CN109999191A

CN109999191A - A kind of chicken virus mycoplasma novel gene engineering subunit vaccine

Info

Publication number: CN109999191A
Application number: CN201910289602.6A
Authority: CN
Inventors: 曹文龙; 孔迪; 滕小锘; 易小萍; 张大鹤
Original assignee: Suzhou Shino Biotechnology Co Ltd
Current assignee: Suzhou Womei Biology Co ltd
Priority date: 2019-04-11
Filing date: 2019-04-11
Publication date: 2019-07-12
Anticipated expiration: 2039-04-11
Also published as: CN109999191B

Abstract

The present invention provides a kind of immune composition and subunit vaccines, the immune composition includes a kind of albumen, described albumen one or more kinds of any combination selected from the following: using the nucleic acid molecules of SEQ ID NO:1 or 3 or 5 or 7 or 9 or the chicken virus mycoplasma GAP-associated protein GAP with the identical nucleic acid molecule encoding of 95% or more nucleotide sequence of SEQ ID NO:1 or 3 or 5 or 7 or 9.The vaccine uses eukaryotic expression; antigenicity, the immunogenicity of product are similar to native protein; expression is higher; immunogenicity is strong; protecting effect is good; there is no pathogenic and of the invention vaccine that can prepare by the extensive serum free suspension culture of bioreactor to chicken, while greatly reducing production of vaccine cost.

Description

A kind of chicken virus mycoplasma novel gene engineering subunit vaccine

Technical field

This application involves animal immune technical field of pharmaceuticals, and in particular to a kind of chicken virus mycoplasma novel gene engineered vaccine Preparation method and application.

Background technique

Chicken virus mycoplasma (MycoplasmaGalliscepticum, MG) infection is one kind of chicken respiratory tract disease, is mainly drawn It plays that chronic respiratory tract disease, air bag be scorching and sinusitis, is clinically mainly shown as cough, has a running nose, when breathing issues rale, when serious Mouth breathing.According to statistics, after mycoplasma gallisepticum infection chicken group, the weak young rate of chick increases by 10% or so, under the laying rate of laying hen 10%-20%, the weight loss 38% of broiler chicken drop, and marketing period extends, and feed conversion rate reduces by 21%, and can cause indirectly big The medication expense of amount is to endanger one of important diseases of poultry husbandry.

Chicken virus mycoplasma (MG) is Mycoplasmas, and Mycoplasmataceae, pathogenic kind of one of Mycoplasma, be that the protokaryon of very little is raw Object, cell-free wall, only cell membrane wraps up.

The prevention and treatment of chicken virus mycoplasma mainly has vaccine prevention and drug therapy at present.The infection characteristic of chicken virus mycoplasma is main It is colonized on the cilium of tunica mucosa tracheae and on air bag, and there is no blood vessel on tracheal cilia end and air bag, and drug is to pass through blood Liquid transport reaches site of action, then by diffusion in virus mycoplasma, therefore drug can only be reduced on tracheal cilia and air bag The virus mycoplasma quantity and mitigation clinical symptoms of field planting, and complete cannot be killed, and mycoplasma gallisepticum infection is generally primary Infection can be in the carrier state of persistent infection throughout one's life, therefore at present, vaccine can effectively provide protection.

Vaccine currently used for preventing, controlling mycoplasma gallisepticum infection is all traditional inactivated vaccine, but chicken virus mycoplasma is trained Difficulty is supported, needs serum, antigenic content is low.Simultaneously in chicken virus mycoplasma incubation, due to cultivation temperature, medium component Etc. conditions change and the continuous duplication of itself, the antigen protein on chicken virus mycoplasma surface is easily mutated and is lacked It loses, inactivated vaccine is caused to there is a problem of that immune protective efficiency is weaker.Therefore, it is imperative to study the strong vaccine of new immunity. Research and develop the dominant direction that the new generation vaccine based on recombinant vaccine is this century.

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, including a hatching egg It is white, described albumen one or more kinds of any combination selected from the following:

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 chicken virus mycoplasma adhesin antibodies MGC1 of nucleic acid molecule encoding；

It is identical using the nucleic acid molecules of SEQ ID NO:3 or with 95% or more the nucleotide sequence of SEQ ID NO:3 The chicken virus mycoplasma adhesin antibodies MGC2 of nucleic acid molecule encoding；

It is identical using the nucleic acid molecules of SEQ ID NO:5 or with 95% or more the nucleotide sequence of SEQ ID NO:5 The chicken virus mycoplasma adhesin antibodies MGC3 of nucleic acid molecule encoding.

It is identical using the nucleic acid molecules of SEQ ID NO:7 or with 95% or more the nucleotide sequence of SEQ ID NO:7 The chicken virus mycoplasma blood clotting GAP-associated protein GAP VLH3 of nucleic acid molecule encoding；

It is identical using the nucleic acid molecules of SEQ ID NO:9 or with 95% or more the nucleotide sequence of SEQ ID NO:9 The chicken virus mycoplasma blood clotting GAP-associated protein GAP VLH5 of nucleic acid molecule encoding.

Preferred technical solution is that the chicken virus mycoplasma adhesin antibodies MGC1 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；

The chicken virus mycoplasma adhesin antibodies MGC2 include SEQ ID NO:4 amino acid sequence or with SEQ ID The identical amino acid sequence of 95% or more full length amino acid sequence of NO:4；

The chicken virus mycoplasma adhesin antibodies MGC3 include SEQ ID NO:6 amino acid sequence or with SEQ ID The identical amino acid sequence of 95% or more full length amino acid sequence of NO:6.

The chicken virus mycoplasma blood clotting GAP-associated protein GAP VLH3 includes the amino acid sequence or and SEQ of SEQ ID NO:8 The identical amino acid sequence of 95% or more full length amino acid sequence of ID NO:8；

The chicken virus mycoplasma blood clotting GAP-associated protein GAP VLH5 includes the amino acid sequence or and SEQ of SEQ ID NO:10 The identical amino acid sequence of 95% or more full length amino acid sequence of ID NO:10.

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 Mycoplasma Gallisepticum Antigen Recognized By Antibody.

Another object of the present invention is to provide immune composition described in one kind for produce for preventing chicken poison branch original The purposes of the medicament of body-sensing dye.

Another object of the present invention is to provide a kind of immune composition, including it is a kind of for encoding chicken virus mycoplasma blood clotting The nucleic acid molecules of GAP-associated protein GAP or adhesin antibodies, the nucleic acid molecules are selected from the following one or more kinds of any Combination:

The sequential nucleotide sequence of SEQ ID NO:1 is identical as 95% or more the nucleotide sequence of SEQ ID NO:1 Sequential nucleotide sequence；

The sequential nucleotide sequence of SEQ ID NO:3 is identical as 95% or more the nucleotide sequence of SEQ ID NO:3 Sequential nucleotide sequence；

The sequential nucleotide sequence of SEQ ID NO:5 is identical as 95% or more the nucleotide sequence of SEQ ID NO:5 Sequential nucleotide sequence；

The sequential nucleotide sequence of SEQ ID NO:7 is identical as 95% or more the nucleotide sequence of SEQ ID NO:7 Sequential nucleotide sequence；

The sequential nucleotide sequence of SEQ ID NO:9 is identical as 95% or more the nucleotide sequence of SEQ ID NO:9 Sequential 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 Mycoplasma Gallisepticum Antigen Recognized By Antibody.

Another object of the present invention is to provide nucleic acid molecules described in one kind for produce it is malicious by chicken for preventing animal The purposes of the medicament of mycoplasma infection.

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

The chicken virus mycoplasma adhesin antibodies MGC1 include SEQ ID NO:2 amino acid sequence or with SEQ ID The identical amino acid sequence of 95% or more full length amino acid sequence of NO:2；

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

The chicken virus mycoplasma adhesin antibodies and blood clotting GAP-associated protein GAP and adjuvant.

Preferred technical solution is that the adjuvant is selected from:

One or more kinds of combinations of white oil (M52), aluminum stearate, department sheet, tween.

The invention discloses a kind of preparation method of the chicken virus mycoplasma recombinant subunit vaccine of Sf9 cell expression and answer With, and proving that the vaccine can generate stronger humoral immunity in chicken body, the chicken after being immunized can resist chicken virus mycoplasma sense Dye, belongs to animal vaccine and veterinary biologics technical field, and its purpose is to provide one kind can large-scale industrial production The preparation method of chicken virus mycoplasma recombinant subunit vaccine: first constructing recombinant shuttle vector, by being cloned respectively comprising blood clotting egg White VLH3 (variably expressed lipoprotein and hemagglutinin lipoproteins 3, it is subsequent The numbers such as 3 are the names of an author, are named according to the cluster of gene and its direction, otherwise referred to as pMGA), VLH5 (variably expressed lipoprotein and hemagglutinin lipoproteins 5, name be same as above) and Adhesin antibodies 1 (Mycoplasma gallisepticum cytadhesin 1, MGC1), adhesin antibodies 2 (Mycoplasma gallisepticum cytadhesin 2, MGC2), 3 (Mycoplasma of adhesin antibodies Gallisepticum cytadhesin3, MGC3) on encoding gene to 1 carrier of shuttle vector pFastBac.Then building weight Group baculoviral strain, including protein SDS-PAGE, WB, Immunofluorescence test, virus titer detection carry out albumen large-scale production Expression and assay；In live chickens virus mycoplasma genome out of office, express this five it is closely related with mycoplasma gallisepticum infection The albumen such as gene of blood clotting GAP-associated protein GAP VLH3, VLH5 and adhesin antibodies MGC1, MGC2 and MGC3 exist it is a large amount of Homologous pseudogene, homologous recombination easily occurs for the true gene pseudogene homologous with it, so the blood clotting in mycoplasma reproductive process Amino acid sequence easily mutates in GAP-associated protein GAP VLH3, VLH5 and adhesin antibodies MGC1, MGC2 and MGC3, leads to one In a group Different Individual or different groups it is wild chicken virus mycoplasma this five Membrane surface proteins it is widely different, present different Group distribution, in order to improve the protecting effect of vaccine as far as possible, offset antigenic difference present in wild chicken virus mycoplasma, The present invention expresses five kinds of antigen proteins and adjuvant is mixed with vaccine.

It is an object of that present invention to provide the chicken virus mycoplasma gene engineered subunits that a kind of good immune effect, technique are safer Vaccine, the present invention use Sf9 cell expression recombination chicken virus mycoplasma blood clotting GAP-associated protein GAP VLH3, VLH5 and adhesin antibodies (MGC1, MGC2 and MGC3).Production process of the present invention is not related to Culture Mycoplasma, carries out protein expression by Sf9 cell, produces Antigenicity, the immunogenicity of product it is similar to native protein and expression antigen there is no mutation and missing the case where, expression It is horizontal higher.Suspend culture, is easy to be mass produced.

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

The immune composition present invention of the invention expresses hemagluttinin proteins VLH3, VLH5 and adhesin antibodies using Sf9 cell (MGC1, MGC2 and MGC3), the antigenicity of product, immunogenicity and function are similar to native protein, and expression is higher, And the antigen of expression, there is no being mutated and lacking, immunogenicity is strong, does not have pathogenic and of the invention epidemic disease to chicken Seedling can be prepared by the extensive serum free suspension culture of bioreactor, while greatly reduce production of vaccine cost.

Present invention uses hemagluttinin proteins VLH3, VLH5 of optimization and adhesin antibodies MGC1, MGC2 and MGC3 sequence, Using suspending, culture Sf9 cell is expressed, and expression is higher, and protein immunogenic is good.Subunit vaccine of the invention can Sufficient for amount with large-scale production, Quality Control is easy；Highly-safe, immunogenicity is good；Stablize between batch；Production cost is low.

