CN110237243A - Duck circovirus genetic engineering subunit vaccine and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of duck circovirus genetic engineering subunit vaccines and its preparation method and application.The present invention uses sequence nucleic acid molecules as shown in SEQ ID NO:1 or the identical nucleic acid molecule encoding duck circovirus outer capsid structural proteins of 95% or more nucleotide sequence with SEQ ID NO:1, immune composition comprising the duck circovirus outer capsid structural proteins can be used for preparing duck circovirus genetic engineering subunit vaccine, the antigenicity of the vaccine, immunogenicity and function are similar to native protein, expression is higher, immunogenicity is strong, it is not pathogenic to duck, and vaccine of the invention can be prepared by the extensive serum free suspension culture of bioreactor, greatly reduce production of vaccine cost.

Description

Duck circovirus genetic engineering subunit vaccine and its preparation method and application

Technical field

The present invention relates to a kind of genetic engineering subunit vaccines, and in particular to a kind of duck circovirus gene engineered subunit Vaccine belongs to animal immune technical field of pharmaceuticals.

Background technique

Duck circovirus disease (Duck circovirus disease, DCVD) is by duck circovirus (Duck Circovirus, DCV) infect a kind of caused inhibitive ability of immunity disease.Each kind and age in days duck can infect, clinical table of falling ill Now for Common Swift is bad, fall off, syntexis, anaemia, slow growth, feed conversion rate reduction etc..

Duck circovirus (DCV) belongs to circovirus section (Circoviridae) Circovirus (Circovirus), virus Particle is rounded or icosahedral symmetry, no cyst membrane, diameter are about 15-16nm, is to be currently known the smallest duck virus.Virus is right External environment resistance is strong, and duck group is difficult to remove after infection.Currently, cannot still cultivate in vitro.DCV single stranded circle DNA Virus, genome are about 2.0kb, have 2 main reading frames, ORFV1 and ORFC1, are separately encoded related with virus replication Rep albumen (Replication Protein) and nucleocapsid structures PROTEIN C ap albumen (Capsid Protein), virus can divide For two different genotype of DCV-1 and DCV-2.

It is depauperation that DCV, which causes the main clinical manifestation of duck morbidity, weight loss and feather are in disorder etc., pathological tissue table Now there is necrosis, lymphocyte reduction and histiocytosis for the bursa of farbricius.The gradually atrophy of the lymphoid tissue of infection animal, causes Immune function decline or immune response inhibit, and then improve double or multiple infection probability.The feature of circovirus infection It is extremely complex with the mixed infection situation of other pathogens or virus.It is worth noting that DCV infection can damage immune system, feel The sick duck of dye DCV is usually occurred together Riemerellosis Anatipestifer, pasteurella multocida, Escherichia coli, duck hepatitis virus, duck plague virus etc. Infection, and after infecting duck circovirus, the probability of these pathogen infections can be significantly improved.Since DCV discovery is later, at present Lack the method for in-vitro multiplication culture DCV again, therefore many difficulties are still had to the research of its pathogenesis and harm, understanding is also It is very shallow.

Market needs a kind of vaccine that can prevent DCV at present.The present invention is therefore.

Summary of the invention

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

To achieve the goals above, according to an aspect of the invention, there is provided a kind of immune composition, includes:

Using sequence nucleic acid molecules as shown in SEQ ID NO:1 or the nucleotide sequence 95% with SEQ ID NO:1 The duck circovirus Cap protein of the above identical nucleic acid molecule encoding.

The further technical solution of the present invention is: the duck circovirus Cap protein includes the amino of SEQ ID NO:2 Acid sequence or the identical amino acid sequence of 95% or more full length amino acid sequence with SEQ ID NO:2.

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 duck circovirus antigen.

Another object of the present invention is to provide immune composition described in one kind for produce it is sick for preventing duck annulus The purposes of the medicament of poison infection.

Another object of the present invention is to provide a kind of nucleic acid molecules, it can be used for encoding duck circovirus Cap protein, Sequential nucleotide sequence comprising SEQ ID NO:1 is identical suitable with 95% or more the nucleotide sequence of SEQ ID NO:1 Sequence nucleotide sequence.

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 duck circovirus antigen.

Another object of the present invention is to provide nucleic acid molecules described in one kind for produce for prevent animal by duck justify The purposes of the medicament of circovirus virus infection.

For example, the medicament can be duck circovirus genetic engineering subunit vaccine.

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

The identical albumen of 95% or more full length amino acid sequence of SEQ ID NO:2 and SEQ ID NO:2.

It is a kind of suitable for generating in animal subject body for duck circovirus another object of the present invention is to provide The immune composition of immune response includes:

The duck circovirus Cap protein and adjuvant.

Another object of the present invention is to the albumen to be used to prepare duck circovirus genetic engineering subunit vaccine side The application in face.

Another object of the present invention is to the preparation methods of the immune composition described in one kind, and the method includes following steps It is rapid:

S1, the encoding gene of duck circovirus outer capsid structural proteins is cloned on shuttle vector, obtains recombination and shuttles Carrier；

S2, recombinant shuttle plasmid is transformed into the DH10Bac bacterium containing Baculovirus Gene group plasmid, recombination, which is shuttled, to be carried Destination gene expression is confined to being inserted into rod-shaped viral genome plasmid in body, obtains the recombination bar containing destination gene expression frame Shape viral genome plasmid；

S3, recombinant baculovirus geneome plasmid transfection insect cell is obtained into recombinant baculovirus；

S4, the recombinant baculovirus of acquisition is inoculated with insect cell, expresses duck circovirus outer capsid structural proteins；

S5, the recombination duck circovirus outer capsid structural proteins that step S4 is obtained are added in adjuvant and are exempted to get described Epidemic disease composition.

In preferred technical solution, the shuttle vector type be selected from pFastBac1, pFastBac HT A/B/C, Any one in pVL1393, but not limited to this.

In preferred technical solution, the insect cell includes Sf9, High Five, any one in S2 or Sf21 cell Kind, but not limited to this.

In preferred technical solution, in step S3, culture medium used in fermented and cultured is secondary culture base.

In preferred technical solution, in step S4, can also after expression obtains duck circovirus outer capsid structural proteins, Carry out polyacrylamide gel electrophoresis, western blot test detection, Electronic Speculum detection and Elisa is quantitative and fine jade to expand detection etc. subsequent Operation.

The invention discloses a kind of preparation method of the duck circovirus recombinant subunit vaccine of Sf9 cell expression and answer With, and proving that the vaccine can generate stronger humoral immunity in duck body, the duck after being immunized can resist duck circovirus Infection, belongs to animal vaccine and veterinary biologics technical field, its purpose is to provide one kind can large-scale industrial production Duck circovirus virus-like particle novel gene engineering subunit vaccine preparation method.The duck circovirus genetic engineering is sub- Subunit vaccine is using Sf9 cell expression recombination duck circovirus Cap protein, the formation VLPs which can be spontaneous The VLPs of (virus like particles), formation have fabulous immune prototype and safety, then make VLPs Vaccine excites stronger more fully antibody protection.

