CN103319576B - Haemophilus parasuis (Hps) immunoprotecive antigen AfuA - Google Patents
Haemophilus parasuis (Hps) immunoprotecive antigen AfuA Download PDFInfo
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Abstract
The invention relates to identification of a Haemophilus parasuis (Hps) immunoprotecive antigen, separation and cloning of a protein gene and application of a protein coded by the Hps immunoprotecive antigen in a vaccine. According to the invention, a new protein AfuA having immunogenicity is separated from Hps (the culture collection number is CVCC3361), and the nucleotide sequence is shown as SEQ ID NO:2 in a sequence table and is formed by coding 346 amino acids. The AfuA is a new protein having immunogenicity and can provide effective immunoprotection for Hps infection in mice. The invention also comprises preparation of an Escherichia coli recombinant bacterium BL21/Hps-afuA expressing the immunogenicity protein gene afuA. The recombinant Hps AfuA protein expressed by the invention has favorable safety and protection efficacy, and the immunoprotection effect is up to 80%.
Description
Technical field
The present invention relates to zoonosis subunit vaccine preparing technical field.Be specifically related to the application of albumen in vaccine of a kind of qualification of haemophilus parasuis immune protective antigen, the separation of protein gene, clone and its coding.
Background technology
Modern proteomic techniques combines with traditional immunization hybridizing method emerging cross discipline---the immunoproteomics (Immunoproteomic) of generation one, and among the immunogenic protein research being promptly applied to people, animals and plants and microorganism, be a strong instrument of Large-scale Screening pathogenic bacteria antigen.This research by two-dimensional electrophoresis and Western blot technology to haemophilus parasuis (Haemophilus parasuis; Hps) secretome is analyzed; filter out the antigen protein that antigenicity is good, protection is strong, to establishing solid basis for the research and development of development serodiagnosis mark and novel subunit vaccine.
Haemophilus parasuis (Haemophilus parasuis, Hps) is a kind of Gram-negative bacteria, is often settled in the upper respiratory tract of pig, is a kind of conditioned pathogen, can causes the Haemophilus parasuis of pig.
this bacterium of reported first in 1910, therefore this disease be also called leather draw Se Shi disease (
disease).Hps infected pigs, can affect the pig at 2 week age to 4 monthly ages, and morbidity is common in pig in 5 ~ 8 week age.Sickness rate is 10% ~ 15%, and time serious, mortality ratio can reach 50%, and to raising pigs, industrial belt carrys out massive losses, and the principal character of this disease is fibrinous pleurisy, peritonitis, sacroiliitis, pericarditis and meningitis.
This test for identification is to the afuA (Iron (Fe with immune protective effect
3+) ABC superfamilyATP binding cassette transporter, binding protein) albumen, this albumen belongs to ABCtransporters family, participate in the transmembrane transport of ferric ion, the process absorbing iron ion with bacterium is closely related, ABC (ATP binding cassette) transporters family, it is one of maximum protein family, the core texture of abc transport albumen is made up of 4 structural domains usually, comprises membrane spaning domain and 2 core former times acid binding domain of 2 very hydrophobic.Each membrane spaning domain is generally made up of 6 α spirals, also exists by 10,17, the membrane spaning domain that forms of 19 α spirals.They form a transmembrane channel to realize the transdermal delivery of substrate molecule, also participate in the recognition process of substrate simultaneously, and colibacillary 5% genome participates in the albumen of this family of coding.It is present in all species, from simple microorganism to the mankind of complexity.In microorganism, ABC transporters absorbs with antibiotics resistance, antimycotic resistance, nutritive substance and metabolite is discharged relevant.This test for identification to the afuA albumen with immune protective effect, and can provide effective immune protective efficiency to mouse infection haemophilus parasuis, can as the candidate antigens of haemophilus parasuis subunit vaccine exploitation.
Summary of the invention
The object of this invention is to provide the albumen and encoding gene thereof that can be used in the candidate antigens preparing Haemophilus parasuis subunit vaccine.
This research by two-dimensional electrophoresis and Western blot law technology to haemophilus parasuis (Haemophilusparasuis; Hps) secretome is analyzed; filter out the antigen protein that antigenicity is good, protection is strong, to establishing solid basis for the research and development of development serodiagnosis mark and novel subunit vaccine.