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 MGC1 gene PCR is carried out gel electrophoresis result, it can be seen that 2.9kbp's Band；Wherein 1 is negative control；2 be MG-MGC1 gene；M is molecular weight marker；

Fig. 2 be multiple MGC1 genetic transformation bacterium colony sample PCR amplification after PCR product carry out gel electrophoresis as a result, 2.9kbp band occurs being positive sample.Wherein 2~7 be MGC1 genetic transformation bacterium colony sample P CR amplification after product, 1 For non-positive sample；M is molecular weight marker；

Fig. 3 is the transfer vector pF-MG-MGC1 map containing target gene of building；

Fig. 4 is that PCR product after the amplification of MGC2 gene PCR is carried out gel electrophoresis result, it can be seen that 0.6kbp's Band；Wherein 1 is negative control；2 be MG-MGC2 gene；M is molecular weight marker；

Fig. 5 be multiple MGC2 genetic transformation bacterium colony sample PCR amplification after PCR product carry out gel electrophoresis as a result, 0.6kbp band occurs being positive sample.Wherein 2~6 be MGC2 genetic transformation bacterium colony sample P CR amplification after product, 1 For non-positive sample；M is molecular weight marker；

Fig. 6 is the transfer vector pF-MG-MGC2 map containing target gene of building；

Fig. 7 is that PCR product after the amplification of MGC3 gene PCR is carried out gel electrophoresis result, it can be seen that 2.7kbp's Band；Wherein 1 is negative control, and 2 be MG-MGC3 gene；M is molecular weight marker；

Fig. 8 be multiple MGC3 genetic transformation bacterium colony sample PCR amplification after PCR product carry out gel electrophoresis as a result, 2.7kbp band occurs being positive sample.Wherein 2~7 be MGC2 genetic transformation bacterium colony sample P CR amplification after product, 1 For non-positive sample；M is molecular weight marker；

Fig. 9 is the transfer vector pF-MG-MGC2 map containing target gene of building；

Figure 10 is that PCR product after the amplification of VLH3 gene PCR is carried out gel electrophoresis result, it can be seen that 1.8kbp's Band；Wherein 1 is negative control, and 2 be MG-VLH3 gene；M is molecular weight marker；

Figure 11 be multiple VLH3 genetic transformation bacterium colony sample PCR amplification after PCR product carry out gel electrophoresis as a result, 1.8kbp band occurs being positive sample.Wherein 2~7 be VLH3 genetic transformation bacterium colony sample P CR amplification after product, 1 For non-positive sample；M is molecular weight marker；

Figure 12 is the transfer vector pF-MG-VLH3 map containing target gene of building；

Figure 13 is that PCR product after the amplification of VLH5 gene PCR is carried out gel electrophoresis result, it can be seen that 1.7kbp's Band；Wherein 1 is negative control, and 2 be MG-VLH5 gene；M is molecular weight marker；

Figure 14 be multiple VLH5 genetic transformation bacterium colony sample PCR amplification after PCR product carry out gel electrophoresis as a result, 1.7kbp band occurs being positive sample.Wherein 2~7 be VLH5 genetic transformation bacterium colony sample P CR amplification after product, 1 For non-positive sample；M is molecular weight marker；

Figure 15 is the transfer vector pF-MG-VLH5 map containing target gene of building；

Figure 16 is that the rBac-MGC1 cell culture supernatant harvested in embodiment 7 carries out the knot of PAGE gel electrophoresis There is purpose band near molecular weight about 106kDa in fruit, wherein 2 be rBac-MGC1 cell culture supernatant；1 is negative right According to；M is molecular weight marker；

Figure 17 is that the rBac-MGC2 cell culture supernatant harvested in embodiment 7 carries out the knot of PAGE gel electrophoresis There is purpose band near molecular weight about 23kDa in fruit, wherein 2 be rBac-MGC2 cell culture supernatant；1 is negative right According to；M is molecular weight marker；

Figure 18 is that the rBac-MGC3 cell culture supernatant harvested in embodiment 7 carries out the knot of PAGE gel electrophoresis There is purpose band near molecular weight about 100kDa in fruit, wherein 2 be rBac-MGC2 cell culture supernatant；1 is negative right According to；M is molecular weight marker；

Figure 19 is that the rBac-VLH3 cell culture supernatant harvested in embodiment 7 carries out the knot of PAGE gel electrophoresis There is purpose band near molecular weight about 67kDa in fruit, wherein 2 be rBac-MGC2 cell culture supernatant；1 is negative right According to；M is molecular weight marker；

Figure 20 is that the rBac-VLH5 cell culture supernatant harvested in embodiment 7 carries out the knot of PAGE gel electrophoresis There is purpose band near molecular weight about 63kDa in fruit, wherein 2 be rBac-VLH5 cell culture supernatant；1 is negative right According to；M is molecular weight marker；

Figure 21 is that the MG-MGC1 recombinant baculovirus cell culture supernatant Western Blot in embodiment 8 detects knot Fruit；Wherein 2 be MG-MGC1 recombinant baculovirus cell culture supernatant；1 is negative control；M is molecular weight marker；

Figure 22 is that the MG-MGC2 recombinant baculovirus cell culture supernatant Western Blot in embodiment 8 detects knot Fruit；Wherein 2 be MG-MGC2 recombinant baculovirus cell culture supernatant；1 is negative control；M is molecular weight marker；

Figure 23 is that the MG-MGC3 recombinant baculovirus cell culture supernatant Western Blot in embodiment 8 detects knot Fruit；Wherein 2 be MG-MGC3 recombinant baculovirus cell culture supernatant；1 is negative control；M is molecular weight marker；

Figure 24 is that the MG-VLH3 recombinant baculovirus cell culture supernatant Western Blot in embodiment 8 detects knot Fruit；Wherein 2 be MG-VLH3 recombinant baculovirus cell culture supernatant；1 is negative control；M is molecular weight marker；

Figure 25 is that the MG-VLH5 recombinant baculovirus cell culture supernatant Western Blot in embodiment 8 detects knot Fruit；Wherein 2 be MG-VLH5 recombinant baculovirus cell culture supernatant；1 is negative control；M is molecular weight marker；

Figure 26 is empty baculoviral Sf9 cell and recombinant baculovirus Sf9 cell fluorescence figure, wherein the empty rod-shaped disease of inoculation Malicious Sf9 cell is vaccinated with recombinant baculovirus Sf9 cell and is able to observe that fluorescence not it is observed that fluorescence.

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 composition, including a kind of albumen, the albumen is selected from the following a kind of or two Kind or more any combination:

It is identical using the nucleic acid molecules of SEQ ID NO:9 or with 95% or more the nucleotide sequence of SEQ ID NO:9 The chicken virus mycoplasma blood clotting GAP-associated protein GAP VLH5 of nucleic acid molecule encoding；

The present invention also relates to it is a kind of induction for Mycoplasma Gallisepticum Antigen Recognized By Antibody 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 from mycoplasma gallisepticum infection, the method includes to institute It states animal subject and applies vaccine of the invention.

The invention further relates to a kind of method protected and diagnosed the animal subject for having mycoplasma gallisepticum infection, the sides Method includes that vaccine of the invention is applied to the animal subject.

The invention also includes the vaccines for being suitable for inducing the immune response for chicken virus mycoplasma.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 may include protein molecular.The protein molecular is selected from one or two kinds of any combination below: including SEQ The albumen of ID NO:2 or 4 or 6 or 8 or 10；In the whole length of the amino acid sequence of SEQ ID NO:2 or 4 or 6 or 8 or 10 95% identical albumen；The segment of SEQ ID NO:2 or 4 or 6 or 8 or 10；With the piece of SEQ ID NO:2 or 4 or 6 or 8 or 10 95% identical albumen of section.

A kind of albumen selected from the group being made up of: (a) SEQ ID NO:2 or 4 or 6 or 8 or 10 is also provided herein； (b) 95% identical on the entire length amino acid sequence of the full length sequence as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 Albumen；(c) 20 or more comprising SEQ ID NO:2 or 4 or 6 or 8 or 10 of SEQ ID NO:2 or 4 or 6 or 8 or 10 The immunogenic fragments of amino acid；And (d) in the whole length of the amino acid sequence of SEQ ID NO:2 or 4 or 6 or 8 or 10 The immunogenic fragments comprising 20 or more amino acid of upper 95% identical albumen.

Vaccine of the invention can be the nucleic acid molecules comprising the sequence for encoding one or more protein moleculars described above. In some embodiments, the nucleic acid molecules include selected from the sequence of group being made up of: SEQ ID NO:1 or 3 or 5 or 7 or 9；The 95% identical nucleic acid sequence in the whole length of the nucleotide sequence of SEQ ID NO:1 or 3 or 5 or 7 or 9；SEQ The segment of ID NO:1 or 3 or 5 or 7 or 9；Nucleotides sequence identical with the segment 95% of SEQ ID NO:1 or 3 or 5 or 7 or 9 Column.

Some aspects of the present invention provide the method for immune response of the induction for chicken virus mycoplasma, and the method includes following Step: to individual application Mycoplasma Gallisepticum Antigen Recognized By Antibody and/or combination thereof object.

The other aspect of the present invention provides the method for protecting individuals from mycoplasma gallisepticum 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 Mycoplasma Gallisepticum Antigen Recognized By Antibody, 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 from mycoplasma gallisepticum infection, the method Including applying nucleic acid molecules of the invention to the animal subject.

Some aspects of the invention provide a kind of method protected and diagnosed the animal subject for having mycoplasma gallisepticum infection, institute The method of stating includes that nucleic acid molecules of the invention are applied to the animal subject.

On the other hand, the present invention provides a kind of albumen selected from the group being made up of: (a) SEQ ID NO:2 or 4 or 6 or 8；(b) the 98% identical albumen in the whole length of the amino acid sequence of SEQ ID NO:2 or 4 or 6 or 8；(c)SEQ The immunogenic fragments comprising 20 or more amino acid of ID NO:2 or 4 or 6 or 8；And (d) in SEQ ID NO:2 or The immunogene comprising 20 or more amino acid of 98% identical albumen in the whole length of 4 or 6 or 8 amino acid sequence Property segment.

Some aspects of the invention provide a kind of suitable for being generated animal subject for the immune anti-of chicken virus mycoplasma 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 can further include one or more nucleic acid molecules as described above and one or more by the core The albumen of acid molecule coding.

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 based on the queue for analyzing specific Mycoplasma Gallisepticum Antigen Recognized By Antibody as used herein Multiple hypotypes polypeptide sequence.The nucleic acid sequence for encoding shared polypeptide sequence can be prepared.Wrap protein-contg vaccine can by with It is immunized to induce for a variety of hypotypes of specific Mycoplasma Gallisepticum Antigen Recognized By Antibody or the extensive of serotype, the vaccine includes to encode these The consensus sequence and/or nucleic acid molecules of albumen.

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 chicken The nucleic acid sequence or part of it of the polypeptide triggered an immune response in the animal of virus mycoplasma antigenic cross-reaction.The segment can To 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 malicious with overall length wild-type strain chicken The polypeptide triggered an immune response in the animal of mycoplasma antigen 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.

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 chicken virus mycoplasma as used herein, host's The activation of immune system (such as immune system of animal).The immune response can be cell effect or humoral response or both Form.

" 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, CMV IE 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 chicken as described herein The amino acid sequence of the amino terminal of virus mycoplasma albumen.Signal peptide/leader sequence is indicated generally at the position of albumen.It is used herein Signal peptide/leader sequence preferably facilitate albumen and secrete from generating in its cell.Signal peptide/leader sequence is usually from albumen Remainder cracking, the albumen from cell after secreting through being commonly referred to as maturation protein.Signal peptide/leader sequence is connected to The N-terminal of the albumen.

" 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 chicken virus mycoplasma, it is intended that chicken virus mycoplasma Genetic mutation a, so that 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

Vaccine of the invention can be designed in control animal subject for one or more chicken virus mycoplasma serotypes The degree or intensity of immune response.The antigen may include the protein epitope for making them particularly effectively be used as immunogene, can Anti- chicken virus mycoplasma immune response is induced for the immunogene.Mycoplasma Gallisepticum Antigen Recognized By Antibody 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 3 or 5 or 7 or 9.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 3 or 5 or 7 or 9.Segment can be with SEQ ID The segment of NO:1 or 3 or 5 or 7 or 9 at least 95%, at least 96%, at least 97%, at least 98% or at least 99% are identical.Segment Can with the segment at least 80% of SEQ ID NO:1 or 3 or 5 or 7 or 9, 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 realities It applies in scheme, segment includes the sequence of encoding leader sequence, such as immunoglobulin leader object, such as IgE lead.In some realities It applies in scheme, segment is free of the coded sequence of encoding leader sequence.In some embodiments, segment is free of encoding leader sequence Column, the such as coded sequence of IgE lead.

Some embodiments are related to the albumen homologous with SEQ ID NO:2 or 4 or 6 or 8 or 10.Some embodiments are related to There is the immunogenic protein of 95% homology with the protein sequence as described in SEQ ID NO:2 or 4 or 6 or 8 or 10.It is some Embodiment is related to having the immune of 96% homology with the protein sequence as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 Immunogenic peptide.Some embodiments are related to having 97% with the protein sequence as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 The immunogenic protein of homology.Some embodiments are related to and the albumen as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 Sequence has the immunogenic protein of 98% homology.Some embodiments are related to and the albumen sequence as described in SEQID NO:2 Arrange the immunogenic protein with 99% homology.