It is an object of that present invention to provide the duck circovirus gene engineered subunits that a kind of good immune effect, technique are safer Vaccine, the present invention use Sf9 cell expression recombination duck circovirus Cap protein.Production process of the present invention is not related to totivirus training Support, by Sf9 cell carry out protein expression, antigenicity, the immunogenicity of product are similar to native protein, expression compared with Height, immunogenicity is strong, does not have pathogenic and of the invention vaccine outstanding by the extensive serum-free of bioreactor to duck Floating culture preparation, greatly reduces production of vaccine cost.

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

Antigenicity, immunogenicity and the function of duck circovirus genetic engineering subunit vaccine provided by the invention and natural Protein is similar, and expression is higher, and immunogenicity is strong, does not have pathogenic and of the invention vaccine that can pass through life to duck The extensive serum free suspension culture preparation of object reactor, while greatly reducing production of vaccine cost.

Detailed description of the invention

The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.In the accompanying drawings:

Fig. 1 is that PCR product after the amplification of DCV Cap protein gene PCR is carried out gel electrophoresis result, it can be seen that 0.8kbp Nearby there is a band；Wherein 1 is DCV-Cap gene, and 2 be negative control, and M is molecular weight marker；

Fig. 2 carries out gel electrophoresis for PCR product after the PCR amplification of the bacterium colony sample of multiple DCV Cap protein genetic transformation As a result, nearby there is positive sample in 0.8kbp band.Wherein 1~5 be DCV Cap protein genetic transformation bacterium colony sample Product after PCR amplification, 6 be non-positive sample, and M is molecular weight marker；

Fig. 3 is the recombinant shuttle vector pF-DCV-Capsid map containing target gene of building；

Fig. 4 is the cell culture supernatant progress PAGE gel electrophoresis harvested in embodiment 3 as a result, DCV Cap There is purpose band near molecular weight about 30kDa in the cell culture of albumen；The wherein 1 cell training to be harvested in embodiment 3 Object supernatant is supported, 2 be negative control, and M is molecular weight marker；

Fig. 5 is the product Western Blot testing result in embodiment 4 after SDS-PAGE electrophoresis；Wherein 1 is recombination Sf9 Cell expresses sample, and 2 be negative control, and M is molecular weight marker；

Fig. 6 is the Cap protein virus-like particle electron microscope of DCV；

Fig. 7 is control group duck feather trophic disturbance, pinna rachis bleeding figure in effect evaluation experiment；

Fig. 8 is to attack control group duck internal anatomy after poison using after vaccine of the present invention.

Specific embodiment

It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another It indicates, all technical and scientific terms that this specification uses have and general technical staff of the technical field of the invention Normally understood identical meanings.

It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to exemplary embodiments of the present invention.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 duck circovirus Cap protein of nucleic acid molecule encoding.

The present invention also relates to it is a kind of induction for duck circovirus antigen immune response method, the method includes to Animal subject applies vaccine of the invention.

The present invention also relates to a kind of method for protecting animal subject to infect from duck circovirus, the method includes to institute It states animal subject and applies vaccine of the invention.

The invention also includes the vaccines for being suitable for inducing the immune response for duck circovirus.Vaccine of the invention can be Plasmid comprising above-mentioned nucleic acid molecules.Nucleic acid molecules can be incorporated into that in virion.Vaccine can also include adjuvant molecules.Adjuvant can For IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNF α, TNF β, GM-CSF, epidermis 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； It and in some embodiments, can be IL-12, IL-15, IL-28 or RANTES.

Vaccine of the invention also includes protein molecular.This specification provides the albumen selected from the group being made up of: including The albumen of SEQ ID NO:2；The 95% identical albumen in the whole length of the amino acid sequence of SEQ ID NO:2；SEQ ID The segment of NO:2；Albumen identical with the segment 95% of SEQ ID NO:2.

This specification also provides a kind of albumen selected from the group being made up of: (a) SEQ ID NO:2；(b) in such as SEQ 95% identical albumen on the entire length amino acid sequence of full length sequence described in IDNO:2；(c) SEQ ID NO:2's includes The immunogenic fragments of 20 or more the amino acid of SEQ ID NO:2；And (d) in the amino acid sequence of SEQ ID NO:2 The immunogenic fragments comprising 20 or more amino acid of 95% identical albumen in the whole length of column.

Vaccine of the invention also includes nucleic acid molecules.The present invention also provides comprising encoding one or more eggs described above The nucleic acid molecules of the sequence of white molecule.In some embodiments, the nucleic acid molecules include selected from the group being made up of Sequence: SEQ ID NO:1；The 95% identical nucleic acid sequence in the whole length of the nucleotide sequence of SEQ ID NO:1；SEQ The segment of ID NO:1；Nucleotide sequence identical with the segment 95% of SEQ ID NO:1.

Some aspects of the present invention provide the method for immune response of the induction for duck circovirus, and the method includes following Step: to individual application duck circovirus antigen and/or combination thereof object.

The other aspect of the present invention provides the method for protecting individuals from duck circovirus infection.The method includes following Step: to the nucleic acid molecules or composition comprising such nucleic acid sequence of the individual application prevention effective dose；The wherein core Acid sequence is expressed in the cell of the individual, and for anti-by the protein induced protective immunity of the nucleic acid sequence encoding It answers.

Some aspects of the invention provide a kind of method for inducing the immune response for duck circovirus antigen, the side Method includes that 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 duck circovirus, the method Including applying nucleic acid molecules of the invention to the animal subject.

Some aspects of the invention provide a kind of suitable for being generated animal subject for the immune anti-of duck circovirus The vaccine answered, the vaccine includes: nucleic acid molecules and adjuvant molecules of the invention.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 further includes one or more nucleic acid molecules as described above and one or more by the nucleic acid The albumen of molecule encoding.

1. defining

Term used in this specification is not intended to limit merely for the sake of the purpose for describing specific embodiment.Such as exist Used in specification and the claim, in addition to the other clear stipulaties of context, singular "one", "an" and " described " includes plural form.

For numberical range cited by this specification, clearly covering is having each insertion between identical precision Number.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 contain Number 6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9 and 7.0 is covered.

Used in this specification " adjuvant " mean to be added to this specification as described in vaccine in enhance by hereafter institute State any molecule of the immunogenicity of the encoded antigen of nucleic acid sequence encoding.

