CN109999191B

CN109999191B - Novel genetic engineering subunit vaccine of mycoplasma gallisepticum

Info

Publication number: CN109999191B
Application number: CN201910289602.6A
Authority: CN
Inventors: 曹文龙; 孔迪; 滕小锘; 易小萍; 张大鹤
Original assignee: Suzhou Shinuo Biotechnology Co ltd
Current assignee: Suzhou Womei Biology Co ltd
Priority date: 2019-04-11
Filing date: 2019-04-11
Publication date: 2022-06-24
Anticipated expiration: 2039-04-11
Also published as: CN109999191A

Abstract

The invention provides an immune composition and a subunit vaccine, wherein the immune composition comprises a protein, and the protein is selected from one or any combination of more than two of the following proteins: a mycoplasma gallisepticum-associated protein encoded by a nucleic acid molecule of SEQ ID No.1 or 3 or 5 or 7 or 9 or a nucleic acid molecule that is 95% identical to or more than 95% of the nucleotide sequence of SEQ ID No.1 or 3 or 5 or 7 or 9. The vaccine uses eukaryotic expression, the antigenicity and immunogenicity of the product are similar to those of natural protein, the expression level is higher, the immunogenicity is strong, the protection effect is good, and the vaccine has no pathogenicity to chickens.

Description

Novel genetic engineering subunit vaccine of mycoplasma gallisepticum

Technical Field

The application relates to the technical field of animal immunity drugs, in particular to a preparation method and application of a novel gene engineering vaccine of mycoplasma gallisepticum.

Background

Mycoplasma Gallisepticum (MG) infection is one of respiratory diseases of chickens, mainly causes chronic respiratory diseases, air sacculitis and sinusitis, and is clinically manifested by cough, rhinorrhea, rales in breathing and mouth opening in severe cases. According to statistics, after the mycoplasma gallisepticum infects a chicken group, the weak brooding rate of chicks is increased by about 10%, the laying rate of laying hens is reduced by 10% -20%, the weight of broilers is reduced by 38%, the slaughtering period is prolonged, the feed conversion rate is reduced by 21%, and a large amount of medicine expense can be indirectly caused, so that the feed additive is one of important diseases harmful to the chicken industry.

Mycoplasma Gallisepticum (MG) is a pathogenic species of Mycoplasmatales, Mycoplasmataceae, Mycoplasma, a very small prokaryotic organism, without cell walls, and only enveloped by cell membranes.

At present, the prevention and treatment of mycoplasma gallisepticum mainly comprise vaccine prevention and drug treatment. The mycoplasma gallisepticum infection is characterized in that the mycoplasma gallisepticum and the air sac are mainly fixedly planted on cilia of a tracheal mucosa, blood vessels do not exist at the tail end of the cilia of the trachea and the air sac, and the medicine is transported to an action part through blood and acts on the mycoplasma gallisepticum through diffusion, so that the medicine can only reduce the number of the mycoplasma gallisepticum and the mycoplasma gallisepticum fixedly planted on the air sac and relieve clinical symptoms, but cannot completely kill the mycoplasma gallisepticum, and the mycoplasma gallisepticum infection generally is a bacterium-carrying state which can lead the mycoplasma gallisepticum to be continuously infected for a lifetime, and therefore, the vaccine can effectively provide protection at present.

The existing vaccines for preventing and controlling mycoplasma gallisepticum infection are traditional inactivated vaccines, but the mycoplasma gallisepticum is difficult to culture, needs serum and has low antigen content. Meanwhile, in the process of culturing the mycoplasma gallisepticum, because conditions such as culture temperature, culture medium components and the like are changed and the mycoplasma gallisepticum is continuously replicated, antigenic proteins on the surface of the mycoplasma gallisepticum are easy to mutate and delete, and the inactivated vaccine has the problem of weak immune protection. Therefore, it is imperative to study new vaccines with strong immunity. Research and development of novel vaccines mainly based on genetic engineering vaccines are the leading direction of this century.

Disclosure of Invention

The present invention is directed to an immunological composition that solves the problems of the prior art.

In order to achieve the above object, according to an aspect of the present invention, there is provided an immunological composition including a protein selected from one or any combination of two or more of:

mycoplasma gallisepticum adhesin protein MGC1 encoded by a nucleic acid molecule of SEQ ID NO.1 or a nucleic acid molecule that is 95% identical or more than 95% identical to the nucleotide sequence of SEQ ID NO. 1;

mycoplasma gallisepticum adhesin protein MGC2 encoded by a nucleic acid molecule of SEQ ID NO. 3 or a nucleic acid molecule that is 95% identical or more than 95% identical to the nucleotide sequence of SEQ ID NO. 3;

mycoplasma gallisepticum adhesin protein MGC3 encoded by a nucleic acid molecule of SEQ ID NO. 5 or a nucleic acid molecule that is 95% or more identical to the nucleotide sequence of SEQ ID NO. 5.

Mycoplasma gallisepticum hemagglutination-associated protein VLH3 encoded by the nucleic acid molecule of SEQ ID No. 7 or a nucleic acid molecule that is 95% or more identical to the nucleotide sequence of SEQ ID No. 7;

mycoplasma gallisepticum hemagglutination-associated protein VLH5 encoded by the nucleic acid molecule of SEQ ID No. 9 or a nucleic acid molecule that is 95% or more identical to the nucleotide sequence of SEQ ID No. 9.

The preferred technical proposal is that the mycoplasma gallisepticum adhesin protein MGC1 comprises an amino acid sequence of SEQ ID NO. 2 or an amino acid sequence which is 95 percent of the same as the full-length amino acid sequence of SEQ ID NO. 2;

the mycoplasma gallisepticum adhesin protein MGC2 comprises an amino acid sequence of SEQ ID NO. 4 or an amino acid sequence which is 95% identical to the full-length amino acid sequence of SEQ ID NO. 4;

the mycoplasma gallisepticum adhesin protein MGC3 comprises an amino acid sequence of SEQ ID NO. 6 or an amino acid sequence which is 95% identical to the full-length amino acid sequence of SEQ ID NO. 6.

The mycoplasma gallisepticum hemagglutination associated protein VLH3 comprises an amino acid sequence of SEQ ID NO. 8 or an amino acid sequence which is more than 95% identical to the full-length amino acid sequence of SEQ ID NO. 8;

the mycoplasma gallisepticum hemagglutination associated protein VLH5 comprises an amino acid sequence of SEQ ID NO. 10 or an amino acid sequence which is more than 95% identical to the full-length amino acid sequence of SEQ ID NO. 10.

It is another object of the present invention to provide a use of said immunological composition for the manufacture of a medicament for inducing an immune response against mycoplasma gallisepticum antigen in a test animal.

It is a further object of the present invention to provide the use of said immunological composition for the manufacture of a medicament for the prevention of mycoplasma gallisepticum infection.

It is still another object of the present invention to provide an immunogenic composition comprising a nucleic acid molecule encoding a mycoplasma gallisepticum haemagglutination-associated protein or an adhesin protein, wherein the nucleic acid molecule is selected from one or any combination of two or more of the following:

1 or a sequence nucleotide sequence which is 95% identical to the nucleotide sequence of SEQ ID NO. 1;

3 or a nucleotide sequence that is 95% identical to the nucleotide sequence of SEQ ID NO. 3;

5 or a nucleotide sequence that is 95% identical to the nucleotide sequence of SEQ ID NO. 5;

7 or a nucleotide sequence which is 95% identical to the nucleotide sequence of SEQ ID NO. 7;

the sequence of SEQ ID NO. 9 or a sequence that is 95% or more identical to the nucleotide sequence of SEQ ID NO. 9.

