CN107513506B

CN107513506B - Mycoplasma hyopneumoniae, vaccine composition and application thereof

Info

Publication number: CN107513506B
Application number: CN201610435285.0A
Authority: CN
Inventors: 田克恭; 金云云; 孙进忠; 张许科
Original assignee: Pulaike Biological Engineering Co Ltd
Current assignee: Pulaike Biological Engineering Co Ltd
Priority date: 2016-06-17
Filing date: 2016-06-17
Publication date: 2021-11-09
Anticipated expiration: 2036-06-17
Also published as: CN107513506A

Abstract

The invention provides a mycoplasma hyopneumoniae GZ strain which has better immunogenicity, the prepared vaccine composition can completely protect pigs, the GZ strain has high titer in amplification preparation, is easy to culture and can produce a large amount of antigens. The prepared antigen can be used together with other antigens, and the respective generated immune efficacies are not influenced mutually.

Description

Mycoplasma hyopneumoniae, vaccine composition and application thereof

Technical Field

The invention relates to mycoplasma hyopneumoniae, a vaccine composition prepared from the mycoplasma hyopneumoniae and application of the vaccine composition, and belongs to the technical field of biology.

Background

Mycoplasma hyopneumoniae (MPS) is a chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae (Mhp), is characterized by high infectivity, high morbidity and low mortality, mainly shows cough and dyspnea, and is mainly characterized by pulmonary sarcoidosis or marbleoid lesion through dissection, particularly by pancreatic and sarcoidosis on two pulmonary heart leaves, middle leaves and sharp leaves. The main harm is the growth retardation and feed conversion rate reduction of pigs. The mycoplasma hyopneumoniae is spread through respiratory tract, and after infecting respiratory tract epithelium, the cilia of the respiratory tract are separated and damaged, epithelial cells are necrotic, the immunologic function of the respiratory tract mucosa is reduced, secondary infection of other respiratory tract pathogens is easily caused, the morbidity symptom is aggravated, and the mortality is increased. Swine enzootic pneumonia is prevalent around the world and is one of the most important diseases causing economic loss in pig raising worldwide.

According to literature data, isolated mycoplasma hyopneumoniae all around the world belong to the same serotype, but antigenicity of isolates is greatly different, and Frey firstly confirms the antigenicity difference between different isolates of the mycoplasma hyopneumoniae in 1992. Mycoplasma hyopneumoniae has extremely high requirements on the culture medium and grows slowly, which greatly increases the production cost of the vaccine. Therefore, the new mycoplasma hyopneumoniae strain which is separated from domestic clinical cases and has better immunogenicity, higher growth speed and higher viable bacteria titer is particularly important for preventing and treating mycoplasma hyopneumoniae in China.

Disclosure of Invention

The invention aims to provide a mycoplasma hyopneumoniae strain which is identified as being better in immunogenicity through separation, wherein the strain is mycoplasma hyopneumoniae GZ (Mycoplasma hyopneumaniae strain GZ) with the deposition number as follows: CCTCC NO: m2016212, preservation unit China center for type culture Collection, preservation address of university of Wuhan, China, and preservation time of 2016, 4 months and 20 days.

Compared with the existing commercial vaccine strain, the strain of the invention has better immunogenicity, and also has the advantages of good culture proliferation titer, easy culture and easy acquisition of a large amount of antigens.

The present invention relates to a vaccine composition, wherein the vaccine composition comprises an immunizing amount of an antigen of a mycoplasma hyopneumoniae strain or a culture thereof and a pharmaceutically acceptable carrier.

The vaccine composition has good immunogenicity, and can completely protect the infection of the mycoplasma hyopneumoniae.

The invention also relates to application of the vaccine composition in preparing a medicament for preventing and treating diseases related to mycoplasma hyopneumoniae infection.

The vaccine strain provided by the invention has high culture titer and good immunogenicity, the use amount is effectively reduced, and the prepared vaccine composition can effectively control the infection of mycoplasma pneumoniae in swinery clinically.

Detailed Description

Hereinafter, embodiments of the present invention will be described.

The invention relates to a mycoplasma hyopneumoniae GZ strain, also called mycoplasma hyopneumoniae GZ, with the preservation number of CCTCC NO: M2016212.

Compared with the existing commercial vaccine strain, the mycoplasma hyopneumoniae GZ strain has better immunogenicity, is easy to culture and is suitable for industrial application. The invention relates to a vaccine composition, wherein the vaccine composition comprises an immunizing dose of inactivated antigen, live whole mycoplasma antigen or subunit antigen of mycoplasma hyopneumoniae GZ strain or culture thereof, and a pharmaceutically acceptable carrier.

"cultures" are subcultures of different generations of the virus, and those skilled in the art know that only minor variations in the gene sequence are possible between generations.

The amount of an ingredient or component of the composition of the present invention is preferably a therapeutically effective amount. The therapeutically effective amount refers to the amount necessary to exert their immunological effects in the host to which the composition is administered without causing undue side effects. The precise amounts of the ingredients used and the composition to be administered will vary depending on factors such as the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.

In one embodiment of the present invention, the culture of mycoplasma hyopneumoniae GZ strain of the present invention is a 1-42 generation culture.

The term "vaccine composition" as used herein refers to a pharmaceutical composition comprising the immunogenicity of mycoplasma hyopneumoniae which induces, stimulates or enhances the immune response of a pig against mycoplasma hyopneumoniae only. The vaccine composition comprises an immunizing amount of an attenuated live vaccine, inactivated vaccine, subunit vaccine or synthetic peptide vaccine of a mycoplasma hyopneumoniae strain.

The term "inactivated vaccine", also known as inactivated vaccine, as used herein, refers to a suspension of inactivated virus used as an antigen to generate immunity. Examples of inactivated vaccines include whole virus vaccines and split vaccines. Inactivated vaccines can be readily produced using known methods. For example, whole virus inactivated vaccines can be obtained by treating the virus with formaldehyde solution. Split vaccines can be prepared from the viral envelope after treatment with ether. For example, the mycoplasma hyopneumoniae GZ strain of the invention can be used for preparing inactivated vaccines by an inactivation method.

In one embodiment of the present invention, in the vaccine composition of the present invention, the inactivated antigen of the mycoplasma hyopneumoniae GZ strain or the culture thereof is an inactivated whole mycoplasma antigen or a lysis antigen thereof, the live whole mycoplasma antigen of the mycoplasma hyopneumoniae GZ strain or the culture thereof is an attenuated live whole mycoplasma antigen, and the subunit antigens of the mycoplasma hyopneumoniae GZ strain or the culture thereof are antigen proteins P97, P110, P46, and P36.

As an embodiment of the present invention, the inactivated whole Mycoplasma hyopneumoniae GZ strain or culture thereof of the present invention can be prepared by an inactivation method known in the art.

As an embodiment of the present invention, the lytic antigen of the Mycoplasma hyopneumoniae GZ strain or culture thereof of the present invention can be treated with a nonionic surfactant to obtain and enrich the antigenic component on the cell membrane.

As an embodiment of the present invention, the antigenic proteins P97, P110, P46 and P36 of the mycoplasma hyopneumoniae GZ strain or the culture thereof of the present invention can be prepared by genetic engineering methods or can be directly synthesized.

In one embodiment of the present invention, the content of the inactivated antigen of the mycoplasma hyopneumoniae GZ strain or the culture thereof in the vaccine composition of the present invention is not less than 10 before inactivation^7.0CCU/ml。

In a preferred embodiment of the present invention, the content of the inactivated antigen of the mycoplasma hyopneumoniae GZ strain or the culture thereof in the vaccine composition of the present invention is 10 before inactivation^7.0～10^10.0CCU/ml. The term "adjuvant" refers to a substance added to the composition of the present invention to increase the immunogenicity of the composition.