As used in this specification " antibody " means the antibody or segment, its piece of type IgG, IgM, IgA, IgD or IgE Section or derivative, including Fab, F (ab') 2, Fd and single-chain antibody, double-chain antibody, bispecific antibody, bifunctional antibody and Its derivative.The antibody can be the antibody isolated from the serum sample of animal, polyclonal antibody, affinity purification antibody Or mixtures thereof, to required epitope or derived from it, sequence shows enough binding specificities to the mixture.

" coded sequence " or " code nucleic acid " as used in this specification mean the nucleotide sequence comprising coding albumen Nucleic acid (RNA or DNA molecular).The coded sequence may further include the initial signal for being operably coupled to controlling element And termination signal, the controlling element include the promoter that expression can be instructed in the individual of administration of nucleic acid or the cell of animal And polyadenylation signal.

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

" shared " or " consensus sequence " as used in this specification mean one based on the specific duck circovirus antigen of analysis The polypeptide sequence of multiple hypotypes of queue.The nucleic acid sequence for encoding shared polypeptide sequence can be prepared.Wrapping protein-contg vaccine can be with It is used to induction to be immunized for a variety of hypotypes of specific duck circovirus antigen or the extensive of serotype, the vaccine includes coding The consensus sequence and/or nucleic acid molecules of these albumen.

" electroporation ", " electricity-permeabilization " or " electronic enhancing " (" EP ") being used interchangeably such as this specification means to make The microcosmic approach (hole) in biomembrane is induced with cross-film electric field pulse；Their presence permission biomolecule such as plasmid, Oligonucleotides, siRNA, drug, ion and water flow to the other side from the side of cell membrane.

" segment " as used in this specification relative to nucleic acid sequence mean coding can with overall length wild-type virus The nucleic acid sequence or part of it of the polypeptide triggered an immune response in the animal of strain duck circovirus antigenic cross-reaction.Described Section can be at least one DNA fragmentation selected from the various nucleotide sequences for encoding protein fragments described below.

For polypeptide sequence, " segment " or " immunogenic fragments " means can be round with overall length wild-type strain duck The polypeptide triggered an immune response in the animal of circovirus virus antigenic cross-reaction.The segment of albumen can wrap it is 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 amino acid or more, at least 40 amino acid or more, at least 50 amino acid or more, at least 60 amino acid or more More, at least 70 amino acid or more, at least 80 amino acid or more, at least 90 amino acid or more, at least 100 ammonia Base 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 ammonia Base 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.

The term as used in this specification " genetic constructs " refer to comprising coding albumen nucleotide sequence DNA or RNA molecule.The coded sequence includes the initial signal and termination signal for being operably coupled to controlling element, the regulation member Part includes 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 Term used in specification " expression-form " refers to gene construct, and the gene construct, which contains, is operably coupled to coding The necessary controlling element of the coded sequence of albumen, so that when in the cell for being present in the individual, coded sequence is by table It reaches.

The term as used in this specification " homology " refers to complementary degree.Homoeology or completely homologous may be present Property (that is, identity).At least partly inhibiting fully-complementary sequence to hybridize in the partial complementarity sequence of target nucleic acids is using function Term " substantially homologous " refers to.When the double-strandednucleic acid sequence about such as cDNA or genomic clone in use, such as this explanation Term used in book " substantially homologous " refers to that probe can hybridize under the conditions of property low strict in the chain of the double-strandednucleic acid sequence.Work as pass In single strand nucleotide sequence in use, the term as used in this specification " substantially homologous " refers to that probe can be in property condition low strict Lower hybridization is in single stranded nucleic acid template sequence (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 in this specification Mean prescribed percentage of the sequence with identical residue in specified region.The percentage can be calculated by following: most Two sequences are compared goodly, compare the number of two sequences, the position for determining residue identical in the two sequences in specified region It measures to generate the quantity of matching position, the total quantity with the quantity of matching position divided by the position in specified region, and will As a result the percentage of sequence identity is generated multiplied by 100.There is different length in two sequences or compare generation one Or in the case that multiple staggered ends and the specified region compared only include unique sequence, the residue of unique sequence is included In the denominator of calculating rather than in molecule.When comparison dna and RNA, thymidine (T) and uracil (U) be may be considered that It is equivalent.Identity can be held manually or by computer sequence algorithm such as BLAST or BLAST 2.0 is used Row.

As used in this specification " immune response " means the introducing that antigen is shared in response to antigen such as duck circovirus, place The activation of main immune system (such as immune system of animal).It is described immune response can be cell effect or humoral response or The form of the two.

" nucleic acid " or " oligonucleotides " as used in this specification or " polynucleotides " mean to be covalently joined together to Few two nucleotide.Single-stranded description also defines the sequence of complementary strand.Therefore, nucleic acid also covers described single-stranded mutual Mend chain.Many variants of nucleic acid can be used for purpose identical with given nucleic acid.Therefore, nucleic acid also covers substantially phase Same nucleic acid and its complement.The probe that single-stranded offer can hybridize under stringent hybridization conditions with target sequence.Therefore, nucleic acid 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.

The expression of gene be the promoter being spatially attached thereto control under carry out.At the control, promoter The upstream 5'(of gene can be positioned in) or the downstream 3'().The distance between the promoter and gene can about with institute It states promoter and its distance between the gene controlled in the promoter therefrom gene of derivation is identical.Such as this field institute Know, the variation of this distance can be adjusted in the case where not losing promoter function.

As used in this specification " promoter " means synthesis or natural source molecule, the molecule can assign, Activation or the expression of the nucleic acid in enhancing cell.Promoter may include one or more specific transcription regulating nucleotide sequences so as into one It walks Enhanced expressing and/or changes the expression in its space and/or the expression of time.Promoter can also include Distal enhancer or resistance Hold back element, they can be located at the almost thousands of pairs of base-pairs since the starting point of transcription.Promoter can be from including It is obtained in virus, bacterium, fungi, plant, insect and the source of animal.Promoter can be thin relative to what is wherein expressed Born of the same parents, tissue 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 Promoter, bacteriophage T3 promoter, SP6 promoter, lactose operon-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter with And CMVIE promoter.

" signal peptide " and " leader sequence ", which is used interchangeably and refers in this specification, can be connected this specification The amino acid sequence of the amino terminal of the duck circovirus albumen.Signal peptide/leader sequence is indicated generally at the position of albumen. Signal peptide/leader sequence used in this specification preferably facilitates albumen and secretes from the cell for generating it.Signal peptide/leading sequence Column are usually cracked from the remainder of albumen, and the albumen from cell after secreting through being commonly referred to as maturation protein.Signal peptide/preceding Lead the N-terminal that sequence is connected to the albumen.