Some embodiments are related to albumen identical with SEQ ID NO:2 or 4 or 6 or 8 or 10.Some embodiments are related to It is long with the entire amino acid sequence in the overall length consensus amino acid sequences as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 Spend the immunogenic protein of upper 80% identical amino acid sequence.Some embodiments are related to having in such as SEQ ID NO:2 or 4 Or 85% identical amino acid sequence on the entire length amino acid sequence of overall length consensus amino acid sequences described in 6 or 8 or 10 The immunogenic protein of column.Some embodiments are related to having in the overall length as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 The immunogenic protein of 90% identical amino acid sequence on the entire length amino acid sequence of consensus amino acid sequences.Some realities The scheme of applying is related to the entire ammonia in the overall length consensus amino acid sequences as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 The immunogenic protein of 91% identical amino acid sequence in base acid sequence length.Some embodiments are related to having in such as SEQ 92% is identical on the entire length amino acid sequence of overall length consensus amino acid sequences described in ID NO:2 or 4 or 6 or 8 or 10 Amino acid sequence immunogenic protein.Some embodiments are related to having in such as SEQ ID NO:2 or 4 or 6 or 8 or 10 The immunogenicity of 93% identical amino acid sequence on the entire length amino acid sequence of the overall length consensus amino acid sequences Albumen.Some embodiments, which are related to having, shares amino acid sequence in the overall length as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 The immunogenic protein of 94% identical amino acid sequence on the entire length amino acid sequence of column.Some embodiments are related to having There is the entire length amino acid sequence in the overall length consensus amino acid sequences as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 The immunogenic protein of upper 95% identical amino acid sequence.Some embodiments be related to having in such as SEQ ID NO:2 or 4 or 96% identical amino acid sequence on the entire length amino acid sequence of overall length consensus amino acid sequences described in 6 or 8 or 10 Immunogenic protein.Some embodiments are related to having total in the overall length as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 There is the immunogenic protein of 97% identical amino acid sequence on the entire length amino acid sequence of amino acid sequence.Some implementations Scheme is related to the entire amino in the overall length consensus amino acid sequences as described in SEQ ID NO:2 or 4 or 6 or 8 or 10 The immunogenic protein of 98% identical amino acid sequence in acid sequence length.Some embodiments are related to having in such as SEQ ID 99% identical ammonia on the entire length amino acid sequence of overall length consensus amino acid sequences described in NO:2 or 4 or 6 or 8 or 10 The immunogenic protein of base acid sequence.

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 or 4 or 6 or 8 or 10 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:2 or 4 or 6 or 8 or 10.In some embodiments In, segment includes leader sequence, such as immunoglobulin leader object, such as IgE lead.In some embodiments, segment Without leader sequence.In some embodiments, segment is free of leader sequence, such as IgE lead.

It can provide the amino acid sequence albumen homologous with the immunogenic fragments of SEQ ID NO:2 or 4 or 6 or 8 or 10 Immunogenic fragments.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% the albumen homologous with SEQ ID NO:2 or 4 or 6 or 8 or 1095%.Some embodiments are related to and this paper egg The immunogenic fragments of Bai Xulie have the immunogenic fragments of 96% homology.Some embodiments are related to and this paper albumen sequence The immunogenic fragments of column have the immunogenic fragments of 97% homology.Some embodiments are related to and this paper protein sequence Immunogenic fragments have the immunogenic fragments of 98% homology.Some embodiments are related to immune with this paper protein sequence Immunogenic fragment has the immunogenic fragments of 99% homology.In some embodiments, segment includes leader sequence, such as Immunoglobulin leader sequence, such as IgE lead.In some embodiments, segment is free of leader sequence.In some embodiment party In case, segment is free of leader sequence, such as IgE lead.

It can provide amino acid sequence albumen identical with the immunogenic fragments of SEQ ID NO:2 or 4 or 6 or 8 or 10 Immunogenic fragments.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% the amino acid sequence described in SEQ ID NO:2 or 4 or 6 or 8 or 10 whole length on 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical albumen.In some embodiment party In case, segment includes leader sequence, such as immunoglobulin leader object, such as IgE lead.In some embodiments, piece Duan Buhan leader sequence.In some embodiments, segment is free of leader sequence, such as IgE lead.

Albumen or nucleic acid molecules can be present in composition with any amount.In immune composition different protein moleculars with Arbitrary proportion is present in composition；Different nucleic acid molecules are present in composition in immune composition with arbitrary proportion.When into Row is in use, apply the nucleic acid molecules or composition comprising such nucleic acid sequence of prevention effective dose to the individual；Wherein institute It states nucleic acid sequence to express in the cell of the individual, and is directed to and is exempted from by the protein induced protectiveness of the nucleic acid sequence encoding Epidemic disease reaction.Term " effective quantity " is the amount for effectively improving the symptom of Animal diseases.

3. vaccine constructs and plasmid

Vaccine may include coding chicken virus mycoplasma blood clotting GAP-associated protein GAP, Mycoplasma Gallisepticum Antigen Recognized By Antibody and chicken virus mycoplasma blood clotting GAP-associated protein GAP/antigen combined nucleic acid construct or plasmid.Provided herein is may include coding chicken poison branch disclosed herein The genetic constructs of the nucleic acid sequence of mycoplasma antigen, the core antigen include protein sequence, with the homologous sequence of protein sequence, The segment of protein sequence and the sequence homologous with the segment of protein sequence.In addition, provided herein is may include that coding is disclosed herein Chicken virus mycoplasma surface antigen (including protein sequence, with the homologous sequence of protein sequence, the segment of protein sequence and and egg The homologous sequence of the segment of Bai Xulie) nucleic acid sequence genetic constructs.The genetic constructs can be used as functional dye Molecule outside colour solid and exist.The genetic constructs can be linear miniature including centromere, telomere or plasmid or clay Chromosome.

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 former in chicken poison branch The downstream of body 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 chicken virus mycoplasma core protein coded sequence or shared chicken virus mycoplasma 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 text.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 664- At 683.Multiple cloning sites are located at base 696-811.Ox GH polyadenylation signal is at base 829-1053.Block that Mycin (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 pcDNA I or pcDNA3 (Invitrogen, SanDiego, Calif.), can be used for generating albumen in zooblast such as Chinese hamster ovary (CHO) cell.The carrier can be logical It crosses routine techniques and is easy available initial substance 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)。

Encode hemagluttinin proteins VLH3.01, the gene order of VLH5.02 and adhesin antibodies (MGC2 and MGC3, MGC1) It can be original series, increase and truncated sequence.The composition of vaccine can be any one albumen or any group Close object, preferably five albumen equal proportion mixing.Baculovirus expression system transfer vector in recombination bacillary viral vector Including but not limited to pFastBac1, Pvl1393 etc., such as can preferably use pFastBac1.Sf9 cell line can be for Sf9, High Five, S2 or Sf21 cell, preferably use Sf9.

1 transfer vector pF-MGC1 of embodiment building and identification

1.MGC1 gene magnification has synthesized the MGC1 gene (SEQ after codon optimization in Nanjing Jin Sirui company with purifying ID NO:1) and be cloned on pUC17 carrier, obtain pUC-MGC1 plasmid vector.Using pUC-MGC1 plasmid as template, MGC1- F, MGC1-R carries out PCR amplification (gene order of MGC1-F, MGC1-R such as SEQ ID NO.11,12 institutes as upstream and downstream primer Show), amplification system is shown in Table 1.

1 MGC1 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 2.9kbp Band, target gene expands successfully, carries out recovery purifying with gel purification kit.

2. pFastBac1 plasmid and MGC1 gene expression frame pcr amplification product are used BamH I, Hind by digestion and purifying III 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 pFastBac1 of gel purification kits digestion respectively Plasmid and MGC1 genetic fragment.

2 MGC1 gene endonuclease reaction system of table

3 pFastbac of table, 1 plasmid enzyme restriction reaction system

3. the pFastBac1 plasmid of double digestion and MGC1 gene digestion products are used 16 DEG C of water of T4DNA ligase by connection Bath connection is overnight.Specific coupled reaction system is shown in Table 4.

4 MGC1 gene of table and pFastBac1 plasmid linked system

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 MGC1-F and MGC1-R The size of verifying purpose gene, as shown in Fig. 2, the sample for 2.9kbp 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 transfer vector pF-MGC1 containing target gene of building Its schematic diagram such as Fig. 3.

2 transfer vector pF-MGC2 of embodiment building and identification

1.MGC2 gene magnification has synthesized the MGC2 gene (SEQ after codon optimization in Nanjing Jin Sirui company with purifying ID NO:3) and be cloned on pUC17 carrier, obtain pUC-MGC2 plasmid vector.Using pUC-MGC2 plasmid as template, MGC2- F, MGC2-R carries out PCR amplification (gene order of MGC2-F, MGC2-R such as SEQ ID NO.13,14 institutes as upstream and downstream primer Show), amplification system is shown in Table 5.

5 MGC2 gene magnification system of table

PCR product is subjected to gel electrophoresis verifying purpose gene size, as shown in figure 4, mesh occurs in the position in 0.6kbp Band, target gene expands successfully, carries out recovery purifying with gel purification kit.

2. pFastBac1 plasmid and MGC2 gene expression frame pcr amplification product are used BamH I, Hind by digestion and purifying III 37 DEG C double digestion 3 hours, specific endonuclease reaction system is shown in Table 6, table 7.

Digestion products are subjected to gel electrophoresis, use the pFastBac1 of gel purification kits digestion respectively Plasmid and MGC2 genetic fragment.

6 MGC2 gene endonuclease reaction system of table

7 pFastbac of table, 1 plasmid enzyme restriction reaction system

3. the pFastBac1 plasmid of double digestion and MGC2 gene digestion products are used 16 DEG C of water of T4DNA ligase by connection Bath connection is overnight.Specific coupled reaction system is shown in Table 8.

8 MGC2 gene of table and pFastBac1 plasmid linked system

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 MGC2-F and MGC2-R The size of verifying purpose gene, as shown in figure 5, the sample for 0.6kbp 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 transfer vector pF-MGC2 containing target gene of building Its schematic diagram such as Fig. 6.

3 transfer vector pF-MGC3 of embodiment building and identification

1.MGC3 gene magnification has synthesized the MGC3 gene (SEQ after codon optimization in Nanjing Jin Sirui company with purifying ID NO:5) and be cloned on pUC17 carrier, obtain pUC-MGC3 plasmid vector.Using pUC-MGC3 plasmid as template, MGC3- F, MGC3-R carries out PCR amplification (gene order of MGC3-F, MGC3-R such as SEQ ID NO.15,16 institutes as upstream and downstream primer Show), amplification system is shown in Table 9.

9 MGC3 gene magnification system of table

PCR product is subjected to gel electrophoresis verifying purpose gene size, as shown in fig. 7, mesh occurs in the position in 2.7kbp Band, target gene expands successfully, carries out recovery purifying with gel purification kit.

2. pFastBac1 plasmid and MGC3 gene expression frame pcr amplification product are used BamH I, Hind by digestion and purifying III 37 DEG C double digestion 3 hours, specific endonuclease reaction system is shown in Table 10, table 11.

Digestion products are subjected to gel electrophoresis, use the pFastBac1 of gel purification kits digestion respectively Plasmid and MGC3 genetic fragment.

10 MGC3 gene endonuclease reaction system of table

11 pFastbac of table, 1 plasmid enzyme restriction reaction system

3. the pFastBac1 plasmid of double digestion and MGC3 gene digestion products are used 16 DEG C of water of T4DNA ligase by connection Bath connection is overnight.Specific coupled reaction system is shown in Table 12.

12 MGC3 gene of table and pFastBac1 plasmid linked system

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 MGC3-F and MGC3-R The size of verifying purpose gene, as shown in figure 8, the sample for 2.7kbp 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 transfer vector pF-MGC3 containing target gene of building Its schematic diagram such as Fig. 9.

4 transfer vector pF-VLH3 of embodiment building and identification

1.VLH3 gene magnification has synthesized the VLH3 gene (SEQ after codon optimization in Nanjing Jin Sirui company with purifying ID NO:7) and be cloned on pUC17 carrier, obtain pUC-VLH3 plasmid vector.Using pUC-VLH3 plasmid as template, VLH3- F, VLH3-R carries out PCR amplification (gene order of VLH3-F, VLH3-R such as SEQ ID NO.17,18 institutes as upstream and downstream primer Show), amplification system is shown in Table 13.

13 VLH3 gene magnification system of table

There is into mesh in the position of 1.8kbp as shown in Figure 10 in PCR product progress gel electrophoresis verifying purpose gene size Band, target gene expands successfully, carries out recovery purifying with gel purification kit.

2. pFastBac1 plasmid and VLH3 gene expression frame pcr amplification product are used BamH I, Hind by digestion and purifying III 37 DEG C double digestion 3 hours, specific endonuclease reaction system is shown in Table 14, table 15.

Digestion products are subjected to gel electrophoresis, use the pFastBac1 of gel purification kits digestion respectively Plasmid and VLH3 genetic fragment.

