CN104306964B - The subunit vaccine for recombinating haemophilus parasuis immune protective antigen HbpA2 purposes and being prepared with it - Google Patents
The subunit vaccine for recombinating haemophilus parasuis immune protective antigen HbpA2 purposes and being prepared with it Download PDFInfo
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Abstract
The invention discloses amino acid sequence such as SEQ ID NO：Purposes of the restructuring HbpA2 albumen in the medicine for preparing prevention Haemophilus parasuis shown in 2, and by amino acid sequence such as SEQ ID NO：Subunit vaccine that restructuring HbpA2 albumen and pharmaceutically acceptable auxiliary material or carrier shown in 2 are prepared from and application thereof.The good immunogenicity of present invention restructuring HbpA2 albumen and protection originality, the HbpA2 specific antibody levels produced after being immunized are high, and the subunit vaccine protective rate of preparation is high, and potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to genetic engineering field, and in particular to one kind restructuring haemophilus parasuis immune protective antigen
HbpA2 purposes and the subunit vaccine prepared with it.
Background technology
It is thermophilic that haemophilus parasuis (Haemophilus parasuis, Hps) belongs to Pasteurella (Pasteurellaceae)
Blood Bacillus (Haemophilus), is the tiny bacillus of Grain-negative, with variform, is colonized in the health pig upper respiratory tract, category
In normal flora, pig Graves disease (Glasser ' s dis-ease, with cellulosic can be caused under the specified conditions such as immunosupress
Polyserositis, meningitis, arthritis are principal character.In the last few years due to the fast development of pig farm large-scale cultivation, especially
It is factory's intensive culture, and pig Graves disease in worldwide prevalence and breaks out, and shows die by visitation of God rate, the incidence of disease and disease
Dead rate is higher, seriously endangers pig industry development, and cause huge economic losses.
China just starts gradually to pay attention to the research to Haemophilus parasuis in recent years.The serotype of haemophilus parasuis is many
It is many, differentiate 15 kinds of serotypes at present, also 15%-41%'s does not identify serotype, wherein 1,5,10,12,13 and 14 is
Velogen strain, and the popular serotype of China is 4 types and 5.The haemophilus parasuis Virulence Difference of different serotypes is very big, and we are right
Its virulence factor and pathogenesis are known little about it, and effective general commodity seedling and sensitive diagnostic method are there is no so far, the disease is still
It is not effectively controlled, huge economic loss is brought to global pig industry.
So far not as hog cholera lapinised virus vaccine, pseudo- mad dog gene-deleted vaccine, ripe vaccine is directed to for China
Haemophilus parasuis, is all at present traditional inactivated vaccine and Attenuate vaccine mostly, lacks good protection, cause clinical case
It is common.And inactivated vaccine there can only be protective effect to the bacterial strain of homologous serotype, lack cross-protection.Haemophilus parasuis
The diversity of serotype and account for the parting bacterial strain that is unable to of significant proportion and have impact on to being ground with high efficiency crossed protection vaccine
System.Subunit vaccine has the advantages that live vaccine immune efficacy is high, cross-protection is strong and inactivated vaccine security is good simultaneously,
With very wide application prospect.Major part is unable to parting bacterial strain haemophilus parasuis and its diversity of serotype have impact on
People are for the development with high efficiency crossed protection vaccine.
Hemopexin (heme-binding protein, HbpA) is a kind of globulin, is widely present in pathogenic
In bacterium.Studies have found that having one or more and haem bonding pad domain in HbpA protein moleculars, with combination, transport and transmission
Ferroheme, participates in the physiological function such as organism metabolism, HbpA albumen is a kind of important virulence factor, with bacterium it is pathogenic it is close not
It can divide.Research on the haemophilus influenzae of Pasteurellaceae hemophilus shows that HbpA albumen is haemophilus influenzae
A kind of important virulence factor, HbpA albumen is relevant with the utilization of ferroheme.At present on haemophilus parasuis HbpA albumen
Function research report it is less, in the haemophilus parasuis SH0165 for carried out gene order-checking find have two HbpA bases
Cause, the size of two genes is 1595bp.Publication No. CN103288934A patent application discloses one kind and uses gene
The mode of engineering recombinantly expresses HbpA genes (Gene ID:7276898) method, still, its restructuring HbpA albumen prepared are made
For subunit vaccine, its immune protective effect only 50% not good to the protection of infection haemophilus parasuis.
The content of the invention
In order to solve the above problems, the invention provides a kind of new subunit's epidemic disease being prepared from by restructuring HbpA eggs
Seedling.