As used in this specification " stringent hybridization conditions " mean condition, i.e., under the described conditions such as in nucleic acid Complex mixture in the first nucleic acid sequence (for example, probe) will hybridize with second nucleotide sequence (for example, target).Strictly Condition be sequence dependent and will be different in different environment.Ionic strength pH of the stringent condition in restriction It can be selected as about 5-10 DEG C lower than the thermal melting point of particular sequence (Tm) down.The Tm can be such temperature ( Under the ionic strength of restriction, pH and nucleic acid concentration), the probe and target sequence with the 50% of target-complementary exist at said temperatures Hybridized (because target sequence is present in excess, at Tm, 50% probe is occupied in the state of the equilibrium) under equilibrium state.Sternly Glazing bar part can be those conditions, i.e., wherein salinity is less than about the sodium ion of 1.0M, the about 0.01- such as at pH 7.0 to 8.3 The Na ion concentration (or other salt) of 1.0M, and temperature is at least about for short probe (for example, about 10-50 nucleotide) 30 DEG C and for long probe (for example, greater than about 50 nucleotide) be at least about 60 DEG C.Stringent condition can also be by adding Destabilizing agent such as formamide is added to realize.For selection or specific hybridization, positive signal can be at least the 2 of background hybridization To 10 times.Illustrative stringent hybridization conditions include the following: 50% formamide, 5x SSC and 1%SDS, incubated at 42 DEG C It educates or 5x SSC, 1%SDS, is incubated at 65 DEG C, is washed at 65 DEG C with 0.2x SSC and 0.1%SDS.

" be substantially complementary " as used in this specification 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, in the region of 360,450,540 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 in this specification " 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, be at least 60% in 100,180,270,360,450,540 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 with The complement of second sequence is substantially complementary, then First ray and the second sequence are also identical in this way.

" hypotype " or " serotype ": being used interchangeably such as this specification and about duck circovirus, it is intended that duck annulus disease The genetic mutation of poison a, so that hypotype is identified and separated from different hypotypes by immune system.

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

" 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.

As used in this specification " carrier " means the nucleic acid sequence containing replication orgin.Carrier can be viral vectors, Bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.Carrier can be DNA or RNA carrier.It is multiple that carrier can be self The outer carrier of the chromosome of system, and preferably DNA vector.

2. vaccine

Vaccine of the invention can be designed in control animal subject for one or more duck circovirus serotypes The degree or intensity of immune response.Vaccine may include that it is inhibited to be integrated into element or reagent in chromosome.Vaccine can be coding The RNA of duck circovirus Cap protein.RNA vaccine can be introduced into cell.Vaccine of the invention may include duck circovirus Cap Albumen.Duck circovirus Cap protein is by inducing 1) cytotoxic T lymphocyte (CTL) reaction, 2) t helper cell reaction And/or 3) B cell is reacted, or preferably all above-mentioned reactions, it is immune-mediated come what is carried out to reach intersection submission The target of virus sweep.

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

Some embodiments are related to the homologous with 95% with this specification nucleic acid coding sequence of encoding immunogenic albumen The nucleic acid molecules of property.What some embodiments were related to encoding immunogenic albumen has 96% with this specification nucleic acid coding sequence The nucleic acid molecules of homology.Some embodiments are related to having with this specification nucleic acid coding sequence for encoding immunogenic albumen The nucleic acid molecules of 97% homology.Some embodiments be related to encoding immunogenic albumen with this specification nucleic acid coding sequence Nucleic acid molecules with 98% homology.Some embodiments be related to encoding immunogenic albumen with this specification nucleic acid encode Sequence has the nucleic acid molecules of 99% homology.In some embodiments, there is the volume with albumen disclosed in this specification The nucleic acid molecules of the coded sequence disclosed in this specification of code sequence homology are connected to coding comprising coding IgE leader sequence The sequence of 5 ' ends of the coded sequence of homologous protein sequence disclosed in this specification.

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.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.Segment can be with the segment at least 95% of SEQ ID NO:1, at least 96%, at least 97%, at least 98% or at least 99% are identical.Segment can be with the segment at least 80% of SEQ ID NO:1, 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% is identical.In some embodiments, segment includes the sequence of encoding leader sequence, such as immune ball Protein leader object, such as IgE lead.In some embodiments, segment is free of the coded sequence of encoding leader sequence.Some In embodiment, 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:2.Some embodiments are related to and such as SEQ ID Protein sequence described in NO:2 has the immunogenic protein of 95% homology.Some embodiments are related to and such as SEQ ID Protein sequence described in NO:2 has the immunogenic protein of 96% homology.Some embodiments are related to and such as SEQ ID Protein sequence described in NO:2 has the immunogenic protein of 97% homology.Some embodiments are related to and such as SEQ ID Protein sequence described in NO:2 has the immunogenic protein of 98% homology.Some embodiments are related to and such as SEQID Protein sequence described in NO:2 has the immunogenic protein of 99% homology.

Some embodiments are related to albumen identical with SEQ ID NO:2.Some embodiments are related to having in such as SEQ 80% identical amino acid sequence on the entire length amino acid sequence of overall length consensus amino acid sequences described in ID NO:2 Immunogenic protein.Some embodiments are related to having the overall length consensus amino acid sequences as described in SEQ ID NO:2 The immunogenic protein of 85% identical amino acid sequence on entire length amino acid sequence.Some embodiments are related to having 90% identical amino acid on the entire length amino acid sequence of the overall length consensus amino acid sequences as described in SEQ ID NO:2 The immunogenic protein of sequence.Some embodiments, which are related to having, shares amino acid in the overall length as described in SEQ ID NO:2 The immunogenic protein of 91% identical amino acid sequence on the entire length amino acid sequence of sequence.Some embodiments are related to With 92% identical on the entire length amino acid sequence of the overall length consensus amino acid sequences as described in SEQ ID NO:2 The immunogenic protein of amino acid sequence.Some embodiments are related to having shared in the overall length as described in SEQ ID NO:2 The immunogenic protein of 93% identical amino acid sequence on the entire length amino acid sequence of amino acid sequence.Some embodiment party Case is related to having 94% on the entire length amino acid sequence of the overall length consensus amino acid sequences as described in SEQ ID NO:2 The immunogenic protein of identical amino acid sequence.Some embodiments are related to having complete as described in SEQ ID NO:2 The immunogenic protein of 95% identical amino acid sequence on the entire length amino acid sequence of long consensus amino acid sequences.It is some Embodiment is related to the entire length amino acid sequence in the overall length consensus amino acid sequences as described in SEQ ID NO:2 The immunogenic protein of upper 96% identical amino acid sequence.Some embodiments are related to having the institute in such as SEQ ID NO:2 The immunogenicity egg of 97% 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 acid in the overall length consensus amino acid sequences as described in SEQ ID NO:2 The immunogenic protein of 98% identical amino acid sequence on sequence length.Some embodiments are related to having in such as SEQ ID 99% identical amino acid sequence exempts from the entire length amino acid sequence of overall length consensus amino acid sequences described in NO:2 Epidemic disease immunogenic peptide.