It is a further object of the present invention to provide a use of said nucleic acid molecule for the manufacture of a medicament for inducing an immune response against mycoplasma gallisepticum antigen in a test animal.

It is a further object of the invention to provide a use of said nucleic acid molecule for the manufacture of a medicament for preventing infection of an animal with mycoplasma gallisepticum.

It is still another object of the present invention to provide a protein composition selected from the group consisting of:

the mycoplasma gallisepticum adhesin protein MGC1 comprises an amino acid sequence of SEQ ID NO. 2 or an amino acid sequence which is 95% identical to the full-length amino acid sequence of SEQ ID NO. 2;

the mycoplasma gallisepticum blood coagulation associated protein VLH5 comprises an amino acid sequence of SEQ ID NO. 10 or an amino acid sequence which is more than 95% identical to the full-length amino acid sequence of SEQ ID NO. 10.

It is still another object of the present invention to provide an immunogenic composition suitable for generating an immune response against mycoplasma gallisepticum in a subject animal, comprising:

the mycoplasma gallisepticum adhesin protein, a protein associated with hemagglutination and an adjuvant.

Preferably, the adjuvant is selected from:

white oil (M52), aluminum stearate, span and tween or a combination of more than two of the white oil, the aluminum stearate, the span and the tween.

The invention discloses a preparation method and application of a recombinant subunit vaccine of mycoplasma gallisepticum expressed by Sf9 cells, and proves that the vaccine can generate stronger humoral immunity in a chicken body, and the immunized chicken can resist mycoplasma gallisepticum infection, belonging to the technical field of animal vaccines and biological products for animals, and aiming at providing a preparation method of the recombinant subunit vaccine of mycoplasma gallisepticum, which can be industrially produced in a large scale: recombinant shuttle vectors were constructed by cloning separately a shuttle vector comprising hemagglutinin protein VLH3 (variant expressed and hemigglutin lipoteins 3, the latter 3 being the name of one of the authors, sometimes also referred to as pMGA), VLH5 (variant expressed and hemigglutin lipoteins 5, named above) and adhesin protein 1(Mycoplasma gallisepticum cytaphthin 1, MGC1), adhesin protein 2(Mycoplasma gallisepticum cyclaphthin 2, MGC2), adhesin protein 3(Mycoplasma gallisepticum cytaphthin 3, MGC3) encoding genes into pFastBac1 vector. Then constructing a recombinant baculovirus strain, including protein SDS-PAGE, WB, immunofluorescence detection and virus titer detection, and carrying out protein large-scale production expression and content determination; in the genome of the wild mycoplasma gallisepticum, a large number of homologous pseudogenes exist for expressing the five proteins closely related to mycoplasma gallisepticum infection, such as the hemagglutinin-related proteins VLH3, VLH5, the adhesin proteins MGC1, MGC2 and MGC3, and the true genes and the homologous pseudogenes thereof are easy to undergo homologous recombination, so that amino acid sequences in the hemagglutinin-related proteins VLH3, VLH5, the adhesin proteins MGC1, MGC2 and MGC3 are easy to mutate in the mycoplasma propagation process, so that the five membrane surface proteins of different individuals or different groups of wild mycoplasma gallisepticum in one group are greatly different and present different group distribution, and in order to improve the protective effect of the vaccine as much as possible and counteract the antigen difference existing in the wild mycoplasma gallisepticum, the five antigen proteins are expressed and mixed with an adjuvant to prepare the vaccine.

The invention aims to provide a mycoplasma gallisepticum genetic engineering subunit vaccine with good immune effect and safer process, and the invention uses Sf9 cells to express recombinant mycoplasma gallisepticum hemagglutination-associated proteins VLH3, VLH5 and adhesin proteins (MGC1, MGC2 and MGC 3). The production process of the invention does not involve mycoplasma culture, protein expression is carried out by Sf9 cells, the antigenicity and the immunogenicity of the product are similar to those of natural protein, and the expressed antigen has no mutation and deletion, and the expression level is higher. Suspension culture and easy large-scale production.

After adopting the scheme, compared with the prior art, the invention has the following outstanding advantages and effects:

the immune composition uses Sf9 cells to express hemagglutinin protein VLH3, VLH5 and adhesin protein (MGC1, MGC2 and MGC3), the antigenicity, the immunogenicity and the functions of the product are similar to those of natural protein, the expression level is higher, the expressed antigen has no mutation and deletion, the immunogenicity is strong, and the product has no pathogenicity to chickens, and the vaccine can be prepared by a bioreactor through large-scale serum-free suspension culture, and the production cost of the vaccine is greatly reduced.

The invention uses optimized sequences of hemagglutinin protein VLH3, VLH5, adhesin protein MGC1, MGC2 and MGC3, uses suspension culture Sf9 cells for expression, and has higher expression level and good protein immunogenicity. The subunit vaccine can be produced in large scale, and has sufficient supply and easy quality control; the safety is high, and the immunogenicity is good; the batch to batch stability; the production cost is low.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. In the drawings:

FIG. 1 shows the result of gel electrophoresis of the PCR product obtained after PCR amplification of MGC1 gene, showing a band of 2.9 kbp; wherein 1 is a negative control; 2 is MG-MGC1 gene; m is a molecular weight marker;

FIG. 2 shows the results of gel electrophoresis of PCR products after PCR amplification of a plurality of colonies transformed with MGC1 gene, and a positive sample appeared as a 2.9kbp band. 2-7 are products obtained after PCR amplification of a colony sample transformed by MGC1 gene, and 1 is a non-positive sample; m is a molecular weight marker;

FIG. 3 is a map of a constructed transfer vector pF-MG-MGC1 containing a target gene;

FIG. 4 shows a 0.6kbp band observed by gel electrophoresis of PCR products obtained by PCR amplification of MGC2 gene; wherein 1 is a negative control; 2 is MG-MGC2 gene; m is a molecular weight marker;

FIG. 5 shows the results of gel electrophoresis of PCR products after PCR amplification of a plurality of colonies transformed with MGC2 gene, and a positive sample appeared as a 0.6kbp band. 2-6 are products obtained after PCR amplification of a colony sample transformed by MGC2 gene, and 1 is a non-positive sample; m is a molecular weight marker;

FIG. 6 is a map of a constructed transfer vector pF-MG-MGC2 containing a target gene;

FIG. 7 shows a 2.7kbp band observed by gel electrophoresis of PCR products obtained after PCR amplification of MGC3 gene; wherein 1 is negative control, and 2 is MG-MGC3 gene; m is a molecular weight marker;

FIG. 8 shows the results of gel electrophoresis of PCR products obtained after PCR amplification of a plurality of colonies transformed with MGC3 gene, and a positive sample appeared in the 2.7kbp band. 2-7 are products obtained after PCR amplification of a colony sample transformed by MGC2 gene, and 1 is a non-positive sample; m is a molecular weight marker;

FIG. 9 is a map of a constructed transfer vector pF-MG-MGC2 containing a target gene;

FIG. 10 shows a 1.8kbp band obtained by gel electrophoresis of a PCR product obtained by PCR amplification of VLH3 gene; wherein 1 is a negative control, and 2 is MG-VLH3 gene; m is a molecular weight marker;