Present invention firstly provides amino acid sequence such as SEQ ID NO：Restructuring HbpA2 albumen shown in 2 is preparing prevention
Purposes in the medicine of Haemophilus parasuis.
Preferably, the medicine includes the restructuring HbpA2 albumen for causing immune effective dose.
Subunit vaccine of the present invention, it is by amino acid sequence such as SEQ ID NO：Restructuring HbpA2 albumen and medicine shown in 2
Acceptable auxiliary material or carrier are prepared from.
Preferably, the vaccine includes the amino acid sequence such as SEQ ID NO for causing immune effective dose：Restructuring shown in 2
HbpA2 albumen and pharmaceutically acceptable auxiliary material or carrier.
Preferably, described cause immune effective dose is to be not less than 0.1 μ g/ml.
Preferably, described auxiliary material or carrier are immunologic adjuvant and/or preservative.
The adjuvant is complete Freund's adjuvant or incomplete Freund's adjuvant.
The preservative is thimerosal.
Present invention also offers purposes of the foregoing vaccine in the medicine of Haemophilus parasuis is prepared.Wherein, it is described
The medicine of prevention Haemophilus parasuis be to be administered by way of injection.
The present invention recombinantly expresses obtained recombinant protein HbpA2 by the way of genetic engineering has good immunogenicity
With protection originality, the HbpA2 specific antibody levels produced after being immunized are high, are made after vaccine, significantly mouse can be protected to resist
The attack of haemophilus parasuis Serotype 5 virulent strain, protective rate is up to 70%, can effectively prevent Haemophilus parasuis,
Potential applicability in clinical practice is good.
The embodiment of form, further specifically to the above work of the present invention by the following examples
It is bright.But the scope that this should not be interpreted as to above-mentioned theme of the invention is only limitted to following embodiment.It is all above-mentioned interior based on the present invention
Hold realized technology and belong to the scope of the present invention.
Brief description of the drawings
The PCR amplification figures of Fig. 1 HbpA2 genes.M:DNA maker；1、2、3：Pcr amplification product；
Fig. 2 induction times optimize.M：Albumen maker；1：pET-39b(BL21)；2~9：Induction time is：0、1h、2h、
3h、4h、5h、6h、7h；
Fig. 3 IPTG induced concentrations optimize.M：Albumen maker；1：pET-39b(BL21)；2~9：IPTG:It is final concentration of：
0mmol/L、0.2mmol/L、0.4mmol/L、0.6mmol/L、0.8mmol/L、1.0mmol/L、1.2mmol/L、1.5mmol/
L；
Fig. 4 inducing temperatures optimize.M：Albumen maker；1、3、5、7：Before pET-39b-HbpA2 (BL21) inductions；2：Induction
Temperature is 25 DEG C；4：Inducing temperature is 30 DEG C；6：Inducing temperature is 37 DEG C；7：Inducing temperature is 42 DEG C；
The form detection of Fig. 5 HbpA2 albumen expression in pET-39b-HbpA2 (BL21).M：Albumen maker；1：pET-
39b(BL21)；2：After pET-39b-HbpA2 (BL21) ultrasonic disruption；3：Supernatant；4：Precipitation；
The SDS-PAGE of Fig. 6 restructuring HbpA2 expression.M：Albumen maker；1：Before pET-39b (BL21) inductions；2：pET-
After 39b (BL21) inductions；3：Before pET-39b-HbpA2 (BL21) inductions；4：After pET-39b-HbpA2 (BL21) inductions；5：
After HbpA2 protein purifications (elution of 100mmol/L imidazoles)；
Fig. 7 recombinant protein Western blotting；
Fig. 8 mouse survivals rate and the relation of time.
Embodiment
Experiment material and reagent：
Haemophilus parasuis CVCC3361, from National Veterinary Culture Collection, bacterium numbering：
CVCC3361.Escherichia coli DH5a, BL21 (DE3) competent cell are purchased from TIANGEN Biotech (Beijing) Co., Ltd..Limitation
Property restriction endonuclease, Ex-Taq archaeal dna polymerases, DNA ligase, DNA glue reclaims kit, plasmid extraction kit, DNA
Marker, pMD19-T simple carriers etc. are purchased from precious biological (Dalian) Engineering Co., Ltd.Bacterial genomes extracts reagent
Box is Omega Products.Pre-dyed albumen Ladder is NEB Products.Mouse reaches the limited public affairs of large experimental animal purchased from Chengdu
Department.