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:2 are provided.Immunogenic fragments may include at least 10%, at least 15%, at least 20%, extremely Few 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:2.In some embodiments, segment includes leader sequence, such as immune Globulin lead, such as IgE lead.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 the amino acid sequence albumen homologous with the immunogenic fragments of SEQ ID NO:2. 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:295% homologous albumen.Some embodiments are related to having with the immunogenic fragments of this specification protein sequence The immunogenic fragments of 96% homology.Some embodiments are related to having with the immunogenic fragments of this specification protein sequence The immunogenic fragments of 97% homology.Some embodiments are related to having with the immunogenic fragments of this specification protein sequence The immunogenic fragments of 98% homology.Some embodiments are related to having with the immunogenic fragments of this specification protein sequence The immunogenic fragments of 99% homology.In some embodiments, segment includes leader sequence, such as immune globulin Cynanchum glaucescens Sequence is led, such as IgE lead.In some embodiments, segment is free of leader sequence.In some embodiments, segment is not Containing 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:2. 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:2,85%, 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.

3. vaccine constructs and plasmid

Vaccine may include coding duck circovirus Cap protein, duck circovirus antigen and duck circovirus Cap protein/ The combined nucleic acid construct or plasmid of antigen.This specification, which provides may include, encodes duck annulus disease disclosed in this specification The genetic constructs of the nucleic acid sequence of malicious antigen, the core antigen include protein sequence and the homologous sequence of protein sequence, egg The segment of Bai Xulie and the sequence homologous with the segment of protein sequence.In addition, this specification offer may include code book explanation Duck circovirus surface antigen disclosed in book (including protein sequence, with the homologous sequence of protein sequence, the segment of protein sequence with And the homologous sequence with the segment of protein sequence) nucleic acid sequence genetic constructs.The genetic constructs can be used as function Can molecule outside sex chromosome and exist.The genetic constructs can be the line including centromere, telomere or plasmid or clay Property 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 carrier may include the heterologous nucleic acids of coding for antigens, and can further include can be in antigen encoding sequence The initiation codon of the upstream of column and can be in the terminator codon in the downstream of antigen encoding sequences.Initiation codon and termination are close Numeral can be with antigen encoding sequences in frame.The carrier also includes the starting for being operably coupled to antigen encoding sequences Son.The promoter for being operably coupled to antigen encoding sequences can be promoter from simian virus 40 (SV40), mouse The long end weight of mammary tumour virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter such as bovine immunodeficiency virus (BIV) Multiple (LTR) promoter, Moloney (Moloney) viral promotors, avian leukosis virus (ALV) promoter, cytomegalovirus (CMV) viral (EBV) promoter of promoter such as CMV immediate early promoter, love bar Er Shi (Epstein Barr) or Lloyd's's meat Tumor virus (RSV) promoter.The promoter can also be the promoter from people's gene, and the people's gene such as people's flesh moves egg White, people's myosin, people's ferroheme, people's muscle creatin or human metal thioalbumen.The promoter can also be tissue specificity Promoter, such as natural or synthetic muscle or skin-specific promoter.

The carrier can also include polyadenylation signal, and the polyadenylation signal can be in duck annulus disease The downstream of malicious core protein coded sequence.It is more that the polyadenylation signal can be SV40 polyadenylation signal, LTR Polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (HGH) (hGH) Polyadenylation letter Number or people's beta-globin polyadenylation signal.The SV40 polyadenylation signal can be from pCEP4 carrier The polyadenylation signal of (Invitrogen, San Diego, CA).

Carrier can also reside in shared duck circovirus core protein coded sequence or shared duck circovirus surface antigen The enhancer of the upstream of albumen coded sequence.The enhancer is necessary for DNA expression.The enhancer can be people's flesh Filamentous actin, people's myosin, people's ferroheme, people's muscle creatin or virus enhancer, such as one from CMV, HA, RSV or EBV Kind enhancer.

Carrier can also include the replication orgin of animal, to maintain carrier outside chromosome and to generate load in cell Multiple copies of body.The carrier can be pVAX1, pCEP4 from Invitrogen (San Diego, CA) or PREP4 may include the replication orgin and nuclear antigen EBNA-1 coding region of love bar Er Shi virus, this can not integrated In the case where generate high copy episomal replication.The carrier can be modified pVAX1 pVax1 variant, such as originally Variant plasmid described in the specification.The variant pVax1 plasmid is Backbone Vector plasmid pVAX1 (Invitrogen, Carlsbad CA 2998 base-pair variants).The CMV promoter is located at base 137-724.T7 promoter/initiation site is located at base At 664-683.Multiple cloning sites are located at base 696-811.Ox GH polyadenylation signal is at base 829-1053. Kanamycins (Kanamycin) resistant gene is at base 1226-2020.PUC starting point is at base 2320-2993.

The carrier can be pSE420 (Invitrogen, San Diego, Calif.), can be used in Escherichia coli (E.coli) albumen is generated in.The carrier can be pYES2 (Invitrogen, San Diego, Calif.), can be used for Albumen is generated in the Wine brewing yeast strain (Saccharomyces cerevisiae strain) of yeast.The carrier may be used also Can be used for the complete baculovirus expression system of MAXBACTM (Invitrogen, San Diego, Calif.) Albumen is generated in insect cell.The carrier can also be pcDNAI or pcDNA3 (Invitrogen, SanDiego, Calif.), can be used for generating albumen in zooblast such as Sf9 cell line.The carrier can be through routine techniques and Available initial substance is easy to generate the expression vector or system of albumen, the technology and substance include Sambrook etc., Molecular Cloning and Laboratory Manual, second edition, ColdSpring Harbor (1989).

The present invention first carries out recombinant shuttle vector building: encoding gene of the clone comprising duck circovirus Cap protein is to wearing On shuttle carrier pFastBac1 carrier.Then recombinant baculovirus strain, including protein SDS-PAGE, WB, immunofluorescence inspection are constructed It surveys, virus titer detection, final protein large-scale production expression and assay prepare the vaccine.The present invention uses The duck circovirus Cap protein sequence of optimization, using suspending, culture Sf9 cell is expressed, and expression is higher, and albumen is exempted from Epidemic focus is good.Duck circovirus Cap protein sequence can be original series, increase and truncated sequence.Recombinant baculovirus Baculovirus expression system shuttle vector in carrier include but is not limited to pFastBac 1, pFastBac HTA/B/C, PVL1393 etc., such as can preferably use pFastBac1.Insect cell line can be as Sf9, High Five, S2 or Sf21 Cell preferably uses Sf9.The invention has the advantages that production process is not related to totivirus, protein expression is carried out by Sf9 cell, The antigenicity of product, immunogenicity and function are similar to native protein, and expression is higher.Suspend culture, is easy to extensive Production.