FIG. 11 shows the results of gel electrophoresis of PCR products obtained after PCR amplification of a plurality of samples of colonies transformed with VLH3 gene, and a 1.8kbp band appeared as a positive sample. 2-7 are products obtained after PCR amplification of colony samples transformed by VLH3 genes, and 1 is a non-positive sample; m is a molecular weight marker;

FIG. 12 is a map of a constructed transfer vector pF-MG-VLH3 containing a target gene;

FIG. 13 shows a 1.7kbp band obtained by gel electrophoresis of a PCR product obtained by PCR amplification of VLH5 gene; wherein 1 is a negative control, and 2 is MG-VLH5 gene; m is a molecular weight marker;

FIG. 14 shows the results of gel electrophoresis of PCR products obtained after PCR amplification of a plurality of samples of colonies transformed with VLH5 gene, and a 1.7kbp band appeared as a positive sample. 2-7 are products obtained after PCR amplification of colony samples transformed by VLH5 genes, and 1 is a non-positive sample; m is a molecular weight marker;

FIG. 15 is a map of the constructed transfer vector pF-MG-VLH5 containing the target gene;

FIG. 16 is a SDS-PAGE gel electrophoresis of the rBac-MGC1 cell culture supernatant harvested in example 7, showing a band of interest around 106kDa molecular weight, where 2 is rBac-MGC1 cell culture supernatant; 1 is negative control; m is a molecular weight marker;

FIG. 17 is a result of SDS-PAGE gel electrophoresis of the rBac-MGC2 cell culture supernatant harvested in example 7, in which a band of interest appears at a molecular weight of about 23kDa, where 2 is rBac-MGC2 cell culture supernatant; 1 is negative control; m is a molecular weight marker;

FIG. 18 is a result of SDS-PAGE gel electrophoresis of the rBac-MGC3 cell culture supernatant harvested in example 7, in which a band of interest appears at a molecular weight of about 100kDa, where 2 is rBac-MGC2 cell culture supernatant; 1 is negative control; m is a molecular weight marker;

FIG. 19 is a result of SDS-PAGE gel electrophoresis of rBac-VLH3 cell culture supernatant harvested in example 7, in which 2 is rBac-MGC2 cell culture supernatant, showing a band of interest around 67kDa in molecular weight; 1 is negative control; m is a molecular weight marker;

FIG. 20 is a result of SDS-PAGE gel electrophoresis of rBac-VLH5 cell culture supernatant harvested in example 7, in which 2 is rBac-VLH5 cell culture supernatant, showing a band of interest around 63kDa in molecular weight; 1 is negative control; m is a molecular weight marker;

FIG. 21 shows Western Blot analysis of supernatant of MG-MGC1 recombinant baculovirus cell culture in example 8; wherein 2 is MG-MGC1 recombinant baculovirus cell culture supernatant; 1 is negative control; m is a molecular weight marker;

FIG. 22 shows Western Blot analysis of supernatant of MG-MGC2 recombinant baculovirus cell culture in example 8; wherein 2 is MG-MGC2 recombinant baculovirus cell culture supernatant; 1 is negative control; m is a molecular weight marker;

FIG. 23 shows Western Blot detection of supernatant of MG-MGC3 recombinant baculovirus cell culture in example 8; wherein 2 is MG-MGC3 recombinant baculovirus cell culture supernatant; 1 is negative control; m is a molecular weight marker;

FIG. 24 shows Western Blot analysis of culture supernatants of MG-VLH3 recombinant baculovirus cells of example 8; wherein 2 is MG-VLH3 recombinant baculovirus cell culture supernatant; 1 is negative control; m is a molecular weight marker;

FIG. 25 shows Western Blot analysis of culture supernatants of MG-VLH5 recombinant baculovirus cells of example 8; wherein 2 is MG-VLH5 recombinant baculovirus cell culture supernatant; 1 is negative control; m is a molecular weight marker;

FIG. 26 is a fluorescent image of empty baculovirus Sf9 cells and recombinant baculovirus Sf9 cells, wherein no fluorescence was observed with inoculated empty baculovirus Sf9 cells, whereas fluorescence was observed with inoculated recombinant baculovirus Sf9 cells.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

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

The invention provides an immune composition, which comprises a protein, wherein the protein is selected from one or any combination of more than two of the following proteins:

mycoplasma gallisepticum hemagglutination-associated protein VLH5 encoded by the nucleic acid molecule of SEQ ID No. 9 or a nucleic acid molecule that is 95% identical or more to the nucleotide sequence of SEQ ID No. 9;

the invention also relates to a method of inducing an immune response against a mycoplasma gallisepticum antigen, said method comprising administering a vaccine of the invention to a subject animal.

The invention also relates to a method of protecting a test animal from infection by mycoplasma gallisepticum comprising administering to said test animal a vaccine of the invention.

The invention further relates to a method of protecting a test animal diagnosed with mycoplasma gallisepticum infection, said method comprising administering to said test animal a vaccine of the invention.

The invention also includes vaccines suitable for inducing an immune response against mycoplasma gallisepticum. The vaccine of the present invention may be a plasmid comprising the above-described nucleic acid molecule. The nucleic acid molecule may be incorporated into a viral particle. The vaccine may further comprise an adjuvant molecule. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof; and in some embodiments, can be IL-12, IL-15, IL-28 or RANTES.

The vaccine may comprise protein molecules. The protein molecule is selected from one or any combination of two of the following: a protein comprising

SEQ ID NO

2 or 4 or 6 or 8 or 10; a protein that is 95% identical over the entire length of the amino acid sequence of

SEQ ID NO

2 or 4 or 6 or 8 or 10; a fragment of

SEQ ID NO

2 or 4 or 6 or 8 or 10; a protein 95% identical to a fragment of

SEQ ID NO

2 or 4 or 6 or 8 or 10.

Also provided herein is a protein selected from the group consisting of: (a) 2 or 4 or 6 or 8 or 10; (b) a protein that is 95% identical over the entire amino acid sequence length of the full-length sequence as set forth in

SEQ ID NO

2 or 4 or 6 or 8 or 10; (c) an immunogenic fragment of

SEQ ID NO

2 or 4 or 6 or 8 or 10 comprising 20 or more amino acids of

SEQ ID NO

2 or 4 or 6 or 8 or 10; and (d) an immunogenic fragment comprising 20 or more amino acids of a protein that is 95% identical over the entire length of the amino acid sequence of

SEQ ID NO

2 or 4 or 6 or 8 or 10.

The vaccine of the invention may be a nucleic acid molecule comprising a sequence encoding one or more protein molecules as described above. In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of seq id no:1 or 3 or 5 or 7 or 9; a nucleic acid sequence which is 95% identical over the entire length of the nucleotide sequence of

SEQ ID NO

1 or 3 or 5 or 7 or 9; 1 or 3 or 5 or 7 or 9; a nucleotide sequence 95% identical to a fragment of

SEQ ID NO

1 or 3 or 5 or 7 or 9.

Some aspects of the invention provide methods of inducing an immune response against mycoplasma gallisepticum, the methods comprising the steps of: administering to the individual a mycoplasma gallisepticum antigen and/or a composition thereof.