The present invention identifies the AfuA albumen with immune protective effect, and can provide effective immune protective efficiency to mouse infection haemophilus parasuis, can as the candidate antigens of Haemophilus parasuis subunit vaccine exploitation.
In order to realize the object of the invention, first the present invention is separated the exocytosis albumen of haemophilus parasuis, then secretome is separated by bidirectional electrophoresis method, and transfer them on nitrocellulose filter, use the rehabilitation serum of pig to carry out western-blot hybridization searching and there is immunogenic albumen, then Mass Spectrometric Identification is carried out by Mass Spectrometric Identification technology to having immunogenic albumen, again clonal expression is carried out to its encoding gene, after the protein purification of expression, carry out Analysis of Immunogenicity again, carry out the Immunoprotection test of mouse simultaneously, result shows that it has certain immanoprotection action.
The present invention protects a kind of method preparing AfuA albumen simultaneously, and described method comprises the recombination bacillus coli cultivated containing AfuA albumen coded sequence, obtains AfuA albumen.The method of the described AfuA of preparation albumen specifically can be: described recombinant bacterium 37 DEG C is cultivated 3h, OD
600when=0.7, add IPTG to final concentration 0.8 μM, go to 37 DEG C and continue to cultivate 4h.
AfuA albumen of the present invention has immunoprotection activity.
AfuA albumen of the present invention can be applied to the preparation of Haemophilus parasuis subunit vaccine.
More specifically, the invention provides the following:
1. a haemophilus parasuis immune protective antigen, its aminoacid sequence is as shown in SEQ ID NO:1.
2. the nucleic acid of the haemophilus parasuis immune protective antigen of coding according to 1, its sequence is as shown in SEQ ID NO:2.
3. recombinant vectors, it comprises the nucleic acid according to 2.
4. comprise the cell of the nucleic acid according to 2 or the recombinant vectors according to 3.
5. the haemophilus parasuis immune protective antigen according to 1, it is used as haemophilus parasuis subunit vaccine.
6. the haemophilus parasuis immune protective antigen according to 1, the nucleic acid according to 2, the recombinant vectors according to 3 and the cell according to 4 are preparing the purposes in haemophilus parasuis subunit vaccine.
7. the haemophilus parasuis immune protective antigen according to 1, the nucleic acid according to 2, the recombinant vectors according to 3 and the cell according to 4 purposes in the medicine of the disease caused by haemophilus parasuis for the preparation of prevention.
8. the purposes according to 7, the wherein said disease caused by haemophilus parasuis is Haemophilus parasuis.
9. vaccine composition, it comprises the haemophilus parasuis immune protective antigen according to 1.
10. prepare a method for haemophilus parasuis immune protective antigen, said method comprising the steps of:
Build the recombinant expression vector of the nucleotide sequence comprised as shown in SEQ ID NO:2;
Described recombinant expression vector is transformed in host cell;
Cultivate the described host cell through conversion and induce it to express described haemophilus parasuis immune protective antigen; With
From haemophilus parasuis immune protective antigen described in the described host cell abstraction and purification through inducing.
11. methods according to 10, wherein said host cell is intestinal bacteria.
Accompanying drawing explanation
Fig. 1: left figure is the bidimensional electrophoretic separation collection of illustrative plates of secretory protein; Right figure is hybridization figure after transferring film.
Hybridization figure after Fig. 2: AfuA transferring film.
Malicious mouse survival rate figure is attacked after Fig. 3: AfuA immunity.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
The discovery of embodiment 1, AfuA protein immunogenic
One, the preparation of secretory protein sample
Haemophilus parasuis (Haemophilus parasuis is taken out from-70 DEG C of refrigerators, Hps) bacterial strain is (purchased from National Veterinary Culture Collection, bacterium numbering: CVCC3361), room temperature is melted, then streak inoculation is in TSA (Becton, Dickinson and Company) solid medium is (containing the horse serum of 10%, the NAD (Reduced nicotinamide-adenine dinucleotide) of 0.01%), cultivate 36h for 37 DEG C, then picking list colony inoculation is in TSB (Becton, Dickinson and Company) liquid nutrient medium is (containing horse serum, NAD containing 0.02%), cultivate 24h for 37 DEG C, then (horse serum is not contained with long-pending the switching in 500mL TSB liquid nutrient medium of bacteria liquid of 1%, NAD containing 0.02%), cultivate about 16h hour for 37 DEG C, bacterium liquid OD is made to reach 1.0.Proteinase inhibitor is added, 4 DEG C of centrifugal 1h of 5000g to bacterial cultures.Get supernatant, with the filter membrane of 0.22 μm residual Hps elimination.100% trichoroacetic acid(TCA) (TCA) of 1/6 volume precooling is added in filtrate, 4 DEG C static 30 minutes for the albumen in precipitation filtrates, centrifugal 30 minutes of 4 DEG C of 12000g afterwards, collecting precipitation, abandon supernatant, then use the acetone of precooling by washing of precipitate three times, fully TCA is dissolved in acetone and washes off.The precipitation finally obtained is exactly the secretory protein of haemophilus parasuis, and room temperature places 10 minutes, acetone is volatilized completely, is dissolved in appropriate lysate (7M urea, 2M thiocarbamide, 4%Chaps).Take out a tubule and carry out determination of protein concentration.