14 VLH3 gene endonuclease reaction system of table

15 pFastbac of table, 1 plasmid enzyme restriction reaction system

3. the pFastBac1 plasmid of double digestion and VLH3 gene digestion products are used 16 DEG C of water of T4DNA ligase by connection Bath connection is overnight.Specific coupled reaction system is shown in Table 16.

16 VLH3 gene of table and pFastBac1 plasmid linked system

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 VLH3-F and VLH3-R The size of verifying purpose gene, as shown in figure 11, the sample for 1.8kbp 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 transfer vector pF-VLH3 containing target gene of building Its schematic diagram such as Figure 12.

5 transfer vector pF-VLH5 of embodiment building and identification

1.VLH5 gene magnification has synthesized the VLH5 gene (SEQ after codon optimization in Nanjing Jin Sirui company with purifying ID NO:9) and be cloned on pUC17 carrier, obtain pUC-VLH5 plasmid vector.Using pUC-VLH5 plasmid as template, VLH5- F, VLH5-R carries out PCR amplification (gene order of VLH5-F, VLH5-R such as SEQ ID NO.19,20 institutes as upstream and downstream primer Show), amplification system is shown in Table 17.

17 VLH5 gene magnification system of table

There is into mesh in the position of 1.7kbp as shown in figure 13 in PCR product progress gel electrophoresis verifying purpose gene size Band, target gene expands successfully, carries out recovery purifying with gel purification kit.

2. pFastBac1 plasmid and VLH5 gene expression frame pcr amplification product are used BamH I, Hind by digestion and purifying III 37 DEG C double digestion 3 hours, specific endonuclease reaction system is shown in Table 18, table 19.

Digestion products are subjected to gel electrophoresis, use the pFastBac1 of gel purification kits digestion respectively Plasmid and VLH5 genetic fragment.

18 VLH5 gene endonuclease reaction system of table

19 pFastbac of table, 1 plasmid enzyme restriction reaction system

3. the pFastBac1 plasmid of double digestion and VLH5 gene digestion products are used 16 DEG C of water of T4DNA ligase by connection Bath connection is overnight.Specific coupled reaction system is shown in Table 20.

20 VLH5 gene of table and pFastBac1 plasmid linked system

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 VLH5-F and VLH5-R The size of verifying purpose gene, as shown in figure 14, the sample for 1.7kbp 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 transfer vector pF-VLH5 containing target gene of building Its schematic diagram such as Figure 15.

Embodiment 6 recombinant baculovirus genome Bac-MGC1, Bac-MGC2, Bac-MGC3, Bac-VLH3 and Bac- VLH5 building

Each 1 μ l pF-MGC1 plasmid, pF-MGC2 plasmid, pF-MGC3 in 1.DH10Bac bacterium conversion difference Example 1-5 Plasmid, pF-VLH3 plasmid and pF-VLH5 plasmid are added in the DH10Bac competent cell of 100 μ l and mix, and ice bath 30 minutes, 42 DEG C water-bath heat shock 90 seconds, then ice bath 2 minutes, each LB liquid medium that 900 μ l are added and are free of Amp, 37 DEG C were cultivated 5 hours. After taking 100 μ l bacterium solutions to dilute 81 times respectively, the 100 diluted bacterium solutions of μ l is taken to be applied to containing gentamicin, kanamycins, Fourth Ring On the LB solid medium of element, X-gal and IPTG, 37 DEG C are cultivated 48 hours.

2. select the white colony that monoclonal uses transfer needle picking big respectively, then containing gentamicin, to block that mould Element, tetracycline, X-gal and IPTG LB solid medium on cross, 37 DEG C are cultivated 48 hours, and then picking single colonie is inoculated with LB liquid medium culture containing gentamicin, kanamycins, tetracycline saves strain, extracts plasmid.It is recombinated respectively Plasmid Bacmid-MGC1, Bacmid-MGC2, Bacmid-MGC3, Bacmid-VLH3 and Bacmid-VLH5.

The transfection of 7 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；Respectively with 100 μ l transfect culture T1 base dilute 3 μ g embodiments 6 in recombination Bacmid-MGC1, Bacmid-MGC2, Bacmid-MGC3, Bacmid-VLH3 and Bacmid-VLH5 plasmid respectively mix diluted transfection reagent and plasmid, gently It blows even, prepares transfection mixture.Above-mentioned transfection composite is added after cell is adherent, 27 DEG C are incubated for 5 hours, supernatant is removed, Add 2ml SF-SFM fresh culture, 27 DEG C of cultures harvest supernatant on the 4th~5.Recombinant baculovirus rBac- is obtained respectively MGC1, rBac-MGC2, rBac-MGC3, rBac-VLH3 and rBac-VLH5 use MTT for recombinant baculovirus to the P1 of harvest Relative effectivenes method detects virus titer, and rBac-MGC1P1 kind poison virus titer is 5.4 × 10⁷Pfu/mL, rBac-MGC2P1 kind Malicious virus titer is 2.3 × 10⁷Pfu/mL, rBac-MGC3P1 kind poison virus titer are 3.4 × 10⁷Pfu/mL, rBac-VLH3P1 The malicious virus titer of kind is 3.7 × 10⁷Pfu/mL, rBac-VLH5P1 kind poison virus titer are 3.9 × 10⁷pfu/mL.Amplification recombination Baculoviral rBac-MGC1, rBac-MGC2, rBac-MGC3, rBac-VLH3 and rBac-VLH5 are spare as kind of poison.

Embodiment 8SDS-PAGE detection

The cell culture harvested in embodiment 7 is subjected to SDS-PAGE detection respectively, while respectively using the empty bar of infection The Sf9 cell of shape virus is as negative control.Concrete operations are as follows: take 40 μ l harvest cell culture, be added 10 μ l 5 × Loading buffer, boiling water bath 5 minutes, 12000r/min was centrifuged 1 minute, and taking supernatant to carry out PAGE gel, (12% is dense Spend gel) electrophoresis, take gel to be dyed after electrophoresis, decolourize after observe purpose band.

As shown in figure 16, there is purpose band near molecular weight about 106kDa in rBac-MGC1 cell culture, negative right Impinging upon corresponding position does not have band.

As shown in figure 17, there is purpose band near molecular weight about 23kDa in rBac-MGC2 cell culture, negative right Impinging upon corresponding position does not have band.

As shown in figure 18, there is purpose band near molecular weight about 100kDa in rBac-MGC3 cell culture, negative right Impinging upon corresponding position does not have band.

As shown in figure 19, there is purpose band near molecular weight about 67kDa in rBac-VLH3 cell culture, negative right Impinging upon corresponding position does not have band.

As shown in figure 20, there is purpose band near molecular weight about 63kDa in rBac-VLH5 cell culture, negative right Impinging upon corresponding position does not have band.

Embodiment 9Western Blot identification

The product after SDS-PAGE electrophoresis in embodiment 8 is transferred on NC (nitrocellulose) film respectively, with 5% degreasing Milk is closed 2 hours, and the anti-MG positive serum of Ji Yuan is incubated for 2 hours, rinsing, and the goat-anti chicken 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.Such as Figure 21 to figure Shown in 25, recombinant baculovirus expression sample has purpose band, and negative control does not have purpose band, and illustration purpose antigen protein exists It is correctly expressed in Sf9 cell.

10 indirect immunofluorescene assay of embodiment

It is added respectively into 96 porocyte culture plates and transfected rBac-MGC1, rBac-MGC2, rBac-MGC3, rBac- The Sf9 cell suspension of VLH3 and rBac-VLH5, (cell concentration is 2.5 × 10 in 100 holes μ l/⁵~4.0 × 10⁵A/ml), inoculation 4 Hole, 27 DEG C stand 15 minutes, and Sf9 cell is made to be affixed on culture plate bottom wall, and then the seed culture of viruses that 10 μ l dilute 10 times is added in every hole.Simultaneously If blanc cell compares.Cell is set in 27 DEG C of constant incubators and is cultivated 72~96 hours after inoculation, abandons culture solution, cold methanol/the third Ketone (1:1) is fixed.It is reacted first with the anti-MG polyvalent antibody of Ji Yuan, is then reacted with the sheep anti-chicken IgG of FITC label, be inverted fluorescence Microscopy results.As shown in figure 26, inoculation sky baculoviral Sf9 cell is not it is observed that fluorescence, and is vaccinated with recombination bar Shape virus Sf9 cell is able to observe that fluorescence, illustration purpose antigen egg are correctly expressed in Sf9 cell, recombinates rod-shaped disease Poison building is correct.

11 antigen protein Blood coagulation test of embodiment

Two albumen of VLH3 and VLH5 have hemagglutinative titer, detect two proteineminas of VLH3 and VLH5 using chicken red blood cell Solidifying potency.Harvest expression VLH3 and VLH5 albuminous cell suspension sample, by sample -80 DEG C of multigelations three times after centrifuging and taking Supernatant is obtained for detecting.On micro plate, from the 1st hole to 12 holes, PBS 0.025mL is added with the every hole of pipettor, uses pipettor Test sample 0.025mL is drawn, from the first hole, successively makees 2 times and is serially diluted, until last 1 hole, discards in pipettor 0.025mL liquid (extension rate is followed successively by 2,4,8,16,32 ...).1% chicken erythrocyte suspension 0.025mL is added in every hole, and If the red blood cell control wells of sample are not added, shaken up on micro plate rocker immediately, set 20~40 DEG C of minutes of room temperature or sets 2~8 DEG C 40~60 minutes, result was determined when the red blood cell in control wells manifests significant button shape.It is aggregated red blood cell completely most High dilution is as judgement emphasis.The hemagglutinative titer of detection display two albumen of VLH3 and VLH5 is respectively 1:32 and 1:32.

The bioreactor serum free suspension culture of 12 insect cell of embodiment and MGC1, MGC2, MGC3, VLH3 and VLH5 The expression of albumen is quantitative 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 3-55 × 10⁶When cell/mL, seed cells into 50L bioreactor, to cell it is long to concentration be 3-55 × 10⁶Cell/mL, is inoculated into 500L bioreactor, reaches 2-85 × 10 to cell concentration⁶When cell/mL, inoculation recombination bar Shape virus rBac-MGC1, 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.Prepare expression MGC2, MGC3, VLH3 and VLH5 egg in the same way simultaneously White vaccinogen liquid.

The protein content of MGC1, MGC2, MGC3, VLH3 and VLH5 use respectively in the above-mentioned vaccine antigen of preparation The detection of Elisa method.Mode of operation is as follows: the use coating buffer dilution anti-chicken virus mycoplasma polyvalent antibody of chicken to suitable concentration, Every 100 μ l of hole, 4 DEG C overnight, and PBST is washed three times, and 1%BSA closes 1h.The antigen standard that various concentration is added (passes through particle Displacement chromatography, hydrophobic chromatography, the albumen that molecular sieve purification obtains) and gradient dilution measuring samples, 37 DEG C are incubated for 1 hour, PBST It washes three times.Detection antibody-MGC1 protein monoclonal antibody is added in every hole, and 37 DEG C are incubated for 1 hour, and PBST is washed three times.Every hole is added The sheep anti-chicken IgG of two anti-i.e. HRP labels, 37 DEG C are incubated for 1 hour, and PBST is washed three times.TMB develops the color 10 minutes, and 2M H2SO4 is terminated Reaction.Microplate reader reading, the amount of MSPA albumen in measuring samples is calculated by standard curve.

Same method detects the content of MGC2, MGC3, VLH3 and VLH5 albumen in measuring samples respectively.

According to embodiment 12, MGC1, MGC2, MGC3, VLH3 and VLH5 albumen of large scale preparation, Elisa detection knot Fruit such as following table, as can be seen from the results in the table that, the average content of five albumen about 163mg/L, 210mg/ respectively in vaccinogen liquid L, 182mg/L, 156mg/L and 231mg/L.

In the vaccine antigen of preparation, expands potency method using fine jade respectively and detect MGC1, MGC2, MGC3, VLH3 and VLH5 egg It is white: to beat plum blossom hole on 5 pieces of agarose gel plates respectively, chicken virus mycoplasma antiserum is added among plum blossom hole, around 5 blocks of plates Be separately added into 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.It sinks The maximum dilution ratio of shallow lake line is that its fine jade expands potency fine jade expansion bioactivity result.MGC1, MGC2, MGC3, VLH3 in vaccinogen liquid And it is respectively 1:16,1:32,1:32,1:16,1:32 that the fine jade of VLH5 albumen, which expands potency,.