Further aspects of the invention provide methods of protecting an individual from infection by mycoplasma gallisepticum. The method comprises the following steps: administering to the individual a prophylactically effective amount of a nucleic acid molecule or composition comprising such a nucleic acid sequence; wherein the nucleic acid sequence is expressed in cells of the individual and induces a protective immune response against a protein encoded by the nucleic acid sequence.

Some aspects of the invention provide a method of inducing an immune response against a mycoplasma gallisepticum antigen, the method comprising administering to a subject animal a nucleic acid molecule of the invention.

Some aspects of the invention provide a method of protecting a test animal from infection by mycoplasma gallisepticum, comprising administering to the test animal a nucleic acid molecule of the invention.

Some aspects of the invention provide a method of protecting a test animal diagnosed with a mycoplasma gallisepticum infection, comprising administering to the test animal a nucleic acid molecule of the invention.

In another aspect, the present invention provides a protein selected from the group consisting of: (a) 2 or 4 or 6 or 8; (b) a protein that is 98% identical over the entire length of the amino acid sequence of

SEQ ID NO

2 or 4 or 6 or 8; (c) an immunogenic fragment comprising 20 or more amino acids of

SEQ ID NO

2 or 4 or 6 or 8; and (d) an immunogenic fragment comprising 20 or more amino acids of a protein that is 98% identical over the entire length of the amino acid sequence of

SEQ ID NO

2 or 4 or 6 or 8.

Some aspects of the invention provide a vaccine suitable for use in generating an immune response against mycoplasma gallisepticum in a test animal, the vaccine comprising: nucleic acid molecules of the invention and adjuvant molecules. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof; and in some embodiments, can be IL-12, IL-15, IL-28 or RANTES.

The vaccine of the present invention may further comprise one or more nucleic acid molecules as described above and one or more proteins encoded by said nucleic acid molecules.

1. And (4) defining.

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

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

As used herein, "adjuvant" means any molecule added to the DNA plasmid vaccines described herein to enhance the immunogenicity of the antigens encoded by the DNA plasmids and encoding nucleic acid sequences described below.

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

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

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

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

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

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

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

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

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

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

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

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

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

"operably linked" as used herein means that expression of a gene is under the control of a promoter spatially linked thereto. Under its control, the promoter may be positioned 5 '(upstream) or 3' (downstream) of the gene. The distance between the promoter and the gene may be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, this change in distance can be adjusted without loss of promoter function.

"promoter" as used herein means a molecule of synthetic or natural origin that is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. The promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or alter spatial and/or temporal expression thereof. A promoter may also contain distal enhancer or repressor elements, which can be located as much as several thousand pairs of base pairs from the start of transcription. Promoters may be obtained from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may regulate expression of a gene component either substantially or differentially with respect to the cell, tissue or organ in which expression occurs or with respect to the developmental stage at which expression occurs or in response to an external stimulus such as a physiological stress, pathogen, metal ion or inducer. Representative examples of promoters include the phage T7 promoter, the phage T3 promoter, the SP6 promoter, the lactose operon-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 late promoter, and the CMVIE promoter.

"Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be attached to the amino terminus of a Mycoplasma gallisepticum protein described herein. The signal peptide/leader sequence is generally indicative of the location of the protein. The signal peptide/leader sequence used herein preferably facilitates secretion of the protein from the cell in which it is produced. The signal peptide/leader sequence is often cleaved from the remainder of the protein, which is often referred to as the mature protein after secretion from the cell. The signal peptide/leader sequence is linked to the N-terminus of the protein.

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

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

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

"subtype" or "serotype": as used interchangeably herein and with mycoplasma gallisepticum, means a genetic variant of mycoplasma gallisepticum such that one subtype is recognized by the immune system and isolated from a different subtype.

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

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

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

2. Vaccine

The vaccines of the present invention can be designed to control the extent or intensity of an immune response in a test animal against one or more mycoplasma gallisepticum serotypes. The antigens may comprise protein epitopes that make them particularly effective as immunogens against which an immune response against mycoplasma gallisepticum can be induced. The mycoplasma gallisepticum antigen may include full-length translation products, variants thereof, fragments thereof, or combinations thereof.

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

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

Some embodiments relate to fragments of

SEQ ID NO

1 or 3 or 5 or 7 or 9. A fragment may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID No.1 or 3 or 5 or 7 or 9. The fragment may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a fragment of

SEQ ID NO

1 or 3 or 5 or 7 or 9. The fragment may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a fragment of SEQ ID No.1 or 3 or 5 or 7 or 9. In some embodiments, a fragment comprises a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, a fragment does not contain a coding sequence that encodes a leader sequence. In some embodiments, the fragment does not contain a coding sequence that encodes a leader sequence, such as, for example, an IgE leader.

Some embodiments relate to proteins homologous to

SEQ ID NO

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having 95% homology to the protein sequence as set forth in

SEQ ID NO

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having 96% homology to the protein sequence as set forth in

SEQ ID NO

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having 97% homology to the protein sequence as set forth in

SEQ ID NO

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having 98% homology to the protein sequence as set forth in

SEQ ID NO

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having 99% homology to the protein sequence as set forth in SEQ ID NO. 2.

Some embodiments relate to the same protein as

SEQ ID NO

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 80% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 85% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 90% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 91% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 92% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical over the entire amino acid sequence length of the full length consensus amino acid sequence as set forth in

SEQ ID NOs

2 or 4 or 6 or 8 or 10.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader. A fragment of a protein may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the protein. Immunogenic fragments of

SEQ ID NO

2 or 4 or 6 or 8 or 10 may be provided. An immunogenic fragment may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID No. 2 or 4 or 6 or 8 or 10. In some embodiments, the fragment includes a leader sequence, such as, for example, an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader.

Immunogenic fragments of proteins having amino acid sequences homologous to the immunogenic fragments of

SEQ ID NO

2 or 4 or 6 or 8 or 10 can be provided. The immunogenic fragment may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the protein homologous to SEQ ID No. 2 or 4 or 6 or 8 or 1095. Some embodiments relate to immunogenic fragments that are 96% homologous to the immunogenic fragments of protein sequences herein. Some embodiments relate to immunogenic fragments that are 97% homologous to the immunogenic fragments of protein sequences herein. Some embodiments relate to immunogenic fragments that are 98% homologous to the immunogenic fragments of protein sequences herein. Some embodiments relate to immunogenic fragments that are 99% homologous to the immunogenic fragments of protein sequences herein. In some embodiments, the fragment comprises a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader.

Immunogenic fragments of proteins having the same amino acid sequence as the immunogenic fragment of

SEQ ID NO

2 or 4 or 6 or 8 or 10 can be provided. The immunogenic fragment may comprise a protein that is 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% identical over the entire length of the amino acid sequence set forth in SEQ ID No. 2 or 4 or 6 or 8 or 10, at least 80%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. In some embodiments, the fragment includes a leader sequence, such as, for example, an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader.

The protein or nucleic acid molecule may be present in the composition in any amount. The different protein molecules in the immunological composition are present in the composition in any ratio; the different nucleic acid molecules in the immunological composition are present in the composition in any ratio. When used, administering to the individual a prophylactically effective amount of a nucleic acid molecule or composition comprising such a nucleic acid sequence; wherein the nucleic acid sequence is expressed in cells of the individual and induces a protective immune response against a protein encoded by the nucleic acid sequence. The term "effective amount" is an amount effective to ameliorate the symptoms of a disease in an animal.