Two, first of secretory protein sample to isoelectrofocusing (IEF)
The secretory protein 600 μ g lysate getting above preparation is diluted to final volume 250 μ L, and carry out isoelectrofocusing by the IPG adhesive tape (Bio-Rad) that pH gradient is pH3 ~ 10, arranging focusing parameter is: temperature 20 DEG C; Maximum current 0.05mA/ adhesive tape, focalizer is: 30V, 12h; 200V, 1h; 500V, 1h; 1000V, 2h; 7000V, 3h; 7000V to 50000Vh.
Three, the second to SDS-PAGE electrophoresis of secretory protein sample
Carefully rinse the covering oil in the adhesive tape focused on deionized water, then balance.First time balances: adhesive tape be placed in level pad I (containing 6M urea, 75mMTris-HCl pH8.8,87% glycerine, 2%SDS, 0.2% tetrabromophenol sulfonphthalein, 0.1g DTT/10mL), incubated at room 10 minutes on horizontal shaker; By deionized water rinsing adhesive tape 4 ~ 5 times.Second time balance: adhesive tape is placed in level pad II (containing 6M urea, 75mMTris-HCl pH8.8,87% glycerine, 2%SDS, 0.2% tetrabromophenol sulfonphthalein, 0.25g IAA/10mL), horizontal shaker is hatched 15 minutes.By the adhesive tape that the 1 × sds gel damping fluid rinse prepared balances.The water-saturated n-butanol covered on acrylamide gel is outwelled, and carefully rinses gel surface with deionized water.The adhesive tape balanced carefully is put into the upper strata of gel, the low melting-point agarose adding melted 0.5% immediately fixes IPG adhesive tape, avoids producing bubble between adhesive tape and gel.In electrophoresis chamber, add electrophoretic buffer, put into by gel glass plate, add upper strata damping fluid, then close the lid, set temperature 12.5 DEG C, electric current 10mA/ glue starts electrophoresis.After electrophoresis 1h, electric current changes 20mA/ glue into, until tetrabromophenol sulfonphthalein band goes to gel stop electrophoresis bottom.Careful taking-up gel, dyes.
Four, secretory protein sample SDS-PAGE glue dyeing
1. fixing: the gel after electrophoresis to be put in stationary liquid (40% ethanol, 10% acetic acid, 50% deionized water), fixedly spend the night.
2. sensitization: outwelled by stationary liquid, adds 10% glacial acetic acid, jog 30 minutes on horizontal shaker.
3. dye: gel is put into R-250 staining fluid, 90 DEG C are heated 30 minutes.
4. decolour: staining fluid is outwelled, adds 10% glacial acetic acid, be placed on horizontal shaker and decolour, until protein site is high-visible.