1 shuttle vector pF-Cap of embodiment building and identification

DCV of the 1.DCV Cap protein gene magnification with purifying after Nanjing Jin Sirui company has synthesized codon optimization Cap protein gene (SEQ ID NO:1) is simultaneously cloned on pUC17 plasmid, obtains pUC-Cap plasmid vector.With pUC-Cap plasmid As template, Cap-F, Cap-R carry out the PCR amplification (gene order of Cap-F, Cap-R such as SEQ ID as upstream and downstream primer NO:3, shown in 4), amplification system is shown in Table 1.

1 Cap protein gene magnification system of table

Reaction condition are as follows: 95 DEG C initial denaturation 5 minutes；94 DEG C be denaturalized 45 seconds, 54 DEG C anneal 45 seconds, 72 DEG C extend 1 minute, 35 A circulation；72 DEG C extend 10 minutes.

PCR product is subjected to gel electrophoresis verifying purpose gene size, as shown in Figure 1, mesh occurs in the position in 0.8kbp Band, target gene expands successfully, carries out recovery purifying with gel purification kit.

2. 1 plasmid of pFastBac and Cap protein gene PCR amplified production are used BamH I, Hind III by digestion and purifying 37 DEG C double digestion 3 hours, specific endonuclease reaction system is shown in Table 2, table 3.

Digestion products are subjected to gel electrophoresis, use the pFastBac 1 of gel purification kits digestion respectively Plasmid and Cap protein gene PCR product.

2 Cap protein gene PCR product endonuclease reaction system of table

3 pFastBac of table, 1 plasmid enzyme restriction reaction system

3. connection connects 1 plasmid of pFastBac of double digestion and Cap protein gene PCR product digestion products using T4DNA Connect 16 DEG C of water-bath connections of enzyme overnight.Specific coupled reaction system is shown in Table 4.

4 Cap protein gene PCR digestion products of table and 1 plasmid enzyme restriction product linked system of pFastBac

4. 10 μ l connection products are added in the DH5 α competent cell of 100 μ l and mix by conversion, and ice bath 30 minutes, 42 DEG C of water Bath heat shock 90 seconds, then ice bath 2 minutes, the LB liquid medium that 900 μ l are free of Amp is added, 37 DEG C are cultivated 1 hour.Take 1.0ml Bacterium solution is condensed into 100 μ l and is coated on the LB solid medium containing Amp, and 37 DEG C are cultivated 16 hours.

5. 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, PCR product is carried out gel electrophoresis as primer progress bacterium colony PCR identification by Cap-F and Cap-R The size of verifying purpose gene, as shown in Fig. 2, the sample for 0.8kbp band occur is positive sample.By the positive bacterium solution of identification Sequencing company is sent to be sequenced, selection is sequenced correct bacterium solution and saves.The shuttle vector pF-Cap containing target gene of building Its schematic diagram such as Fig. 3.

2 recombinant baculovirus genome Bac-Cap of embodiment building

The DH10Bac competence of 100 μ l is added in 1 μ l pF-Cap plasmid in 1.DH10Bac bacterium conversion difference Example 1 Mixed in cell, ice bath 30 minutes, 42 DEG C water-bath heat shock 90 seconds, then ice bath 2 minutes, the LB liquid that 900 μ l are free of Amp is added Culture medium, 37 DEG C are cultivated 5 hours.After taking 100 μ l bacterium solutions to dilute 81 times, the 100 diluted bacterium solutions of μ l are taken to be applied to big mould containing celebrating Element, kanamycins, tetracycline, X-gal and IPTG LB solid medium on, 37 DEG C are cultivated 48 hours.

2. selecting the monoclonal white colony big using transfer needle picking, then containing gentamicin, kanamycins, four It crosses on the LB solid medium of ring element, X-gal and IPTG, 37 DEG C are cultivated 48 hours, and then picking single colonie inoculation contains The LB liquid medium culture of gentamicin, kanamycins, tetracycline saves strain, extracts plasmid.Obtain recombinant plasmid Bacmid-Cap plasmid.

The transfection of 3 recombinant baculovirus of embodiment

Each hole inoculation 0.8 × 10 in six orifice plates⁶A Sf9 cell, cell confluency degree are 50~70%.For each hole It prepares compound below: diluting the Cellfectin transfection reagent of 4 μ l, of short duration whirlpool shake with 100 μ l transfection media T1 It swings；The recombination Bacmid-Cap plasmid in culture T1 base 3 μ g embodiments 2 of dilution is transfected with 100 μ l, respectively tries diluted transfection Agent and plasmid mixing, gently blow even, prepare transfection mixture.Above-mentioned transfection composite is added after cell is adherent, 27 DEG C incubate It educates 5 hours, removes supernatant, add 2ml SF-SFM fresh culture, 27 DEG C of cultures harvest supernatant on the 4th~5.It is rod-shaped to obtain recombination Viral rBac-Cap detects virus titer, rBac-Cap using MTT relative effectivenes method for recombinant baculovirus to the P1 of harvest P1 kind poison virus titer is 3.4 × 10⁷pfu/mL.It is spare as kind of poison to expand recombinant baculovirus rBac-Cap.

4 SDS-PAGE of embodiment detection

The cell culture harvested in embodiment 3 is subjected to SDS-PAGE detection, while using the empty baculoviral of infection Sf9 cell is as negative control.Concrete operations are as follows: 5 × loading buffer of 10 μ l is added in the cell culture for taking 40 μ l to harvest Liquid (loadingbuffer), boiling water bath 5 minutes, 12000r/min was centrifuged 1 minute, and supernatant is taken to carry out PAGE gel (12% Concentration gel) electrophoresis, take gel to be dyed after electrophoresis, decolourize after observe purpose band.

As shown in figure 4, there is purpose band, negative control near molecular weight about 30kDa in rBac-Cap cell culture There is no band in corresponding position.

5 Western Blot of embodiment identification

The product after SDS-PAGE electrophoresis in embodiment 4 is transferred on NC (nitrocellulose) film respectively, with 5% degreasing Milk is closed 2 hours, and the anti-DCV positive serum in duck source is incubated for 2 hours, rinsing, and the goat-anti duck polyclonal antibody secondary antibody of HRP label is incubated It educates 2 hours, rinses, enhanced chemical luminous fluorescent substrate is then added dropwise, is taken pictures using chemiluminescence imaging instrument.As shown in figure 5, Recombinant baculovirus expression sample has purpose band, and negative control does not have purpose band, and illustration purpose antigen protein is in Sf9 cell In correctly expressed.