The preparation of 13 vaccine of embodiment

The five kinds of vaccinogen liquids expressed in Example 12 are mixed, so that this 5 each antigens of antigen protein is dense Degree reach 30mg/mL, then by the vaccinogen liquid mixed and oil adjuvant according to the proportional arrangement of 2:3 at oil emulsion vaccine.Tool Body, white oil 1429g is added in the polyvalent vaccine stoste of every 1L, takes charge of this 70.2g, aluminum stearate 8.43g and tween 53.3g.So Oil emulsion adjuvant inactivated vaccine is prepared into the broken crusher machine emulsification of emulsification afterwards.Simultaneously preparation contain only respectively MGC1, MGC2, MGC3, The vaccine of one of VLH3 and VLH5 albumen, each antigen concentration are 30mg/mL.

14 immunization experiment of embodiment

With 21 age in days SPF chickens 70, it is divided into 7 groups, every group 10, first group is negative control group, injects PBS；Second group For vaccine group, inject the vaccine that five antigens are mixed with, third is control group to the 7th group, respectively injection contain only MGC1, The vaccine of one of five albumen of MGC2, MGC3, VLH3 and VLH5.Vaccine is prepared in each neck subcutaneous injection embodiment 13 0.3ml (a plumage part).After 21 days, the 2nd inoculation is carried out with same dose and approach.21 days after 2nd inoculation, all chickens are adopted Blood, detection blood clotting inhibit (HI) antibody titer, will adopt SN1 plants of 600ml of chicken spray attacks chicken virus mycoplasma velogen strain of blood (10⁸-10⁹Ccu/ml), the spraying duration is no less than 5 minutes, and droplet is at 2 μm or so.Observation 14 days, dissect observe airsac disease Become, and score to lesion, the number of the chicken of 2 points and the above lesion occurs in statistics, calculates protection efficiency.HI potency and Protective rate see the table below, and 3 points or more lesions all occur in 10 chickens of negative control group, wherein 84 points of air bags occur disease seriously occurs The chicken of change, disease incidence 100%；All chicken airsac diseases of vaccine immunity group of five antigen preparation become smaller in 2 points, wherein not having completely The number for having the chicken of lesion is 8, and the number for the chicken of 1 point of lesion occur is 2, protective rate 100%.Remaining five contain only one The vaccine immunity group of antigen, protective rate are that 70%-80% is differed, and some has also appeared the more serious lesion that lesion degree is 4 points, Vaccine effectiveness wants poor compared with vaccine immunity group prepared by five antigens.Illustrate that vaccine air bag protective rate prepared by the present invention is 100%, Vaccine potency is qualified.

21 chicken virus mycoplasma recombinant vaccine of table attacks toxic effect force inspecting result

A:HI potency is the average value calculated.

B: giving a mark according to standard below to air bag lesion degree,

0 point --- normal air bag, clean transparent and it is thin；

1 point --- air bag slightly thickens and slight haziness, locally there is a small number of grey or yellow exudate spot；

2 points --- there are visible grey and yellow exudate in part air bag region, while thickening with air bag moderate；

3 points --- large stretch of air bag is covered with the cheesy exudate of yellow；

4 points --- entire air bag is covered with the cheesy exudate of yellow, and air bag follows the string；

C: protective rate calculation formula

The number of the chicken of protective rate=chicken of the air bag lesion degree less than 2 points number/total.

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>a kind of chicken virus mycoplasma novel gene engineering subunit vaccine

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2949

<212> DNA

<213>artificial sequence (artificial sequence)

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cgctggagga atagcaattt cacctccctg tctattgacg gtacaaaccc tggagctctg 180

gtgcttaccg gttccaagtc catctcccgc atcgacctgt acggtaacgt gatctggacc 240

ttcgaccctg gtaacaccaa cgaccttact ggtaaggtgg gcttctacga cgctaacaat 300

cgcctgactg ccttctccgg cgacgttccc ttcaacgtga gtgacctgtc ctctaaaacc 360

gtggtcgagg ccacccaaga ccaagaggac cccaatgtgt tttacctgct gctgatcccc 420

gacgccgctg tgcagcaaga acagaagacc aaagaccagg tgttcgagaa ctacttcatg 480

agtgacgctc ctgccaccgg tgacacctcc gctgaaggta gcgctacccc cgccggtggt 540

ggttccagtt cctccgctgc tggaggaggt gctgtggctc ctgctgctgc ttcttccacc 600

gctcgcctcg ttgaggaagg taacagcgct ggtatgggta ctatgactcc tactgcttcc 660

acctccgaga ccgtgatcga ttataattcc gatcaaaaca aaatccctaa gcctaagacc 720

ctgctggaca gtagcgaatc ctccgaaagc atcaatggcg gtcgcaccta cgctaacatc 780

aacacgcaaa ataaccttca aggagtgatc gtcaaggtga acgaaaacct gttcaactcc 840

gaaaacccct tcgctgtgga gaacatggcg ttcatcaagc ctaaggacat ggtggacaac 900

tacccttcca cctggaccca gggttccgct aacggtaaga tgaccaacgt gctgcagttc 960

tacaagcacg ataatcctaa cgcggtgaac aacagatttt acagggctaa atactaccct 1020

aaacgcctgg agacccaaac taccaccccc ctgatcgact cctccttttc tccttacgag 1080

caccccgaat ggtacgagga taatcagttc gtgatgccct ggatgcaata catcaccaac 1140

ctcggtggac tgtacgccaa ggacggtatg gtgtatctgt tcggtggtaa cggcacctgg 1200

gtgaacaacg agagcgccct gtccatcggt gtgtttcgca ccaagttcga aaaccgcacc 1260

gctgaggctc ctggtaacac taagaccgtg ggttaccctt acggtatcct gctgtccgct 1320

atctcctttg acgctactcg caacggcctg gccctcgctc ccgctagtct gggtcaggac 1380

gtgggttatc atttcgtgcc ccgcctggct gtgggtggtg tgtcctctcc tcgcggtgcc 1440

aacggtaaca tcttcctggg cagtgctatc acctggggta ctaacggtgg taattttctg 1500

gacaccaagt ggcattctcc tgctgtgatt gaggatgctc cgaccacctt cgtgactgtg 1560

aactcctccg gtgctttgca gaatagcggt aaccctcagc ctactagcac ccctatgcct 1620

aactccaacg gcaacgaatc tattccctat cgctggacca actcctatga ttacaattcc 1680

gtgcgcttcg ctgctctcat ctcaaagccc gccggtggca acaccaagca agtggagagc 1740

ctgttcacca ccgccctcaa gctggacacc ctgaactccc tgcccaacaa gttcacccag 1800

gagaacaaca tcttcttctc ctacgccatg ctggacggcc gccaatggtc cctgggcacc 1860

cgtaaagact ccgcttggct caccaccaac accatcaaca acttcaccta caacacccaa 1920

cagcaactgg cctccaccgt ggctggcgag aacgctaacc cccgcaacat cctgaacgct 1980

ctgaccaccg ctaagggttt cgaccgccgc gacatcggca acgtggtgta cacctactcc 2040

aacaacacca acaaattcac ctactattac caggtgggtg gagcaatcac cacatggcct 2100

gaagtgcagg tcaattacaa gacctccgct aacatcacat actataacct gacccgcacc 2160

gacttcggaa gcaccacccc tgccacccaa gatgctaata ctgtctcctc caagctcaac 2220

ggtgcctatc tgtcctccac cggtgaccag caaggctggt acaacggttc catctacgtg 2280

aagaaggcct ccttcacccc ttcctcccag ggttacacct ggcaggactt caaaggtctg 2340

accaccaccg cttccaacgc tgtgatctcc aactggacca aagcaggtta ctccattcgc 2400

cccgacgacg acaccgtctt caacgtctct aagatccctt tcgagaagga gattaccgcc 2460

gccgtgaatg tccgctccct ggactcctac tacgtgcagt tgaatggcga gacctccgta 2520

aacaccgtgg cccgcgtgag ccccgactcc tctgctctcg ctctgaaccc caaccgtatc 2580

accaaccctc tcatgaaccg cgacaacgtg attggccagg gcgctttcat ctccaggaac 2640

gacattccct cctccttctt tgagaacaag attaacgata tcgtgaccac cgaggctgac 2700

ggcaaagaag tgctggactc caagtatatc aactccattt accgctacac tcccccccaa 2760

aacaaccctg acatccgcct gaggctgctg gtgattgaca gatccagagc taccaacgac 2820

ttcatcaagc tcctgcctca ggtgctcgtg gacggtgagt atgtggctgt ccctcaagca 2880

aacagcgtgt tcgtgtccga ccaagagttt accggattcg acgcactgcc cggttattaa 2940

tgaaagctt 2949

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<212> PRT

<213>chicken virus mycoplasma (Mycoplasma gallisepticum)