Five, the Western blotting analysis of secretory protein sample
The half-dried mode that turns is adopted to carry out transfer printing by second of secretory protein sample to SDS-PAGE glue Western blotting conveniently.Transfer printing 80 minutes, according to 0.8mA/cm
2setting electric current.After transfer printing terminates, pvdf membrane (Pall Corparation) ponceau dyeing, marks several markup points with syringe needle, preservation of taking pictures.Close for 4 DEG C and spend the night, confining liquid is the skimming milk of 5%.PBST washes 3 times, each 10 minutes.Primary antibodie is that (choose 4-6 age in week, ELISA antibody test is negative sodium selenite to Hps pig rehabilitation serum, with 5 × 10
7cFU/mL Hps (CVCC3361) attacks poison, raises after 30 days, and the blood sample extracting survival piglet prepares rehabilitation serum, packing in laboratory, frozen) (1: 200), hatch 1h for 37 DEG C.PBST washes 3 times, each 10 minutes.Two resist anti-(Sigma) (1: 5000) of the anti-pig of rabbit two for horseradish peroxidase HRP mark, hatch 1h for 37 DEG C.PBST washes 3 times, each 10 minutes, and last chemoluminescence colouring reagents box (the biological company limited of sky root) develops the color.According to the characteristic protein point that prior ponceau dyeing picture and syringe needle mark, the gel of the coomassie brilliant blue staining of correspondence is found out corresponding protein site.As Fig. 1, find 9 immunoreactive albumen occurs altogether.
Six, mass spectroscopy
The protein site the found out 200 μ L rifle head stamps cut are taken off, then puts into clean centrifuge tube, carry out mark, be sent to Beijing Proteome Research Center and carry out mass spectroscopy.Through identifying that No. 8 points are Iron (Fe
3+) ABC superfamily ATP binding cassette transporter, bindingprotein (AfuA), massfraction is 667, and molecular weight is 38595Da (aminoacid sequence is as shown in SEQID NO:1).
The expression of embodiment 2, AfuA albumen
One, the extraction of Hps STb gene
By centrifugal 1 minute of the incubated overnight bacterium liquid 12000rpm of 1mL Hps (CVCC3361), abandon supernatant.40 μ L DB liquid (TIANamp Bacteria DNA Kit, the biological company limited of sky root) are added, 160 μ L N,O-Diacetylmuramidases and 8 μ LRNaseA in bacterial sediment.Concuss, mixes.37 DEG C of temperature are bathed 30 ~ 60 minutes, constantly put upside down centrifuge tube for several times.Add 200 μ L DLT liquid (TIANamp Bacteria DNA Kit, the biological company limited of sky root) and 25 μ L Proteinase Ks (TIANamp Bacteria DNAKit, the biological company limited of sky root), leniently put upside down mixing immediately.To put in 65 DEG C of water-baths at least 30 minutes, constantly put upside down centrifuge tube for several times.Centrifugal 3 ~ 5 minutes of 12000rpm, draws whole supernatant in a clean centrifuge tube with pipettor.Add 200 μ L dehydrated alcohols, all suck adsorption column after mixing, 12000rpm, centrifugal 30 seconds.Outwell the liquid in collection tube, put back to adsorption column.Add 500 μ L W1 liquid (TIANamp Bacteria DNAKit, the biological company limited of sky root), leave standstill 1 minute, 12000rpm, centrifugal 30 seconds.Outwell the liquid in collection tube, put back to adsorption column.Add 500 μ LW1 liquid, 12000rpm, centrifugal 30 seconds.Outwell the liquid in collection tube, put back to adsorption column.12000rpm, centrifugal 1 minute.Adsorption column is put into a 1.5mL centrifuge tube.100 μ L T1 liquid (TIANamp Bacteria DNAKit, sky root biological company limited) are added, 65 DEG C of water-baths 5 minutes, centrifugal 1 minute of 12000rpm in adsorption film central authorities.
Two, the preparation of afuA gene
According to the nucleotide sequence (as shown in the SEQ ID NO:2 of sequence table) of afuA gene, design primer pair is as follows:
Forward primer: 5 '-ATA
cATATGaAAAAATTGCAATTAAAAAAATG-3 '
Reverse primer: 5 '-ACT
gGATCCtCAATTCGATAATTTGAT-3 '
The underscore part of forward primer is the restriction enzyme site of NdeI, and the underscore part of reverse primer is BamHI restriction enzyme site.
With the STb gene of Hps for template, carry out pcr amplification with the primer pair of design.
Amplification system:
PCR reaction conditions: 94 DEG C of denaturations 5 minutes, then 94 DEG C of sex change-52 DEG C of annealing in 30 seconds 30 seconds-72 DEG C extend 30 circulations in 1 minute, last 72 DEG C of extensions 10 minutes.
PCR primer 1% agarose gel electrophoresis detects output and specificity, and with DNA purification kit (ultrathin centrifugal column type, the production of Tian Gen company) purifying.The PCR primer of purifying checked order, result shows to obtain sequence for the DNA fragmentation shown in SEQ ID NO:2 in such as sequence table, and it is the gene of coding haemophilus parasuis AfuA albumen.