3. Vaccine constructs and plasmids

Vaccines can include nucleic acid constructs or plasmids encoding mycoplasma gallisepticum hemagglutination-associated protein, mycoplasma gallisepticum antigen, and combinations of mycoplasma gallisepticum hemagglutination-associated proteins/antigens. Provided herein are genetic constructs that can comprise a nucleic acid sequence encoding a mycoplasma gallisepticum antigen disclosed herein, the core antigen comprising a protein sequence, a sequence homologous to the protein sequence, a fragment of the protein sequence, and a sequence homologous to a fragment of the protein sequence. Additionally, provided herein are genetic constructs that can comprise a nucleic acid sequence encoding a mycoplasma gallisepticum surface antigen (including protein sequences, sequences homologous to protein sequences, fragments of protein sequences, and sequences homologous to fragments of protein sequences) disclosed herein. The genetic construct may be present as a functional extrachromosomal molecule. The genetic construct may be a linear minichromosome comprising a centromere, telomere or plasmid or cosmid.

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

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

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

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

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

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

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

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

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

The gene sequences coding for the hemagglutinin proteins VLH3.01, VLH5.02 and adhesin proteins (MGC2 and MGC3, MGC1) may be native, added and truncated sequences. The composition of the vaccine can be any one protein or any combination thereof, and preferably five proteins are mixed in equal proportion. Baculovirus expression system transfer vectors in recombinant baculovirus vectors include, but are not limited to, pFastBac1, Pvl1393, etc., and for example, pFastBac1 can be preferably used. Sf9 cell line can be Sf9, High Five, S2 or Sf21 cells, preferably Sf 9.

Example 1 construction and characterization of the transfer vector pF-MGC1

MGC1 gene amplification and purification codon optimized MGC1 gene (SEQ ID NO:1) was synthesized in Nanjing Kinshire and cloned into pUC17 vector to obtain pUC-MGC1 plasmid vector. PCR amplification was performed using pUC-MGC1 plasmid as template and MGC1-F, MGC1-R as upstream and downstream primers (the gene sequences of MGC1-F, MGC1-R are shown in SEQ ID NO.11 and 12), and the amplification system is shown in Table 1.

TABLE 1 MGC1 Gene amplification System

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

The PCR product was subjected to gel electrophoresis to verify the size of the target gene, and as shown in FIG. 1, a band of interest appeared at a position of 2.9kbp, and the target gene was successfully amplified and recovered and purified using a gel recovery and purification kit.

2. Enzyme digestion and purification the pFastBac1 plasmid and MGC1 gene expression frame PCR amplification product were digested simultaneously for 3 hours at 37 ℃ with BamHI and HindIII, and the specific digestion reaction system is shown in tables 2 and 3.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBac1 plasmid and the MGC1 gene fragment by using a gel recovery and purification kit respectively.

TABLE 2 MGC1 Gene digestion reaction System

TABLE 3 pFastbac1 plasmid cleavage reaction System

3. Ligation the double-digested pFastBac1 plasmid and the MGC1 gene digestion product were ligated using T4DNA ligase in a water bath at 16 ℃ overnight. The specific ligation reaction system is shown in Table 4.

TABLE 4 connection System of MGC1 Gene and pFastBac1 plasmid

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

5. Colony PCR and sequencing identification single colonies on the selected plate are respectively inoculated into an LB liquid culture medium, cultured for 2 hours at 37 ℃, and subjected to colony PCR identification by taking a bacterial liquid as a template and MGC1-F and MGC1-R as primers, and a PCR product is subjected to gel electrophoresis to verify the size of a target gene, as shown in figure 2, a sample with a 2.9kbp band is a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector pF-MGC1 containing the target gene is shown in FIG. 3.

Example 2 construction and characterization of the transfer vector pF-MGC2

MGC2 gene amplification and purification codon optimized MGC2 gene (SEQ ID NO:3) was synthesized in Nanjing Kinshire and cloned into pUC17 vector to obtain pUC-MGC2 plasmid vector. PCR amplification was performed using pUC-MGC2 plasmid as template and MGC2-F, MGC2-R as upstream and downstream primers (the gene sequences of MGC2-F, MGC2-R are shown in SEQ ID NO.13 and 14), and the amplification system is shown in Table 5.

TABLE 5 MGC2 Gene amplification System

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 4, the target gene was successfully amplified by the occurrence of a band of 0.6kbp, and was recovered and purified by a gel recovery and purification kit.

2. Enzyme digestion and purification the pFastBac1 plasmid and MGC2 gene expression frame PCR amplification product were digested simultaneously for 3 hours at 37 ℃ with BamHI and HindIII, and the specific digestion reaction system is shown in tables 6 and 7.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBac1 plasmid and the MGC2 gene fragment by using a gel recovery and purification kit respectively.

TABLE 6 MGC2 Gene digestion reaction System

TABLE 7 pFastbac1 plasmid cleavage reaction system

3. Ligation the double-digested pFastBac1 plasmid and the MGC2 gene digestion product were ligated using T4DNA ligase in a water bath at 16 ℃ overnight. The specific ligation reaction system is shown in Table 8.

TABLE 8 connection System of MGC2 Gene and pFastBac1 plasmid

5. Colony PCR and sequencing identification Single colonies on the selected plate are respectively inoculated into an LB liquid culture medium, cultured for 2 hours at 37 ℃, and bacterial liquid is used as a template, MGC2-F and MGC2-R are used as primers to carry out colony PCR identification, and a PCR product is subjected to gel electrophoresis to verify the size of a target gene, as shown in figure 5, a sample with a band of 0.6kbp is a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector pF-MGC2 containing the target gene is shown in FIG. 6.

Example 3 construction and characterization of the transfer vector pF-MGC3

MGC3 gene amplification and purification codon optimized MGC3 gene (SEQ ID NO:5) was synthesized in Nanjing Kinshire and cloned into pUC17 vector to obtain pUC-MGC3 plasmid vector. PCR amplification was performed using pUC-MGC3 plasmid as template and MGC3-F, MGC3-R as upstream and downstream primers (the gene sequences of MGC3-F, MGC3-R are shown in SEQ ID NO.15 and 16), and the amplification system is shown in Table 9.

TABLE 9 MGC3 Gene amplification System

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 7, a band of interest appeared at a position of 2.7kbp, and the target gene was successfully amplified and recovered and purified using a gel recovery and purification kit.

2. Enzyme digestion and purification the pFastBac1 plasmid and MGC3 gene expression frame PCR amplification product were digested simultaneously for 3 hours at 37 ℃ with BamHI and HindIII, and the specific digestion reaction system is shown in tables 10 and 11.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBac1 plasmid and the MGC3 gene fragment by using a gel recovery and purification kit respectively.

TABLE 10 MGC3 Gene restriction system

TABLE 11 pFastbac1 plasmid cleavage reaction system

3. Ligation the double-digested pFastBac1 plasmid and the MGC3 gene digestion product were ligated using T4DNA ligase in a water bath at 16 ℃ overnight. The specific ligation reaction system is shown in Table 12.

TABLE 12 connection System of MGC3 Gene and pFastBac1 plasmid

5. Colony PCR and sequencing identification Single colonies on the selected plate are respectively inoculated into an LB liquid culture medium, cultured for 2 hours at 37 ℃, and bacterial liquid is used as a template, MGC3-F and MGC3-R are used as primers to carry out colony PCR identification, and a PCR product is subjected to gel electrophoresis to verify the size of a target gene, as shown in figure 8, a sample with a band of 2.7kbp is a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector pF-MGC3 containing the target gene is shown in FIG. 9.