<400> 2

Met His His His His His His Val Ser Gly Ala Lys Pro Asn Asn Leu

1 5 10 15

Lys Pro Val Asn Gln Val Gly Glu Met Asn Ser Gln Gly Gln Ser Asn

20 25 30

Leu Leu Glu Lys Ala Arg Arg Trp Arg Asn Ser Asn Phe Thr Ser Leu

35 40 45

Ser Ile Asp Gly Thr Asn Pro Gly Ala Leu Val Leu Thr Gly Ser Lys

50 55 60

Ser Ile Ser Arg Ile Asp Leu Tyr Gly Asn Val Ile Trp Thr Phe Asp

65 70 75 80

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

85 90 95

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

100 105 110

Asp Leu Ser Ser Lys Thr Val Val Glu Ala Thr Gln Asp Gln Glu Asp

115 120 125

Pro Asn Val Phe Tyr Leu Leu Leu Ile Pro Asp Ala Ala Val Gln Gln

130 135 140

Glu Gln Lys Thr Lys Asp Gln Val Phe Glu Asn Tyr Phe Met Ser Asp

145 150 155 160

Ala Pro Ala Thr Gly Asp Thr Ser Ala Glu Gly Ser Ala Thr Pro Ala

165 170 175

Gly Gly Gly Ser Ser Ser Ser Ala Ala Gly Gly Gly Ala Val Ala Pro

180 185 190

Ala Ala Ala Ser Ser Thr Ala Arg Leu Val Glu Glu Gly Asn Ser Ala

195 200 205

Gly Met Gly Thr Met Thr Pro Thr Ala Ser Thr Ser Glu Thr Val Ile

210 215 220

Asp Tyr Asn Ser Asp Gln Asn Lys Ile Pro Lys Pro Lys Thr Leu Leu

225 230 235 240

Asp Ser Ser Glu Ser Ser Glu Ser Ile Asn Gly Gly Arg Thr Tyr Ala

245 250 255

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

260 265 270

Glu Asn Leu Phe Asn Ser Glu Asn Pro Phe Ala Val Glu Asn Met Ala

275 280 285

Phe Ile Lys Pro Lys Asp Met Val Asp Asn Tyr Pro Ser Thr Trp Thr

290 295 300

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

305 310 315 320

His Asp Asn Pro Asn Ala Val Asn Asn Arg Phe Tyr Arg Ala Lys Tyr

325 330 335

Tyr Pro Lys Arg Leu Glu Thr Gln Thr Thr Thr Pro Leu Ile Asp Ser

340 345 350

Ser Phe Ser Pro Tyr Glu His Pro Glu Trp Tyr Glu Asp Asn Gln Phe

355 360 365

Val Met Pro Trp Met Gln Tyr Ile Thr Asn Leu Gly Gly Leu Tyr Ala

370 375 380

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

385 390 395 400

Asn Glu Ser Ala Leu Ser Ile Gly Val Phe Arg Thr Lys Phe Glu Asn

405 410 415

Arg Thr Ala Glu Ala Pro Gly Asn Thr Lys Thr Val Gly Tyr Pro Tyr

420 425 430

Gly Ile Leu Leu Ser Ala Ile Ser Phe Asp Ala Thr Arg Asn Gly Leu

435 440 445

Ala Leu Ala Pro Ala Ser Leu Gly Gln Asp Val Gly Tyr His Phe Val

450 455 460

Pro Arg Leu Ala Val Gly Gly Val Ser Ser Pro Arg Gly Ala Asn Gly

465 470 475 480

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

485 490 495

Phe Leu Asp Thr Lys Trp His Ser Pro Ala Val Ile Glu Asp Ala Pro

500 505 510

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

515 520 525

Asn Pro Gln Pro Thr Ser Thr Pro Met Pro Asn Ser Asn Gly Asn Glu

530 535 540

Ser Ile Pro Tyr Arg Trp Thr Asn Ser Tyr Asp Tyr Asn Ser Val Arg

545 550 555 560

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

565 570 575

Glu Ser Leu Phe Thr Thr Ala Leu Lys Leu Asp Thr Leu Asn Ser Leu

580 585 590

Pro Asn Lys Phe Thr Gln Glu Asn Asn Ile Phe Phe Ser Tyr Ala Met

595 600 605

Leu Asp Gly Arg Gln Trp Ser Leu Gly Thr Arg Lys Asp Ser Ala Trp

610 615 620

Leu Thr Thr Asn Thr Ile Asn Asn Phe Thr Tyr Asn Thr Gln Gln Gln

625 630 635 640

Leu Ala Ser Thr Val Ala Gly Glu Asn Ala Asn Pro Arg Asn Ile Leu

645 650 655

Asn Ala Leu Thr Thr Ala Lys Gly Phe Asp Arg Arg Asp Ile Gly Asn

660 665 670

Val Val Tyr Thr Tyr Ser Asn Asn Thr Asn Lys Phe Thr Tyr Tyr Tyr

675 680 685

Gln Val Gly Gly Ala Ile Thr Thr Trp Pro Glu Val Gln Val Asn Tyr

690 695 700

Lys Thr Ser Ala Asn Ile Thr Tyr Tyr Asn Leu Thr Arg Thr Asp Phe

705 710 715 720

Gly Ser Thr Thr Pro Ala Thr Gln Asp Ala Asn Thr Val Ser Ser Lys

725 730 735

Leu Asn Gly Ala Tyr Leu Ser Ser Thr Gly Asp Gln Gln Gly Trp Tyr

740 745 750

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

755 760 765

Gly Tyr Thr Trp Gln Asp Phe Lys Gly Leu Thr Thr Thr Ala Ser Asn

770 775 780

Ala Val Ile Ser Asn Trp Thr Lys Ala Gly Tyr Ser Ile Arg Pro Asp

785 790 795 800

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

805 810 815

Thr Ala Ala Val Asn Val Arg Ser Leu Asp Ser Tyr Tyr Val Gln Leu

820 825 830

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

835 840 845

Ser Ala Leu Ala Leu Asn Pro Asn Arg Ile Thr Asn Pro Leu Met Asn

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

Arg Tyr Thr Pro Pro Gln Asn Asn Pro Asp Ile Arg Leu Arg Leu Leu

915 920 925

Val Ile Asp Arg Ser Arg Ala Thr Asn Asp Phe Ile Lys Leu Leu Pro

930 935 940

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

945 950 955 960

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

965 970 975

Tyr

<210> 3

<211> 657

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 3

ggatccatgc atcaccatca ccatcacgct aagaaaaagg agaggatgat gatccaggag 60

agggaggagc accagaagat ggtggagagc ctgggaatca tcgaggagca gaacaagaca 120

gaggcaatcg agccaacaga ggaggtgaac acacaggagc ccactcagcc ggcaggagtg 180

aatgtggcaa acaacccaca gatgggaatc aaccagcctc agatcaaccc ccaattcggt 240

cctaaccctc agcagagaat caacccccag tgcttcggtg gccccatgca gcctaatcaa 300

atgggtatgc gccccggttt caatcagatg cccccccaaa tgggcggtat gcctcctaac 360

caaatgggta tgagacccgg tttcaaccag atgcctcctc agatgggtgg tatgcctccg 420

cgccccaatt tccctaacca gatgcccaac atgaaccagc ctaggcccgg tttccgcccc 480

cagcctggtg gaggtgtgcc tatgggtaac aaagctggtg gtggttttaa ccatcctggt 540

actcctatgg gtcccaaccg catgaacttc ccgaaccaag gaatgaatca gcctcctcac 600

atggctggtc ctcgcgcagg tttccccccc caaaacggtc ctcgctaatg aaagctt 657

<210> 4

<211> 213

<212> PRT

<213>chicken virus mycoplasma (Mycoplasma gallisepticum)

<400> 4

Met His His His His His His Ala Lys Lys Lys Glu Arg Met Met Ile

1 5 10 15

Gln Glu Arg Glu Glu His Gln Lys Met Val Glu Ser Leu Gly Ile Ile

20 25 30

Glu Glu Gln Asn Lys Thr Glu Ala Ile Glu Pro Thr Glu Glu Val Asn

35 40 45

Thr Gln Glu Pro Thr Gln Pro Ala Gly Val Asn Val Ala Asn Asn Pro

50 55 60

Gln Met Gly Ile Asn Gln Pro Gln Ile Asn Pro Gln Phe Gly Pro Asn

65 70 75 80

Pro Gln Gln Arg Ile Asn Pro Gln Cys Phe Gly Gly Pro Met Gln Pro

85 90 95

Asn Gln Met Gly Met Arg Pro Gly Phe Asn Gln Met Pro Pro Gln Met

100 105 110

Gly Gly Met Pro Pro Asn Gln Met Gly Met Arg Pro Gly Phe Asn Gln

115 120 125

Met Pro Pro Gln Met Gly Gly Met Pro Pro Arg Pro Asn Phe Pro Asn

130 135 140

Gln Met Pro Asn Met Asn Gln Pro Arg Pro Gly Phe Arg Pro Gln Pro

145 150 155 160

Gly Gly Gly Val Pro Met Gly Asn Lys Ala Gly Gly Gly Phe Asn His

165 170 175

Pro Gly Thr Pro Met Gly Pro Asn Arg Met Asn Phe Pro Asn Gln Gly

180 185 190

Met Asn Gln Pro Pro His Met Ala Gly Pro Arg Ala Gly Phe Pro Pro

195 200 205

Gln Asn Gly Pro Arg

210

<210> 5

<211> 2739

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 5

ggatccatga agcagtccga caagtccaac gacaacacac agctggtgaa ccaggcaagg 60

acactggacg ccaactccgt gaggctggcc ggcctgggac aaaacggaag cctgttcaac 120

accgtgctgc gcgacgtgga cgacaacttc atcacagccg ctaacggcac cattatcaag 180

ctggactcct tcaccaagcc cctctacggt ctcgacctct ccgatgactt cgctggctac 240

aaggtgaaac aaatcgtgag cgactacacc acctcccgca acagattcga ccagagacaa 300

acccgcgcct actacgccct gctggtgaac gacgaagcta acgtgcactt gaaacgcatc 360

aacaccaact ccaaccgcat cggcaaccgc aacaacaact ccaagttcgt catcggcggc 420

gtggataacc ctgctcacgt cattaggttc accgatgacg gaaccaagtt caacttcacc 480

aaacaaaccc aaggtgagat tgtcaacgac ttcatcctcg acgctcctat cctgcctaag 540

gacctgcacc ctgactggta caacctctac atccaacgca agatcctccc caacgacgtg 600

aacaccgctg tggtgccctg gcccgttggt cgcgtgtccg gtactaacgc tgacgacgga 660

atgttcgact tcggtaacgg ccaaatcacc aacaccgacc ctatcgccca aacaaagacc 720

accaccgata accaaaaccc ttccaccttc aactccggcg ccatgcctgg agctaacaac 780

cgctacgact cccaactcaa cgtcaaacac cgcatcaaaa cctccttcca actcgacgaa 840

aagttcgtgt accctgagtg gaccggctcc gaggagaaca agaacattac ccgcctggct 900

accggaagcc tgccttccaa cgaacgctac tggatcttgg acatccccgg cactcctcaa 960

gtgactctga aagaggactc cgtgaacgtg ttctcccgcc tgtacctgaa ctccgtgaac 1020

tcattgtcct tcatcggcga ctccatctac atcttcggaa cctccgagct gccttccctg 1080

tggtactact ccttccctac ccgcctctcc gacctcaccg ctctgaacca ggttaaaacc 1140

gacgacatcg aggcttcctc caccgacaac ggtactacca ccaacggtac tactaccacc 1200

accgacacct cctccggctc caccggtgct ggcaccggta acaccaccaa cacctcccaa 1260

accgtcagca accccaccct caacacctac cgctccttcg gtatcgactc caaacccacc 1320

tccgctaaca agatcgacga aaccaactgg gccgacccca acgtgatcga ggcccgcatc 1380

tacgccgaat accgcctcgg catccagaac gagatcccta tcaccaacgc cggcaacttc 1440

attcgcaaca ccatcggagg cgtgggcttc acctccaccg gctcccgtgt ggtgctgcgc 1500

gcttcctaca acggtgacca gagacccacc ggcaacttcc aacccttcct gtacgtcttc 1560

ggttacctgg gttaccagca gacacgcacc ggtacattct ggtacggtac ttacaagttg 1620

ctcaacaaca gcccttacga cgtcctggat gctgcccgcg tcggtactga gaccaaccag 1680

ttccgccgta cctccctcac ataccccgtg atgggtggtt acctgaccga ggagggtgcc 1740

cgctcattct ccaacacccc ctacattcgt gctcaaggtg atactcctga aagccgctcc 1800

atcttccaat ccggttactc cgataacacc tacgaataca tccaatccgt gctcggtttc 1860

gacggtatcc gcaacaacct caacgtggga gtcaaggcta gctccttcct gaactccaac 1920

cgtcccaacc ctaacggcct ggagatgatc gctgctacca cctacctgcg ctctcagatc 1980

ggtctggctc gcacctccgg actgcctaac caacaaccct tcggcaccac ccaccaagtg 2040

atctccgtca gccctggtga tcagttctcc tccatcaaga acatccgcac catcttcccc 2100

ggtaaccaac tgtggtactt cctcttcacc aacgagaaca acaaaagcag cgtctacacc 2160

ctgagactgg ctgactcctc caaccccgac gctagctcct ccttctcccc cacctcactg 2220

atcgacgtca acgagatcgg cgtcatcctg cctctgctgg acaactcctt ctacaccgtg 2280

aacgctgctg gcaacgtcgc cctcttctcc tccaaccctg gttcccctgg ttcctacacc 2340

gctgtgaaca ctttcaacca gaacctctcc gacatcgctt tcgagggttc cggcgctaaa 2400

tacacctctg acttctgggg tactatccaa ttcaagcctg acgaatacct gatccaaaac 2460

ggtttcacct cccaagtcgc tcgtaacttc gtgaccaacc agtccttcct gaacagcctg 2520

gtggacttca cccctgctaa cgcaggtact aactacaggg tggtggttga ccccgacggt 2580

aacctgacca accagaacct gcccctcaag gtgcagatcc agtacctcga cggaaaatac 2640

tacgacgcca aactgaaaaa caacaacctg gtgacattct cctacaacaa cttcgctgcc 2700

ctgccctcct gggtggttcc taccgcctaa tgaaagctt 2739

<210> 6

<211> 907

<212> PRT

<213>chicken virus mycoplasma (Mycoplasma gallisepticum)