Three, the structure of recombinant expression vector
1, by the correct PCR primer NdeI of order-checking and BamHI double digestion, agarose electrophoresis reclaims digestion products.
2, by plasmid pET28a (article No. 69864-3, Novagen) BamHI and NdeI double digestion, agarose electrophoresis reclaims digestion products.
3, the digestion products of step 1 is connected with the digestion products of step 2, obtains recombinant plasmid.
Recombinant plasmid is checked order.Result shows, inserts the gene of sequence for the coding haemophilus parasuis AfuA albumen shown in sequence table 2, by this recombinant plasmid called after pET28a-afuA between BamHI and the NdeI restriction enzyme site of pET28a.
Four, the preparation of engineering bacteria
With the electroporated e. coli bl21 of pET28a-afuA (DE3) (article No. CB105, TIANGEN) LB coated after containing 50 μ g/ml kantlex is dull and stereotyped, 37 DEG C of incubated overnight, obtain the recombinant bacterium containing pET28a-afuA, are engineering bacteria.
Replace pET28a-afuA with pET28a, transformation of E. coli BL21 (DE3), step is the same, obtains the recombinant bacterium containing pET28a, in contrast bacterium.
Five, the preparation of AfuA albumen and purifying
By the recombinant of preparation in above four in containing in the LB substratum of 50 μ g/ml kantlex, cultivate 3h for 37 DEG C; OD
600when=0.7, add IPTG to final concentration 0.8 μM, go to 37 DEG C and continue to cultivate 4h.
5000rpm, 10 minutes collected by centrifugation thalline, be suspended in PBS solution, ultrasonication in ice bath (300w, 10 minutes; Ultrasonic 3s, stops 5s), 15000rpm centrifugal 10 minutes collecting precipitations afterwards, by precipitation 30ml solution A (NaH
2pO
4100mmol/L, Tris-HCl10mmol/L, urea 8mol/L, adjust pH to 8.0) dissolve, can of short duration ultrasonication dissolution.Add the resin (Ni-NTA His-Bind Resin, Novagen) that 2mL handles well in advance, 4 DEG C of low-speed oscillations make resin fully be combined with target protein in 30 minutes, in conjunction with complete, add in adsorption column.Treat that liquid flows to end, add 10mL solution B (NaH
2pO
4100mmol/L, Tris-HCl10mmol/L, urea 8mol/L, adjust pH to 6.3) wash 6 times.Add 2mL solution C (NaH
2pO
4100mmol/L, Tris-HCl10mmol/L, urea 8mol/L, adjust pH to 5.9) wash 1 time.Add 10mL solution D (NaH
2pO
4100mmol/L, Tris-HCl10mmol/L, urea 8mol/L, adjust pH to 4.5), press often pipe 1mL and collect elutriant.
The molecular weight of the AfuA albumen of SDS-PAGE electrophoresis showed purifying is about 43kDa (comprising histidine-tagged), and basic coincidence theory is inferred.Collect the fraction containing AfuA albumen.
Adopt identical step to carry out cultivating and purifying the contrast bacterium of preparation in above four, the solution obtained is enzyme liquid in contrast.
Six, the Western blotting qualification of AfuA albumen
Western blotting analysis is carried out to the restructuring AfuA albumen of purifying in above five, first prepares the polyacrylamide gels of 12%, AfuA albumen loading is carried out SDS-PAGE; After electrophoresis terminates, soak balance 15min in electrophoresis liquid (39mM glycine, 48mM Tris alkali, 0.037%SDS, 20% methyl alcohol); Cut out the nitrocellulose filter slightly larger than gel, be positioned over electrophoresis liquid and be dipped to completely moistening; Cut out two thick filter paper, upper strata filter paper is slightly smaller than gel, and lower floor's filter paper less times greater than nitrocellulose filter, and will be immersed in electrophoresis liquid to completely moistening; Be added on Graphite Electrodes according to the order of filter paper-film-glue-filter paper, drive bubble away with glass stick; Switch on power, 15v constant voltage, transfer printing 30min.Nitrocellulose filter is taken off after transfer printing terminates; Film is loaded in little valve bag, add 5% skimming milk 4 DEG C close spend the night; Take out film, wash 5 times with PBST, each 10min; Then add the primary antibodie (pig rehabilitation serum) that 5% skimming milk 1: 200 dilutes, hatch 1h for 37 DEG C; Take out film, wash 5 times with PBST, each 10min; Then add two anti-(the anti-pig two of rabbit of HRP mark resists) that 5% skimming milk 1: 5000 dilutes, hatch 1h for 37 DEG C; Take out film, wash 5 times again with PBST, each 10min; Then with the colour developing of ECL chemical luminescence reagent kit, result shows that the protein band of AfuA albumen and expection is in the same size, as shown in Figure 2.