Example 4 construction and characterization of the transfer vector pF-VLH3

VLH3 gene amplification and purification codon-optimized VLH3 gene (SEQ ID NO:7) was synthesized in Nanjing Kinsley and cloned into pUC17 vector to obtain pUC-VLH3 plasmid vector. PCR was performed using pUC-VLH3 plasmid as template and VLH3-F, VLH3-R as upstream and downstream primers (the gene sequences of VLH3-F, VLH3-R are shown in SEQ ID NO.17 and 18), and the amplification system is shown in Table 13.

TABLE 13 VLH3 Gene amplification System

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 10, the target gene was successfully amplified by the occurrence of a band of interest at a position of 1.8kbp, and was recovered and purified by a gel recovery and purification kit.

2. Digestion and purification the PCR amplification products of pFastBac1 plasmid and VLH3 gene expression cassette were digested simultaneously for 3 hours at 37 ℃ with BamHI and HindIII, and the specific digestion reaction systems are shown in tables 14 and 15.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBac1 plasmid and the VLH3 gene fragment by using a gel recovery and purification kit respectively.

TABLE 14 restriction reaction system for VLH3 gene

TABLE 15 pFastbac1 plasmid cleavage reaction System

3. Ligation the double-digested pFastBac1 plasmid and the VLH3 gene digestion product were ligated using T4DNA ligase in a 16 ℃ water bath overnight. The specific ligation reaction system is shown in Table 16.

TABLE 16 connection System of VLH3 Gene and pFastBac1 plasmid

5. Colony PCR and sequencing identification Single colonies picked on the plate were inoculated respectively to LB liquid medium, cultured at 37 ℃ for 2 hours, colony PCR identification was performed using the bacterial solution as template and VLH3-F and VLH3-R as primers, the PCR product was subjected to gel electrophoresis to verify the size of the target gene, as shown in FIG. 11, and the sample with a 1.8kbp band appeared was a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector pF-VLH3 containing the target gene is shown in FIG. 12.

Example 5 construction and characterization of the transfer vector pF-VLH5

VLH5 gene amplification and purification codon-optimized VLH5 gene (SEQ ID NO:9) was synthesized in Nanjing Kinsley and cloned into pUC17 vector to obtain pUC-VLH5 plasmid vector. PCR amplification was performed using pUC-VLH5 plasmid as template and VLH5-F, VLH5-R as upstream and downstream primers (the gene sequences of VLH5-F, VLH5-R are shown in SEQ ID NO.19 and 20), and the amplification system is shown in Table 17.

TABLE 17 VLH5 Gene amplification System

The size of the target gene was verified by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 13, a band of interest appeared at a position of 1.7kbp, and the target gene was successfully amplified and recovered and purified using a gel recovery and purification kit.

2. Digestion and purification the PCR amplification products of pFastBac1 plasmid and VLH5 gene expression cassette were digested simultaneously for 3 hours at 37 ℃ with BamHI and HindIII, and the specific digestion reaction systems are shown in tables 18 and 19.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBac1 plasmid and the VLH5 gene fragment by using a gel recovery and purification kit respectively.

TABLE 18 restriction reaction system for VLH5 gene

TABLE 19 pFastbac1 plasmid cleavage reaction system

3. Ligation the double-digested pFastBac1 plasmid and the VLH5 gene digestion product were ligated using T4DNA ligase in a 16 ℃ water bath overnight. The specific ligation reaction system is shown in Table 20.

TABLE 20 ligation System of VLH5 Gene and pFastBac1 plasmid

5. Colony PCR and sequencing identification Single colonies picked on the plate were inoculated respectively to LB liquid medium, cultured at 37 ℃ for 2 hours, colony PCR identification was carried out using the bacterial solution as template and VLH5-F and VLH5-R as primers, the PCR product was subjected to gel electrophoresis to verify the size of the target gene, as shown in FIG. 14, and the sample with a 1.7kbp band appeared was a positive sample. And (4) sending the bacteria liquid with positive identification to a sequencing company for sequencing, and selecting the bacteria liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector pF-VLH5 containing the target gene is shown in FIG. 15.

Example 6 construction of recombinant baculovirus genomes Bac-MGC1, Bac-MGC2, Bac-MGC3, Bac-VLH3 and Bac-VLH5

1, transforming DH10Bac bacteria, respectively taking 1 mul pF-MGC1 plasmid, pF-MGC2 plasmid, pF-MGC3 plasmid, pF-VLH3 plasmid and pF-VLH5 plasmid in each example 1-5, adding 100 mul DH10Bac competent cells, mixing uniformly, ice-bathing for 30 minutes, water-bath heat shock for 90 seconds at 42 ℃, ice-bathing for 2 minutes, adding 900 mul LB liquid culture medium without Amp, and culturing for 5 hours at 37 ℃. After 100. mu.l of each of the bacterial solutions was diluted 81 times, 100. mu.l of the diluted bacterial solutions were applied to LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37 ℃ for 48 hours.

2. And selecting single colonies, respectively using an inoculating needle to select large white colonies, streaking on an LB solid culture medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, culturing at 37 ℃ for 48 hours, selecting single colonies, inoculating an LB liquid culture medium containing gentamicin, kanamycin and tetracycline, culturing, preserving strains, and extracting plasmids. Recombinant plasmids Bacmid-MGC1, Bacmid-MGC2, Bacmid-MGC3, Bacmid-VLH3 and Bacmid-VLH5 were obtained, respectively.

Example 7 recombinant baculovirus transfection

Six well plates were seeded 0.8X 10 per well⁶The confluency of Sf9 cells is 50-70%. The following complexes were prepared for each well: diluting 4. mu.l of Cellffectin transfection reagent with 100. mu.l of transfection medium T1, and shaking briefly with vortex; mu.g of the recombinant Bacmid-MGC1, Bacmid-MGC2, Bacmid-MGC3, Bacmid-VLH3 and Bacmid-VLH5 plasmids from example 6 were diluted with 100. mu.l of transfection medium T1, respectively, and the diluted transfection reagents and plasmids were mixed, gently and evenly blown, to prepare a transfection mixture. And adding the transfection compound after the cells adhere to the wall, incubating for 5 hours at 27 ℃, removing the supernatant, adding 2ml of SF-SFM fresh culture medium, and culturing for 4-5 days at 27 ℃ to obtain the supernatant. Recombinant baculovirus rBac-MGC1, rBac-MGC2, rBac-MGC3, rBac-VLH3 and rBac-VLH5 are obtained respectively, the virus titer of the harvested P1 generation recombinant baculovirus is detected by using an MTT relative efficacy method, and the titer of the rBac-MGC1P1 virus is 5.4 x 10⁷pfu/mL, rBac-MGC2P1 virus titers are 2.3X 10⁷pfu/mL, rBac-MGC3P1 virus titer 3.4 × 10⁷pfu/mL, rBac-VLH3P1 virus titer 3.7X 10⁷pfu/mL，rBac-VLThe titer of H5P1 virus is 3.9X 10⁷pfu/mL. The amplified recombinant baculovirus rBac-MGC1, rBac-MGC2, rBac-MGC3, rBac-VLH3 and rBac-VLH5 are used as seed viruses for standby.

