Phe Asn Leu Thr Val Thr Asp Glu

65 70 75 80

Tyr Ile Gln Thr Arg Met Asp Lys Val Gln Ile Asn Cys Leu Gln Tyr

85 90 95

Val Cys Gly Asn Ser Leu Asp Cys Arg Asp Leu Phe Gln Gln Tyr Gly

100 105 110

Pro Val Cys Asp Asn Ile Leu Ser Val Val Asn Ser Ile Gly Gln Lys

115 120 125

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

130 135 140

Phe Asn Thr Pro Phe Leu Ser Asn Val Ser Thr Gly Glu Phe Asn Ile

145 150 155 160

Ser Leu Leu Leu Thr Thr Pro Ser Ser Pro Arg Arg Arg Ser Phe Ile

165 170 175

Glu Asp Leu Leu Phe Thr Ser Val Glu Ser Val Gly Leu Pro Thr Asp

180 185 190

Asp Ala Tyr Lys Asn Cys Thr Ala Gly Pro Leu Gly Phe Leu Lys Asp

195 200 205

Leu Ala Cys Ala Arg Glu Tyr Asn Gly Leu Leu Val Leu Pro Pro Ile

210 215 220

Ile Thr Ala Glu Met Gln Thr Leu Tyr Thr Ser Ser Leu Val Ala Ser

225 230 235 240

Met Ala Phe Gly Gly Ile Thr Ala Ala Gly Ala Ile Pro Phe Ala Thr

245 250 255

Gln Leu Gln Ala Arg Ile Asn His Leu Gly Ile Thr Gln Ser Leu Leu

260 265 270

Leu Lys Asn Gln Glu Lys Ile Ala Ala Ser Phe Asn Lys Ala Ile Gly

275 280 285

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

290 295 300

Gln Asp Val Val Asn Lys Gln Ser Ala Ile Leu Thr Glu Thr Met Ala

305 310 315 320

Ser Leu Asn Lys Asn Phe Gly Ala Ile Ser Ser Val Ile Gln Glu Ile

325 330 335

Tyr Gln Gln Leu Asp Ala Ile Gln Ala Asn Ala Gln Val Asp Arg Leu

340 345 350

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

355 360 365

Ala Glu His Ile Arg Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys

370 375 380

Ile Asn Glu Cys Val Lys Ser Gln Ser Ile Arg Tyr Ser Phe Cys Gly

385 390 395 400

Asn Gly Arg His Val Leu Thr Ile Pro Gln Asn Ala Pro Asn Gly Ile

405 410 415

Val Phe Ile His Ser Ser Tyr Thr Pro Asp Ser Phe Val Asn Val Thr

420 425 430

Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn Ala Ser Gln Tyr Ala

435 440 445

Ile Val Pro Ala Asn Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Ser

450 455 460

Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met Pro Arg Ala Ile Thr Ala

465 470 475 480

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

485 490 495

Asn Lys Thr Val Ile Thr Thr Phe Val Asp Asn Asp Asp Phe Asp Phe

500 505 510

Asn Asp Glu Leu Ser Lys Trp Trp Asn Asp Thr Lys His Glu Leu Pro

515 520 525

Asp Phe Asp Lys Phe Asn Tyr Thr Val Pro Ile Leu Asp Ile Asp Ser

530 535 540

Glu Ile Asp Arg Ile Gln Gly Val Ile Gln Gly Leu Asn Asp Ser Leu

545 550 555 560

Ile Asp Leu Glu Lys Leu Ser Ile Leu Lys Thr Tyr Ile Lys Trp Pro

565 570 575

His His His His His His

580

<210> 5

<211> 3489

<212> DNA/RNA

<213>artificial sequence (artificial sequence)