The qualification of embodiment 3, AfuA protein immunization protectiveness
1, Hps is to kunming mice LD
50mensuration
By Hps (purchased from National Veterinary Culture Collection, bacterium numbering: CVCC3361) be inoculated in TSB liquid nutrient medium (containing 10% horse serum, 0.01%NAD) 37 DEG C of 200rpm/min cultivate 14 ~ 16 hours, then be coated with TSA solid medium (containing 10% horse serum, 0.01%NAD) 37 DEG C next day and cultivate 24 ~ 36h.Wash lower lawn with PBS, be diluted to 5 × 10
8cFU/mL (OD
600be about 1.0), then 2 times concentrate step by step, are concentrated into required dosage (as shown in table 1) backward injected in mice.Female KM mouse in 8 ~ 10 week age (being purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center) is divided into 5 groups, often organizes 10.What every mouse peritoneal injected 100 μ L attacks poisonous substance, to attack after poison Continuous Observation 5 days, notes down each group of death condition.According to Kou Shi formulae discovery LD
50.Formula is as follows:
LD
50＝/g
-1{Xm-i〔∑P-(3-Pm-Pn)/4〕}
Xm: the logarithmic value of maximal dose group dosage
I: the difference of two adjacent groups dosage (d) logarithmic value
∑ P: the summation of each treated animal mortality ratio
P: each treated animal mortality ratio, represents decimally
Pm: maximum dose level treated animal mortality ratio, represents decimally
Pn: minimum dose treated animal mortality ratio, represents decimally
N: every treated animal number
Attack poison after 5 days, the 5th group dead 10, the 1st group dead 2.Each group of concrete mortality ratio is as shown in the table.
Table 1
LD is drawn by Kou Shi formulae discovery
50=1.46 × 10
9cFU.
2, mouse immunity with attack poison
With the kunming mice (being purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center) in 4 ~ 6 week age of AfuA proteantigen immunity according to embodiment 2 purifying, often organize 10.Control group does not carry out immunity.First time immunity, 100 μ g/ only, mix with Freund's complete adjuvant 1: 1, back multi-point injection immune mouse.Interval is after two weeks, carry out second time immunity, 100 μ g/ only, mix with freund 's incomplete adjuvant 1: 1, back multi-point injection immune mouse, after 10 days, ELISA detects antibody horizontal, the goat against murine (Sigma) of HRP mark is two anti-(1: 4000), and two exempt from after 10 days through abdominal injection Hps (culture presevation numbering: CVCC3361), attack the LD that toxic agent amount is above mensuration
505 times, to observe in 5 days the death condition of each group mouse, as shown in Figure 3, AfuA immune group has the survival rate of 80%.
Claims (4)
1. the haemophilus parasuis immune protective antigen of aminoacid sequence as shown in SEQ ID NO:1, the nucleic acid of sequence as shown in SEQ ID NO:2, comprise as described in the recombinant vectors of nucleic acid and haemophilus parasuis immune protective antigen as described in comprising or as described in the cell of nucleic acid preparing the purposes in haemophilus parasuis subunit vaccine.
2. the haemophilus parasuis immune protective antigen of aminoacid sequence as shown in SEQ ID NO:1, the nucleic acid of sequence as shown in SEQ ID NO:2, comprise as described in the recombinant vectors of nucleic acid and haemophilus parasuis immune protective antigen as described in comprising or as described in the cell of nucleic acid for the preparation of the purposes of preventing in the medicine of the disease caused by haemophilus parasuis.
3. purposes according to claim 2, the wherein said disease caused by haemophilus parasuis is Haemophilus parasuis.
4. vaccine composition, it comprises the haemophilus parasuis immune protective antigen of aminoacid sequence as shown in SEQ ID NO:1.
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