Example 8SDS-PAGE detection

The cell cultures harvested in example 7 were each subjected to SDS-PAGE detection, while each empty baculovirus-infected Sf9 cells were used as negative controls. The specific operation is as follows: mu.l of the harvested cell culture was taken, 10. mu.l of 5 × loading buffer was added, the mixture was centrifuged in a boiling water bath for 5 minutes at 12000r/min for 1 minute, the supernatant was subjected to SDS-PAGE gel (12% strength gel) electrophoresis, and the gel was stained and decolored after electrophoresis to observe the band.

As shown in FIG. 16, the rBac-MGC1 cell culture showed a band of interest around a molecular weight of about 106kDa, and the negative control showed no band at the corresponding position.

As shown in FIG. 17, the rBac-MGC2 cell culture showed a band of interest around a molecular weight of about 23kDa, and the negative control showed no band at the corresponding position.

As shown in FIG. 18, the rBac-MGC3 cell culture showed a band of interest around a molecular weight of about 100kDa, and the negative control showed no band at the corresponding position.

As shown in FIG. 19, the rBac-VLH3 cell culture showed a band around 67kDa in molecular weight, and the negative control showed no band at the corresponding position.

As shown in FIG. 20, the rBac-VLH5 cell culture showed a band of interest around a molecular weight of about 63kDa, and the negative control showed no band at the corresponding position.

Example 9Western Blot identification

The products of example 8 after SDS-PAGE electrophoresis were transferred to NC (nitrocellulose) membranes, blocked with 5% skim milk for 2 hours, incubated with chicken-derived anti-MG positive serum for 2 hours, rinsed, incubated with HRP-labeled goat-anti-chicken polyclonal antibody for 2 hours, rinsed, and then added dropwise with an enhanced chemiluminescent fluorogenic substrate, and photographed using a chemiluminescent imager. As shown in FIGS. 21 to 25, the recombinant baculovirus expression sample had a band of interest, and the negative control had no band of interest, indicating that the antigen protein of interest was correctly expressed in Sf9 cells.

Example 10 Indirect immunofluorescence assay

Sf9 cell suspensions transfected with rBac-MGC1, rBac-MGC2, rBac-MGC3, rBac-VLH3 and rBac-VLH5 were added to 96-well cell culture plates, respectively, at a cell concentration of 2.5X 10/well⁵～4.0×10⁵One/ml), 4 wells were inoculated, left at 27 ℃ for 15 minutes, Sf9 cells were attached to the bottom wall of the plate, and 10. mu.l of a 10-fold diluted seed was added to each well. Meanwhile, a blank cell control is set. After inoculation, the cells are placed in a constant-temperature incubator at 27 ℃ for culture for 72-96 hours, the culture solution is discarded, and cold methanol/acetone (1:1) is used for fixation. Firstly reacting with chicken source anti-MG multi-antiserum, then reacting with FITC labeled goat anti-chicken IgG, and observing the result by an inverted fluorescence microscope. As shown in fig. 26, no fluorescence could be observed with the inoculated empty baculovirus Sf9 cells, whereas fluorescence could be observed with the inoculated recombinant baculovirus Sf9 cells, indicating that the target antigen was correctly expressed in Sf9 cells and that the recombinant baculovirus was correctly constructed.

Example 11 antigen protein hemagglutination assay

Both VLH3 and VLH5 proteins had hemagglutination titers, which were measured using chicken erythrocytes for both VLH3 and VLH5 proteins. Samples of cell suspensions expressing VLH3 and VLH5 proteins were harvested, frozen and thawed three times at-80 ℃ and centrifuged to obtain supernatants for testing. On the microplate, from the 1 st well to the 12 th well, 0.025mL of PBS was added to each well by a pipette, 0.025mL of the test sample was aspirated by a pipette, and from the first well, serial dilutions were performed by 2-fold in order to the last 1 well, and 0.025mL of the liquid in the pipette was discarded (dilution by 2,4,8,16,32 … … in order). 0.025mL of 1% chicken erythrocyte suspension is added into each hole, an erythrocyte control hole without a sample is arranged, the hole is immediately shaken on a micro-plate shaker and is placed at room temperature for 20-40 minutes or 2-8 ℃ for 40-60 minutes, and the result is judged when the erythrocyte in the control hole is in a significant button shape. The highest dilution at which erythrocytes were completely agglutinated was regarded as the determination point. The detection shows that the hemagglutination titers of the two proteins VLH3 and VLH5 are 1:32 and 1:32 respectively.

Example 12 bioreactor serum-free suspension culture of insect cells and quantification of expression of MGC1, MGC2, MGC3, VLH3, and VLH5 proteins and agar titer determination

Aseptically culturing Sf9 insect cells in 1000ml shake flask for 3-4 days until the concentration reaches 3-5X 10⁶cell/mL, when the activity is more than 95%, inoculating the cells into a 5L bioreactor, wherein the inoculation concentration is 3-8 × 10⁵cell/mL. When the cell concentration reaches 3-55X 10⁶At cell/mL, cells were seeded into a 50L bioreactor until the cells grew to a concentration of 3-55X 10⁶cell/mL, inoculating into 500L bioreactor until cell concentration reaches 2-85 × 10⁶When cell/mL, the recombinant baculovirus rBac-MGC1 is inoculated, and the culture conditions of the reactor are that the pH value is 6.0-6.5, the temperature is 25-27 ℃, the dissolved oxygen is 30-80 percent, and the stirring speed is 100-180 rpm. In view of the optimum conditions for cell culture, it is preferable to set pH6.2, the temperature at the stage of cell culture at 27 ℃, the dissolved oxygen at 50%, and the stirring speed at 100-180 rpm. Culturing for 5-9 days after infection, adding one-thousandth final concentration BEI, acting at 37 deg.C for 48 hr, adding two-thousandth final concentration Na₂S₂O₃The inactivation is terminated. Cell culture supernatant is obtained by centrifugation or hollow fiber filtration, and the vaccine stock solution is stored at 2-8 ℃. Vaccine stocks expressing MGC2, MGC3, VLH3 and VLH5 proteins were also prepared in the same manner.

The protein content of MGC1, MGC2, MGC3, VLH3 and VLH5 in the prepared vaccine antigen is detected by an Elisa method respectively. The operation mode is as follows: the chicken anti-mycoplasma gallisepticum polyclonal antiserum was diluted with coating buffer to appropriate concentration, 100 μ l per well, overnight at 4 ℃, washed three times with PBST, and blocked with 1% BSA for 1 h. Adding antigen standard substances (protein obtained by particle exchange chromatography, hydrophobic chromatography and molecular sieve purification) with different concentrations and diluting the sample to be detected in a gradient manner, incubating for 1 hour at 37 ℃, and washing with PBST for three times. The detection antibody-MGC 1 protein monoclonal antibody is added into each well, incubated for 1 hour at 37 ℃, and washed by PBST three times. A secondary antibody, i.e., HRP-labeled goat anti-chicken IgG, was added to each well, incubated at 37 ℃ for 1 hour, and washed three times with PBST. TMB was developed for 10 min and the reaction was stopped with 2M H2SO 4. Reading by a microplate reader, and calculating the amount of MSPA protein in the sample to be detected through a standard curve.

The content of MGC2, MGC3, VLH3 and VLH5 protein in the sample to be detected is detected respectively by the same method.