As one embodiment of the present invention, in the vaccine composition of the present invention, the pharmaceutically acceptable carrier includes an adjuvant; the adjuvant comprises: (1) alumino-gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide and Gel adjuvant.

Preferably, the saponin is Quil A, QS-21, GPI-0100.

Preferably, the emulsion is an SPT emulsion, an MF59 emulsion, or an emulsion formed from an oil in combination with an emulsifier, the emulsion may be based on light liquid paraffin oil, isoprenoid oil resulting from the oligomerization of olefins (such as squalane or squalene oil, oil resulting from the oligomerization of olefins, in particular isobutene or decene), linear alkyl-containing esters of acids or alcohols (more particularly vegetable oil, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate), esters of branched fatty acids or alcohols (in particular isostearate); the emulsifier is a nonionic surfactant (especially esters of polyoxyethylated fatty acids (e.g. oleic acid), sorbitan, mannide (e.g. anhydrous mannitol oleate), aliphatic diols, glycerol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which may be ethoxylated, ethers of fatty alcohols and polyhydric alcohols (e.g. oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers (especially

And particularly L121)).

Preferably, the polymer of acrylic or methacrylic acid is a crosslinked polymer of acrylic or methacrylic acid, in particular a compound carbomer crosslinked with polyalkenyl ethers or polyalcohols of sugars, preferably carbopol 974P, 934P and 971P.

Preferably, the copolymer of maleic anhydride and alkenyl derivative is a copolymer EMA of maleic anhydride and ethylene.

Preferably, the adjuvant comprises Gel01, ISA206, ISA760VG, carbomer, aluminum hydroxide.

The concentration of the adjuvant ranges from 10% to 70% V/V, preferably 10% V/V.

Known adjuvants include, but are not limited to: (1) aluminium hydroxide, saponins (saponin) (e.g. QuilA), alfuzidine, DDA, (2) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivatives, or (3) vaccines can be made in the form of oil-in-water, water-in-oil or water-in-oil-in-water emulsions.

In particular, the emulsion may be based on light liquid paraffin oil, isoprenoid oil, such as squalane or squalene; oils resulting from the oligomerization of olefins, in particular isobutene or decene, esters of acids or alcohols with linear alkyl groups, more in particular vegetable oils, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate), propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of polyoxyethylated fatty acids (e.g.oleic acid), sorbitan, mannitol (e.g.anhydromannitol oleate), glycerol, polyglycerol, propylene glycol and optionally ethoxylated oleic acid, isostearic acid, ricinoleic acid, hydroxystearic acid, ethers of fatty alcohols and polyols (e.g.oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic R, especially L121 (see Hunter et al, 1995, "The Theory and Practical applications of Advances" (Steward-Tull, D.E.S. eds.) John Wiley and sons, NY, 51-94; Todd et al, Vaccine, 1997, 15, 564 + 570).

In particular, the acrylic or methacrylic acid polymers are crosslinked by polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers.

The adjuvant used in the invention also comprises one or a combination of more of Gel01 (SEPPIC, France), ISA206 (SEPPIC, France) and ISA760VG (SEPPIC, France).

Preferably, the vaccine adjuvant according to the present invention is Carbomer (Carbomer) (trade name Carbopol), Gel01 (SEPPIC, france), aluminum hydroxide.

Most preferably, the vaccine adjuvant of the invention is Gel01 (SEPPIC, france).

The concentration of the adjuvant in the final vaccine composition ranges from 10% to 70% V/V, preferably 10% V/V.

As an embodiment of the present invention, the vaccine composition of the present invention further comprises a drug, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant and a preservative.

Preferably, the immunostimulant comprises alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor and interleukin 2. The vaccine composition of the present invention may further comprise other agents added to the composition of the present invention. To prepare such compositions, methods well known in the art may be used. As an embodiment of the present invention, the vaccine composition of the present invention further comprises other pathogenic antigens, including hog cholera virus antigen, porcine pseudorabies virus antigen, porcine circovirus antigen, haemophilus parasuis antigen, streptococcus suis antigen, swine influenza antigen, porcine contagious pleuropneumonia antigen, swine pasteurella multocida antigen, bordetella suis antigen, porcine reproductive and respiratory syndrome virus antigen, salmonella choleraesuis antigen, porcine parvovirus antigen, and/or porcine encephalitis b virus antigen. As an embodiment of the invention, the vaccine composition further comprises an immunizing amount of any one antigen selected from the group consisting of: porcine circovirus antigen, haemophilus parasuis antigen.

As an embodiment of the present invention, the porcine circovirus antigen includes a porcine circovirus type 2 ZJ/H strain antigen, a porcine circovirus type ii DBN-SX07 strain antigen, a porcine circovirus type 2SD strain antigen, a porcine circovirus type 2 ZJ/C strain antigen, a porcine circovirus type 2 strain PCV2SD strain antigen, a porcine circovirus type 2SH strain antigen, or a porcine circovirus type ii PCV2/HZ09 strain antigen; the Haemophilus parasuis antigens comprise a serum 13 type Haemophilus parasuis GX0905 strain antigen, a serum 5 type Haemophilus parasuis JX1002 strain antigen, a serum 4 type Haemophilus parasuis HN1009 strain antigen, a serum 4 type Haemophilus parasuis YBH04 strain antigen, a serum 5 type Haemophilus parasuis YBH05 strain antigen, a serum 13 type Haemophilus parasuis YBH13 strain antigen, a serum 1 type Haemophilus parasuis LC strain antigen, a serum 12 type Haemophilus parasuis SHCM10 strain antigen, a serum 5 type Haemophilus parasuis JYZ 10 strain antigen, a serum 13 type Haemophilus parasuis FJMH10 strain antigen, a serum 4 type Haemophilus parasuis FS0307 strain antigen, a serum 5 type Haemophilus parasuis XX0306 strain antigen, a serum 5 type Haemophilus parasuis LZ-20100109 strain antigen, a serum 5 type Haemophilus parasuis JS-5 strain antigen, a serum 5 type Haemophilus parasuis JS-JS-4 strain antigen, a serum type ZJ 5 strain antigen, Haemophilus parasuis serotype 12 HeB strain antigen or a combination thereof.

The porcine circovirus type 2 ZJ/H strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.6391 and disclosed in China patent application CN 102787100A; the porcine circovirus type II DBN-SX07 strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.3064 and disclosed in the Chinese patent application CN 101549155A; the porcine circovirus type 2SD strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.5774 and disclosed in China patent application CN 102732486A; porcine circovirus type 2 ZJ/C strains are preserved in China center for type culture Collection with the preservation number of CCTCC NO. V201251 and disclosed in China patent application CN 103285385A; the porcine circovirus type 2 strain PCV2SD is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.7707 and disclosed in China patent application CN 103421748A; the porcine circovirus type 2SH strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.2389 and is disclosed in China patent application CN 101240264A; the porcine circovirus type II PCV2/HZ09 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO. V201312 and disclosed in China patent application CN 103436498A.