<400> 6

Met Lys Gln Ser Asp Lys Ser Asn Asp Asn Thr Gln Leu Val Asn Gln

1 5 10 15

Ala Arg Thr Leu Asp Ala Asn Ser Val Arg Leu Ala Gly Leu Gly Gln

20 25 30

Asn Gly Ser Leu Phe Asn Thr Val Leu Arg Asp Val Asp Asp Asn Phe

35 40 45

Ile Thr Ala Ala Asn Gly Thr Ile Ile Lys Leu Asp Ser Phe Thr Lys

50 55 60

Pro Leu Tyr Gly Leu Asp Leu Ser Asp Asp Phe Ala Gly Tyr Lys Val

65 70 75 80

Lys Gln Ile Val Ser Asp Tyr Thr Thr Ser Arg Asn Arg Phe Asp Gln

85 90 95

Arg Gln Thr Arg Ala Tyr Tyr Ala Leu Leu Val Asn Asp Glu Ala Asn

100 105 110

Val His Leu Lys Arg Ile Asn Thr Asn Ser Asn Arg Ile Gly Asn Arg

115 120 125

Asn Asn Asn Ser Lys Phe Val Ile Gly Gly Val Asp Asn Pro Ala His

130 135 140

Val Ile Arg Phe Thr Asp Asp Gly Thr Lys Phe Asn Phe Thr Lys Gln

145 150 155 160

Thr Gln Gly Glu Ile Val Asn Asp Phe Ile Leu Asp Ala Pro Ile Leu

165 170 175

Pro Lys Asp Leu His Pro Asp Trp Tyr Asn Leu Tyr Ile Gln Arg Lys

180 185 190

Ile Leu Pro Asn Asp Val Asn Thr Ala Val Val Pro Trp Pro Val Gly

195 200 205

Arg Val Ser Gly Thr Asn Ala Asp Asp Gly Met Phe Asp Phe Gly Asn

210 215 220

Gly Gln Ile Thr Asn Thr Asp Pro Ile Ala Gln Thr Lys Thr Thr Thr

225 230 235 240

Asp Asn Gln Asn Pro Ser Thr Phe Asn Ser Gly Ala Met Pro Gly Ala

245 250 255

Asn Asn Arg Tyr Asp Ser Gln Leu Asn Val Lys His Arg Ile Lys Thr

260 265 270

Ser Phe Gln Leu Asp Glu Lys Phe Val Tyr Pro Glu Trp Thr Gly Ser

275 280 285

Glu Glu Asn Lys Asn Ile Thr Arg Leu Ala Thr Gly Ser Leu Pro Ser

290 295 300

Asn Glu Arg Tyr Trp Ile Leu Asp Ile Pro Gly Thr Pro Gln Val Thr

305 310 315 320

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

325 330 335

Val Asn Ser Leu Ser Phe Ile Gly Asp Ser Ile Tyr Ile Phe Gly Thr

340 345 350

Ser Glu Leu Pro Ser Leu Trp Tyr Tyr Ser Phe Pro Thr Arg Leu Ser

355 360 365

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

370 375 380

Ser Thr Asp Asn Gly Thr Thr Thr Asn Gly Thr Thr Thr Thr Thr Asp

385 390 395 400

Thr Ser Ser Gly Ser Thr Gly Ala Gly Thr Gly Asn Thr Thr Asn Thr

405 410 415

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

420 425 430

Ile Asp Ser Lys Pro Thr Ser Ala Asn Lys Ile Asp Glu Thr Asn Trp

435 440 445

Ala Asp Pro Asn Val Ile Glu Ala Arg Ile Tyr Ala Glu Tyr Arg Leu

450 455 460

Gly Ile Gln Asn Glu Ile Pro Ile Thr Asn Ala Gly Asn Phe Ile Arg

465 470 475 480

Asn Thr Ile Gly Gly Val Gly Phe Thr Ser Thr Gly Ser Arg Val Val

485 490 495

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

500 505 510

Pro Phe Leu Tyr Val Phe Gly Tyr Leu Gly Tyr Gln Gln Thr Arg Thr

515 520 525

Gly Thr Phe Trp Tyr Gly Thr Tyr Lys Leu Leu Asn Asn Ser Pro Tyr

530 535 540

Asp Val Leu Asp Ala Ala Arg Val Gly Thr Glu Thr Asn Gln Phe Arg

545 550 555 560

Arg Thr Ser Leu Thr Tyr Pro Val Met Gly Gly Tyr Leu Thr Glu Glu

565 570 575

Gly Ala Arg Ser Phe Ser Asn Thr Pro Tyr Ile Arg Ala Gln Gly Asp

580 585 590

Thr Pro Glu Ser Arg Ser Ile Phe Gln Ser Gly Tyr Ser Asp Asn Thr

595 600 605

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

610 615 620

Leu Asn Val Gly Val Lys Ala Ser Ser Phe Leu Asn Ser Asn Arg Pro

625 630 635 640

Asn Pro Asn Gly Leu Glu Met Ile Ala Ala Thr Thr Tyr Leu Arg Ser

645 650 655

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

660 665 670

Gly Thr Thr His Gln Val Ile Ser Val Ser Pro Gly Asp Gln Phe Ser

675 680 685

Ser Ile Lys Asn Ile Arg Thr Ile Phe Pro Gly Asn Gln Leu Trp Tyr

690 695 700

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

705 710 715 720

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

725 730 735

Ser Leu Ile Asp Val Asn Glu Ile Gly Val Ile Leu Pro Leu Leu Asp

740 745 750

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

755 760 765

Ser Asn Pro Gly Ser Pro Gly Ser Tyr Thr Ala Val Asn Thr Phe Asn

770 775 780

Gln Asn Leu Ser Asp Ile Ala Phe Glu Gly Ser Gly Ala Lys Tyr Thr

785 790 795 800

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

805 810 815

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

820 825 830

Ser Phe Leu Asn Ser Leu Val Asp Phe Thr Pro Ala Asn Ala Gly Thr

835 840 845

Asn Tyr Arg Val Val Val Asp Pro Asp Gly Asn Leu Thr Asn Gln Asn

850 855 860

Leu Pro Leu Lys Val Gln Ile Gln Tyr Leu Asp Gly Lys Tyr Tyr Asp

865 870 875 880

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

885 890 895

Ala Ala Leu Pro Ser Trp Val Val Pro Thr Ala

900 905

<210> 7

<211> 1884

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 7

ggatccatgt cctgcaccac ccccacccct aaccctaacc ctccttccgg tggtatgaac 60

ggtggtgaca ccaaccctgg tgacggtcag ggtatgatga acgctgcttc ccaggagctg 120

gctgctgctc gtatgggcct caccaccgta ttcgactcca aggctaagaa cctcggtctg 180

tacgtggact acaagaagac acaggacact ctgaccaaag cctatgacgc cgcaaaaacc 240

gtactggaca actcctcctc caccacccag aacctcaacg aagccaaaac ccgcctcgaa 300

accgctatcc gcaccgccgc cacctccaaa caaaccttcg acgaacaaca cgccgaactc 360

gtcaaagtct acgaagaact caaaaccacc ctctccaacg aaaccgccac cctcgccccc 420

tacgccgact cacaatacgc cggcatcaaa atgcacctgt ccggcctcta cgacgctgga 480

aaagctatca ccaccaaaac cctcgaaccc gtcgaaggtg accctctgac cgctgacgtg 540

gtcatgatgg ctaacaccaa aatcgtggag gctatcaagg atgaagtgct gaaccctcag 600

aaagagaacg caaccaaact ggctgactcc ttcgtgaagc aagttctggt taaggagaag 660

atcacgggtg tggaggaggc tcataacaag gctcagcctg ctaattacag cttcgtcgga 720

tattccgtag acatcactgg tactgtgacc ggtcaaacct cgattcctaa ctgggactac 780

gctcagagga caattttcac caacggcgat gaaccccgtt ccatctccaa cacccctgct 840

gacggtcaga caatggttca acctctgagc aacgtgtctt ggatctacag cctggccggt 900

acaggagcca agtacaccct ggagttcacc tattatggtc cctccaccgg ctacctgtat 960

tttccctaca agctggtaaa cacctccgac cagatgaagc tgggcctgga atataagctg 1020

aacgacgcca ccgaacctag tgctatcacc ttcggttccg agcagactat gaacggtaag 1080

acccccaccg tcaacgacat caacgtggct aaggtaaccc tggctaacct gaaatttggt 1140

tccaacaaga tcgaattttc cgtgcctgct gagaaggtgt cccctatgat cggtaacatg 1200

tacctgtcct cctcccccaa caattggaat aagatctacg atgacatctt cggcaattcc 1260

gtgaccaccg agaacaacag gaccatcatt tccgtggacg ctctgaacgg ttactccctc 1320

gcttccgact ggtccaccta catcgctgag tactccggtg ctggcctcac cctcaacgac 1380

caagctaagc ccaacgagaa gtactacctg atcggatatg tgggcggtac tggtgctcgt 1440

aacgacatga tggtgcctaa gaataacgtg cagaagttcc ctctggctaa caacacctcc 1500

aatcgcaact acgtgttcta cgtgaacgct cctaaggctg gtgactacta tatcaaaggt 1560

gtgttcgcta gcggtgtgca ctccgatctc aaattctcca ctggtgacat gtcctccaac 1620

aacgtcaccg tgaagcagct gttcactggt aaccttacta ccaccctgcg cacctttgat 1680

acctccgcca ctaccgagtc cacccgcgtg accaccgacc ctaccaacaa gaaaacactg 1740

acactggtgg agggtctcaa caaaatcgtg gtctccggca ccaccgaaaa catcggtgcc 1800

cctaatttcg gctatctcga atttatcctc aacgagaccc agcccgaaac aaccaacgtc 1860

tccaatccta gttaatgaaa gctt 1884

<210> 8

<211> 622

<212> PRT

<213>chicken virus mycoplasma (Mycoplasma gallisepticum)