<400> 5

acgcgtggag ctagttatta atagtaatca attacggggt cattagttca tagcccatat 60

atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 120

ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgtcaat agggactttc 180

cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 240

tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 300

tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cctattagtc 360

atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 420

gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttgcacc 480

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 540

gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg 600

cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc 660

tccgcggatt cgaatcccgg ccgggaacgg tgcattggaa cgcggattcc ccgtgccaag 720

agtgacgtaa gtaccgccta tagagtctat aggcccacaa aaaatgcttt cttcttttaa 780

tatacttttt tgtttatctt atttctaata ctttccctaa tctctttctt tcagggcaat 840

aatgatacaa tgtatcatgc ctctttgcac cattctaaag aataacagtg ataatttctg 900

ggttaaggca atagcaatat ttctgcatat aaatatttct gcatataaat tgtaactgat 960

gtaagaggtt tcatattgct aatagcagct acaatccagc taccattctg cttttatttt 1020

atggttggga taaggctgga ttattctgag tccaagctag gcccttttgc taatcatgtt 1080

catacctctt atcttcctcc cacagctcct gggcaacgtg ctggtctgtg tgctggccca 1140

tcactttggc aaagaattgg gattcgaacg ttaacgccgc caccatggaa acagatacac 1200

tcctcctctg ggtgctgctc ctctgggtgc caggatctac aggcagcatc accgagaacg 1260

tggccaattg tccttacgtg tcttatggca agttctgcat caagccagac ggctccatcg 1320

ctaccatcgt gcccaagcag ctggagcagt tcgtggcccc tctgctgaac gtgacagaga 1380

atgtgctgat cccaaacagc tttaatctga ccgtgacaga cgagtacatc cagacacgga 1440

tggataaggt gcagatcaac tgtctgcagt acgtgtgcgg caattctctg gactgtagag 1500

atctgtttca gcagtacggc ccagtgtgcg acaacatcct gagcgtggtg aactccatcg 1560

gccagaagga ggatatggag ctgctgaact tctattccag caccaagccc gccggcttca 1620

acacaccttt tctgtccaat gtgagcaccg gcgagtttaa tatctccctg ctgctgacca 1680

caccatcttc ccccaggagg aggtccttca tcgaggacct gctgtttaca tctgtggagt 1740

ccgtgggact gccaaccgac gatgcttaca agaactgtac agccggccct ctgggcttcc 1800

tgaaggatct ggcctgcgct cgagagtata atggcctgct ggtgctgccc cctatcatca 1860

ccgctgagat gcagaccctg tacacaagct ctctggtggc tagcatggcc ttcggcggaa 1920

tcaccgctgc tggagctatc ccctttgcca cacagctgca ggccaggatc aaccacctgg 1980

gcatcaccca gagcctgctg ctgaagaacc aggagaagat cgccgcttct ttcaataagg 2040

ctatcggccg catgcaggag ggctttaggt ctacatccct ggccctgcag cagatccagg 2100

acgtggtgaa caagcagtcc gctatcctga ccgagacaat ggccagcctg aacaagaatt 2160

ttggcgctat ctccagcgtg atccaggaga tctatcagca gctggacgcc atccaggcca 2220

atgctcaggt ggatcgcctg atcaccggca ggctgtcttc cctgtccgtg ctggcttccg 2280

ccaagcaggc tgagcatatc cgggtgagcc agcagagaga gctggccacc cagaagatca 2340

acgagtgcgt gaagagccag tctatccgct actctttctg cggcaatggc aggcacgtgc 2400

tgacaatccc tcagaacgct ccaaatggca tcgtgtttat ccatagctct tatacccccg 2460

attctttcgt gaacgtgaca gccatcgtgg gcttttgcgt gaagccagcc aatgcttccc 2520

agtacgctat cgtgccagct aacggaaggg gcatcttcat ccaagtgaat ggctcttact 2580

atatcaccgc tcgggacatg tatatgccta gagctatcac agccggcgat atcgtgaccc 2640

tgacatcttg ccaggccaac tacgtgtccg tgaataagac cgtgatcacc acatttgtgg 2700

acaacgacga tttcgacttt aatgatgagc tgtctaagtg gtggaacgat accaagcacg 2760

agctgcctga cttcgataag tttaattaca cagtgccaat cctggacatc gattccgaga 2820

tcgacagaat ccagggcgtg atccagggcc tgaacgacag cctgatcgat ctggagaagc 2880

tgtctatcct gaagacctac atcaagtggc ctcaccacca ccatcatcac tgatgaatcg 2940

atacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac 3000

tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt 3060

gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa 3120

gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc 3180

ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga 3240

gttgttggga ttccaggctt ggatgaccag gctcagctaa tttttgtttt tttggtagag 3300

acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc 3360

accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct 3420

tctgattttg taggtaacca cgtgcgtatc gagtagcccc tcactttggc aaagaattgg 3480

gattcgaac 3489

<210> 6

<211> 32

<212> DNA/RNA

<213>artificial primer (artificial sequence)

<400> 6

ataggtaccg ccgccaccat ggaaacagat ac 32

<210> 7

<211> 46

<212> DNA/RNA

<213>artificial primer (artificial sequence)

<400> 7

atactcgagt catcagtgat ggtgatggtg atggccattg gtgatc 46

<210> 8

<211> 35

<212> DNA/RNA

<213>artificial primer (artificial sequence)

<400> 8

acgcgtggag ctagttatta atagtaatca attac 35

<210> 9

<211> 31

<212> DNA/RNA

<213>artificial primer (artificial sequence)

<400> 9

ggggctactc gatacgcacg tggttaccta c 31

Claims (10)

1. a kind of immune composition, includes:

Using SEQ ID NO:1 nucleic acid molecules or with the identical nucleic acid of 95% or more nucleotide sequence of SEQ ID NO:1 The avian infectious bronchitis virus of molecule encoding recombinates S1 albumen；

And SEQ ID NO:2 nucleic acid molecules or the identical nucleic acid of 95% or more nucleotide sequence with SEQ ID NO:2 The avian infectious bronchitis virus of molecule encoding recombinates S2 albumen.

2. immune composition according to claim 1, the avian infectious bronchitis virus recombinant protein includes:

The amino acid sequence of SEQ ID NO:3 or the identical ammonia of 95% or more full length amino acid sequence with SEQ ID NO:3 Base acid sequence；

The amino acid sequence of SEQ ID NO:4 or the identical ammonia of 95% or more full length amino acid sequence with SEQ ID NO:4 Base acid sequence.

3. immune composition described in claim 1 is directed to avian infectious bronchus for inducing in animal subject for producing The purposes of the medicament of the immune response of scorching viral antigen.

4. immune composition described in claim 1 is used to prepare chicken infectious bronchitis virogene engineering subunit vaccine In application.

5. a kind of nucleic acid molecules composition, can be used for being separately encoded avian infectious bronchitis virus recombinant protein, includes The sequential nucleotide sequence of SEQ ID NO:1 or the identical order core of 95% or more nucleotide sequence with SEQ ID NO:1 Nucleotide sequence；And SEQ ID NO:2 sequential nucleotide sequence or 95% or more nucleotide sequence with SEQ ID NO:2 Identical sequential nucleotide sequence.

6. nucleic acid molecules composition described in claim 5 is directed to avian infectious branch for inducing in animal subject for producing The purposes of the medicament of the immune response of bronchitis virus antigen.

7. nucleic acid molecules composition described in claim 5 is for producing for preventing animal by avian infectious bronchitis virus The purposes of the medicament of infection.

8. a kind of protein composition, selected from the group being made up of:

The amino acid sequence of SEQ ID NO:3 or the identical ammonia of 95% or more full length amino acid sequence with SEQ ID NO:3 Base acid sequence；

The amino acid sequence of SEQ ID NO:4 or the identical ammonia of 95% or more full length amino acid sequence with SEQ ID NO:4 Base acid sequence.

9. a kind of chicken infectious bronchitis virogene engineering subunit vaccine is, characterized by comprising: pharmaceutical effective amount Protein composition according to any one of claims 8；

Adjuvant.

10. subunit vaccine according to claim 9, which is characterized in that the adjuvant is selected from: white oil (M52), stearic acid One or more kinds of combinations of aluminium, department sheet, tween.