6 indirect immunofluorescene assay of embodiment

It is added into 96 porocyte culture plates and transfected the Sf9 cell suspension of rBac-Cap, (cell concentration is in 100 holes μ l/ 2.5×10⁵~4.0 × 10⁵A/ml), 4 holes are inoculated with, 27 DEG C stand 15 minutes, so that Sf9 cell is affixed on culture plate bottom wall, then often The seed culture of viruses that 10 μ l dilute 10 times is added in hole.Blanc cell control is set simultaneously.Cell is set in 27 DEG C of constant incubators and is cultivated after inoculation 72~96 hours, culture solution is abandoned, cold methanol/acetone (1:1) is fixed.Reacted first with the anti-DCV polyvalent antibody in duck source, then with The goat-anti duck IgG reaction of FITC label, inverted fluorescence microscope observe result.Inoculation sky baculoviral Sf9 cell cannot be observed To fluorescence, and it is vaccinated with recombinant baculovirus Sf9 cell and is able to observe that fluorescence, illustration purpose antigen egg obtain in Sf9 cell To correct expression, recombinant baculovirus building is correctly.

The detection of 7 expression product Electronic Speculum of embodiment

By recombinant baculovirus cell culture ultrasonication, 12000r/min is centrifuged 30 minutes, takes supernatant, 0.22 μm of filter Film filtering, removes impurity, is concentrated 10 times using the super filter tube that molecular cut off is 3kDa.It is 40% that concentration, which is added, in each centrifuge tube Sucrose solution 10ml, then be added 2.0ml be concentrated by ultrafiltration sample, 29000r/min ultracentrifugation 2 hours, abandon supernatant, precipitating It is resuspended with 2.0ml PBS.Then by suspension through over-richness be respectively 50%, 60%, 70%, 80% gradient concentration sucrose from The heart 29000r/min ultracentrifugation 2 hours, then collects the band for being located at 60%~70% concentration intersection.It will be a small amount of pure DCV virus-like particle sample after changing concentration is added drop-wise on the copper mesh of carbon film, and after natural air drying, it is molten that 2% sodium phosphotungstate is added dropwise Liquid carries out negative staining, carries out Electronic Speculum observation after negative staining.As shown in fig. 6, the Cap protein of DCV can be spontaneous be assembled into diameter About 20nm virus-like particle.

The bioreactor serum free suspension culture of 8 insect cell of embodiment and the expression of Cap protein quantify and fine jade expands Titration

Insect cell 3-4 days sterile culture Sf9 in 1000ml shaking flask, grow to 3-5 × 10 to concentration⁶Cell/mL, vigor It when greater than 95%, seeds cells into the bioreactor of 5L, inoculum density is 3-8 × 10⁵cell/mL.Work as cell concentration Reach 3-5.5 × 10⁶When cell/mL, seed cells into 50L bioreactor, to cell it is long to concentration be 3-5.5 × 10⁶Cell/mL, is inoculated into 500L bioreactor, reaches 2-8.5 × 10 to cell concentration⁶When cell/mL, inoculation recombination bar Shape virus rBac-Cap, bioreactor culture condition are pH value 6.0-6.5,25-27 DEG C of temperature, dissolved oxygen 30-80%, mixing speed 100-180rpm.In view of the optimum condition of cell culture, preferred pH6.2, cell culture phase temperature set 27 DEG C, dissolved oxygen 50%, mixing speed 100-180rpm.Continue after infection culture 5-9 days after, addition one thousandth final concentration BEI, 37 DEG C After acting on 48h, add 2/1000ths final concentration Na₂S₂O₃Terminate inactivation.Cell is harvested by the method for centrifugation or hollow fibre filtering Culture supernatant sets 2-8 DEG C of preservation vaccinogen liquid.

Cap protein content is detected using Elisa method in the above-mentioned vaccine antigen of preparation.Mode of operation is as follows: with coating Buffer dilutes the anti-DCV polyvalent antibody of duck to suitable concentration, and every 100 μ l of hole, 4 DEG C overnight, and PBST is washed three times, 1%BSA closing 1h.Be added various concentration antigen standard (being chromatographed by exchange of particles, hydrophobic chromatography, the albumen that molecular sieve purification obtains) and Gradient dilution measuring samples, 37 DEG C are incubated for 1 hour, and PBST is washed three times.Detection antibody-Cap protein monoclonal antibody is added in every hole, 37 DEG C are incubated for 1 hour, and PBST is washed three times.The goat-anti duck IgG of two anti-i.e. HRP labels is added in every hole, and 37 DEG C are incubated for 1 hour, PBST It washes three times.TMB develops the color 10 minutes, 2M H₂SO₄Terminate reaction.Microplate reader reading calculates Cap in measuring samples by standard curve The amount of albumen.

According to embodiment 7, the Cap protein of large scale preparation, Elisa testing result is as follows, and albumen is flat in vaccinogen liquid Equal content about 165mg/L respectively.

In the vaccine antigen of preparation, expands potency method using fine jade and detect Cap protein: beating plum blossom on 5 pieces of agarose gel plates Hole, is added duck circovirus antiserum among plum blossom hole, be separately added into around 5 blocks of plates dilute 20,1,2,3,4,5 powers 5 kinds of vaccinogen liquids.It is inverted after being incubated for 72h and observes precipitation line.The maximum dilution ratio for precipitation line occur is that its fine jade expands potency fine jade Expand bioactivity result such as following table.It is 1:64 that the fine jade of Cap protein, which expands potency, in vaccinogen liquid.

The preparation of 9 vaccine of embodiment

Vaccinogen liquid is expressed in Example 7, uses normal saline dilution that the concentration of antigen protein is made to reach 50 μ g/ ML, then by vaccinogen liquid and oil adjuvant according to the proportional arrangement of 2:3 at oil emulsion vaccine.Specifically, the vaccinogen liquid of every 1L White oil 1429g is added, takes charge of this 70.2g, aluminum stearate 8.43g and tween 53.3g.Then with the broken crusher machine emulsification system of emulsification For at oil emulsion adjuvant inactivated vaccine.

The experiment of 10 effect evaluation of embodiment

7 age in days duckling 30 is taken, is randomly divided into two groups, every group 15, respectively immune group and control group.By embodiment 9 In the duck circovirus inactivated vaccine for preparing respectively with 0.3ml leg muscle injecting immune group duck, the same sample prescription of control group duck Formula injects equivalent sterilizing white-oil adjuvant.21 days after immune, each group test duck carries out leg muscle injection with duck circovirus and attacks Poison, every 0.2ml (includes 10⁵ELD₅₀) NJ plants of velogen strain, isolated rearing.After observation 14 days, discovery immune group duck is showed no bright Aobvious clinical symptoms, no death, immune protective rate 100%；There is feather trophic disturbance, pinna rachis bleeding successively in control group duck The phenomenon that (Fig. 7), gradually body condition is thin dead, and dead 12.Dissect dead and not dead control group duck discovery, dead duck Capillary injection, air bag have the cheesy object of yellow, liver surface exudation tissue fluid and have diffusivity blood spots, and similar duck is infected Property scrositis symptom bursa of farbricius atrophy it is downright bad phenomena such as (Fig. 8), not dead duck do not occur obvious lesion.The results show that this hair The inactivated vaccine immune efficacy of bright embodiment preparation is qualified.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Sequence table