The serum 13 type haemophilus parasuis GX0905 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2014125 and is disclosed in China patent application CN 104498384A; the serum 5 type haemophilus parasuis JX1002 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2014127 and is disclosed in China patent application CN 104388340A; the serotype 4 haemophilus parasuis HN1009 strain is deposited in China center for type culture Collection with the preservation number of CCTCC NO. M2014126 and disclosed in China patent application CN 104312964A; serum 4 type haemophilus parasuis YBH04 strain, serum 5 type haemophilus parasuis YBH05 strain and serum 13 type haemophilus parasuis YBH13 strain are preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number YBH04 strain is CGMCC NO.5479, the preservation number YBH05 strain is CGMCC NO.5480 and the preservation number YBH13 strain is CGMCC NO.5501, which are disclosed in Chinese patent application CN 102499982A; the serum 1 type haemophilus parasuis LC strain is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.5257 and disclosed in the China patent application CN 102399724A; the serum 12 type haemophilus parasuis SHCM10 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2014261 and disclosed in China patent application CN 104450556A; the serum 5 type haemophilus parasuis JSYZ10 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2014260 and is disclosed in China patent application CN 104450557A; the serum 13 type haemophilus parasuis FJMH10 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2014262 and is disclosed in Chinese patent application CN 104450555A; the serum 4 type haemophilus parasuis FS0307 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2013094 and disclosed in China patent application CN 103194413A; the serotype 5 haemophilus parasuis XX0306 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2013095 and is disclosed in China patent application CN 103194412A; the haemophilus parasuis LZ-20100109 strain of serum 5 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.5802 and disclosed in the China patent application CN 102851249A; the serum 5 type haemophilus parasuis LX-5 strain is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.10230 and disclosed in the China patent application CN 104611274A; the JS strain of haemophilus parasuis serotype 4, the ZJ strain of haemophilus parasuis serotype 5 and the HeB strain of haemophilus parasuis serotype 12 are preserved in the China center for type culture Collection, the JS strain with the preservation number of 4 is CCTCC NO. M2011172, the ZJ strain with the preservation number of 5 is CCTCC NO. M2011173, the HeB strain with the preservation number of 12 is CCTCC NO. M2011174, and the JS strain with the preservation number of 12 is disclosed in China patent application CN 102908615A.

In one embodiment of the present invention, in the vaccine composition of the present invention, the porcine circovirus antigen is an inactivated porcine circovirus antigen or subunit antigen, and the content of the inactivated porcine circovirus is 5 × 10 before inactivation^5.0TCID₅₀The porcine circovirus subunit antigen content is 20 mu g/ml; the haemophilus parasuis antigen is inactivated haemophilus parasuis antigen, and the content of the inactivated haemophilus parasuis antigen is 2 multiplied by 10 before inactivation⁹CFU/ml。

In an embodiment of the present invention, in the vaccine composition of the present invention, the porcine circovirus antigen is an inactivated porcine circovirus SH strain antigen, and the inactivated haemophilus parasuis antigen is an inactivated haemophilus parasuis serotype 4 JS strain antigen and an inactivated haemophilus parasuis serotype 5 ZJ strain antigen. In one embodiment of the present invention, the vaccine composition of the present invention comprises an immunizing amount of whole mycoplasma hyopneumoniae GZ strain inactivated antigen and whole virus porcine circovirus SH strain inactivated antigen, wherein the content of the whole mycoplasma hyopneumoniae GZ strain inactivated antigen is 5 × 10 before inactivation⁸CCU/ml, the content of the inactivated porcine circovirus is 5 multiplied by 10 before inactivation^5.0TCID₅₀/ml。

In one embodiment of the present invention, the vaccine composition of the present invention comprises an immunizing amount of inactivated whole mycoplasma antigen of mycoplasma hyopneumoniae GZ strain and subunit antigen of porcine circovirus, wherein the content of the inactivated whole mycoplasma antigen of mycoplasma hyopneumoniae GZ strain is 5 × 10 before inactivation⁸CCU/ml, and the content of the porcine circovirus subunit antigen is 20 mu g/ml.

In one embodiment of the present invention, the vaccine composition according to the present invention comprises an immunizing amount of a whole mycoplasma antigen inactivated by mycoplasma hyopneumoniae GZ strain, a whole virus antigen inactivated by porcine circovirus SH strain, a whole bacterium antigen inactivated by JS strain type 4 haemophilus parasuis serotype, and a whole bacterium antigen inactivated by haemophilus parasuis serotype 5ZJ strain inactivated whole antigen, wherein the content of the inactivated mycoplasma hyopneumoniae GZ strain whole mycoplasma antigen is 5 multiplied by 10 before inactivation⁸CCU/ml, the content of the inactivated porcine circovirus is 5 multiplied by 10 before inactivation^5.0TCID₅₀The inactivated haemophilus parasuis serotype 4 JS strain antigen content is 2 multiplied by 10 before inactivation⁹CFU/ml, the content of the inactivated haemophilus parasuis serum 5 type ZJ strain antigen is 2 multiplied by 10 before inactivation⁹CFU/ml。

In one embodiment of the present invention, the vaccine composition of the present invention comprises an immunizing amount of whole mycoplasma antigen inactivated by mycoplasma hyopneumoniae GZ strain, porcine circovirus subunit antigen, whole mycoplasma antigen inactivated by Haemophilus parasuis serotype 4 JS strain, and whole mycoplasma antigen inactivated by Haemophilus parasuis serotype 5 ZJ strain, wherein the content of the whole mycoplasma antigen inactivated by the inactivated mycoplasma hyopneumoniae GZ strain is 5 × 10 before inactivation⁸CCU/ml, the porcine circovirus subunit antigen content is 20 mu g/ml, and the inactivated haemophilus parasuis serotype 4 JS strain antigen content is 2 x10 before inactivation⁹CFU/ml, the content of the inactivated haemophilus parasuis serum 5 type ZJ strain antigen is 2 multiplied by 10 before inactivation⁹CFU/ml。

The invention also relates to application of the vaccine composition in preparing a medicament for preventing and treating swine mycoplasma pneumonia and diseases related to swine mycoplasma pneumonia infection. The term "preventing" means blocking or delaying by the symptoms of its infection or disease associated with mycoplasma hyopneumoniae; the term "treatment" refers to a process by which the symptoms of an infection or disease associated with mycoplasma hyopneumoniae are alleviated or completely eliminated.

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 isolation and identification of Mycoplasma hyopneumoniae GZ Strain

1. Origin of disease material

20 portions of the pathological materials are derived from suspected mycoplasma hyopneumoniae lung lesion collected from a certain pig farm in Henan province in 2016.

2. Multiplex PCR identification of disease material

2.1 primer design and Synthesis

Multiplex PCR (Mycoplasma hyopneumoniae, Mycoplasma hyofloccosum and Mycoplasma hyorhinis) primer references (T.stakenbory et al. A multiplex PCR to identity Port Mycoplasma presentment in Broth Cultures) were synthesized:

mhp-f：5’-TTCAAAGGAGCCTTCAAGCTTC-3’；

mhr-f：5’-GGGAAGAAAAAAATTAGGTAGGG-3’；

mfl-f：5’-CGGGATGTAGCAATACATTCAG-3’；

m-r：5’-AGAGGCATGATGATTTGACGTC-3’；

the lengths of the amplified fragments of the mycoplasma hyopneumoniae, the mycoplasma hyorhinotracheae and the mycoplasma hyofloccosum are estimated to be 1000bp, 1129bp and 754bp respectively.

2.2DNA template extraction and PCR identification

Aseptically taking lung tissues at the junction of the disease and the health, extracting a DNA template by using an animal tissue genome extraction kit, and carrying out PCR amplification according to the following reaction system: 2 mul of template DNA, 10nmol/L of d NTP, 5.0 mul of 10 x buffer, 8pmol of upstream primer, 12pmol of downstream primer and 0.5 mul of Ex Tag polymerase, and adding double distilled water to 50 mul; the PCR reaction conditions are as follows: 5min at 95 ℃; amplifying for 30 cycles at 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1 min; extension at 72 ℃ for 10 min; storing at 2-8 deg.C. And (3) carrying out 1.0% agarose gel electrophoresis detection on the PCR product, and screening 5 parts of diseased lung tissues which are positive to the mycoplasma hyopneumoniae and free from the pollution of the mycoplasma hyorhinis and the mycoplasma hyofloccosum.

3. Isolation and culture of strains

Collecting lung with PCR (polymerase chain reaction) positive mycoplasma hyopneumoniae and without mycoplasma hyorhinis and mycoplasma hyofloccosum pollution, shearing the marginal tissue of diseased part in superclean bench with sterile surgical scissors, placing into sterile plate, shearing the diseased lung tissue into 1-2mm pieces, inoculating Friis broth, culturing at 37 deg.C, observing culture every dayThe pH of the liquid changes. When the culture solution changes color, 0.5ml of the first generation culture filtered by a 0.45 mu m filter is taken and inoculated into 1.5ml Friis broth culture medium containing 2000IU/ml of penicillin, the culture is continuously cultured at 37 ℃, after the culture medium turns yellow, the culture is directly taken for transplantation and subculture, if the culture changes color, smear is carried out, and gram staining and Switzerland staining and microscopic examination are respectively carried out. When no bacteria were found in gram stain and mycoplasma-like bacteria were found in Sweden stain, 0.2m1 culture was spread on the surface of Fris plate solid medium at 37 deg.C and 5% CO₂The culture was carried out under the conditions, and the presence or absence of mycoplasma-like colonies was observed daily. Inoculating single colony of mycoplasma to Fris broth, spreading 0.2m1 culture on the surface of Fris plate solid medium after the medium turns yellow, and placing at 37 deg.C and 5% CO₂The purification culture was performed under the conditions, and thus the purification culture was performed 2 times. And inoculating the mycoplasma-like colony grown by the last purification culture to a Fris liquid culture medium, and preserving the strain after the color of the culture medium turns yellow.

4. Biological characteristics of mycoplasma hyopneumoniae GZ strain

4.1 culture Properties

The GZ strain grows well in a Fris liquid medium (pH value is 7.4-7.6), the culture is cultured for 3-5 days at 37 ℃, and the culture p H is reduced to 6.8-7.0, so that the strain is slightly uniformly turbid. The titer of live bacteria in the culture can reach 10¹⁰～10¹¹CCU/ml (CCU is the unit of color change). Inoculating a solid culture medium, culturing at 37 ℃ for 3-5 days, and allowing the bacterial colony to grow into the culture medium, wherein the bacterial colony is in a typical fried egg shape and conforms to the bacterial colony morphology of mycoplasma.

4.2 morphological and biochemical Properties

The bacterium is gram-negative, and is in the shape of a ring, a sphere, a filiform, a dot, a rod or a dipolar object under the oil-scope observation after being stained by a bacterium liquid smear giemsa, and the like, and has no cell wall.

Biochemical tests were performed in the literature (Cao 28557zee. veterinary microbiology and immunology [ M ]. Beijing: Beijing university of agriculture publishers 1992.49-50, 126-129, 371-373.). Mainly carries out digitonin sensitivity test, urease test, glucose decomposition test, arginine hydrolysis test, triphenyltetrazolium chloride (TTC) reduction test, esculin hydrolysis test, mannitol decomposition test and film and spot formation test, and the results are shown in Table 1.

TABLE 1 Biochemical test results of Mhp GZ strain

Note + positive, -negative; in the digitonin sensitivity test, mycoplasma is the inhibitor with the size of more than 1 mm; TTC reduction test is negative for mycoplasma hyopneumoniae and positive for mycoplasma hyorhinis; (Cao 28557, ze. veterinary microbiology and immunology [ M ] Beijing: Beijing university of agriculture publishers 1992.49-50, 126-129, 371-373.).

4.3 serological Properties

Fris plates 0.1ml of log phase Mycoplasma hyopneumoniae culture, and 25. mu.l of undiluted rabbit anti-Mycoplasma hyopneumoniae serum was adsorbed onto a sterilized 6mm filter paper sheet, which was attached to the surface of the plate and cultured until the colonies were visible. Normal rabbit serum treated paper sheets served as negative controls. Culturing and observing the generation of colony inhibition rings around the circular paper sheets. The inhibition width reaches 2.42mm, and the mycoplasma hyopneumoniae is judged to be positive.

5. Isolation of pathogenicity of the Strain

Injecting 3 healthy susceptible pigs of 1-2 weeks old with 5ml (10 ml) of isolated strain culture via trachea⁸CCU/ml). 3 control pigs with the same conditions were placed and each trachea was injected with 5ml of medium as a blank control. The test pigs and the control pigs are separately fed. Feeding the chickens after the toxin is eliminated according to a conventional method, and the feed does not contain antibiotics. Body temperature was measured daily for 28 days. And 7, performing an autopsy 28 days after the challenge injection, and scoring the pulmonary disease change of the test pig according to the swine mycoplasma pneumonia pulmonary disease index scoring standard. The test group and the control group are subjected to lung lesion index difference analysis. Separating mycoplasma hyopneumoniae of the test pigs and the control pigs, and carrying out PCR identification on the separated strains.

The clinical symptom observation result shows that the test pigs continuously have the symptoms of cough, asthma and the like after 10 days of toxin attack, and the control pigs do not have the symptoms; after 28 days of virus challenge, the lung of the test pig has lesions of different degrees through the autopsy, and the control pig has no abnormality. The clinical observation of symptoms and the results of lung lesions are shown in Table 2.

And (3) separating mycoplasma hyopneumoniae of 3 pigs in the test group, carrying out PCR identification on the mycoplasma hyopneumoniae specific P46 gene amplified by the color-changing liquid culture of the pigs, and amplifying a 1200bp target band as a result to prove that the separated pathogen is the mycoplasma hyopneumoniae.

TABLE 2 results of the pathogenicity test of Mhp GZ strain on piglets

Note that in statistical analysis of differences, when compared among groups, the ones with the same letter indicate no significant difference, and the ones with different capital letters indicate very significant difference (P < 0.01).

The method is characterized in that the suspected mycoplasma hyopneumoniae microorganisms are separated from diseased lungs which have typical mycoplasma hyopneumoniae pneumonia lesions, are positive in mycoplasma hyopneumoniae through multiple PCR detection of the mycoplasma hyopneumoniae and are free from the pollution of the mycoplasma hyopneumoniae and the mycoplasma hyopneumoniae, the characteristics of the mycoplasma hyopneumoniae are met through various identifications, and growth inhibition tests, PCR identifications and sequencing results show that the microorganisms belong to the mycoplasma hyopneumoniae and are named as mycoplasma hyopneumoniae GZ strains. The culture content of the mycoplasma hyopneumoniae GZ strain in the Friis culture medium can reach 10¹⁰～10¹¹CCU has strong toxicity to healthy and susceptible pigs, and can cause typical clinical symptoms of mycoplasma pneumonia such as cough, asthma, dyspnea and the like and lung lesions.

Example 2 preparation of inactivated vaccine against Mycoplasma hyopneumoniae GZ Strain

1. Preparation of production strains

After the freeze-dried strain of the mycoplasma hyopneumoniae GZ strain is unsealed, inoculating a liquid culture medium according to the inoculation amount of 10%, culturing for 3-5 days at 37 ℃, harvesting when the pH value is reduced to 6.8-7.0, and performing pure inspection to obtain the first-level production seeds. Inoculating the first-stage seeds with a liquid nutrient medium according to the inoculation amount of 5%, culturing at 37 ℃ for 3-5 days, harvesting when the pH value is reduced to 6.8-7.0, and passing the seeds through pure inspection to serve as second-stage production seeds.

The liquid culture medium comprises 300ml of cattle heart extract (BD company), 360m of double distilled water 1, pH adjusted to 7.4, sterilized at 121 deg.C for 15 min, and the following components for filtration and sterilization: hank's balanced salt solution (10X) 40m1,0.25(W/V) phenol red 10m1, horse serum 200m1, 5% (W/V) hydrolyzed milk protein 100m1, 25% W/V yeast extract 20m1, 10000IU/ml penicillin 10 ml.

2. Culture preparation of bacterial liquid

Respectively inoculating qualified secondary seeds of the mycoplasma hyopneumoniae GZ strain into a liquid culture medium according to 5% (v/v), culturing for 3-5 days at 37 ℃, and harvesting when the pH value is reduced to 6.8-7.0.

3. Mycoplasma hyopneumoniae viable count assay

Subpackaging liquid culture medium containing phenol red indicator into 10ml penicillin bottles, arranging two rows, wherein each row comprises 13 penicillin bottles and 1.8ml penicillin bottles, inoculating 0.2ml of culture to be detected to the 1 st penicillin bottle, uniformly mixing, sequentially diluting by 10 times until the culture is diluted to the 12 th penicillin bottle, taking the 13 th bottle as a blank control, culturing at 37 ℃, recording the maximum number of bottles with color change till 21 days to judge the CCU titer, taking the average value of the two rows of results, and obtaining the result that the viable bacteria titer is 10¹¹CCU/ml。

4. Inactivation of bacterial liquid

And (3) carrying out pure inspection on the obtained bacterial liquid, counting viable bacteria, adding thimerosal with the final concentration of 0.01%, uniformly mixing, inactivating at 2-8 ℃ for 12 hours, and shaking for 3 times.

5. Inactivation test and sterility test

Inactivation test: inoculating 1ml of the inactivated bacterial liquid into 50ml of liquid culture medium, culturing at 37 ℃, transplanting once on each of days 5 and 10, continuously culturing and observing for 11 days after the last transplantation, wherein the pH value of the culture medium is not reduced, and the color of the culture medium is not changed.

And (4) sterile inspection: the test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the mixed bacteria do not grow.

6. Vaccine formulation

Mixing the qualified mycoplasma hyopneumoniae GZ inactivated antigen solution with Gel01 adjuvant at a ratio of 90:10(V/V), supplementing the volume with sterile PBS (pH 7.2), stirring at 300rpm/min for 30min, and preparing into 3 kinds of inactivated vaccines with different antigen contents, wherein the specific formula and content of the vaccine are shown in Table 3.

TABLE 3 inactivated vaccine formula and content of Mycoplasma hyopneumoniae

Example 3 examination of inactivated vaccine against porcine Mycoplasma pneumoniae GZ Strain

1. Character testing

The vaccine is a light yellow aqueous solution, a small amount of precipitate appears after long-term storage, and the vaccine is a uniform suspension after shaking.

2. Sterility testing

The bacteria-free growth is carried out according to the examination of the appendix of the current Chinese veterinary pharmacopoeia.

3. Safety inspection

3.1BALB/c mouse safety test

30 mice 16-18 g BALB/c are divided into 3 groups and 10 mice per group, the 1 st group, the 2 nd group and the 3 rd group are injected with 0.5ml of vaccine 1, vaccine 2 and vaccine 3 respectively, after immunization, the mice are observed for 14 days, and whether the mice are healthy or not is observed, including food intake, drinking water, mental status, behavior status and the like.

The results show that 3 groups of BALB/c mice immunized by the 3 vaccines respectively have good mental state, normal food intake and drinking, no adverse reaction and no death. The inactivated vaccine for the mycoplasma hyopneumoniae is proved to be safe for mice.

3.2 safety testing of piglets

Selecting 15 healthy piglets of 14-21 days old mycoplasma hyopneumoniae negative pigs, dividing the piglets into 3 groups and 5 piglets/group, wherein the 1 st group, the 2 nd group and the 3 rd group are respectively injected with vaccine 1, vaccine 2 and vaccine 34 ml through neck muscle of each piglet, all piglets are continuously observed for 14 days after immunization, including whether the mental state, the food intake state, the drinking water state, the behavior state and the like are abnormal, whether the vaccine injection part is red and swollen and the like are observed, sampling the injection part after 14 days, making tissue slices, and observing whether inflammation, vaccine residue, granuloma and the like are existed. The result shows that no adverse reaction is seen on the vaccine immunized piglets within 14 days, the mental state is good, the food intake and the water drinking are normal, and no death condition exists. After the killing, all organs do not have abnormal changes, vaccine residues do not exist in the vaccine injection part in the autopsy, and abnormal conditions such as inflammation, granuloma, necrosis and the like do not exist in the tissue section observation. The inactivated vaccine for the mycoplasma hyopneumoniae with different antigen contents has better safety for piglets.

4. Efficacy test

4.1 efficacy testing of piglets

Selecting 20 healthy susceptible pigs with 14-21 days old mycoplasma hyopneumoniae ELISA antibody negatives and PRRSV antigen negatives, and randomly dividing the pigs into 4 groups. 1(1ml) of vaccine for intramuscular injection of neck in group 1, 2(1ml) of vaccine for intramuscular injection of neck in group 2, 2(1ml) of vaccine for intramuscular injection of neck in group 3, and 4 immunization as challenge control, and isolated breeding under the same conditions. After 35 days of immunization, injecting CVCC354 strain tissue virulent strain (which is a strain for the efficacy test of the mycoplasma pneumoniae vaccine preserved in the Chinese veterinary medicine supervision department) into all pig air tubes at a rate of 5 ml/head (100MID), observing for 28 days, dissecting and killing to obtain lungs, scoring the asthma pneumonia lesions of the test pigs according to a 28-division method, and calculating the pneumonia lesion reduction rate according to the following formula.

The pneumonia lesion reduction rate is (average score of virus attacking control pig pneumonia lesions-average score of immune pig pneumonia lesions)/average score of virus attacking control pig pneumonia lesions is multiplied by 100%

The protection results are shown in Table 4. The results show that compared with the challenge control group, the vaccine 1, the vaccine 2 and the vaccine 3 have very obvious difference in average lung lesion between the immune group and the control group, and show that the mycoplasma hyopneumoniae GZ strain has good immunogenicity.