<400> 8

Met Ser Cys Thr Thr Pro Thr Pro Asn Pro Asn Pro Pro Ser Gly Gly

1 5 10 15

Met Asn Gly Gly Asp Thr Asn Pro Gly Asp Gly Gln Gly Met Met Asn

20 25 30

Ala Ala Ser Gln Glu Leu Ala Ala Ala Arg Met Gly Leu Thr Thr Val

35 40 45

Phe Asp Ser Lys Ala Lys Asn Leu Gly Leu Tyr Val Asp Tyr Lys Lys

50 55 60

Thr Gln Asp Thr Leu Thr Lys Ala Tyr Asp Ala Ala Lys Thr Val Leu

65 70 75 80

Asp Asn Ser Ser Ser Thr Thr Gln Asn Leu Asn Glu Ala Lys Thr Arg

85 90 95

Leu Glu Thr Ala Ile Arg Thr Ala Ala Thr Ser Lys Gln Thr Phe Asp

100 105 110

Glu Gln His Ala Glu Leu Val Lys Val Tyr Glu Glu Leu Lys Thr Thr

115 120 125

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

130 135 140

Ala Gly Ile Lys Met His Leu Ser Gly Leu Tyr Asp Ala Gly Lys Ala

145 150 155 160

Ile Thr Thr Lys Thr Leu Glu Pro Val Glu Gly Asp Pro Leu Thr Ala

165 170 175

Asp Val Val Met Met Ala Asn Thr Lys Ile Val Glu Ala Ile Lys Asp

180 185 190

Glu Val Leu Asn Pro Gln Lys Glu Asn Ala Thr Lys Leu Ala Asp Ser

195 200 205

Phe Val Lys Gln Val Leu Val Lys Glu Lys Ile Thr Gly Val Glu Glu

210 215 220

Ala His Asn Lys Ala Gln Pro Ala Asn Tyr Ser Phe Val Gly Tyr Ser

225 230 235 240

Val Asp Ile Thr Gly Thr Val Thr Gly Gln Thr Ser Ile Pro Asn Trp

245 250 255

Asp Tyr Ala Gln Arg Thr Ile Phe Thr Asn Gly Asp Glu Pro Arg Ser

260 265 270

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

275 280 285

Asn Val Ser Trp Ile Tyr Ser Leu Ala Gly Thr Gly Ala Lys Tyr Thr

290 295 300

Leu Glu Phe Thr Tyr Tyr Gly Pro Ser Thr Gly Tyr Leu Tyr Phe Pro

305 310 315 320

Tyr Lys Leu Val Asn Thr Ser Asp Gln Met Lys Leu Gly Leu Glu Tyr

325 330 335

Lys Leu Asn Asp Ala Thr Glu Pro Ser Ala Ile Thr Phe Gly Ser Glu

340 345 350

Gln Thr Met Asn Gly Lys Thr Pro Thr Val Asn Asp Ile Asn Val Ala

355 360 365

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

370 375 380

Ser Val Pro Ala Glu Lys Val Ser Pro Met Ile Gly Asn Met Tyr Leu

385 390 395 400

Ser Ser Ser Pro Asn Asn Trp Asn Lys Ile Tyr Asp Asp Ile Phe Gly

405 410 415

Asn Ser Val Thr Thr Glu Asn Asn Arg Thr Ile Ile Ser Val Asp Ala

420 425 430

Leu Asn Gly Tyr Ser Leu Ala Ser Asp Trp Ser Thr Tyr Ile Ala Glu

435 440 445

Tyr Ser Gly Ala Gly Leu Thr Leu Asn Asp Gln Ala Lys Pro Asn Glu

450 455 460

Lys Tyr Tyr Leu Ile Gly Tyr Val Gly Gly Thr Gly Ala Arg Asn Asp

465 470 475 480

Met Met Val Pro Lys Asn Asn Val Gln Lys Phe Pro Leu Ala Asn Asn

485 490 495

Thr Ser Asn Arg Asn Tyr Val Phe Tyr Val Asn Ala Pro Lys Ala Gly

500 505 510

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

515 520 525

Lys Phe Ser Thr Gly Asp Met Ser Ser Asn Asn Val Thr Val Lys Gln

530 535 540

Leu Phe Thr Gly Asn Leu Thr Thr Thr Leu Arg Thr Phe Asp Thr Ser

545 550 555 560

Ala Thr Thr Glu Ser Thr Arg Val Thr Thr Asp Pro Thr Asn Lys Lys

565 570 575

Thr Leu Thr Leu Val Glu Gly Leu Asn Lys Ile Val Val Ser Gly Thr

580 585 590

Thr Glu Asn Ile Gly Ala Pro Asn Phe Gly Tyr Leu Glu Phe Ile Leu

595 600 605

Asn Glu Thr Gln Pro Glu Thr Thr Asn Val Ser Asn Pro Ser

610 615 620

<210> 9

<211> 1752

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 9

ggatccatgg actctaaccc taacaacgga caaacccaac tgcaggctgc tcgcatggaa 60

ctgaccgacc tgattaacgc taaagctagg accctggctt ccctgcagga ctacgctaag 120

attgaagcta gcctgtcctc cgcctacatc gaagctgaaa ccgtgaataa caacctgaac 180

gccactctgg agcaactgaa catggcaaaa accaacctcg aaagcgctat caatcaggcc 240

aacaccgaca aaaccacctt cgacaacgag catcctaacc tggtggaagc ctacaaggct 300

ctgaaaacca ccctcgaaca gagagccacc aacctcgaag gcctcgcctc caccgcctac 360

aaccaaatcc gcaacaacct ggtggacctc tacaacaaag ctagctccct catcaccaaa 420

accctggacc ctctgaacgg cggaaccctg ctcgactcca acgagatcac caccgctaac 480

aaaaacatca acaacaccct gtccaccatc aacgaacaga aaaccaacgc cgacgccctc 540

gccaactcct tcatcaagga agtgatccaa aacaacaaac aatccttcgt gggcatgttc 600

accaacacca acgtgcaacc ctccaactac tccttcgtgg ccttctccgc tgacgtgacc 660

cccgtgaact acaaatacgc tcgccgcacc gtgtggaacg gtgacgaacc atcctcccgc 720

atcctggcta ataccaactc catcaccgac gtgtcttgga tctactctct ggctggcacc 780

aatacaaagt accagttttc tttctccaat tatggtcctt ccaccggtta cctgtatttc 840

ccttataaac tggtgaagac agctgacgct aacaatatcg gtctccaata caaactcaac 900

aacggtaacg tccagcaggt ggagttcgct acctccactt ccgagaacaa caccaccgct 960

aatcccaccc ctgccgtgga cgagatcaaa gtggccaaag tgaccctctc caacctgaag 1020

ttcggctcca acaccatcga attttccgtc cctaccggtg agggcaacat gaacaaagtc 1080

gctcctatga tcggcaacat gtacatcacc tccagcaacg ccgaggctaa caaaaagcag 1140

atctacgact ccatcttcgg taacacctcc tcccagaccg catcccagac ctccgtgtcc 1200

gtcgacctcc tgaaaggcta cagcctcgcc acctcctctc gcacctacat ccgccaattc 1260

accggtctca ccgacaacgg cgtgcagacc tccgaccctg tctacctgat cggtctcatc 1320

ggtggtcacc aggaccgcac cgtggctacc ggtcctacta acatccagaa ctcccctaac 1380

gtggacaacg acaaccgcac cttcaccatc tacgtcaatg cccctgtcaa cggtaactac 1440

catatctccg gagcttacct gcagggcact cgcaccgccc gctccttgaa gttcagcacc 1500

ggcacatcct cctccaacaa cgaggtcacc gtgttgggcc tggagcaacg cgactggacc 1560

atcctgggtc atttcgacac caagatggac ggcaccacca ccatctcctg gacaaacacc 1620

gcctccaagc gtactctgac cctcaacaag ggtttgaaca agatcattgt gagcggcggc 1680

acccaggaca acacaaacgc ccctttcatc ggcaacctga ccttcaccct gcacctgacc 1740

taatgaaagc tt 1752

<210> 10

<211> 578

<212> PRT

<213>chicken virus mycoplasma (Mycoplasma gallisepticum)

<400> 10

Met Asp Ser Asn Pro Asn Asn Gly Gln Thr Gln Leu Gln Ala Ala Arg

1 5 10 15

Met Glu Leu Thr Asp Leu Ile Asn Ala Lys Ala Arg Thr Leu Ala Ser

20 25 30

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

35 40 45

Glu Ala Glu Thr Val Asn Asn Asn Leu Asn Ala Thr Leu Glu Gln Leu

50 55 60

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

65 70 75 80

Asp Lys Thr Thr Phe Asp Asn Glu His Pro Asn Leu Val Glu Ala Tyr

85 90 95

Lys Ala Leu Lys Thr Thr Leu Glu Gln Arg Ala Thr Asn Leu Glu Gly

100 105 110

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

115 120 125

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

130 135 140

Gly Gly Thr Leu Leu Asp Ser Asn Glu Ile Thr Thr Ala Asn Lys Asn

145 150 155 160

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

165 170 175

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

180 185 190

Ser Phe Val Gly Met Phe Thr Asn Thr Asn Val Gln Pro Ser Asn Tyr

195 200 205

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

210 215 220

Ala Arg Arg Thr Val Trp Asn Gly Asp Glu Pro Ser Ser Arg Ile Leu

225 230 235 240

Ala Asn Thr Asn Ser Ile Thr Asp Val Ser Trp Ile Tyr Ser Leu Ala

245 250 255

Gly Thr Asn Thr Lys Tyr Gln Phe Ser Phe Ser Asn Tyr Gly Pro Ser

260 265 270

Thr Gly Tyr Leu Tyr Phe Pro Tyr Lys Leu Val Lys Thr Ala Asp Ala

275 280 285

Asn Asn Ile Gly Leu Gln Tyr Lys Leu Asn Asn Gly Asn Val Gln Gln

290 295 300

Val Glu Phe Ala Thr Ser Thr Ser Glu Asn Asn Thr Thr Ala Asn Pro

305 310 315 320

Thr Pro Ala Val Asp Glu Ile Lys Val Ala Lys Val Thr Leu Ser Asn

325 330 335

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

340 345 350

Gly Asn Met Asn Lys Val Ala Pro Met Ile Gly Asn Met Tyr Ile Thr

355 360 365

Ser Ser Asn Ala Glu Ala Asn Lys Lys Gln Ile Tyr Asp Ser Ile Phe

370 375 380

Gly Asn Thr Ser Ser Gln Thr Ala Ser Gln Thr Ser Val Ser Val Asp

385 390 395 400

Leu Leu Lys Gly Tyr Ser Leu Ala Thr Ser Ser Arg Thr Tyr Ile Arg

405 410 415

Gln Phe Thr Gly Leu Thr Asp Asn Gly Val Gln Thr Ser Asp Pro Val

420 425 430

Tyr Leu Ile Gly Leu Ile Gly Gly His Gln Asp Arg Thr Val Ala Thr

435 440 445

Gly Pro Thr Asn Ile Gln Asn Ser Pro Asn Val Asp Asn Asp Asn Arg

450 455 460

Thr Phe Thr Ile Tyr Val Asn Ala Pro Val Asn Gly Asn Tyr His Ile

465 470 475 480

Ser Gly Ala Tyr Leu Gln Gly Thr Arg Thr Ala Arg Ser Leu Lys Phe

485 490 495

Ser Thr Gly Thr Ser Ser Ser Asn Asn Glu Val Thr Val Leu Gly Leu

500 505 510

Glu Gln Arg Asp Trp Thr Ile Leu Gly His Phe Asp Thr Lys Met Asp

515 520 525

Gly Thr Thr Thr Ile Ser Trp Thr Asn Thr Ala Ser Lys Arg Thr Leu

530 535 540

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

545 550 555 560

Asp Asn Thr Asn Ala Pro Phe Ile Gly Asn Leu Thr Phe Thr Leu His

565 570 575

Leu Thr

<210> 11

<211> 41

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 11

ataggatcca tgcatcacca tcaccatcac gtgtctggtg c 41

<210> 12

<211> 37

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 12

ataaagcttt cattaataac cgggcagtgc gtcgaat 37

<210> 13

<211> 42

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 13

ataggatcca tgcatcacca tcaccatcac gctaagaaaa ag 42

<210> 14

<211> 38

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 14

ataaagcttt cattagcgag gaccgttttg ggggggga 38

<210> 15

<211> 34

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 15

ataggatcca tgaagcagtc cgacaagtcc aacg 34

<210> 16

<211> 35

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 16

ataaagcttt cattaggcgg taggaaccac ccagg 35

<210> 17

<211> 38

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 17

ataggatcca tgtcctgcac cacccccacc cctaaccc 38

<210> 18

<211> 42

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 18

ataaagcttt cattaactag gattggagac gttggttgtt tc 42

<210> 19

<211> 34

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 19

ataggatcca tggactctaa ccctaacaac ggac 34

<210> 20

<211> 34

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 20

ataaagcttt cattaggtca ggtgcagggt gaag 34

Claims

1. a kind of immune composition, including a kind of albumen, one or more kinds of any groups selected from the following of the albumen It closes:

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 chicken virus mycoplasma adhesin antibodies MGC1 of molecule encoding；

Using SEQ ID NO:3 nucleic acid molecules or with the identical nucleic acid of 95% or more nucleotide sequence of SEQ ID NO:3 The chicken virus mycoplasma adhesin antibodies MGC2 of molecule encoding；

Using SEQ ID NO:5 nucleic acid molecules or with the identical nucleic acid of 95% or more nucleotide sequence of SEQ ID NO:5 The chicken virus mycoplasma adhesin antibodies MGC3 of molecule encoding.

Using SEQ ID NO:7 nucleic acid molecules or with the identical nucleic acid of 95% or more nucleotide sequence of SEQ ID NO:7 The chicken virus mycoplasma blood clotting GAP-associated protein GAP VLH3 of molecule encoding；

Using SEQ ID NO:9 nucleic acid molecules or with the identical nucleic acid of 95% or more nucleotide sequence of SEQ ID NO:9 The chicken virus mycoplasma blood clotting GAP-associated protein GAP VLH5 of molecule encoding.

2. immune composition according to claim 1, the chicken virus mycoplasma adhesin antibodies MGC1 includes SEQ ID The amino acid sequence of NO:2 or the identical amino acid sequence of 95% or more full length amino acid sequence with SEQ ID NO:2；

The chicken virus mycoplasma adhesin antibodies MGC2 include SEQ ID NO:4 amino acid sequence or with SEQ ID NO: The 4 identical amino acid sequence of 95% or more full length amino acid sequence；

The chicken virus mycoplasma adhesin antibodies MGC3 include SEQ ID NO:6 amino acid sequence or with SEQ ID NO: The 6 identical amino acid sequence of 95% or more full length amino acid sequence.

The chicken virus mycoplasma blood clotting GAP-associated protein GAP VLH3 include SEQ ID NO:8 amino acid sequence or with SEQ ID The identical amino acid sequence of 95% or more full length amino acid sequence of NO:8；

The chicken virus mycoplasma blood clotting GAP-associated protein GAP VLH5 include SEQ ID NO:10 amino acid sequence or with SEQ ID The identical amino acid sequence of 95% or more full length amino acid sequence of NO:10.

3. immune composition described in claim 1 is directed to Mycoplasma Gallisepticum Antigen Recognized By Antibody for inducing in animal subject for producing Immune response medicament purposes.

4. immune composition described in claim 1 is for producing for preventing animal by the use of the medicament of mycoplasma gallisepticum infection On the way.

5. a kind of immune composition, including a kind of for encoding the core of chicken virus mycoplasma blood clotting GAP-associated protein GAP or adhesin antibodies Acid molecule, described nucleic acid molecules one or more kinds of any combination selected from the following:

The sequential nucleotide sequence of SEQ ID NO:1 is identical suitable with 95% or more the nucleotide sequence of SEQ ID NO:1 Sequence nucleotide sequence；

The sequential nucleotide sequence of SEQ ID NO:3 is identical suitable with 95% or more the nucleotide sequence of SEQ ID NO:3 Sequence nucleotide sequence；

The sequential nucleotide sequence of SEQ ID NO:5 is identical suitable with 95% or more the nucleotide sequence of SEQ ID NO:5 Sequence nucleotide sequence；

The sequential nucleotide sequence of SEQ ID NO:7 is identical suitable with 95% or more the nucleotide sequence of SEQ ID NO:7 Sequence nucleotide sequence；

The sequential nucleotide sequence of SEQ ID NO:9 is identical suitable with 95% or more the nucleotide sequence of SEQ ID NO:9 Sequence nucleotide sequence.

6. immune composition described in claim 5 is directed to Mycoplasma Gallisepticum Antigen Recognized By Antibody for inducing in animal subject for producing Immune response medicament purposes.

7. immune composition described in claim 5 is for producing for preventing animal by the use of the medicament of mycoplasma gallisepticum infection On the way.

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

The amino acid sequence of SEQ ID NO:2 or the identical ammonia of 95% or more full length amino acid sequence with SEQ ID NO:2 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；

The amino acid sequence of SEQ ID NO:6 or the identical ammonia of 95% or more full length amino acid sequence with SEQ ID NO:6 Base acid sequence；

The amino acid sequence of SEQ ID NO:8 or the identical ammonia of 95% or more full length amino acid sequence with SEQ ID NO:8 Base acid sequence；

The amino acid sequence of SEQ ID NO:10 is identical with 95% or more the full length amino acid sequence of SEQ ID NO:10 Amino acid sequence.

9. a kind of immune composition suitable for generating the immune response for chicken virus mycoplasma in animal subject body, includes:

Protein composition and adjuvant according to any one of claims 8.

10. immune composition according to claim 9, which is characterized in that the adjuvant is selected from: