CN102988978A

CN102988978A - Vaccine composition containing porcine circovirus type 2 antigen and haemophilus parasuis antigen, as well as preparation method and application thereof

Info

Publication number: CN102988978A
Application number: CN2012101808420A
Authority: CN
Inventors: 张许科; 孙进忠; 白朝勇
Original assignee: Pulaike Biological Engineering Co Ltd
Current assignee: Pulaike Biological Engineering Co Ltd
Priority date: 2011-08-01
Filing date: 2012-06-04
Publication date: 2013-03-27
Anticipated expiration: 2032-06-04
Also published as: CN105327344A; CN105327344B; CN102988978B

Abstract

The invention relates to a polyvalent vaccine composition for porcine, and in particular relates to a vaccine composition capable of resisting infection of porcine circovirus type 2 (PCV 2) and haemophilus parasuis (HPS) at the same time. The vaccine composition comprises at least one PCV2 antigen, at least one HPS, as well as vector, an excipient and an adjuvant available in the field of veterinary medicine. The vaccine composition can be used for preventing and treating PCV2 related diseases and HPS diseases.

Description

The vaccine combination and preparation method thereof and application that contain porcine circovirus 2 type antigen and haemophilus parasuis antigen

Technical field

The present invention relates to a boar vaccine combination, be specifically related to a kind of vaccine combination of resisting simultaneously porcine circovirus 2 type (PCV2) and haemophilus parasuis (HPS) infection and preparation method thereof and application.

Background technology

Pig circular ring virus (PCV) is the animal virus of a kind of minimum of finding so far.Existing known PCV has two serotypes, i.e. PCV1 and PCV2.PCV1 is the virus of non-pathogenic.PCV2 is pathogenic virus, and it is the main pathogen of pmws (PMWS).The Clinical symptoms of PMWS is become thin, palor, dyspnea, diarrhoea, yellow cellulitis etc.Pathological change visible lymph nodes of body as a whole enlargement, particularly inguinal lymph nodes enlargement can reach 5～10 times; Lung mainly is dispersivity, interstitial pneumonia changes, and quality is hard such as rubber, and the surface generally is dun mottling outward appearance; The variation of kidney is more, and visible cortex and medullary substance are dispersed in white necrosis region not of uniform size, present wax sample outward appearance owing to edema causes it; The spleen silght enlargement; The liver of most sick pig has atrophy, fibrosis in various degree.Except PMWS, Corii Sus domestica scorching with nephrotic syndrome (PDNS), hypertrophy necrotizing pneumonia (PNP), porcine respiratory disease complex (PRDC), breeding difficulty, congenitally tremble, the disease such as enteritis also has important related with the PCV2 infection.The swine diseases that PCV2 is relevant, mortality rate reaches 10%～30% not to be waited, and more serious pig farm death rate when breaking out causes serious economic loss up to 40% to pig industry.

Haemophilus parasuis claims again multiple fiber disposition oromeningitis and arthritis, also claims GlasserShi sick.This disease is to be caused by haemophilus parasuis.A haemophilus parasuis infected pigs, can affect from the young pig at 2 age to 4 monthly ages in week, mainly in wean front and back and the morbidity of child care stage, usually see the pig in 5～8 ages in week, sickness rate is generally 10%～15%, mortality rate can reach 50% clinical symptoms and depends on inflammation part when serious, comprises heating, dyspnea, arthroncus, limping, skin and mucosa cyanosis, dysstasia even paralysis, cad pig or death.The sow morbidity can be miscarried, and boar has limping.The limping of nursing sow may cause the extreme reduction of maternal instinct.Body surface is blue when dead, and belly is large, and a large amount of yellow ascites are arranged, and has a large amount of celluloses to ooze out on the mesentery, and especially liver is whole is wrapped, the interstitial edema of lung.

Lot of documents report, porcine circovirus 2 type and haemophilus parasuis mixed infection very general (Lu Xinghua etc., Chinese veterinary's magazine, 2010, (03): 46～48; Jia Songtao etc., Chinese animal and veterinary, 2008, (08): 99～100; Etc.).Therefore, need to resist simultaneously the vaccine combination that PCV2 infects and haemophilus parasuis infects.

Bao Chunxing etc. " reason and the countermeasure of the failure of simple analysis vaccine immunity " (animal and veterinary scientific and technological information 2010, (02) 8～12), the blanking time of two kinds of vaccinations of report is inadequate, or some raiser is loaded down with trivial details because of the epidemic prevention task, in order to save trouble, two kinds of vaccines are inoculated simultaneously, after vaccine enters body, may the inhibition of competing property between the vaccine antigen, cause immuning failure.For fear of immuning failure, the strategy of the difference immunity of PCV2 vaccine and haemophilus parasuis vaccine is generally taked on the pig farm, the interval of two kinds of vaccine immunities is more than 15 days, to avoid two kinds of interference between the antigen, this just causes repeatedly duplicate injection, strengthened Animal stress and improved the epidemic prevention cost, the epidemic prevention effect is also imprecise.

Summary of the invention

The present invention is by a large amount of screening studies, and the vaccine combination that the unexpected antigen of finding porcine circovirus 2 type (PCV2) and haemophilus parasuis prepares, two kinds of antigen be mutual interfere with or compromise mutually; On the contrary; when the antigen composition of porcine circovirus 2 type (PCV2) and haemophilus parasuis and vaccine combination not only can produce better protective immune response; also the unexpected haemophilus parasuis of finding has strengthened the immune effect of porcine circovirus 2 type antigen (PCV2); particularly with the situation of haemophilus parasuis combination under, still can keep the immune effect of PCV2 even PCV2 antigen amount reduces by half.Therefore a first aspect of the present invention is to provide the vaccine combination of prevention or treatment porcine circovirus 2 type (PCV2) antigen and the infection of haemophilus parasuis antigen.

To achieve these goals, the antigen composition that the present invention's prevention or treatment porcine circovirus 2 type (PCV2) and haemophilus parasuis infect, it comprises at least a porcine circovirus 2 type antigen and at least a haemophilus parasuis antigen.

Porcine circovirus 2 type antigen of the present invention refers to contain any compositions of at least a PCV2 antigen form, and the immunne response that opposing PCV2 infects can be induced, stimulates or be strengthened to described antigen inoculation pig.Preferably, described PCV2 antigen is the PCV2 totivirus, contains the embedded virus of PCV2 immunogenicity aminoacid sequence (such as ORF2 albumen) that any other contains polypeptide or subunit or other compositions of PCV2 immunogenicity aminoacid sequence at least.PCV2 antigen can also comprise any antigen of following compositions: such as the Circovac of Cimmeria company (Merial) The Suvaxyn of Pfizer (Pfizer)

PCV2; The porcine circovirus 2 type inactivated vaccine (LG strain) of Harbin biotechnology development company of dimension section; The porcine circovirus 2 type inactivated vaccine of Fuzhou Dabeinong Bioisystech Co., Ltd (DBN-SX07 strain) and genetic engineering subunit vaccine are (such as the Ingelvac CircoFLEX of Boehringer Ingelheim company (Boehringer-Ingelheim)

)

Preferably, porcine circovirus 2 type antigen is the PCV2 totivirus antigen of deactivation among the present invention, wherein preferably uses PCV2 SH strain, and preserving number is CGMCC No.23890, is disclosed in patent documentation CN101240264A.

Because the PCV2 Genome Size is 1767bp or 1768bp; nucleotide sequence homology is higher between the different strains; all more than 90%, so PCV2SH strain (the GenBank accession number is AY686763), PCV2LG strain (the GenBank accession number is HM038034), PCV2 DBN-SX07-2(GenBank accession number are HM641752) antigen composition or vaccine combination all within protection domain of the present invention.

Preferably, every dosage or every part (2ml) contain PCV2 totivirus antigen deactivation provirus content and are at least 10 in the vaccine of the present invention ^5.5TCID ₅₀More preferably 4.5 * 10 ^5.0TCID ₅₀～10 ^6.0TCID ₅₀Preferred every dosage or every part (2ml) contain PCV2 virus 9 * 10 again ^5.0TCID ₅₀

Preferably, porcine circovirus 2 type antigen is polypeptide or subunit or other compositions that contains PCV2 immunogenicity aminoacid sequence among the present invention, and more preferably, porcine circovirus 2 type antigen is the PCV2 subunit antigen, most preferably, porcine circovirus 2 type antigen is Porcine circovirus type 2 ORF2 protein.Wherein, PCV2 ORF2 albumen of the present invention, the albumen of preferred sequence table SEQNO1.DNA sequential coding or the albumen of sequence table SEQ NO2. aminoacid sequence or with its homology at more than 75%, preferred more than 80%, the more preferably albumen of the aminoacid sequence more than 90%, for reaching the protein sequence of the object of the invention.The present invention has no particular limits PCV2 ORF2 albumen, as long as it has the immunogenicity that keeps PCV2, any PCV2 ORF2 albumen all can be used as the source of PCV2 ORF2DNA described herein and/or polypeptide.

Preferably, in the vaccine combination of the present invention, the content of described PCV2 ORF2 protein biology is 0.5 ~ 20 μ g/ head part, more preferably 5 ~ 10 μ g/ head parts

Haemophilus parasuis antigen refers to contain any compositions of at least a haemophilus parasuis antigen form, and the immunne response that the opposing haemophilus parasuis infects can be induced, stimulates or be strengthened to described haemophilus parasuis antigen inoculation pig.Haemophilus parasuis is the street strain that comprises clinical separation well known to those skilled in the art, comprises 1～15 serotype of having identified at present.Preferably, described haemophilus parasuis antigen is complete haemophilus parasuis, is preferably the deactivation form, and more preferably, the serotype that described haemophilus parasuis antigen is deactivation is the hybrid antigen of 4 types and 5 types; Most preferably be the haemophilus parasuis serum 4 type JS strains of deactivation, and the haemophilus parasuis Serotype 5 ZJ strain of deactivation.Wherein, haemophilus parasuis serum 4 type JS strains (Haemophilus parasuis Serotype 4, Strain JS) preserving number is: CCTCC NO:M 2011172; The preserving number of haemophilus parasuis Serotype 5 ZJ strain (Haemophilus parasuisSerotype 5, Strain ZJ) is: CCTCC NO:M 2011173; Preservation date is on May 18th, 2011, and the preservation address is: Wuhan, China, Wuhan University, Chinese Typical Representative culture collection center; Depositary institution is Chinese Typical Representative culture collection center (being called for short CCTCC).

Haemophilus parasuis antigen of the present invention can also comprise any antigen of following compositions: such as the Ingelvac HP-1 of Boehringer Ingelheim company; The Hiprasuis-Glasser of Hai Bolai company (Hipra); The haemophilus parasuis inactivated vaccine of Wuhan Keqian Animal Biological Products Co., Ltd. (4 type MD0322 strains+5 type SH0165 strains).

Just as is known to the person skilled in the art, the open-air separated strain of different microorganisms has small variation in gene order, yet, when variation does not affect its protein synthesis, structure or Main Function functional areas, can't be absolutely identical even he of this microorganism plants open-air separated strain gene order, its basic physiological function can't change to some extent.Therefore, often use in the world at present 16S Ribosomal RNA(16S rRNA) carry out the resolution of bacteria type, therefore can compare and obtain its homology with 16S rRNA relatively going up of similarity.So, having at 16S rRNA under the prerequisite of homology, haemophilus parasuis antigen used in the present invention is not limited to open-air separated strain used in the present invention,

16s rRNA gene is the corresponding DNA sequence of coding rRNA on the bacterial chromosome, is present in the germy chromogene group of institute.16S rRNA has conservative and specificity and this gene order long enough (comprising about 50 functional domains) of height

Therefore, the present invention includes and haemophilus parasuis two strain bacterial strain (JS strains, the ZJ strain) bacterial strain of the homology of 16SrRNA more than on 80%, more preferably with JS strain or ZJ strain 16SrRNA homology more than at least 90%, preferably more than at least 95%, the haemophilus parasuis bacterial strain more than at least 95%～99% more preferably, namely do not change haemophilus parasuis two strain bacterial strain (JS strains, the ZJ strain) under the prerequisite of 16S rRNA, those skilled in the art can be by simple screening or mutation haemophilus parasuis of the present invention, the bacterial strain of acquisition and haemophilus parasuis 16S rRNA of the present invention height homology, and obtain correspondingly to have same or similar immunogenic antigen composition.

Preferably, the 4 type haemophilus parasuises of serum described in the vaccine combination of the present invention are the JS strain, and preserving number is CCTCC No.M2011172.

Preferably, content is 1 * 10 before before the 4 type haemophilus parasuis JS strain deactivations of serum described in the vaccine combination of the present invention ⁹CFU/ head part～8 * 10 ⁹CFU/ head part; More preferably, content is 2 * 10 before the described haemophilus parasuis JS strain deactivation ⁹CFU/ head part, again content 1 * 10 before the preferred described serum 4 type haemophilus parasuis JS strain deactivations ⁹CFU/ head part.

Preferably, Serotype 5 haemophilus parasuis described in the vaccine combination of the present invention is the ZJ strain, and preserving number is CCTCC No.M2011173.

Preferably, content is 1 * 10 before the ZJ of Serotype 5 haemophilus parasuis described in the vaccine combination of the present invention strain deactivation ⁹CFU/ head part～8 * 10 ⁹CFU/ head part; More preferably, content is 1 * 10 before the described haemophilus parasuis ZJ strain deactivation ⁹CFU/ head part, again content 2 * 10 before the preferred described Serotype 5 haemophilus parasuis ZJ strain deactivation ⁹CFU/ head part.

Optimal way of the present invention relates to the adjuvant of vaccine combination, is preferably aluminium hydroxide gel, mineral oil, carbomer, Montanide ^TMGel, propolis, ISA206, ISA760VG, aluminium hydroxide, aluminum phosphate, saponin, vegetable oil, ethyl oleate, two (caprylic/capric) propylene glycol ester, three (caprylic/capric) glyceride, two oleic acid propylene glycol esters, isostearate, sorbitan, mannide, glycerol, polyglycereol, propylene glycol, oleic acid, isostearic acid, castor oil acid, hydroxy stearic acid ester, polyoxypropylene, polyoxyethylene block copolymer, the acrylic or methacrylic acid polymer, maleic anhydride and thiazolinyl derivant copolymer, the one or more combination thing of one or more of the polymer of the acrylic or methacrylic acid that the polyalkenyl ether of sugar or polyhydric alcohol is crosslinked.

Preferably, the vaccine combination of prevention provided by the invention and treatment pig circular ring virus relevant disease and Haemophilus parasuis contains PCV210 ^6.0TCID ₅₀/ head part, serum 4 type haemophilus parasuises 4 * 10 ⁹CFU/ head part contains Serotype 5 haemophilus parasuis 4 * 10 ⁹CFU/ head part, vaccine adjuvant are 10%(V/V) Montanide ^TMThe Gel adjuvant.

Vaccine combination can also comprise the additional antigen of the pathogen of other pig.Antigen, mycoplasma hyopneumoniae antigen, Actinobacillus pleuropneumoniae (APP) antigen, PRV (Pseudorabies virus) (PRV) antigen, pig bordetella bacilli antigen, pig pasteurella multocida antigen, swine influenza virus (SIV) antigen and their combination of the additional preferred porcine reproductive and respiratory syndrome virus of antigen (PRRSV).

Therefore, the vaccine combination that prevention and treatment porcine circovirus 2 type and haemophilus parasuis infect among the present invention, not only do not produce the mutual immune interference of two kinds of antigen compositions, but also the unexpected haemophilus parasuis of finding has strengthened the immune effect of porcine circovirus 2 type antigen.As one embodiment of the present invention, in the vaccine combination of the present invention, described PCV2 antigen is PCV2 ORF2 albumen.Described haemophilus parasuis antigen is the full bacterium antigen of deactivation.

Preferably, in the vaccine combination of the present invention, the content of described Porcine circovirus type 2 ORF2 protein biology is 0.5 ~ 20 μ g/ head part, more preferably 5 ~ 10 μ g/ head parts.

Preferably, in the vaccine combination of the present invention, described haemophilus parasuis inactivation antigen is the inactivation antigen of haemophilus parasuis JS strain and the inactivation antigen of haemophilus parasuis ZJ strain.

Preferably, in the vaccine combination of the present invention, the content of described haemophilus parasuis JS strain inactivation antigen is 1 * 10 ⁹~ 8 * 10 ⁹The content of CFU/ head part and haemophilus parasuis ZJ strain inactivation antigen is 1 * 10 ⁹～8 * 10 ⁹CFU/ head part.

Preferably, in the vaccine combination of the present invention, described vaccine adjuvant is aluminium hydroxide, aluminum phosphate, saponin, vegetable oil, ethyl oleate, two (caprylic/capric) propylene glycol ester, three (caprylic/capric) glyceride, two oleic acid propylene glycol esters, isostearate, sorbitan, mannide, glycerol, polyglycereol, propylene glycol, oleic acid, isostearic acid, castor oil acid, hydroxy stearic acid ester, polyoxypropylene, polyoxyethylene block copolymer, the acrylic or methacrylic acid polymer, maleic anhydride and thiazolinyl derivant copolymer, one or more of the polymer of the acrylic or methacrylic acid that the polyalkenyl ether of sugar or polyhydric alcohol is crosslinked.

Preferably, in the vaccine combination of the present invention, described vaccine adjuvant is one or both of carbomer, chitosan.

The vaccine combination of another kind of prevention and treatment pig circular ring virus relevant disease and Haemophilus parasuis is provided to provide as another embodiment of the present invention, wherein, contain the PCV2 totivirus of deactivation and serum 4 types and the 5 type haemophilus parasuises of deactivation, vaccine adjuvant and other excipient.

Preferably, the virus of PCV2 described in the vaccine combination of the present invention is the PCV2SH strain.

Preferably, every dosage or every part (2ml) contain PCV2 totivirus antigen deactivation provirus content 2.2 * 10 in the vaccine of the present invention ^5.0TCID ₅₀～10 ^6.0TCID ₅₀More preferably contain PCV2 virus-4 .5 * 10 for every dosage or every part (2ml) ^5.0TCID ₅₀

Preferably, content is 1 * 10 before the 4 type haemophilus parasuis JS strain deactivations of serum described in the vaccine combination of the present invention ⁹CFU/ head part～4 * 10 ⁹CFU/ head part; More preferably, content is 1 * 10 before the described haemophilus parasuis JS strain deactivation ⁹CFU/ head part, again content 2 * 10 before the preferred described serum 4 type haemophilus parasuis JS strain deactivations ⁹CFU/ head part.Preferably, Serotype 5 haemophilus parasuis described in the vaccine combination of the present invention is the ZJ strain, and preserving number is CCTCC No.M2011173.

Preferably, content is 1 * 10 before the ZJ of Serotype 5 haemophilus parasuis described in the vaccine combination of the present invention strain deactivation ⁹CFU/ head part～4 * 10 ⁹CFU/ head part; More preferably, content is 1 * 10 before the described haemophilus parasuis ZJ strain deactivation ⁹CFU/ head part, again content 2 * 10 before the preferred described Serotype 5 haemophilus parasuis ZJ strain deactivation ⁹CFU/ head part.

Optimal way of the present invention relates to the adjuvant of vaccine combination, is preferably the one or more combination thing of aluminium hydroxide gel, mineral oil, carbomer, GEL, propolis, ISA206, ISA760VG.

Preferably, the vaccine combination of prevention provided by the invention and treatment pig circular ring virus relevant disease and Haemophilus parasuis contains PCV29 * 10 ^5.0TCID ₅₀/ head part, serum 4 type haemophilus parasuises 2 * 10 ⁹CFU/ head part contains Serotype 5 haemophilus parasuis 2 * 10 ⁹CFU/ head part, vaccine adjuvant are 10%V/V GEL adjuvant.

Excipient of the present invention refers to replenish the liquid of vaccine volume, includes but not limited to normal saline, PBS liquid.

The preparation method of the vaccine combination of second aspect present invention may further comprise the steps:

Cultivate haemophilus parasuis 4 types and 5 type bacterial strains, and deactivation, concentrated;

Cultivate PCV2, and deactivation, concentrated;

Three kinds of antigenic components are mixed in proportion, are aided with adjuvant and are prepared into vaccine.

Preferably, the preparation method of vaccine combination of the present invention, described method for concentration is ultrafiltration, concentrated 3 times.

Preferably, in the preparation method of the present invention, ablation method is the formalin-inactivated method; More preferably, the final concentration of formalin of the present invention is 0.1～0.3%(v/v), and inactivation time is 24～48h.

Preferably, in the preparation method of the present invention, the mixed proportion of three kinds of antigens is that equivalent volumes is mixed.

Bacterial strain information of the present invention is as follows:

Classification And Nomenclature: haemophilus parasuis serum 4 type JS(Haemophilus parasuis JS),

Preserving number is: CCTCC NO:M 2011172;

Preservation date: on May 18th, 2011,

Preservation address: Wuhan, China, Wuhan University, Chinese Typical Representative culture center;

Depositary institution: Chinese Typical Representative culture center (being called for short CCTCC).

Haemophilus parasuis Serotype 5 ZJ strain:

Classification And Nomenclature: haemophilus parasuis Serotype 5 ZJ(Haemophilus parasuis ZJ),

Preserving number is: CCTCC NO:M 2011173;

Preservation date: on May 18th, 2011,

Preservation address: Wuhan, China, Wuhan University, Chinese Typical Representative culture center

Therefore the present invention has following advantage at least:

1) inventor has solved the difficulty of prior art, has changed the understanding prejudice of distich Seedling first with porcine circovirus 2 type antigen and the antigen combined use of haemophilus parasuis, and before the present invention, is considered to this two kinds of antigens meeting phases mutual interference always;

2) inventor finds porcine circovirus 2 type of the present invention and haemophilus parasuis antigen composition and uses the prevention of said composition preparation and treat porcine circovirus 2 type and the vaccine combination of haemophilus parasuis infection, not only can not produce the mutual immune interference of two kinds of antigen compositions, but also the unexpected haemophilus parasuis antigen of finding has strengthened the immune effect of porcine circovirus 2 type antigen, and prove such as subsequent embodiment of the present invention, in the situation that has haemophilus parasuis antigen, still can keep the immune effect of PCV2 even PCV2 antigen amount reduces by half, and this exceeds the expectation of field of veterinary those of ordinary skill.

3) inventor is surprised to find that above-mentioned prevention compares with the effectiveness of the univalent vaccine of porcine circovirus 2 type or haemophilus parasuis with the vaccine combination that treatment porcine circovirus 2 type and haemophilus parasuis infect, in to pig injecting immune process, the stress of pig body is unexpectedly little, therefore, vaccine combination safety of the present invention is better, the untoward reaction that can avoid repeatedly immunoprophylaxis to occur.

4) inventor is surprised to find that the vaccine combination that above-mentioned prevention and treatment porcine circovirus 2 type and haemophilus parasuis infect not only can be the PCV2 virus antigen of deactivation and the vaccine combination of the haemophilus parasuis antigen composition of deactivation, can also be that the PCV2 subunit protein is the vaccine combination of the haemophilus parasuis antigen composition of antigen and deactivation, namely PCV2 ORF2 albumen be the vaccine combination of the haemophilus parasuis antigen composition of antigen and deactivation;

4) in addition, the vaccine combination of the present invention's prevention and treatment pig circular ring virus relevant disease and Haemophilus parasuis, preparation method is simple, the content height of tiring of vaccine, and immunity is convenient and swift, with gradation immunity of the prior art, at least need to make a call to 2 pins or 3 pins and could prevent and treat vaccine and the immunization method thereof of above two kinds of diseases and compare, reduced immune cost, saved immune programme for children, reliably and by the user accept more economically, have commercial successful basis.

Description of drawings

Fig. 1 is PCV2-ORF2 purpose clip size of the present invention;

Fig. 2 is Recombinant Swine circovurus type 2 ORF2 protein SDS-PAGE electrophoretogram of the present invention;

Fig. 3 is restructuring Porcine circovirus type 2 Cap protein Western blotting figure of the present invention;

Fig. 4 is the preparation flow figure of embodiments of the invention 1 described vaccine combination.

The specific embodiment

Selected porcine circovirus 2 type (Porcine circovirus type 2 in the embodiments of the invention, PCV2) strain is the PCV2SH strain, carry out preservation at China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date: on March 4th, 2008, preserving number is CGMCC No.23890.This strain is open at the open CN101240264 of Chinese patent.

Selected serum 4 type haemophilus parasuis (Haemophilus parasuis in the embodiments of the invention, HPS) bacterial strain is haemophilus parasuis JS strain, carry out preservation at Chinese Typical Representative culture collection center, preservation date: on May 18th, 2011, preserving number is CCTCC No.M2011172.

Selected Serotype 5 haemophilus parasuis (Haemophilus parasuis in the embodiments of the invention, HPS) bacterial strain is haemophilus parasuis ZJ strain, carry out preservation at Chinese Typical Representative culture collection center, preservation date: on May 18th, 2011, preserving number is CCTCC No.M2011173.

In the embodiments of the invention, the production of PCV2SH strain is easy with the preparation of poison, and tiring higher is 10 ^6.0TCID ₅₀More than/the ml, the content of PCV2SH strain is 10 ^6.0TCID ₅₀/ head part vaccine (1ml).The present invention has also tested content and has been lower than 10 ^6.0TCID ₅₀The vaccine combination of/head part, but evidence needs to reach 10 at least ^5.5TCID ₅₀/ head part just can have better immune effect.

Inactivator of the present invention is formalin, and the final concentration of its use is 0.1～0.3%(v/v), and inactivation time is 24～48h.Preferably, used the formalin-inactivated PCV2 SH strain of final concentration 0.2% volume in the embodiment of the invention, inactivation time is 18 hours; Having used final concentration is formalin deactivation haemophilus parasuis bacterial strain JS strain, the ZJ strain of 0.3% volume, and inactivation time is 24 hours.

Used Mi Libo (Millipore company) film bag (the molecular retention amount is 300Kda) to do 3 times in the embodiments of the invention and concentrated, proved such as subsequent embodiment, the method and concentration can make last acquisition vaccine combination obtain better vaccine effect; The present invention can also use other hyperfiltration process, such as cellulose ultrafiltration and the method such as ultrafiltration repeatedly.

The term of invention " head part " refers to the amount of vaccine of every pig injection, is equal to " dosage ".

Preferably, porcine circovirus 2 type antigen is the immunogenicity aminoacid sequence that contains PCV2 among the present invention, and more preferably, porcine circovirus 2 type antigen is the PCV2 subunit antigen, and most preferably, porcine circovirus 2 type antigen is PCV2 ORF2 albumen.Wherein, PCV2 ORF2 albumen of the present invention, the albumen of preferred sequence table SEQNO1.DNA sequential coding or the albumen of sequence table SEQ NO2. aminoacid sequence or with the albumen of its homology more than 75%.The present invention has no particular limits PCV2 ORF2 albumen, as long as it has the immunogenicity that keeps PCV2, any PCV2 ORF2 albumen all can be used as the source of PCV2 ORF2 DNA described herein and/or polypeptide.The preference of the preferred PCV2 ORF2 of other that use in embodiment of the invention albumen can be enumerated the disclosed SEQ ID of W02006-072065 NO.3, SEQ ID NO.5, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.9, and its instruction and content all are incorporated herein for referencial use.Those skilled in the art know can reach in above-mentioned sequence the change of the sequence homology of 6-10%, but still keeps the antigenicity of its albumen, thereby can be used for giving expression to antigenicity in combined antigen of the present invention and the vaccine combination.The PCV2 ORF2 albumen of modified antigen and polynucleotide sequence SEQID NO:3 or SEQ ID NO:4 coding is compared, and can give at least 70%, preferred 80%, more preferably 90% protective immunity, just thinks that this modified antigen has still kept antigenicity.

Porcine circovirus 2 type antigenic component amount PCV2 ORF2 albumen is 0.1～60 microgram/head part, preferred 0.5～20 microgram head part in the embodiment of the invention; More preferably 5～10 micrograms/dosage, 8 micrograms/head part most preferably

" haemophilus parasuis antigen " refers to contain any antigen composition of the antigen form of at least a haemophilus parasuis in the embodiment of the invention, the immunne response that the opposing haemophilus parasuis infects can be induced, stimulates or be strengthened to described antigen when to the pig administration.Preferably, described haemophilus parasuis antigen is complete Haemophilus parasuis poison, is preferably the deactivation form, more preferably, serotype is the hybrid antigen of 4 types and 5 types, and most preferably being haemophilus parasuis 4 type bacterial strains is the JS strain, and haemophilus parasuis 5 type bacterial strains are the ZJ strain.

Haemophilus parasuis 4 type JS strains in the embodiment of the invention and haemophilus parasuis 5 type ZJ strains, prove by test discovery and subsequent embodiment test example, this two strains haemophilus parasuis inactivation antigen and PCV2 ORF2 cooperatively interact can have good prevention and therapeutic effect to the present popular Haemophilus parasuis of China and the morbidity of uniting of pig circular ring virus 2, particularly can solve existing vaccine therapy poor effect, the problem that the multiple injection cost is too high, program is complicated.

Haemophilus parasuis antigen amount in the embodiment of the invention is: haemophilus parasuis 4 type content are at least 2 * 10 ⁹CFU/ head part, more preferably at least 4 * 10 ⁹CFU/ head part; Most preferably 8 * 10 ⁹CFU/ head part; Haemophilus parasuis 5 type content are at least 2 * 10 ⁹CFU/ head part, more preferably at least 4 * 10 ⁹CFU/ head part; Most preferably 8 * 10 ⁹CFU/ head part.

Used " adjuvant " in the embodiment of the invention can comprise aluminium hydroxide and aluminum phosphate, saponin such as Quil A, Q; The acid or pure ester, more specifically vegetable oil, ethyl oleate, two (caprylic/capric) propylene glycol ester, three (caprylic/capric) glyceride or the two oleic acid propylene glycol esters that contain straight chained alkyl; The ester of branched chain fatty acid or alcohol, specifically isostearate.With oil and emulsifying agent coupling to form emulsion.The preferred nonionic surfactant of emulsifying agent, the ester (optional ethoxylation) of sorbitan, mannide (such as oleic acid dehydration Nitranitol), glycerol, polyglycereol, propylene glycol and oleic acid, isostearic acid, castor oil acid or hydroxy stearic acid specifically, and polyoxypropylene. polyoxyethylene block copolymer, specifically pluronic product, especially L121.

It is to be selected from the chemical compound acrylic or methacrylic acid polymer or maleic anhydride and thiazolinyl derivant copolymer that the embodiment of the invention has also been used another example of adjuvant.That adjuvant compound is preferably is crosslinked, especially with the polymer of the crosslinked acrylic or methacrylic acid of the polyalkenyl ether of sugar or polyhydric alcohol.These chemical compounds are called carbomer (carbomer).

Other suitable adjuvant includes but not limited to: RIBI adjuvant system (Ribi Inc.), block copolymer (CytRx; Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, avridine (Avridine) lipid. the thermal instability enterotoxin of amine adjuvant, escherichia coli (E.coli) (restructuring or other), cholera toxin, IMS1314 or muramyldipeptide etc.

The preferred every dosage of the adjuvant of the embodiment of the invention (head part) adds the adjuvant of about 100 micrograms～10 milligram quantities.More preferably every dosage adds the adjuvant of about 500 micrograms-5 milligram quantities.More preferably every dosage adds the adjuvant of about 750 micrograms-2.5 milligram quantities.Most preferably every dosage adds the adjuvant of about 1 milligram quantities

In another technical scheme, it is polysaccharide that bigeminy thing adjuvant can also add, polysaccharide includes but not limited to: astragalus polysaccharides, Radix Et Caulis Acanthopanacis Senticosi polysaccharide, lentinan, chitosan, cyclodextrin, dextrin and SL-dextrin, preferred chitosan is the chitosan addition wherein, preferred 0.1--10mg/ dosage (head part), more preferably 0.1～5mg/ dosage, most preferably 1mg/ dosage.

The below further specifies the present invention with specific embodiment:

Preparation and the check of the vaccine combination of embodiment 1 pig circular ring virus relevant disease and Haemophilus parasuis

1.1 produce the preparation of planting with bacterium (poison)

The preparation of porcine circovirus 2 type SH: seed culture of viruses PCV2SH strain is done the 1:9 dilution with MEM fluid medium (using the MEM dehydrated medium by specification preparation available from American I nvitrogen company), then be inoculated in PK15(ATCC by 5% of cell culture fluid volume, preserving number is CCL-33) cell monolayer, 37 ℃ adsorbed 30 minutes, add cell maintenance medium (adding the D-glucosamine hydrochloric acid of 4% calf serum and 2mmol/L in the MEM fluid medium), cultivated 4 for 37 ℃, freeze thawing 2 ~ 3 times, results virus, virus titer is 10 ^6.5TCID ₅₀/ ml.

The preparation of haemophilus parasuis strain: with haemophilus parasuis 4 type JS strains and haemophilus parasuis 5 type ZJ strain streak inoculations in containing 5% new-born calf serum and 0.005% nicotinamide adenine dinucleotide (NAD, U.S. BBI company) Trypsin soy agar (TSA, available from U.S. company BD) dull and stereotyped (it is dull and stereotyped to be called for short TSA/NAD), cultivated 24～48 hours for 37 ℃, respectively select 5 above colonies typicals, purely after the assay was approved, as first order seed.The single bacterium colony of first order seed picking, access contains 5% new-born calf serum and 0.005% nicotinamide adenine dinucleotide (NAD, U.S. BBI company) Trypsin soybean broth (TSA, available from U.S. company BD) (be called for short TSB/NAD fluid medium), 37 ℃ of shaking table 180rpm shaken cultivation 12 hours, the Gram’s staining of taking a sample simultaneously, it is even to examine under a microscope ne ar, meet the morphological characteristic of haemophilus parasuis, without any varied bacteria growing, as secondary seed

Wherein: the preparation method that contains the TSA/NAD flat board of 5% serum: Trypsin soy agar (Tryptic Soy Agar, TSA, BD Difico product) 40 grams add the 945ml distilled water, fully shake up post-heating to fully dissolving, 121 ℃ of high pressure steam sterilization 15min, temperature is down to about 60 ℃, adds the Ox blood serum of 50ml filtration sterilization, the 1%NAD of 5ml filtration sterilization is down flat ware after fully shaking up.

The TSB/NAD fluid medium that contains 5% new-born calf serum: pancreas soybean protein meat soup (Tryptic Soy Broth, TSB, BD Difico product) 30 grams add the 940ml distilled water, fully shake up post-heating to fully dissolving, 121 ℃ of high pressure steam sterilization 15min, the Ox blood serum that adds the 50ml filtration sterilization, the 0.01%NAD of 10ml filtration sterilization.

1.2 the cultivation of virus liquid or bacterium liquid preparation

The preparation of porcine circovirus 2 type SH strain virus liquid: use the rolling bottle cell culture method.To cover with the PK15 cell of monolayer, remove cell culture fluid (adding the D-glucosamine hydrochloric acid of 6% calf serum and 2mmol/L in the MEM fluid medium), seed culture of viruses liquid volume ratio is pressed 0.1～0.2TCID ₅₀The inoculum concentration an of/cell is inoculated on the PK15 cell, and in rotation cell 2 weeks of bottle, 37 ℃ of absorption 30 minutes adds cell maintenance medium (step 1.1 is described), puts 37 ℃ of rotations (10 ~ 12 turn/hour) cultivation.Observe every day 1 ~ 2 time, Growth of Cells is good, cultivates harvesting on the 4th and Cell sap for 37 ℃, and freeze thawing 3 times is put below-20 ℃ and preserved.

The preparation of haemophilus parasuis (4 type JS strains and 5 type ZJ strains) bacterium liquid: with the secondary seed of the haemophilus parasuis 4 type JS strains of accreditation and haemophilus parasuis 5 type ZJ strains respectively by 1:100(v/v) be inoculated in TSB/NAD fluid medium (step 1.1 is described), put 37 ℃ of shaking table 200rpm and cultivate.Be cultured to 12～16 hours, results.

1.3 the processing of virus liquid or bacterium liquid

The processing of porcine circovirus 2 type SH strain virus liquid: the virus liquid of step 1.2 is filtered post (Millipore company aperture 10 μ m and 0.45 μ m) with doughnut filter, remove cell debris.

The processing of haemophilus parasuis (4 type JS strains and 5 type ZJ strains) bacterium liquid: just two of step 1.2 kinds of bacterium liquid (haemophilus parasuis ZJ strain and JS strain) centrifugal with continuous centrifuge (10000 rev/mins) respectively after again take the PBS(pH value as 7.2～7.4) redissolve volume before centrifugal.

Use respectively Mi Libo (Millipore company) film bag (the molecular retention amount is 300Kda) work 3 times (volume ratios) concentrated porcine circovirus 2 type SH, haemophilus parasuis 4 type JS strains, haemophilus parasuis 5 type ZJ strains 1.4 concentrate.

1.5 assay

1.5.1 pig circular ring virus SH strain virus assay

Virus liquid is made 10 times of serial dilutions with MEM fluid medium (step 1.1 is described) by those skilled in the art's universal method, get 10 ^-5, 10 ^-6, 10 ^-73 dilution factors, each dilution factor are inoculated respectively 96 well culture plate PK15 cell monolayers, 4 holes, and every hole 0.1ml sets up negative control simultaneously, contains 5%CO at 37 ℃ ₂Incubator in continue to cultivate 24 hours, change cell maintenance medium, continue to cultivate 24 hours; Use the cold acetone fixed cell, measure each dilution factor with indirect immunofluorescence assay (IFA) and contain the PCV2 positive cell hole count of (being green), calculate viral TCID according to the KarberShi method ₅₀

1.5.2 haemophilus parasuis ZJ strain and JS strain viable count are measured

The sampling of bacterium liquid is made 10 times of serial dilutions by method well known to those skilled in the art, gets 10 ^-6, 10 ^-72 dilution factors are inoculated in aforementioned TSA/NAD solid medium, and each plating 0.1ml cultivates after 24 hours for 37 ℃, selects the flat board of clump count between 30 and 300 to carry out colony counting.

1.6 virus liquid or bacterium liquid hold-up measurement result:

Three batches of virus liquids or bacterium liquid are cultivated respectively in porcine circovirus 2 type SH strain, haemophilus parasuis 4 type JS strains and haemophilus parasuis 5 type ZJ strains, are respectively K001, K002 and K003 and criticize.Virus liquid or bacterium liquid hold-up see Table 1.

Table 1 assay

1.7 the deactivation of virus liquid or bacterium liquid

The deactivation of porcine circovirus 2 type SH strain virus liquid: the virus liquid that the step 1.3 that is up to the standards is processed adds formalin (Luoyang City's chemical reagent factory analytical pure, content is 37%～40%), the final concentration that makes formalin is 0.2%(V/V), 37 ℃ of deactivations 18 hours, stirred 1 time in per 4 hours, each 10min, deactivation is put 2 ~ 8 ℃ of preservations with inactivation of viruses liquid after finishing.

The deactivation of haemophilus parasuis (4 type JS strains and 5 type ZJ strains) bacterium liquid: with the bacterium liquid of two kinds of different serotypes in the step 3, add formalin, the final concentration that makes formalin is 0.3%(V/V), 37 ℃ of deactivations 24 hours, stirred 1 time every 4 hours therebetween, each 10min, deactivation is put 2 ~ 8 ℃ of preservations with inactivation of viruses liquid after finishing.

1.8 inactivating efficacy is measured

1.8.1 the deactivation of porcine circovirus 2 type SH strain virus liquid check: the inactivation of viruses liquid that takes a morsel inoculation has grown up to the PK15 cell of monolayer, 37 ℃ of absorption were abandoned virus liquid after 1 hour, add new cell maintenance medium, cultivated 2 for 37 ℃, answer acellular pathological changes (CPE), blind passage is 3 times continuously, change cell maintenance medium into after growing up to cell monolayer, cultivated 2 for 37 ℃, detect with indirect immunofluorescence (IFA), redgreen PCV2 positive cell produces.

1.8.2 haemophilus parasuis deactivation check: the TSA/NAD solid medium of 6 plates of preparation, the sterile working drips 1 on 3 TSA/NAD solid medium plates therein with the bacterium liquid of deactivation, rule with inoculating loop, put 37 ℃ of common incubators and cultivate, set up simultaneously 3 TSA/NAD solid mediums that do not connect bacterium to compare.Observe after 24 hours, plate must be without any bacterial growth, and simultaneously contrast does not connect 2 culture medium of bacterium also should be without bacterial growth, in hours 6 plates of continuation observed result to 48 all without bacterial growth.

1.8.3 deactivation result

The deactivation assay of 3 crowdes of porcine circovirus 2 type SH strain, haemophilus parasuis 4 type JS strains and haemophilus parasuis 5 type JS strains sees Table, and deactivation is thoroughly, sees Table 2.

The deactivation test effect of 3 batches of antigens of table 2

1.9 join Seedling prescription such as table 3, with three kinds of antigen liquids after concentrated by 1: 1: 1(V/V) mix, then mixed liquor and GEL adjuvant (France match Bick SEPPIC company produces) are by 90:10(V/V) mix, got final product in 30 minutes with 300 rev/mins of stirrings.

The prescription of table 3 vaccine and content

Below for preparing the method for inspection of vaccine combination:

1.10 character check

The canescence suspension, leaving standstill for a long time the bottom has a small amount of precipitation.Be even suspension after the vibration.

1.11 steriling test

Undertaken by 169～171 pages of " People's Republic of China's veterinary drug allusion quotation " (version in 2005) appendix, answer asepsis growth.

Character and steriling test the results are shown in down

The results are shown in Table 4, three batches of vaccine lot numbers is Y001, Y002, Y003, is prepared by poison (bacterium) the strain antigen of above-mentioned lot number K001, K002, K003, and through steriling test and character check, three batches of vaccine finished products all are qualified as a result respectively.

The character of 3 batches of vaccines of table 4 and steriling test result

1.12 safety verification

With 5 of the negative piglets of 28～35 age in days Haemophilus parasuis antibody and porcine circovirus 2 type antibody, each musculi colli vaccinate compositions 4ml observed 14, annotated the Seedling part without severe reaction, and all strong living is qualified.

The results are shown in down:

Lot number is three batches of vaccines of Y001, Y002, Y003, passes through respectively safety verification, and three batches of vaccine finished products all are qualified as a result, and injection site, whole body, internal organs are showed no unusually, the results are shown in Table 5.

The safety verification of 3 batches of vaccines of table 5

It below is the efficacy test of vaccine combination

1.13 efficacy test

1.13.1 porcine circovirus 2 type efficacy test part

With 15 of the healthy susceptible piglets of 14～21 ages in days, be divided into 3 groups, every group 5, the 1st group of every incidence intramuscular injection vaccine 1ml, vaccine uses above-mentioned lot number to be three batches of vaccines of Y001, Y002, Y003, carries out the 2nd inoculation by identical approach and dosage after two weeks, does nonimmune counteracting toxic substances contrast for the 2nd group, make blank (nonimmune, non-counteracting toxic substances) for the 3rd group, all isolated rearing is observed.5 weeks all pigs were weighed after head exempts from, the 1st, 2 group each (contains 10 with the PCV2SH strain ^6.0TCID ₅₀/ ml) collunarium 1ml, intramuscular injection 2ml, isolated rearing.Counteracting toxic substances the 4th, 7 days, respectively in the oxter, both sides of every pig and the both sides buttocks totally 4 points to the keyhole hemocyanin (KLH/ICFA of all pigs inoculations with the incomplete Freunds adjuvant emulsifyings, 0.5mg/ml), each some inoculation 1ml(4ml/ head), while intraperitoneal inoculation thioglycollate medium, the 10ml/ head; Intraperitoneal inoculation thioglycollate medium again on the the 11st, 19 behind the counteracting toxic substances, the 10ml/ head.Continuous Observation is 25 behind the counteracting toxic substances, slaughters after weighing on 25th, cuts open inspection.Judge according to body temperature, relative daily gain and virus antigen detection result.The counteracting toxic substances matched group is should at least 4 hairs sick, and immune group should at least 4 head protections.

1.13.2 haemophilus parasuis efficacy test part: with 10 of 28～35 age in days Haemophilus parasuis negative antibody piglets, divide 2 groups, 5 every group, every incidence intramuscular injection vaccine 1ml, two exempt from every incidence intramuscular injection vaccine 1ml after 2 weeks.Exempt from rear 14 days for every group 2 together with 5 of contrast pigs, serum 4 type JS strains and 5 each 3ml of type ZJ strain bacterium liquid of 1 fatal dose of intraperitoneal injection observed 14 respectively.Every group of immune swine protected 4 at least; Contrast at least 3 death of pig or 4 hairs disease are qualified.

Efficacy test the results are shown in down:

Above-mentioned lot number is Y001, Y002, three batches of vaccines of Y003;, passing through respectively efficacy test, three batches of vaccines are all to porcine circovirus 2 type SH, serum 4 type JS strains and the protection of 5 type ZJ strain bacterial strain Immunizations as a result; three batches of vaccines all show good immune effect as a result, the results are shown in Table 6.

The efficacy test result of 3 batches of vaccines of table 6

Embodiment 2: the preparation of porcine circovirus 2 type antigen, haemophilus parasuis bivalence (serum 4 types+Serotype 5) antigen vaccine compositions

Adopt the inactivated bacterial liquid of embodiment 1 preparation (be that K001 criticizes antigen, see Table 2) porcine circovirus 2 type inactivation of viruses liquid and haemophilus parasuis (serum 4 type JS strains and Serotype 5 ZJ strain), prepare vaccine by following prescription:

(deactivation provirus content is 10 to the PCV2 virus liquid of deactivation ^6.0TCID ₅₀/ ml) 45ml

(the pathogenic bacteria liquid hold-up is 4.5 * 10 to the haemophilus parasuis 4 type bacterium liquid of deactivation before the deactivation ^9.0Cfu/ml) 22.5ml

(the pathogenic bacteria liquid hold-up is 4.5 * 10 to the haemophilus parasuis 5 type bacterium liquid of deactivation before the deactivation ^9.0Cfu/ml) 22.5ml

Montanide ^TM Gel 10ml

The bacterium liquid (embodiment 1 preparation) of inactivation of viruses liquid and deactivation is put into container according to the above ratio, put into again Montanide ^TMThen Gel uses IKA digital display Eurostar agitator (500～800r/min) stirring and evenly mixings, 5～10min.

Embodiment 3:PCV2 inactivated whole virus antigen vaccine to the vaccine combination of PCV2 counteracting toxic substances immune effect and PCV2 inactivation of viruses antigen and haemophilus parasuis inactivation antigen to PCV2 counteracting toxic substances immune effect relatively

Preparing antigen with embodiment 1 (is that K001 criticizes antigen, see Table 2) prepare respectively porcine circovirus 2 type, haemophilus parasuis (serum 4 types+Serotype 5) vaccine combinations (V1) and (V2), pig circular ring virus vaccine (V3) and (V4), it is as follows specifically to fill a prescription.

(1) porcine circovirus 2 type, haemophilus parasuis (serum 4 types+Serotype 5) vaccine combinations (V1)

The PCV2 virus liquid of deactivation (contains 10 ^6.0TCID ₅₀/ ml) 45ml

The haemophilus parasuis 4 type bacterium liquid of deactivation (contain 4.5 * 10 ^9.0Cfu/ml) 22.5ml

The haemophilus parasuis 5 type bacterium liquid of deactivation (contain 4.5 * 10 ^9.0Cfu/ml) 22.5ml

Montanide ^TM Gel 10ml

(2) porcine circovirus 2 type, haemophilus parasuis (serum 4 types+Serotype 5) vaccine combinations (V2) cumulative volume 100ml

The PCV2 virus liquid of deactivation (contains 10 ^6.0TCID ₅₀/ ml) 22.5ml

The haemophilus parasuis 4 type bacterium liquid of deactivation (contain 4.5 * 10 ^9.0Cfu/ml) 11.25ml

The haemophilus parasuis 5 type bacterium liquid of deactivation (contain 4.5 * 10 ^9.0Cfu/ml) 11.25ml

0.85% normal saline 45ml

Montanide ^TMGel 10ml

(3) porcine circovirus 2 type inactivated vaccine (V3) cumulative volume 100ml:

The PCV2 virus liquid of deactivation (contains 10 ^6.0TCID ₅₀/ ml) 45ml

0.85% normal saline 45ml

Montanide ^TM Gel 10ml

(4) porcine circovirus 2 type inactivated vaccine (V4) cumulative volume 100ml

The PCV2 virus liquid of deactivation (contains 10 ^6.0TCID ₅₀/ ml) 22.5ml

0.85% normal saline 67.5ml

Montanide ^TM Gel 10ml

Select 30 of 21～28 age in days ablactational baby pig, be divided into 6 groups, 5 every group (seeing Table 7); The 1st group of every pig difference musculi colli vaccine (V1) 2ml, 21d inoculates 2ml again after the immunity; The 2nd group of every pig difference musculi colli vaccinate (V2) 2ml, 21d inoculates 2ml again after the immunity; The 3rd group of every pig difference musculi colli vaccinate (V3) 2ml, 21d inoculates 2ml again after the immunity; The 4th group of every pig difference musculi colli vaccinate (V4) 2ml, 21d inoculates 2ml again after the immunity; The 5th as the counteracting toxic substances matched group, and the 6th group as the blank group.35d after first immunisation, the 1st, 2,3,4,5 group each (contains 10 with the PCV2SH strain ^6.0TCID ₅₀/ ml) collunarium 1ml, intramuscular injection 2ml, the 4th group of counteracting toxic substances not, behind the counteracting toxic substances the 4th, 7 day, respectively in the oxter, both sides of the 1st, 2,3,4,5,6 group of every pig and the both sides buttocks totally 4 points to the keyhole hemocyanin (KLH/ICFA of all pigs inoculations with the incomplete Freunds adjuvant emulsifyings, 0.5mg/ml), each some inoculation 1ml(4ml/ head), while intraperitoneal inoculation thioglycollate medium (Thio), 10ml/ head; Intraperitoneal inoculation thioglycollate medium again on the the 11st, 19 behind the counteracting toxic substances, the 10ml/ head.Continuous Observation is 25 behind the counteracting toxic substances, slaughters after weighing in 25th behind the counteracting toxic substances, cuts open inspection.Detecting PCV2 virus antigen result according to body temperature, tissue injury and immunohistochemistry (IHC) judges.

Table 7 test grouping and processing

The body temperature production sees Table 8, as seen behind the pig PCV2 counteracting toxic substances of vaccine combination (V2) immunity that reduces by half of vaccine combination (V1) and antigenic content almost without fervescence, and 1～2 day of short duration fervescence is arranged mostly more than 40.5 ℃ behind the pig PCV2 counteracting toxic substances of the V4 vaccine immunity that porcine circovirus 2 type inactivated vaccine (V3) and PCV2 antigenic content reduce by half, wherein 1 has reached 3 days.

Respectively organize the natural law comparison that test pig body temperature surpasses 40.5 ℃ behind the table 8 PCV2 counteracting toxic substances

Tissue injury and SABC detectable antigens situation see Table 9, as seen behind the pig PCV2 counteracting toxic substances of vaccine combination (V1) immunity without the pathology damage and do not detect PCV2 antigen, and 5 pigs of pig of porcine circovirus 2 type inactivated vaccine (V2) immunity have 1 to detect PCV2 antigen and it has the distinctive pathology damage of pathology porcine circovirus (appearance of interstitial pneumonia, lymph node lymphocyte damage and multinucleated giant cell and mononuclear phagocyte infiltration etc.).

Respectively organizing test pig tissue injury and IHC behind the table 9 PCV2 counteracting toxic substances detects

The result of this research is unexpected: when using porcine circovirus 2 type (PCV2) with the combination of haemophilus parasuis antigen, not only can be used for immune swine so that good immune effect to be provided, and kept the good immune efficacy of former PCV2 antigen composition, its effect also is better than independent porcine circovirus 2 type inactivated vaccine, especially in the situation that antigenic content reduces by half, still had alone PCV antigen and the effect that is beyond one's reach has been held the immune effect of porcine circovirus 2 type (PCV2) antigen, two kinds of antigen to PCV2 immunity played synergism, because the adjuvant kind is all identical with amount, obvious this synergism is that two kinds of antigens cause together simultaneously.It is the immune effect that the immune effect of the antagonism PCV2 of vaccine combination of the present invention is better than using separately PCV2 antigen.Especially still kept the immune effect of porcine circovirus 2 type (PCV2) antigen in the situation that PCV2 antigen amount reduces by half, the above results exceeds those skilled in the art's expectation.Embodiment 4: the vaccine combination of haemophilus parasuis inactivation antigen vaccine combination and PCV2 inactivation of viruses antigen, haemophilus parasuis inactivation antigen is to the comparison of haemophilus parasuis counteracting toxic substances protection effect

This research design is in order to confirm that combination product and independent haemophilus parasuis inactivated vaccine do not disturb.

Criticize antigen with the antigen K001 of embodiment 1 preparation and prepare respectively porcine circovirus 2 type, haemophilus parasuis (serum 4 types+Serotype 5) vaccine combinations (V1), haemophilus parasuis inactivated vaccine (serum 4 types+Serotype 5) (V2) by embodiment 2 methods, it is as follows specifically to fill a prescription.

(1) porcine circovirus 2 type, haemophilus parasuis (serum 4 types+Serotype 5) vaccine combinations (V1):

The PCV2 virus liquid of deactivation (contains 10 ^6.0TCID ₅₀/ ml) 45ml

Montanide ^TM Gel 10ml

(2) haemophilus parasuis inactivated vaccine (serum 4 types+Serotype 5) inactivated vaccines (V2):

0.85% normal saline 45ml

Montanide ^TMGel 10ml

Select 35 of 21～28 age in days ablactational baby pig, be divided into 7 groups, 5 every group (seeing Table 10); 1st, 2 groups of every pig difference musculi collis are injected haemophilus parasuis (4 types+5 types), porcine circovirus 2 type bivalent inactivated vaccine (V1) 2ml, and 2ml was inoculated in immunity in rear 21 days again; 3rd, 4 groups of every pig difference musculi colli injection Haemophilus parasuis inactivated vaccine (4 types+5 types) 2ml, 2ml was inoculated in immunity in rear 21 days again; 5th, do not inoculate for 6,7 groups.After first immunisation 35 days, the 1st, 3,5 group of every pig observed day by day respectively through the serum 4 type JS strain bacterium liquid 3ml of 1 fatal dose of intraperitoneal injection, observed to the counteracting toxic substances to slaughter in 10th and cutd open inspection; 2nd, 4,6 groups of every pigs are respectively through the Serotype 5 ZJ of 1 fatal dose of intraperitoneal injection strain bacterium liquid 3ml, observe day by day, observe to the counteracting toxic substances to slaughter in 10th and cut open inspection; The 7th group not counteracting toxic substances make blank.

Table 10 test grouping and handling procedure

Group	D0	D21	D35	D45
						1	V1, the 2ml/ head	V1, the 2ml/ head	JS strain 3ml(contains 7 * 10 ⁹CFU) attack	Slaughter
2	V1, the 2ml/ head	V1, the 2ml/ head	ZJ strain 3ml(contains 6 * 10 ⁹CFU) attack	Slaughter
						3	V2, the 2ml/ head	V2, the 2ml/ head	JS strain 3ml(contains 7 * 10 ⁹CFU) attack	Slaughter
4	V2, the 2ml/ head	V2, the 2ml/ head	ZJ strain 3ml(contains 6 * 10 ⁹CFU) attack	Slaughter
						5	Not immune	Not immune	JS strain 3ml(contains 7 * 10 ⁹CFU) attack	Slaughter
6	Not immune	Not immune	ZJ strain 3ml(contains 6 * 10 ⁹CFU) attack	Slaughter
					7	Not immune	Not immune	Counteracting toxic substances not	Slaughter

Result of the test such as table 11; protection is respectively 4/5(80% to vaccine combination (V1) to the counteracting toxic substances of 4 type JS strains and 5 type ZJ strains), 5/5(100%), haemophilus parasuis vaccine (serum 4 types+Serotype 5) is 4/5(80% to the counteracting toxic substances protection of 4 type JS strains and 5 type ZJ strains), 5/5(100%).

Respectively organize comparative result behind the table 11 haemophilus parasuis counteracting toxic substances

Group	Size of animal	Morbidity quantity	Dead quantity	Protective rate (morbidity number/number of animals)
					V1	5	1	0	4/5
V1	5	0	0	5/5
					V2	5	1	0	4/5
V2	5	0	0	5/5
					4 type counteracting toxic substances matched groups	5	5	3	/
5 type counteracting toxic substances matched groups	5	5	4	/
					The blank group	5	0	0	/

The result of this research is unexpected: use the combination of porcine circovirus 2 type of the present invention (PCV2) and haemophilus parasuis antigen to make vaccine and mixes the good immune efficacy that can keep former haemophilus parasuis antigen composition when using, this understanding and understanding with those skilled in the art is distinguished very large.

Embodiment 3 and embodiment 4 proofs, porcine circovirus 2 type of the present invention (PCV2) is made combined vaccine with the combination of haemophilus parasuis antigen, haemophilus parasuis antigen is to the immune effect of porcine circovirus 2 type (PCV2) antigen, and haemophilus parasuis antigen itself is unaffected, obtain useful effect, had significant progress.

Embodiment 5: haemophilus parasuis (4 types+5 types), porcine circovirus 2 type bivalent inactivated vaccine compare with the immune effect that uses separately two kinds of vaccines (haemophilus parasuis inactivated vaccine or porcine circovirus 2 type inactivated vaccine) and immune side effect

1 material

Haemophilus parasuis (4 types+5 types) antigen, porcine circovirus 2 type antigen vaccine compositions, (every ml vaccine contains that PCV2 is 5 * 10 before the deactivation to select laboratory products (V1) among the embodiment 3 ⁵TCID ₅₀, haemophilus parasuis 4 types and 5 type antigenic contents are 10 ⁹Cfu, every part consumption is 1ml, twice of immunity); Porcine circovirus 2 type inactivated vaccine (SH strain) (V2), Pulaike Biological Engineering Co., Ltd. produces that (lot number is 100903, and every ml vaccine contains that PCV2 is 5 * 10 before the deactivation ⁵TCID ₅₀Every part consumption is 1ml, twice of immunity); Haemophilus parasuis inactivated vaccine (4 type MD0322 strains+5 type SH0165 strains) (V3), (lot number is 100306 to biological product company limited before the section of Wuhan, and every ml vaccine contains that PCV2 is 5 * 10 before the deactivation ⁵TCID ₅₀, haemophilus parasuis 4 types and 5 type antigenic contents are 2 * 10 ⁹Cfu; Every part consumption is 1ml, twice of immunity).

The design of 2 animal experiments

Select 50 of 21～28 age in days ablactational baby pig, be divided into 10 groups, 5 every group (seeing Table 12); 1st, 2,3 groups of every pig difference musculi collis are injected haemophilus parasuis (4 types+5 types), porcine circovirus 2 type bivalent inactivated vaccine (lot number 001) 1ml, and 14d inoculates 1ml again after the immunity; 4th, 5,6 groups of every pigs difference left side musculi colli injection Haemophilus parasuis inactivated vaccine (4 type MD0322 strains+5 type SH0165 strains) 1ml, right side musculi colli injection porcine circovirus 2 type inactivated vaccine (SH strain) 1ml, 14d inoculates each 1ml again after the immunity; 7th, do not inoculate for 8,9,10 groups.35d after first immunisation, the 1st, 4,7 group of every pig through the serum 4 type JS strain bacterium liquid 3ml of 1 fatal dose of intraperitoneal injection, observed respectively day by day, observes to the counteracting toxic substances to slaughter in 14th and cuts open inspection; 2nd, 5,8 groups of every pigs are respectively through the Serotype 5 ZJ of 1 fatal dose of intraperitoneal injection strain bacterium liquid 3ml, observe day by day, observe to the counteracting toxic substances to slaughter in 14th and cut open inspection; 3rd, 6,9 groups respectively (contain 10 with PCV2 SH strain ^6.0TCID ₅₀/ ml) collunarium 1ml, intramuscular injection 2ml, the 10th group of counteracting toxic substances not, behind the counteracting toxic substances the 4th, 7 day, respectively in the oxter, both sides of the 3rd, 6,9,10 group of every pig and the both sides buttocks totally 4 points to the keyhole hemocyanin (KLH/ICFA of all pigs inoculations with the incomplete Freunds adjuvant emulsifyings, 0.5mg/ml), each some inoculation 1ml(4ml/ head), while intraperitoneal inoculation thioglycollate medium, 10ml/ head; Intraperitoneal inoculation thioglycollate medium again on the the 11st, 19 behind the counteracting toxic substances, the 10ml/ head.Continuous Observation is 25 behind the counteracting toxic substances, slaughters after weighing in 25th behind the counteracting toxic substances, cuts open inspection.Judge according to body temperature, relative daily gain and virus antigen detection result.

KLH/ICFA is the keyhole hemocyanin (KLH) (containing KLH is 0.5mg/ml) of incomplete Freunds adjuvant (ICFA) emulsifying, and KLH and ICFA are all available from Sigma company; Thio is thioglycollate medium, available from Sigma company.

Table 12 test grouping and processing

Result of the test such as table 13; protection is 4/5(80% to vaccine combination to the counteracting toxic substances of 4 type JS strains and 5 type ZJ strains), 5/5(100%); counteracting toxic substances protection to PCV2 SH strain is 4/5(80%); two kinds of single Seedling couplings are 4/5(80% to the counteracting toxic substances protection of 4 type JS strains and 5 type ZJ strains), 4/5(80%), the counteracting toxic substances protection of PCV2SH strain is 4/5(80%).

The controlled trial of table 13 vaccine combination and univalent vaccine

Annotate: untoward reaction shows as injection site redness, fervescence, anorexia, lassitude.By demonstrating as seen from Table 13 good immune protective effect, show that the immune effect of vaccine combination of the present invention and single Seedling is substantially suitable; From dosage of inoculation comparatively speaking, vaccine combination is played 2 pins, is total to 2ml, and two kinds of single Seedling couplings need be played 4 pins, is total to 4ml, and apparent effect is that the vaccine combination use is more convenient, time saving and energy saving; From the view of safety, the safety of vaccine combination is comparatively safe, and single Seedling coupling has 1/5 test pig that untoward reaction is arranged, and vaccine combination will increase by 1 times because the dosage of injection compares; Comprehensively state it, vaccine combination is not only easy to use, stress be little.

Embodiment 6 Porcine circovirus type 2 Cap proteins and preparation method thereof

(1) clone pig circovurus type 2 ORF2 gene obtains recombinant yeast expression vector pPIC9K-ORF2 in yeast expression vector pPIC9K.

1. the design of primer

Take the PCV2-ORF2 gene order as reference, use Oligo6.0 primer-design software design primer,

P1 upstream: 5'-AGG

GGCATCTTCAACACC-3'

P2 downstream: 5'-CC

TTAGGGGTTAAGTGGGG-3'

Add respectively nucleic acid restriction endonuclease BamH I and EcoRI recognition sequence (inclination underscore part) at upstream and downstream primer 5' end, the purpose fragment is about 702bp.

2. the extraction of PCV2 viral nucleic acid

1mlDNAzol Reagent(Invitrogen

) add to turn upside down in the centrifuge tube of the PCV2-SH virus liquid contain 0.1ml and shake up for 3 times; Leave standstill 5min under the room temperature; After 4 ℃ centrifugal (10,000rpm, 10min), supernatant is moved to new centrifuge tube; To wherein adding the 0.5ml dehydrated alcohol, leave standstill 5min under the room temperature behind the mixing that turns upside down, after 4 ℃ centrifugal (4000rpm, 2 min), leave standstill 1min, then suck liquid in pipe; Add 1ml75% ethanol in the test tube, behind the mixing 5 times of turning upside down, leave standstill 1min, suck ethanol, after this step repeats twice, allow sample natural drying (5min) in air; In centrifuge tube, slowly add 30 μ l8mmol/LNaOH dissolving DNAs, then for subsequent use-20 ℃ of lower storages.

3. the pcr amplification of PCV2-ORF2 gene and connect into cloning vehicle pMD18-T(available from Takara company, article No.: D101A)

Take step 2. in the genomic DNA of PCV2 of preparation as template, with step 1. in primer P1, the P2 of design genes of interest ORF2 is carried out pcr amplification.The PCV2-ORF2 gene is connected in the pMD18-T carrier called after pMD18-T-ORF2 plasmid.Identify and the order-checking evaluation with nucleic acid restriction endonuclease BamH I and EcoRI double digestion.PCR the results are shown in Figure 1.The 1st hole is PCV2-ORF2 purpose fragment, and size is 702bp, and the 2nd hole is DL2000Marker.By electrophoresis pattern and sequencing result as can be known, the PCV2-ORF2 protein gene contains SEQ ID NO1 sequence in the sequence table in the restructuring connection carrier.

4. Expression vector pPIC9K-ORF2 plasmid construction

With nucleic acid restriction endonuclease BamH I and EcoRI double digestion pMD18-T-ORF2 plasmid, reclaim test kit with glue and reclaim the PCV2-ORF2 genetic fragment.Be connected construction of expression vector pPIC9K-ORF2 through BamH I with the glue recovery purpose fragment of EcoRI double digestion pPIC9K plasmid (available from Pharmacia company) with same.With BamH I and EcoRI double digestion and PCR evaluation, the employing PCR method is analyzed, with boiling-freeze-the standby template of boiling, take universal primer 5 ' AOX1 and 3 ' AOX1 as primer, its sequence is that 5 '-GACTGGTTCCAATTGACAAGC-3 ' and 5 '-GCAAATGGCATTCTGACATCC-3 ' carries out PCR and is accredited as positive recombiant plasmid.

(2) restructuring positive plasmid pPIC9K-ORF2 transformed yeast GS115 bacterial strain is (available from Life Technologies company, article No.: C18100)

After the positive recombinant expression plasmid pPIC9K-ORF2 linearisation after identifying, electricity transforms Pichia pastoris GS115, obtains positive gene engineering bacteria GS115/pPIC9K-ORF2.This genetic engineering bacterium is accredited as the restructuring yeast strains that contains 3 copy expression cassettes through G418 resistant panel, PCR and Southern-blot, carries out strain with 15% glycerol and preserves.

(3) Pichia anomala expression Porcine circovirus type 2 Cap protein

1. the evaluation of the abduction delivering of positive strain and restructuring Porcine circovirus type 2 Cap protein

With the negative contrast of the saccharomycetic positive recombinant yeast of pPIC9k Plasmid Transformation GS115, the picking restructuring yeast strains is in BMGY (Buffered glycerol-complex medium oil buffering co-induction culture medium) culture medium simultaneously, being cultured to OD600 is 3 o'clock, the centrifugal 15min of 5000rpm collects thalline, go to erlenmeyer flask, adding the BMMY culture medium, to be diluted to OD600 be 1, and with 8 layers of sterile gauzes sealing, 30 ℃ of 250rpm shaking tables are cultivated 72h.In order to keep abduction delivering, adding 100% methanol to final concentration every 24h is 0.5%, get the 1.5ml culture in the sterilization centrifuge tube respectively at 24h, 48h, 72h, in the centrifugal 15min of 12000rpm, collect respectively supernatant, supernatant filters by the filter membrane of 1 μ m, carries out the SDS-PAGE electrophoresis after the filtration, the results are shown in Figure 2.The 1st hole is protein Marker; The 2nd hole is the culture supernatant of gathering in the crops behind the pPIC9K-ORF2 recombinant yeast pichia pastoris 24h; The 3rd hole is the culture supernatant of gathering in the crops behind the pPIC9K-ORF2 recombinant yeast pichia pastoris 48h; The 4th hole is the culture supernatant of gathering in the crops behind the pPIC9K-ORF2 recombinant yeast pichia pastoris 72h; The 5th hole is the cell negative control.As seen from the figure, restructuring Porcine circovirus type 2 Cap protein size is consistent with predicted protein.Through order-checking as can be known, Recombinant Swine circovurus type 2 ORF2 protein sequence is the SEQ ID NO.2 sequence in the sequence table.

2. the recombinate Immunity identification of Porcine circovirus type 2 Cap protein

Behind the SDS-PAGE electrophoresis, carry out again transferring film, the protein delivery of the results on the gel to nitrocellulose filter, after the confining liquid sealing, is added the monoclonal antibody for Porcine circovirus type 2 Cap protein, add two anti-with label.Then judge Recombinant Swine circovurus type 2 ORF2 protein expression.The results are shown in accompanying drawing 3, wherein, the 1st hole is that blueness is dyed low molecular weight protein Marker in advance; The 2nd hole is culture supernatant and the porcine circovirus 2 type positive serum reaction result of gathering in the crops behind the 24h; The 3rd hole is culture supernatant and the porcine circovirus 2 type positive serum reaction result of gathering in the crops behind the 48h, and the 4th hole is culture supernatant and the porcine circovirus 2 type positive serum reaction result of gathering in the crops behind the 72h, and the 5th hole is and the negative serum reaction result.Analyze the restructuring Porcine circovirus type 2 Cap protein of expressing with Western blotting.Resisting porcine circovirus 2 type hyper-immune serums with pig detect the restructuring Porcine circovirus type 2 Cap protein.The result shows, on nitrocellulose filter, only observe a molecular size range and expection specific band of the same size, illustrate that the restructuring Porcine circovirus type 2 Cap protein of expressing can with the porcine circovirus 2 type antibody specific binding, prove that the restructuring Porcine circovirus type 2 Cap protein has special porcine circovirus 2 type immunogenicity.

3. gather in the crops and the purification of Recombinant Porcine circovirus type 2 Cap protein

With the culture of 72h abduction delivering through the centrifugal 20min of 10000rpm, precipitation separation and supernatant, precipitation discards, supernatant filters by the filter membrane of 1 μ m, then by Mi Libo (Millipore company) ultrafiltration and concentration purification, sample solution behind the purification carries out determining the protein quantity by ultraviolet spectrophotometer well known in the art, and expression is 55mg/L.Adding the divinyl imines behind the purification, to make ultimate density be 7mmol/L divinyl imines (BEI) deactivation recombination expression product, and adding final concentration behind the 48h is the sodium thiosulfate neutralization of 7mmol/L.

The preparation of the vaccine combination of embodiment 7, pig circular ring virus antigen, haemophilus parasuis antigen

Antigen: adopt porcine circovirus 2 type (PCV2) the ORF2 proteantigen of embodiment 6 preparations and the haemophilus parasuis antigen of embodiment 1 (k001 criticize carry out 3 times concentrated, haemophilus parasuis 4 type inactivation antigens and haemophilus parasuis 5 type inactivation antigen bacterial concentrations are deactivation front 13.5 * 10 ⁹CFU/ml),

Carbomer solution preparation: with carbomer Carbomer 934P(Qingdao Tian Liyuan bio tech ltd) is dissolved in the deionized water, makes 12 ℃ of 30min autoclavings of stock solution of 25mg/ml, 4 ℃ of preservations

The preparation of the chitosan solution of 2%W/V: the 0.5M sodium acetate solution uses the 4.lg sodium acetate (available from Sigma Chemical Co., St.Louis, MO) be dissolved in the 50ml deionized water, with about 7ml glacial acetic acid pH is transferred to 4.5, add again the adding of 1.5ml glacial acetic acid compensation poly-glucosamine to the impact of pH value of solution.Overall solution volume transfers to l00ml with deionized water, and 2g chitosan (it wins biological product company limited product, the degree of polymerization 88.5% Shanghai) slowly adds above-mentioned sodium acetate solution and constantly stirred 2-3 hour until the chitosan dissolving.0.22 μ m membrane filtration is removed impurity, 112 ℃ of 30min autoclavings, 4 ℃ of preservations.

The vaccine combination test example chief component of table 14, Porcine circovirus desease, Haemophilus parasuis (every part vaccine is 2ml)

Preparation method:

Proportioning according to table 14, the Porcine circovirus type 2 Cap protein, haemophilus parasuis 4 type JS bacterial strain inactivation antigens, the haemophilus parasuis 5 type ZJ bacterial strain inactivation antigens that in sterile chamber, add successively above-mentioned content ratio, carbomer solution and chitosan solution, with PBS liquid (pH value is 7.2～7.4) mix and blend 30 minutes both the vaccine combination of pig circular ring virus antigen, haemophilus parasuis antigen.

Vaccine combination immunological testing and the challenge test of embodiment 8 pig circular ring virus antigens, haemophilus parasuis antigen

The vaccine immunity 25 age in days PCV2 negative antibody piglets Animal House of (Pulaike Biological Engineering Co., Ltd. raise), are established not vaccination not 8 of counteracting toxic substances negative control pigs and 8 of vaccination counteracting toxic substances contrast pigs not simultaneously by 8 every group.Immune group is carried out primary immune response, and immune bigeminy thing adopts the vaccine combination of embodiment 3 preparations.Rear 21 days of immunity to immune group and counteracting toxic substances contrast carry out virus attack, strain is selected strong poison (the strong poison 10 of PCV2-SH strain ^6.0TCID50/ml), every pig collunarium 1ml, musculi colli injection 2ml, isolated rearing.Behind the counteracting toxic substances the 4th, 7 day respectively at keyhole hemocyanin (4mg/ml, 0.5ml) and the lumbar injection thioglycollate medium 10ml of two oxters and two buttock injections incomplete Freunds adjuvant emulsifyings.At the 11st day and 19 days lumbar injection thioglycollate medium 10ml.The not independent isolated rearing of counteracting toxic substances negative control group of not vaccination.Cutd open extremely in 28 days behind the counteracting toxic substances.

Before the 1st immunity, before the counteracting toxic substances and before cuing open extremely, all pigs are collected blood sample, carry out the analysis of PCV2 serum antibody by the ELISA method.Result of the test sees Table 3.Cut open the whole pigs of inspection in 28 days behind the counteracting toxic substances, observe each histoorgan pathological change, Taking Pictures recording.Meet any 2 in following 3, can be judged to morbidity.

A. clinical symptoms: piglet fervescence (〉=40 ℃), should continue 3 at least, occur obvious loss of appetite, spirit depressed, thick disorderly by hair, become thin and the speed of growth is slowed down;

B.. pathological change: groin and lymphoglandulae tracheales edema, lungs Mild edema, kidney are turned to be yellow or spotty necrosis are arranged.Histologic lesion is that lymph has obvious lymphocyte intrusion, or multinucleated giant cell is arranged;

C. virus detects: detect lymph node tissue with PCR, detect PCV2.The immune result of each test group sees Table 15 in the table 14.

Table 15 is respectively organized test pig PCV2 serum antibody analysis result

Table 16. is respectively organized experimental animal morbidity result of determination

Interpretation of result: detect judged result according to experimental animal clinical symptoms, the PCR that cuts open inspection pathological change detection and cause of disease, wherein Immunization matched group piglet does not all fall ill, and all test group are not all fallen ill.

The immune side effect of embodiment 9, porcine circovirus 2 type antigen, haemophilus parasuis antigen vaccine compositions and prior art vaccine relatively

1 material

Haemophilus parasuis, porcine circovirus type 2 vaccines compositions are selected laboratory products test example 3 goods (V1) and test example 1 goods (V2) among the embodiment 7; Porcine circovirus 2 type inactivated vaccine (SH strain), Pulaike Biological Engineering Co., Ltd. produces (lot number is 100903) (V3); Haemophilus parasuis inactivated vaccine (4 type MD0322 strains+5 type SH0165 strains), biological product company limited (lot number is 100306) (V4) before the section of Wuhan.

The design of 2 animal experiments

Select 60 of 21～28 age in days ablactational baby pig, be divided into 12 groups, 5 every group; 1st, 2,3 groups of every pig difference musculi collis are injected porcine circovirus 2 type antigen, haemophilus parasuis antigen vaccine compositions embodiment 7 test examples 5) 1ml, 4th, 5,6 groups of every pig difference musculi collis are injected haemophilus parasuis, porcine circovirus type 2 vaccines compositions embodiment 7 test examples 8) 1ml, 7,8,9 groups of every pigs difference left side musculi colli injection Haemophilus parasuis vaccine (4 type MD0322 strains+5 type SH0165 strains) 1ml, right side musculi colli injection porcine circovirus type 2 vaccines (SH strain) 1ml, 14d inoculates each 1ml again after the immunity; 10th, do not inoculate for 11,12,13 groups.35d after first immunisation, the 1st, 4,7,10 group of every pig through the serum 4 type JS strain bacterium liquid 3ml of 1 fatal dose of intraperitoneal injection, observed respectively day by day, observes to the counteracting toxic substances to slaughter in 14th and cuts open inspection; 2nd, 5,8,11 groups of every pigs are respectively through the Serotype 5 ZJ of 1 fatal dose of intraperitoneal injection strain bacterium liquid 3ml, observe day by day, observe to the counteracting toxic substances to slaughter in 14th and cut open inspection; 3rd, 6,9,12 groups respectively (contain 10 with the PCV2SH strain ^6.0TCID ₅₀/ ml) collunarium 1ml, intramuscular injection 2ml, the 13rd group of counteracting toxic substances not, Continuous Observation is 25 behind the counteracting toxic substances, slaughters after weighing in 25th behind the counteracting toxic substances, cuts open inspection.Judge according to body temperature, relative daily gain and virus antigen detection result.

The controlled trial of table 17, vaccine combination and univalent vaccine

Annotate: untoward reaction shows as injection site redness, fervescence, anorexia, lassitude.

By demonstrating as seen from Table 17 good immune protective effect, show that the immune effect of vaccine combination of the present invention and single Seedling is substantially suitable; From dosage of inoculation comparatively speaking, vaccine combination is played 1 pin, is total to 1ml, and two kinds of single Seedling couplings need be played 4 pins, is total to 4ml, and apparent effect is that the vaccine combination use is more convenient, time saving and energy saving; From the view of safety, the safety of vaccine combination is comparatively safe, and single Seedling coupling has 1/5 test pig that untoward reaction is arranged, and vaccine combination will increase by 4 times because the dosage of injection compares; Comprehensively state it, vaccine combination is not only easy to use, and is safer.

PCV2 counteracting toxic substances protection effect relatively behind embodiment 10:PCV2 ORF2 recombinant protein antigen, the haemophilus parasuis antigen vaccine compositions immunity piglet

This research design is in order to confirm vaccine combination in the identical situation of adjuvant (PCV2 recombiant protein subunit antigen+haemophilus parasuis) and the independent immune effect of porcine circovirus 2 type recombiant protein subunit vaccine in identical adjuvant situation

Such as previous embodiment 1 preparation haemophilus parasuis inactivation antigen, embodiment 6 preparation reach Porcine circovirus type 2 ORF2 protein, vaccine combination according to table 18 preparation pig circular ring virus antigen, haemophilus parasuis antigen, in sterile chamber, add successively pig circular ring virus ORF2 albumen, haemophilus parasuis 5 type antigens, haemophilus parasuis 4 type antigens, adding vaccine adjuvant, is 7.2～7.4 with PBS(phosphate buffer pH) liquid adjustment volume.

The prescription of each experimental vaccine of table 18 (every part vaccine is 2ml)

Select 30 of 21～28 age in days ablactational baby pig, be divided into 6 groups, 5 every group (seeing Table 19); The 1st group of every pig difference musculi colli vaccine (V1) 2ml, 21d inoculates 2ml again after the immunity; The 2nd group of every pig difference musculi colli vaccinate (V2) 2ml, 21d inoculates 2ml again after the immunity; The 3rd group of every pig difference musculi colli vaccinate (V3) 2ml, 21d inoculates 2ml again after the immunity; The 4th group of every pig difference musculi colli vaccinate (V4) 2ml, 21d inoculates 2ml again after the immunity; The 5th as the counteracting toxic substances matched group, and the 6th group as the blank group.35d after first immunisation, the 1st, 2,3,4,5 group each (contains 10 with the PCV2SH strain ^6.0TCID ₅₀/ ml) collunarium 1ml, intramuscular injection 2ml, the 4th group of counteracting toxic substances not, behind the counteracting toxic substances the 4th, 7 day, respectively in the oxter, both sides of the 1st, 2,3,4,5,6 group of every pig and the both sides buttocks totally 4 points to the keyhole hemocyanin (KLH/ICFA of all pigs inoculations with the incomplete Freunds adjuvant emulsifyings, 0.5mg/ml), each some inoculation 1ml(4ml/ head), while intraperitoneal inoculation thioglycollate medium (Thio), 10ml/ head; Intraperitoneal inoculation thioglycollate medium again on the the 11st, 19 behind the counteracting toxic substances, the 10ml/ head.Continuous Observation is 25 behind the counteracting toxic substances, slaughters after weighing in 25th behind the counteracting toxic substances, cuts open inspection.Carry out immune effect relatively according to microcosmic Histopathologic change and immunohistochemistry (IHC) detection PCV2 virus antigen result.

Table 19 test grouping and processing

Tissue injury and SABC detectable antigens situation see Table 20, as seen behind the pig PCV2 counteracting toxic substances of vaccine combination (V1) immunity without the pathology damage and do not detect PCV2 antigen, and 5 pigs of pig of porcine circovirus 2 type inactivated vaccine (V2) immunity have 1 to detect pcv2 antigen and it has the distinctive pathology damage of pathology porcine circovirus (appearance of interstitial pneumonia, lymph node lymphocyte damage and multinucleated giant cell and mononuclear phagocyte infiltration etc.).

Respectively organizing test pig tissue injury and IHC behind the table 20 PCV2 counteracting toxic substances detects

The vaccine combination of subunit ORF2 vaccine and other antigens is thought in this area always, because the easy degeneration of albumen or combine with other antigens, may deleterious.But the result of this research is unexpected: when using porcine circovirus 2 type (PCV2) ORF2 albumen as PCV2 antigen, the vaccine combination of the combined antigen preparation of itself and haemophilus parasuis antigen has shown the identical or essentially identical immune effect of vaccine combination that deactivation PCV2 totivirus antigen and the combined antigen of haemophilus parasuis antigen prepare.

In addition, vaccine combination of the present invention not only can be used for immune swine so that good immune effect to be provided, and kept the good immune efficacy of former PCV2 antigen composition, its effect also is better than independent Porcine circovirus type 2 Cap protein subunit vaccine, and namely the immune effect of the antagonism PCV2 of vaccine combination of the present invention is better than using separately the immune effect of PCV2 ORF2 protein subunit vaccine.Especially still kept the immune effect of porcine circovirus 2 type (PCV2) antigen in the situation that PCV2 antigen amount reduces by half, the above results also exceeds those skilled in the art's expectation.

Embodiment 11:PCV2ORF2 recombinant protein antigen, haemophilus parasuis antigen vaccine compositions, with the immunity of haemophilus parasuis inactivated vaccine after haemophilus parasuis counteracting toxic substances protection effect relatively

Press embodiment 1 preparation haemophilus parasuis antigen, by preparing Porcine circovirus type 2 Cap protein among the embodiment 6, vaccine combination according to table 21 preparation pig circular ring virus antigen, haemophilus parasuis antigen, in sterile chamber, add successively pig circular ring virus ORF2 albumen, haemophilus parasuis 5 type antigens, haemophilus parasuis 4 type antigens, add vaccine adjuvant, adjust volume with PBS liquid (pH value 7.2～7.4).

The prescription of each experimental vaccine of table 21 (every part vaccine is 2ml)

Select 35 of 21～28 age in days ablactational baby pig, be divided into 7 groups, 5 every group (seeing Table 22); 1st, 2 groups of every pig difference musculi collis are injected haemophilus parasuis (4 types+5 types), porcine circovirus 2 type bivalent inactivated vaccine (V1) 2ml, and 2ml was inoculated in immunity in rear 21 days again; 3rd, 4 groups of every pig difference musculi colli injection Haemophilus parasuis inactivated vaccine (4 types+5 types) 2ml, 2ml was inoculated in immunity in rear 21 days again; 5th, do not inoculate for 6,7 groups.After first immunisation 35 days, the 1st, 3,5 group of every pig observed day by day respectively through the serum 4 type JS strain bacterium liquid 3ml of 1 fatal dose of intraperitoneal injection, observed to the counteracting toxic substances to slaughter in 10th and cutd open inspection; 2nd, 4,6 groups of every pigs are respectively through the Serotype 5 ZJ of 1 fatal dose of intraperitoneal injection strain bacterium liquid 3ml, observe day by day, observe to the counteracting toxic substances to slaughter in 10th and cut open inspection; The 7th group not counteracting toxic substances make blank.

Table 22 test grouping and handling procedure

Result of the test such as table 23, vaccine combination (V1) is 5/5(100% to the counteracting toxic substances protection of 4 type JS strains and 5 type ZJ strains), haemophilus parasuis vaccine (serum 4 types+Serotype 5) (V2) is 5/5(100% to the counteracting toxic substances protection of 4 type JS strains and 5 type ZJ strains).

Respectively organize comparative result behind the table 23 haemophilus parasuis counteracting toxic substances

Group	Size of animal	Morbidity quantity	Dead quantity	Protective rate (morbidity number/number of animals)
					V1	5	0	0	5/5
V1	5	0	0	5/5
					V2	5	0	0	5/5
V2	5	0	0	5/5

[0301]

4 type counteracting toxic substances matched groups	5	5	3	/
					5 type counteracting toxic substances matched groups	5	5	4	/
The blank group	5	0	0	/

The result of this research is unexpected equally: it has been generally acknowledged that the vaccine combination of subunit ORF2 vaccine and other antigens, because albumen combines with other antigens easily, may cause the deleterious of other vaccines.But the result of this research is unexpected: at the vaccine combination that uses the preparation of haemophilus parasuis inactivation antigen and porcine circovirus 2 type (PCV2) ORF2 albumen, the good immune efficacy that has still kept the haemophilus parasuis composition, and can infect the immune effect that provides good for resisting porcine circovirus 2 types (PCV2) and haemophilus parasuis simultaneously.Prior art thinks that then protein ingredient can disturb the effect of haemophilus parasuis antigen usually, unites and uses the usually immunogenicity of meeting partial offset haemophilus parasuis vaccine.

Embodiment 12: the evaluation of haemophilus parasuis

(1) PCR identifies

Will be for inspection JS strain bacterium liquid ZJ strain bacterium liquid and 10, the centrifugal 5min of 000r/min, abandon that supernatant suspends with sterilized water or with the bacterial strain of separation and purification with 2 bacterium colony eluting of inoculating loop scraping in the centrifuge tube that contains 100 μ l sterilized water, vortex, in boiling water, behind the water-bath 10min, place-20 ℃ again and freeze.After the freeze thawing 10, the centrifugal 5min of 000r/min gets supernatant and makes template.

Reference literature (Oliveira S, Galina L, Pijoan C.Development of a PCR test to diagnose Haemophilus parasuis infections.Vet Diagn Invest. PCR method that is used for the infection of diagnosis haemophilus parasuis is set up) synthetic by the prompt base in the English Weihe River (Shanghai) trade Co., Ltd, sequence is respectively forward primer: 5 '-GGC TTC GTC ACC CTC TGT-3 '; Downstream primer: 5 '-GTG ATG AGG AAG GGT GGT GT-3 '.This primer amplification fragment is the dna fragmentation of coding 16S rRNA, and length is about 822bp.

Reaction system (25 μ l): 10 * PCR Buffer, 2.5 μ l, 25mmol/L MgCl ₂1.5 μ l, 2.5m mol/L dNTPs 2.0 μ l, each 0.8 μ l of 10 μ mol/L upstream and downstream primers, 5U/ μ l Ex-Taq 0.1 μ l, sterilized water 15.3 μ l, template 2 μ l.

Reaction condition: 94 ℃ of denaturation 5min, 94 ℃ of degeneration 30s, 60 ℃ of annealing 30s, totally 30 circulations, last 72 ℃ are extended 10min.PCR sample after the amplification is in 1.0% agarose gel electrophoresis.Gel dyes with GelRed, the ultraviolet (uv) transmission observation analysis.The haemophilus parasuis reference culture is the fragment of 822bp size according to haemophilus parasuis 16S rRNA primers through the product of pcr amplification available from Hua Zhong Agriculture University.Product confirms that through agarose gel electrophoresis its size is all consistent with positive control.

(2) Serotype Identification

With reference to Kielstein-Rapp-Gabrielson(KRG) (KIELSTEIN P, RAPP-GABR IELSON V J, JOURNAL OF CLINICAL MICROBIOLOGY, Apr.1992, method 30:862-865), 4 types and 5 type haemophilus parasuis standard antigens and standard positive serum available from Wuhan section before the animal biological product company limited, as a result JS strain is serum 4 types, the ZJ strain is Serotype 5.

The above only is preferred embodiment of the present invention; not in order to limit the present invention; within the spirit and principles in the present invention all; any modification of doing, be equal to alternative and dilute before use merging etc. and other and improve etc. as using the similar strain of other gene or two kinds of business-like vaccines instead, all should be included within protection scope of the present invention.

Claims

1. a vaccine combination that prevents or treat porcine circovirus 2 type (PCV2) and haemophilus parasuis to infect is characterized in that it comprises at least a porcine circovirus 2 type antigen and at least a haemophilus parasuis antigen.

2. vaccine combination according to claim 1 is characterized in that, described porcine circovirus 2 type antigen is selected from totivirus antigen, attenuated live virus antigen, subunit antigen, live recombinant vectors and the dna vector antigen of deactivation; Haemophilus parasuis antigen is selected from the antigen of the full bacterium antigen of deactivation, attenuated live antigen, subunit antigen, live recombinant vectors and dna vector.

3. vaccine combination according to claim 2 is characterized in that, described porcine circovirus 2 type antigen is subunit antigen; Described haemophilus parasuis antigen is the full bacterium antigen of deactivation.

4. vaccine combination according to claim 3 is characterized in that, described porcine circovirus 2 type antigen is PCV2 ORF2 albumen; The haemophilus parasuis ZJ strain of the haemophilus parasuis JS strain that described haemophilus parasuis antigen is deactivation and deactivation.

5. vaccine combination according to claim 4 is characterized in that, the content of described PCV2 ORF2 albumen is 0.5 ~ 20 μ g/ head part; The content of the haemophilus parasuis JS strain antigen of described deactivation is 1 * 10 ⁹~ 8 * 10 ⁹CFU/ head part, the content of the haemophilus parasuis ZJ strain antigen of described deactivation is 1 * 10 ⁹~ 8 * 10 ⁹CFU/ head part.

6. vaccine combination according to claim 2 is characterized in that, the totivirus antigen that described porcine circovirus 2 type antigen is deactivation; Haemophilus parasuis antigen is the full bacterium antigen of deactivation.

7. vaccine combination according to claim 6, it is characterized in that, the porcine circovirus 2 type PCV2 SH strain that described porcine circovirus 2 type antigen is deactivation, the haemophilus parasuis ZJ strain of the haemophilus parasuis JS strain that described haemophilus parasuis antigen is deactivation and deactivation.

8. vaccine combination according to claim 7 is characterized in that, described haemophilus parasuis JS strain content is 1 * 10 ⁹CFU/ head part, described haemophilus parasuis ZJ strain content is 1 * 10 ⁹CFU/ head part.

9. vaccine combination according to claim 7 is characterized in that, contains PCV2 4.5 * 10 in the described antigen composition ^5.0TCID ₅₀/ head part, serum 4 type haemophilus parasuises 1 * 109CFU/ head part contains Serotype 5 haemophilus parasuis 1 * 10 ⁹CFU/ head part, vaccine adjuvant are 10%V/V GEL adjuvant.

10. the described vaccine combination of any one is characterized in that according to claim 1～9, also can comprise the additional antigen of other porcine pathogen.

11. antigen composition according to claim 10, it is characterized in that described other the additional antigen of porcine pathogen is selected from: porcine reproductive and respiratory syndrome virus (PRRSV) antigen, mycoplasma hyopneumoniae antigen, Actinobacillus pleuropneumoniae (APP) antigen, PRV (Pseudorabies virus) (PRV) antigen, pig bordetella bacilli antigen, pig pasteurella multocida antigen, swine influenza virus (SIV) antigen or their combination.

12. according to right 12 described vaccine combinations, it is characterized in that described adjuvant is selected from: aluminium hydroxide, aluminum phosphate, saponin such as Quil A and QS-21, water in oil emulsion, oil in water emulsion, W/O/W Emulsion, carbomer, Montanide ^TMGel.

13. according to right 12 described vaccine combinations, it is characterized in that described adjuvant is preferably Montanide ^TMGel.

14. according to claim 1-13 application of the described vaccine combination of any one in preparation prevention and treatment pig circular ring virus relevant disease and Haemophilus parasuis medicine.

15. a haemophilus parasuis is characterized in that, it is characterized in that, described haemophilus parasuis is the 4 type JS strains of haemophilus parasuis serum, and preserving number is CCTCC No.M2011172; With haemophilus parasuis Serotype 5 ZJ strain, preserving number is CCTCC No.M2011173.