TABLE 4 piglet efficacy testing and challenge protection results

Group of	Vaccine	Number of animals	Mean lung lesion index ± standard deviation	Reduction rate of pneumonia lesions
					Group 1	Vaccine 1	5	2.8±1.48^Bb	79％
Group 2	Vaccine 2	5	1.8±1.30^Bb	84.2％
					Group 3	Vaccine 3	5	0.8±0.84^Bc	92.9％
Group 4	Control of offensive toxin	5	11.4±5.12^Aa	/

Note: in the difference statistical analysis, compared among groups, the difference is not significant when the letters are the same, the difference is extremely significant when the capital letters are different (P < 0.01), and the difference is significant when the lowercase letters are different (P < 0.05)

Example 4 comparison of the efficacy of inactivated Mycoplasma hyopneumoniae vaccine (strain GZ) with imported vaccines

5 healthy piglets of 14-21 days old are immunized by the vaccine 1 prepared in the example 2, 1ml of the vaccine is used for one piglet, and the mycoplasma hyopneumoniae import inactivated vaccine 4(P strain, 7.5 multiplied by 10)⁸CCU/head) is used for immunizing 5 healthy piglets of 14-21 days old, 2 ml/head, 5 piglets are not immunized and are used as an offensive toxin control group, and isolated feeding is carried out under the same condition. After 35 days of immunization, injecting CVCC354 strain tissue virulent strain (which is a strain for the efficacy test of the mycoplasma pneumoniae vaccine preserved in the Chinese veterinary medicine supervision department) into all pig air tubes at a rate of 5 ml/head (100MID), observing for 28 days, dissecting and killing to obtain lungs, scoring the asthma pneumonia lesions of the test pigs according to a 28-division method, and calculating the pneumonia lesion reduction rate according to the following formula.

The protection results against toxic challenge are shown in Table 5. The result shows that compared with the vaccine 1 and the imported vaccine immunization group which attacks the virus control group, the average lung lesion of the immunization group and the average lung lesion of the control group have very obvious difference, and the protective effect of the vaccine 1 and the imported vaccine immunization group is equivalent and slightly higher than that of the imported vaccine. However, the content of the antigen used for achieving the above effects is far lower than that of the control vaccine group, which further indicates that the vaccine strain of the present invention has good immunogenicity.

TABLE 5 piglet immunoprotection results

Example 5 preparation of porcine circovirus type 2 antigen

1. Preparation of bacterial (toxin) species for production

The strain PCV2SH was diluted 1:9 with MEM liquid medium (prepared as described in the specification using MEM dry powder medium available from Invitrogen, USA), inoculated on PK15(ATCC, accession number CCL-33) monolayer at 5% of the cell culture volume, adsorbed at 37 ℃ for 30 minutes, and added with cell vitaminsHolding liquid (4% calf serum and 2mmol/L D-glucosamine hydrochloride are added into MEM liquid culture medium), culturing for 4 days at 37 ℃, freezing and thawing for 2-3 times, and harvesting virus with virus titer of 10^6.5TCID₅₀/ml。

2. Culture and preparation of virus liquid

The spinner flask cell culture method is used. Pouring out a monolayer of PK15 cells, pouring out a cell culture solution (6% calf serum and 2mmol/L D-glucosamine hydrochloride are added into MEM liquid culture medium), and mixing the volume ratio of venom solution according to 0.1-0.2 TCID₅₀The inoculation amount of each cell is inoculated on PK15 cells, the cells are adsorbed for 30 minutes at 37 ℃ by rotating a cell bottle for 2 weeks, and a cell maintaining solution is added and placed at 37 ℃ for rotary culture (10-12 r/h). Observing for 1-2 times every day, wherein the cell growth is good, culturing at 37 ℃ for 4 days, harvesting cells and cell sap, freezing and thawing for 3 times, and storing at the temperature below-20 ℃.

3. Treatment and concentration of virus liquid

The virus solution was filtered through a hollow fiber filter column (Millipore, Inc. pore size: 10 μm and 0.45 μm), and cell debris was removed and then concentrated 5-fold (volume ratio) with a Millipore (Millipore, Inc.) membrane module (molecular cut-off: 300 Kda).

4. Content determination of porcine circovirus SH strain virus liquid

Diluting the virus solution with MEM liquid culture medium by 10 times, and collecting 10 times^-5、10^-6、10^-73 dilutions were used, each dilution was inoculated into a monolayer of PK15 cells in 96-well culture plates (0.1 ml per well), and negative controls were set at 37 deg.C and 5% CO₂Continuously culturing for 24 hours in the incubator, changing the cell maintenance liquid, and continuously culturing for 24 hours; cells were fixed with cold acetone, the number of wells containing PCV2 positive cells (in green) at each dilution was determined by the indirect immunofluorescence assay (IFA), and the virus TCID was calculated according to the Karber method₅₀The result was 5X 10^6.0TCID₅₀/ml。

5. Inactivation of virus liquid and determination of inactivation effect

Adding the qualified virus liquid into a formaldehyde solution to ensure that the final concentration of the formaldehyde solution is 0.2% (V/V), inactivating the virus liquid at 37 ℃ for 18 hours, stirring the virus liquid for 1 time every 4 hours for 10min every time, and storing the inactivated virus liquid at 2-8 ℃ after inactivation is finished.

Inoculating a small amount of inactivated virus liquid to PK15 cells which grow into a monolayer, adsorbing the inactivated virus liquid for 1 hour at 37 ℃, then abandoning the virus liquid, adding new cell maintenance liquid, culturing the cells at 37 ℃ for 2 days without cytopathic effect (CPE), continuously conducting blind propagation for 3 times, changing the cells into the cell maintenance liquid after growing into the monolayer, culturing the cells at 37 ℃ for 2 days, detecting by using an indirect Immunofluorescence (IFA) method, generating no green PCV2 positive cells, and indicating that the inactivation is thorough.

Example 6 preparation of porcine circovirus type 2 subunit antigen

PCV2 protein virus-like particle antigen was prepared according to patent CN103173470A at a content of 1.3 mg/ml.

Example 7 preparation of Haemophilus parasuis antigens

1. Preparation of bacterial (toxin) species for production

A JS strain of Haemophilus parasuis 4 and a ZJ strain of Haemophilus parasuis 5 are streaked and inoculated on a tryptic soy agar (TSA, purchased from American BD company) plate (TSA/NAD plate for short) containing 5 percent of newborn bovine serum and 0.005 percent of nicotinamide adenine dinucleotide (NAD, American BBI company), the plate is cultured for 24 to 48 hours at 37 ℃, more than 5 typical colonies are respectively selected and used as a first-class seed after pure inspection is qualified. The primary seed was inoculated into a tryptic soy broth (TSA, obtained from BD, USA) (TSB/NAD broth for short) containing 5% of newborn bovine serum and 0.005% of nicotinamide adenine dinucleotide (NAD, BBI, USA), cultured with shaking at 180rpm in a shaker at 37 ℃ for 12 hours,

and simultaneously, sampling and gram staining are carried out, the shape of the bacteria is uniform when observed under a microscope, the morphology accords with the morphological characteristics of haemophilus parasuis, and no mixed bacteria grow and are used as secondary seeds.

2. Preparation of Haemophilus parasuis (JS type 4 and ZJ type 5) bacterial liquid

And respectively inoculating the qualified secondary seeds of the JS strain of the haemophilus parasuis 4 type and the ZJ strain of the haemophilus parasuis 5 type into a TSB/NAD liquid culture medium according to a ratio of 1:100(v/v), and culturing the secondary seeds by a shaker at 37 ℃ and 200 rpm. Culturing for 12-16 hours, and harvesting.

3. Treatment of bacterial liquid

The haemophilus parasuis type 4 ZJ strain and the type 5 JS strain are respectively centrifuged by a continuous centrifuge (10000rpm/min), then dissolved again by PBS (pH value is 7.2-7.4) to the volume before centrifugation, and concentrated by 5 times (volume ratio) by a membrane package (molecular cut-off is 300KDa) of Millipore (Millipore company).