<110>Suzhou Shi Nuo Bioisystech Co., Ltd

<120>duck circovirus genetic engineering subunit vaccine and its preparation method and application

<160> 4

<170> SIPOSequenceListing 1.0

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<212> DNA

<213>duck circovirus (Duck circovirus)

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ggatccatgc gcggccgtac ctaccgccgt gcttacaggg gtaggagaaa gcgccgtggc 60

ctgaggagac gcttccgtag gagacgcctg aggatcgcca gaccacgtag gagattctcc 120

gtggtcacct acaaggtgac cagacacact gtcttcggct tcttcggaag ccagaccgga 180

cctactgctg ccggcaagtg gcagtctctg tcactggagg acggagctca gtacaccgac 240

cctcccgctc gcggtaacaa catctgcgga ctgaacatgc gttgggctat gttcggcgac 300

actaactcct acatgaccgg aagcactccc ttctaccact acccatacga ctactacatg 360

atcaagggtg tggctatcac cctgcgtccc gcctacaaca tctaccagaa gtctaagacc 420

cagggttcaa ctgtgatcga caaggacggc cagatcgtca agacttccac cactggatgg 480

agcatcgacc cttacggttc tacctccagc cgccgtactt gggacccctc aagggtccac 540

aggagatact tcatcccaaa gcctatcatc cagggagccg gagagggcac caagcactct 600

actttcttcc tgggtggcaa gaacttcacc tggatcaact gcactcagga ccaggtggtc 660

cactacggaa tgggaatgtc cctgcgcaag cctgacaaca ccactggtgt gaacgctcag 720

tacgacatcg aagcccagtt caccttctac atcaagttcg gacagttcac tggcttccat 780

caccatcacc atcactaatg aaagctt 807

<210> 2

<211> 263

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 2

Met Arg Gly Arg Thr Tyr Arg Arg Ala Tyr Arg Gly Arg Arg Lys Arg

1 5 10 15

Arg Gly Leu Arg Arg Arg Phe Arg Arg Arg Arg Leu Arg Ile Ala Arg

20 25 30

Pro Arg Arg Arg Phe Ser Val Val Thr Tyr Lys Val Thr Arg His Thr

35 40 45

Val Phe Gly Phe Phe Gly Ser Gln Thr Gly Pro Thr Ala Ala Gly Lys

50 55 60

Trp Gln Ser Leu Ser Leu Glu Asp Gly Ala Gln Tyr Thr Asp Pro Pro

65 70 75 80

Ala Arg Gly Asn Asn Ile Cys Gly Leu Asn Met Arg Trp Ala Met Phe

85 90 95

Gly Asp Thr Asn Ser Tyr Met Thr Gly Ser Thr Pro Phe Tyr His Tyr

100 105 110

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

115 120 125

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

130 135 140

Asp Lys Asp Gly Gln Ile Val Lys Thr Ser Thr Thr Gly Trp Ser Ile

145 150 155 160

Asp Pro Tyr Gly Ser Thr Ser Ser Arg Arg Thr Trp Asp Pro Ser Arg

165 170 175

Val His Arg Arg Tyr Phe Ile Pro Lys Pro Ile Ile Gln Gly Ala Gly

180 185 190

Glu Gly Thr Lys His Ser Thr Phe Phe Leu Gly Gly Lys Asn Phe Thr

195 200 205

Trp Ile Asn Cys Thr Gln Asp Gln Val Val His Tyr Gly Met Gly Met

210 215 220

Ser Leu Arg Lys Pro Asp Asn Thr Thr Gly Val Asn Ala Gln Tyr Asp

225 230 235 240

Ile Glu Ala Gln Phe Thr Phe Tyr Ile Lys Phe Gly Gln Phe Thr Gly

245 250 255

Phe His His His His His His

260

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<213>artificial primer (artificial sequence)

<400> 3

ataggatcca tgcgcggccg tacctaccgc cgtgc 35

<210> 4

<211> 47

<212> DNA

<213>artificial primer (artificial sequence)

<400> 4

ataaagcttt cattagtgat ggtgatggtg atggaagcca gtgaact 47

Claims (10)

1. a kind of immune composition, it is characterised in that include:

Using sequence nucleic acid molecules as shown in SEQ ID NO:1 or with 95% or more the phase of nucleotide sequence of SEQIDNO:1 The duck circovirus outer capsid structural proteins of same nucleic acid molecule encoding.

2. immune composition according to claim 1, which is characterized in that the duck circovirus outer capsid structural proteins Amino acid sequence comprising SEQ ID NO:2 or the identical amino of 95% or more full length amino acid sequence with SEQIDNO:2 Acid sequence.

3. the preparation method of immune composition as claimed in claim 1 or 2, which is characterized in that the described method comprises the following steps:

S1, the encoding gene of duck circovirus outer capsid structural proteins is cloned on shuttle vector, obtains recombinant shuttle vector；

S2, recombinant shuttle plasmid is transformed into the DH10Bac bacterium containing Baculovirus Gene group plasmid, in recombinant shuttle vector Destination gene expression is confined to being inserted into Baculovirus Gene group plasmid, and it is rod-shaped to obtain the recombination containing destination gene expression frame Viral genome plasmid；

S3, recombinant baculovirus geneome plasmid transfection insect cell is obtained into recombinant baculovirus；

S4, the recombinant baculovirus of acquisition is inoculated with insect cell, expresses duck circovirus outer capsid structural proteins；

S5, the recombination duck circovirus outer capsid structural proteins that step S4 is obtained are added in adjuvant to get the immune group Close object.

4. preparation method according to claim 3, which is characterized in that the shuttle vector be selected from pFastBac1, Any one in pFastBacHTA/B/C, pVL1393.

5. preparation method according to claim 3, which is characterized in that the insect cell includes Sf9, HighFive, S2 Or any one in Sf21 cell.

6. preparation method according to claim 3, which is characterized in that in step S3, culture medium used in fermented and cultured For secondary culture base.

7. a kind of nucleic acid molecules are used to encode duck circovirus outer capsid structural proteins, the order core comprising SEQ ID NO:1 Nucleotide sequence or the identical sequential nucleotide sequence of 95% or more nucleotide sequence with SEQ ID NO:1.

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

The amino acid sequence of SEQ ID NO:2 or the identical amino of 95% or more full length amino acid sequence with SEQIDNO:2 Acid sequence.

9. immune composition of any of claims 1 or 2 is directed to duck circovirus for inducing in animal subject for producing The purposes of the medicament of the immune response of antigen.

10. immune composition of any of claims 1 or 2 is for producing the medicament infected for preventing animal by duck circovirus Purposes.