According to example 12, the results of Elisa tests on large scale preparations of MGC1, MGC2, MGC3, VLH3 and VLH5 proteins are shown in the following table, and it can be seen from the results in the table that the average contents of five proteins in the vaccine stock solution are about 163mg/L, 210mg/L, 182mg/L, 156mg/L and 231mg/L, respectively.

In the prepared vaccine antigens, MGC1, MGC2, MGC3, VLH3 and VLH5 proteins are detected by using an agar titer method respectively: respectively punching plum blossom holes on 5 agarose gel plates, adding mycoplasma gallisepticum antiserum in the middle of the plum blossom holes, and respectively adding 2 diluted stock solutions of 5 vaccines of 0, 1, 2, 3, 4, and 5 times at the periphery of 5 agarose gel plates. After incubation in an inverted position for 72h, the line of precipitation was observed. The maximum dilution ratio of the precipitation line is the detection result of the agar-agar titer and the agar-agar titer. The agar titer of MGC1, MGC2, MGC3, VLH3 and VLH5 proteins in the vaccine stock solution is 1:16, 1:32, 1:16 and 1:32 respectively.

EXAMPLE 13 preparation of the vaccine

Five vaccine stock solutions expressed in example 12 were mixed so that the concentration of each antigen of the 5 antigen proteins reached 30mg/mL, and then the mixed vaccine stock solutions were mixed with an oil adjuvant according to a ratio of 2: 3 is prepared into oil emulsion vaccine. Specifically, 1429g of white oil, 70.2g of span, 8.43g of aluminum stearate and 53.3g of tween are added into 1L of mixed vaccine stock solution. Then crushing and emulsifying by an emulsifying crusher to prepare the oil emulsion adjuvant inactivated vaccine. Vaccines were prepared containing only one of MGC1, MGC2, MGC3, VLH3 and VLH5 proteins, respectively, at a concentration of 30mg/mL per antigen.

Example 14 immunization experiment

Injecting PBS into 70 SPF chickens of 21 days old, wherein the SPF chickens are divided into 7 groups of 10 chickens, and the first group is a negative control group; the second group was a vaccine group to which a vaccine prepared by mixing five antigens was injected, and the third to seventh groups were control groups to which a vaccine containing only one of five proteins MGC1, MGC2, MGC3, VLH3 and VLH5 was injected, respectively. Subcutaneous injection of each neck 0.3ml (one wing) of the vaccine prepared in example 13. After 21 days, the 2 nd vaccination was performed at the same dose and route. 21 days after the 2 nd inoculation, all chickens were bled, Hemagglutination Inhibition (HI) antibody titers were measured, and the bled chickens were challenged with a sprayMycoplasma gallisepticum virulent strain SN1 strain 600ml (10)⁸-10⁹ccu/ml), the spraying duration is not less than 5 minutes, and the fog drops are about 2 mu m. Observing for 14 days, performing a autopsy, observing balloon lesions, scoring the lesions, counting the number of chickens with lesions of 2 points or more, and calculating the protection efficiency. The HI titer and the protection rate are shown in the table, 10 chickens in the negative control group have lesions of more than 3 scores, 8 chickens with 4 scores of severe lesions appear, and the incidence rate is 100%; the vaccine prepared by five antigens has less than 2 scores of all chicken ballooning lesions, wherein the number of chickens with no lesions at all is 8, the number of chickens with 1 score of lesions is 2, and the protection rate is 100 percent. The protection rate of the rest five vaccine immune groups containing only one antigen is different from 70 percent to 80 percent, and some vaccine immune groups have serious pathological changes with 4 grades of pathological changes, and the protection efficacy is poorer than that of the vaccine immune groups prepared by five antigens. The vaccine prepared by the invention has the air sac protection rate of 100 percent and qualified vaccine efficacy.

TABLE 21 test results of virus challenge efficacy of mycoplasma gallisepticum genetic engineering vaccines

HI titers are calculated averages.

b: the degree of air bag disease progression is scored according to the following criteria,

0 min-normal air bag, clean, transparent and thin;

score 1-balloon slightly thickened and slightly turbid, with a few blotches of grey or yellow exudate locally;

2 points-some balloon regions had visible gray and yellow exudates with moderate thickening of the balloon;

3 minutes-a large number of air bags are full of yellow cheese-like exudates;

4 minutes, the whole air bag is full of yellow cheese-like exudates, and the air bag loses elasticity;

c: formula for calculating protection rate

The protection rate is the number of chickens with balloon lesion degree less than 2 points/total number of chickens.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Sequence listing

<110> Suzhou Shino Biotechnology Ltd

<120> a novel genetic engineering subunit vaccine of mycoplasma gallisepticum

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2949

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

ggatccatgc atcaccatca ccatcacgtg tctggtgcta agcctaataa tctgaagcct 60

gtgaatcaag tgggtgagat gaatagccag ggtcagtcca atctgctgga gaaggctagg 120

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

<210> 2

<211> 977

<212> PRT

<213> Mycoplasma gallisepticum (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> Mycoplasma gallisepticum (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> Mycoplasma gallisepticum (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> Mycoplasma gallisepticum (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> Mycoplasma gallisepticum (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. An immunological composition comprising:

mycoplasma gallisepticum adhesin protein MGC1 encoded with the nucleic acid molecule of SEQ ID NO. 1;

mycoplasma gallisepticum adhesin protein MGC2 encoded with the nucleic acid molecule of SEQ ID NO. 3;

mycoplasma gallisepticum adhesin protein MGC3 encoded with the nucleic acid molecule of SEQ ID NO. 5;

mycoplasma gallisepticum hemagglutination-associated protein VLH3 encoded by the nucleic acid molecule of SEQ ID NO. 7;

the mycoplasma gallisepticum hemagglutination associated protein VLH5 encoded by the nucleic acid molecule of SEQ ID NO 9.

2. The immunogenic composition of claim 1, wherein the Mycoplasma gallisepticum adhesin protein MGC1 has the amino acid sequence shown in SEQ ID NO. 2;

the amino acid sequence of the mycoplasma gallisepticum adhesin protein MGC2 is shown in SEQ ID NO. 4;

the amino acid sequence of the mycoplasma gallisepticum adhesin protein MGC3 is shown in SEQ ID NO. 6;

the amino acid sequence of the mycoplasma gallisepticum hemagglutination associated protein VLH3 is shown in SEQ ID NO. 8;

the amino acid sequence of the mycoplasma gallisepticum hemagglutination associated protein VLH5 is shown in SEQ ID NO 10.

3. Use of the immunogenic composition of claim 1 for the manufacture of a medicament for inducing an immune response in a test animal against a mycoplasma gallisepticum antigen.

4. Use of the immunogenic composition of claim 1 for the manufacture of a medicament for preventing infection of an animal by mycoplasma gallisepticum.

5. A protein composition, comprising:

2, the amino acid sequence of SEQ ID NO;

amino acid sequence of SEQ ID NO 4

6, the amino acid sequence of SEQ ID NO;

the amino acid sequence of SEQ ID NO 8;

10, SEQ ID NO.

6. An immunogenic composition suitable for use in generating an immune response against mycoplasma gallisepticum in a subject animal, comprising:

the protein composition of claim 5, and an adjuvant.

7. The immunological composition of claim 6 wherein said adjuvant is selected from the group consisting of:

white oil M52, aluminum stearate, span and Tween or a combination of more than two of the white oil M52, the aluminum stearate, the span and the Tween.