4. Determination of viable count of bacterium liquid of haemophilus parasuis type 4 ZJ strain and type 5 JS strain

Sampling bacterial liquid, diluting 10 times in series according to method known by skilled person, and taking 10 times^-6、10^-7Inoculating 2 dilutions into the above TSA/NAD solid culture medium, inoculating 0.1ml of each plate, culturing at 37 deg.C for 24 hr, selecting the plate with colony number between 30 and 300, and counting bacterial strains of type 4 ZJ strain and type 5 JS strain by 5 × 10⁹CFU/ml。

5. Inactivation of bacterial liquid and inactivation test

And adding the two prepared bacterial liquids of different serotypes into a formaldehyde solution to enable the final concentration of the formaldehyde solution to be 0.3% (V/V), inactivating the formaldehyde solution for 24 hours at 37 ℃, stirring the solution for 1 time every 4 hours every time, and storing the inactivated bacterial liquid at 2-8 ℃ after inactivation is finished.

Preparing 6 plates of TSA/NAD solid culture medium, dropwise adding 1 drop of inactivated bacteria liquid on 3 plates of TSA/NAD solid culture medium in aseptic operation, streaking with an inoculating ring, culturing in a common incubator at 37 ℃, and simultaneously setting up 3 non-inoculated TSA/NAD solid culture medium as a control. After 24 hours, the plates were observed to have no bacterial growth, while the control, 2 media without inoculation, had no bacterial growth, and continued observation until 6 plates had no bacterial growth at 48 hours, indicating complete inactivation.

EXAMPLE 8 preparation of vaccine composition of porcine circovirus antigen, Haemophilus parasuis antigen, Mycoplasma hyopneumoniae antigen

Taking the mycoplasma hyopneumoniae antigen, the PCV2 antigen, the PCV2 subunit antigen and the haemophilus parasuis antigen prepared in the embodiments 2, 4, 5 and 6, preparing four antigen solutions into mixed antigen solutions according to the final antigen content of combined vaccine or directly preparing the antigen solutions, then mixing the antigen solutions with a Gel01 adjuvant (produced by SEPPIC, Sebec, France) according to a ratio of 90:10(V/V), supplementing the volume with PBS solution with a pH of 7.2, stirring at 500rpm/min for 30min, and respectively preparing 5 different inactivated vaccines, wherein the specific formula and content of the vaccines are shown in Table 6.

TABLE 6 vaccine composition formulations and amounts

The 5 prepared vaccines are respectively qualified in character, sterility and safety inspection and can be used for subsequent tests.

Example 9 test of the immunological Effect of vaccine compositions comprising different porcine circovirus type 2 antigen, Haemophilus parasuis antigen, and Mycoplasma hyopneumoniae antigen on pigs

1. Vaccine for test immunization of piglets

100 piglets of 14-21 years old are selected, 20 groups are selected, and 5 piglets are selected. Immunization group 15, wherein group 8, group 9, group 10, group 11, group 12, group 13, and group 14 immunization vaccines 5, group 15, group 16, group 17, and group 18 immunization vaccines 8, group 19, group 20, group 21, and group 22 immunization vaccines 9; and a control group 5, wherein the 23 rd, 24 th, 25 th and 26 th groups are challenge controls, and the 27 th group is a normal control. Specific immunization groups are shown in table 7.

TABLE 7 immunization groups

2. Attacking poison after immunization of piglets

2.1PCV2 challenge

At 35d after immunization, the immunized group was collected20 pigs in 4 groups of 8 th group, 10 th group, 15 th group and 19 th group, 5 pigs in 1 group of 23 th group control group, and PCV2SH strain (containing 10) respectively used for piglets in immune group and piglets in control group^6.0TCID₅₀(ml) 1 ml/head of nasal drip, 2 ml/head of intramuscular injection, 4 th and 7 th days after challenge, inoculating all pigs with keyhole limpet hemocyanin (KLH/ICFA, 0.5mg/ml) emulsified with Freund's incomplete adjuvant at 4 points on both axilla and both buttocks of each challenge pig, inoculating 1ml (4 ml/head) of each point, and simultaneously inoculating thioglycolic acid culture medium to the abdominal cavity, 10 ml/head; the thioglycollic acid culture medium is inoculated to the abdominal cavity again at the 11 th day and the 19 th day after the challenge, and the culture medium is 10 ml/head. Continuously observing for 25 days after challenge, weighing for killing on 25 th day after challenge, and performing autopsy. And judging according to the body temperature, the relative daily gain and the detection result of the virus antigen.

2.2 Haemophilus parasuis type 4, 5 counteracting toxic substances

At 35d after immunization, 15 piglets of 12 th group, 16 th group and 20 th group of 3 groups of the immunization group and 5 piglets of 1 group of 24 th control group of piglets are taken, 4-type strains are used for counteracting the toxin, 3ml of bacterial liquid is injected into the abdominal cavity, and the counteracting dose is 4 type 9.0 multiplied by 10⁹CFU/head, type 5, 6.0X 10⁹CFU/head, observing the clinical expression after attacking the toxin, and observing the test pig which is killed after 14 days to carry out pathological observation; taking 15 piglets of 13 th group, 17 th group and 21 st group, 3 piglets of 1 piglet group and 5 piglets of 25 th control group, performing virus killing with 5-type strain, and performing intraperitoneal injection with 3ml of bacterial liquid, wherein the virus killing dose is 5 type 6.0 × 10⁹CFU/head, observing the clinical manifestations after attacking, observing the test pigs killed 14 days later, and carrying out pathological observation.

2.3 Swine pneumonia Mycoplasma attacking toxin

At 35d after immunization, 25 piglets of the 9 th group, the 11 th group, the 14 th group, the 18 th group and the 22 th group of the 5 th group and 5 piglets of the 26 th group of the 5 th group of the immune group are taken to carry out a mycoplasma hyopneumoniae challenge test, the piglets of the immune group and the control group are injected with 5 ml/head (100MID) of CVCC354 strain (purchased from China veterinary medicine institute, the strain is a strain for swine mycoplasma pneumoniae vaccine efficacy test preserved in China veterinary medicine institute) through air pipes, after the challenge, the piglets are observed for 28 days, lungs are cut and killed, asthma pneumonia lesions of the test pigs are scored according to a 28-division method, and the pneumonia lesion reduction rate is calculated according to the following formula.

3. Toxic pathogen attacking the body

3.1PCV2 challenge

After PCV2 challenge, the body temperature change condition is shown in Table 8, only individual pigs of the vaccine 5, the vaccine 6, the vaccine 8 and the vaccine 9 immune group piglets have a transient body temperature rise phenomenon, the body temperature rises for one day and then returns to normal quickly, and no other clinical symptoms exist; after the control group pigs are detoxified, the body temperature of all pigs is raised by more than 40.5 ℃ for 3-5 days, and the appetite is reduced, the spirit is depressed, the hair is rough, the weight is reduced and the growth speed is slowed down.

The onset and protection after the attack of toxic materials are shown in tables 9 and 10. After the vaccine 5, the vaccine 6, the vaccine 8 and the vaccine 9 are used for immunizing piglets, no obvious clinical symptoms exist, no specific pathological change is observed, the antigen PCR detection is negative, and the protection effect reaches 5/5; the piglets in the control group are all attacked, the clinical symptoms and the pathological changes are obvious, and the pathogen PCR detection is positive. And when the challenge observation is finished, the average daily gain of the piglets immunized by the vaccine 5, the vaccine 6, the vaccine 8 and the vaccine 9 has no significant difference, and compared with a control group, the average daily gain of the piglets immunized by the vaccine 5, the vaccine 6, the vaccine 8 and the vaccine 9 is significantly higher than that of the control group. The vaccine 5, the vaccine 6, the vaccine 8 and the vaccine 9 have good virus attack protection effect on PCV2 after immunization.

TABLE 8 comparison of days after PCV2 challenge when the temperatures of various test pigs exceeded 40.5 deg.C

TABLE 9 results of disease determination of groups of test animals after PCV2 challenge

PCV2 accords with any 2 items in the following 3 items after being attacked by toxin, and then the disease can be judged to be the onset of disease.

A: clinical symptoms: the body temperature of the piglets is increased (more than or equal to 40 ℃), and the piglets are kept for at least 3 days, so that the appetite is obviously reduced, the spirit is greatly depressed, the fur is rough and disordered, the weight is reduced, and the growth speed is slowed down;

b: pathological changes are as follows: inguinal and tracheal lymph node edema, mild edema of the lung, yellow or somewhat necrotic kidneys.

The histological lesion is that lymph nodes have obvious lymphocyte invasion or multinucleated giant cells;

c: and (3) virus detection: lymph node tissue was detected by PCR and PCV2 was detected.

TABLE 10 post-challenge protection of immunized piglets PCV2

Group of	Number of piglets	Protective rate of offensive	Average daily gain (kg)
				Group 8	5	5/5	0.0251^Bb
Group 10	5	5/5	0.0255^Bb
				Group 15	5	5/5	0.0249^Bb
Group 19	5	5/5	0.0253^Bb
				Group 23	5	0/5	0.0149^Aa

3.2 Haemophilus parasuis attacking toxin

After the haemophilus parasuis type 4, 5 strains were challenged, the test results are shown in table 11. The results showed that the challenge protection of vaccine 3, vaccine 4 and vaccine 5 against JS strain type 4 and ZJ strain type 5 was 4/5 (80%) to 5/5 (100%), respectively.

TABLE 11 protection of immunized piglets after challenge with Haemophilus parasuis

Group of	Number of animals (head)	Virus attacking protection rate of type 4 JS strain	Type 5 ZJ strain toxicity attacking protection rate
				Group 12	5	5/5	/
Group 13	5	/	4/5
				Group 16	5	5/5	/
Group 17	5	/	5/5
				Group 20	5	5/5	/
Group 21	5	/	5/5
				Group 24	5	0/5	/
Group 25	5	/	0/5

Note: the pathogenic standard of haemophilus parasuis disease: the sick pigs have clinical symptoms of fever (body temperature of more than 40.5 ℃ and lasting for 1-5 days), listlessness, cough, dyspnea, emaciation, lameness, rough fur and the like. The autopsy of the dying pig shows the pathological changes of multiple serositis (pleuritis, pericarditis and peritonitis), arthritis, meningitis and the like, and serous or cellulosic exudates appear on each serosal surface (joint capsule, pericardium, pleura and peritoneum).

3.3 Swine pneumonia mycoplasma counteraction

The protective results of the mycoplasma hyopneumoniae challenge are shown in table 12. The results show that compared with the challenge control group, the average lung lesion of the immune group and the average lung lesion of the control group of each vaccine composition have very significant difference.

TABLE 12 piglet efficacy testing challenge protection results

Group of	Number of animals	Mean lung lesion index ± standard deviation	Reduction rate of pneumonia lesions
				Group 9	5	2.4±1.14^Bb	85.6％
Group 11	5	2.0±0.71^Bb	85.0％
				Group 14	5	2.2±0.84^Bb	84.3％
Group 18	5	1.8±1.30^Bb	84.7％
				Group 22	5	1.6±1.14^Bb	86.4％
Group 26	5	11.8±3.96^Aa	/

In conclusion, from the virus attack protection results of the different vaccine compositions on PCV2, the JS strain of Haemophilus parasuis 4 type, the ZJ strain of Haemophilus parasuis 5 type and the mycoplasma hyopneumoniae, the vaccine compositions can achieve better protection effects, and the result shows that the immune protection effect of a single vaccine can be still achieved or exceeded when the new separated mycoplasma hyopneumoniae GZ strain, the PCV2 antigen, the PCV2 subunit antigen, the JS strain of Haemophilus parasuis 4 type and the ZJ strain antigen of the invention are prepared into different vaccine compositions, and the vaccine compositions are not influenced by other antigens.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The mycoplasma hyopneumoniae GZ strain has the preservation number of CCTCC NO: M2016212.

2. A vaccine composition comprising an immunizing amount of the inactivated antigen of mycoplasma hyopneumoniae GZ strain of claim 1 or culture thereof, and a pharmaceutically acceptable carrier.

3. The vaccine composition according to claim 2, wherein the culture of the mycoplasma hyopneumoniae GZ strain is a 1-42 generation culture; the inactivated antigen of the mycoplasma hyopneumoniae GZ strain or the culture thereof is inactivated whole mycoplasma antigen or a split antigen thereof.

4. The vaccine composition according to claim 2, wherein the content of inactivated antigen of the mycoplasma hyopneumoniae GZ strain or the culture thereof is not less than 10 before inactivation^7.0CCU/ml。

5. The vaccine composition according to claim 2, wherein the content of inactivated antigen of the mycoplasma hyopneumoniae GZ strain or the culture thereof is 10 before inactivation^7.0～10^10.0CCU/ml。

6. The vaccine composition according to claim 2, wherein the pharmaceutically acceptable carrier comprises an adjuvant, and the adjuvant is a Gel adjuvant.

7. The vaccine composition of claim 6, wherein the concentration of the adjuvant ranges from 10% to 70% V/V.

8. The vaccine composition of claim 7, wherein the concentration of the adjuvant is 10% V/V.

9. The vaccine composition of claim 2, wherein the vaccine composition further comprises an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative.

10. The vaccine composition of claim 9, wherein the immunostimulant comprises alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor, and interleukin 2.

11. The vaccine composition of claim 2, wherein the vaccine composition further comprises other pathogenic antigens, including classical swine fever virus antigen, porcine pseudorabies virus antigen, porcine circovirus antigen, haemophilus parasuis antigen, streptococcus suis antigen, swine influenza antigen, porcine contagious pleuropneumonia antigen, pasteurella suicidae antigen, bordetella suis antigen, porcine reproductive and respiratory syndrome virus antigen, salmonella choleraesuis antigen, porcine parvovirus antigen, and/or porcine encephalitis b virus antigen.

12. The vaccine composition of claim 11, wherein the porcine circovirus antigen comprises a porcine circovirus type 2SH strain antigen;

the haemophilus parasuis antigen comprises a JS strain antigen of haemophilus parasuis serum type 4, a ZJ strain antigen of haemophilus parasuis serum type 5, a HeB strain antigen of haemophilus parasuis serum type 12 or a combination thereof.

13. The vaccine composition according to claim 11, wherein the porcine circovirus antigen is an inactivated porcine circovirus antigen or subunit antigen, and the inactivated porcine circovirus content is 5 x10 prior to inactivation^5.0TCID₅₀The porcine circovirus subunit antigen content is 20 mu g/ml; the haemophilus parasuis antigen is inactivated haemophilus parasuis antigen, and the content of the inactivated haemophilus parasuis antigen is 2 multiplied by 10 before inactivation⁹CFU/ml。

14. The vaccine composition of claim 11, wherein the porcine circovirus antigen is an inactivated porcine circovirus SH strain antigen, and the inactivated haemophilus parasuis antigen is an inactivated haemophilus parasuis serotype 4 antigen JS strain antigen and an inactivated haemophilus parasuis serotype 5 ZJ strain antigen.

15. Use of a vaccine composition according to any one of claims 2 to 14 in the manufacture of a medicament for the prevention of mycoplasma hyopneumoniae and diseases associated with mycoplasma hyopneumoniae infection.