The formula of LB culture mediums is as follows：Tryptone (Tryptone) 10g/L, yeast extract (Yeast extract)
5g/L, sodium chloride (NaCl) 10g/L, the pH of the culture medium is adjusted with NaOH, 7.2-7.4, high pressure steam sterilization is reached
30min。
Loading buffer formula of liquid：50mM NaH2PO4,300mM NaCl,5mM imidazole,pH8.0.
100mM imidazole elutions：50mM NaH2PO4,300mM NaCl,100mM imidazole,pH 8.0.
The haemophilus parasuis immune protective antigen HbpA2 of the present invention of embodiment 1 preparation
1st, recombinantly express
The clone of 1.1HbpA2 genes and the structure of expression vector
1.1.1 design of primers and synthesis
According to haemophilus parasuis HbpA2 gene orders (Gene ID：7278927) biosoftware Premier, is used
Primer 5.0 designs l to primer, expands HbpA2 genes.
Sense primer：5’-AGTACTCCGACAAATACATTGGTCAACTGT-3’；
Anti-sense primer：5’-CTCGAGTTAAGGCTTCAGACTTACGCCATA-3’；
1.1.2HbpA2 the PCR amplifications of gene
Using haemophilus parasuis CVCC3361 genomic DNAs template, performing PCR amplification, amplification system are entered according to a conventional method
25 μ L, 4 repetitions.Amplification system：
| Material | Volume |
| Genomic templates | 1μL |
| Primer (upstream and downstream) | 2μL |
| EX-Taq MasterMix | 12.5μL |
| ddH2O | 9.5μL |
| Cumulative volume | 25μL |
Loop parameter：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 100s, 30 are followed
Ring；72 DEG C of extension 5min, 4 DEG C of preservations.PCR primer carries out 1% agarose gel electrophoresis detection, and with DNA glue reclaim kits
Glue reclaim purifying (being operated according to Omega glue reclaim kit specifications) is carried out to the PCR fragment after electrophoresis, will after purification
PCR fragment be sequenced.
Nucleotide sequence (the SEQ ID NO of target gene fragment after purification:1)：
The mode of synthesis can also be taken directly to synthesize target gene fragment.
1.1.3 the structure of expression vector
Purpose and genetic fragment (PCR recovery products) are cloned into carrier T and obtain pMD19-T-HbpA2, carrier T clone
Linked system：
| Material agents | Volume |
| PCR recovery products | 3μL |
| PMD19-T carriers | 1μL |
| T4DNA Ligase | 0.5μL |
| 10×Buffer | 1μL |
| ATP | 1μL |
| ddH2O | 3.5μL |
| Cumulative volume | 10μL |
16 DEG C of connections are stayed overnight, PCR Rapid identification connection products, the product CaCl after connection2Method conversion enters competence
Cell E. coli DH5a, its step is as follows：(1) -70 DEG C of μ L of the competent cell frozen DH5a 50 are taken with liquid-transfering gun on ice
Dissolving, adds the μ L of pMD19-T-HbpA2 plasmids 10 and mixes, -20 DEG C of ice bath 30min, heat shock 90s in 42 DEG C of water-baths, rapidly -
20 DEG C of ice bath 5min.(2) sample through above-mentioned processing is added to the LB culture mediums of 600 μ L preheatings, 37 DEG C, 180 revs/min of cultures
1.5h.(3) take out 100 μ L bacterium solutions to be coated on the LB solid mediums of the resistance containing Amp, in 37 DEG C of 12~24h of insulating box culture.
Positive transformant is screened, the doubtful bacterium colony of picking enters performing PCR, identifies the recon containing pMD19-T-HbpA2 and expand culture,
Extract plasmid and carry out sequencing identification.
Correct pMD19-T-HbpA2 is sequenced with XhoI and ScaI directed clonings into prokaryotic expression carrier pET-39b, structure
Recombinant expression carrier pET-39b-HbpA2 is built, its step is as follows：(1) with XhoI and ScaI to pMD19-T-HbpA2 plasmids and
PET-39b plasmids carry out double digestion, 37 DEG C of digestion 3h.(2) digestion products are carried out after 1% agarose gel electrophoresis with DNA glue time
Kit is received (to grasp the purpose fragment progress glue reclaim purifying after electrophoresis according to Omega glue reclaim kit specifications
Make), -20 DEG C save backup.(3) purpose fragment after digestion is attached conversion and obtains recombinant expression carrier pET-39b-
HbpA2 (method of connection conversion is cloned with carrier T), performing PCR of going forward side by side identification, digestion identification, sequencing identification.
1.1.4 the structure of recombinant bacterium
The recombinant expression carrier pET-39b-HbpA2 built is finally transformed into expressive host bacterium BL21 (DE3), produced
Recombinant bacterium.
The induced expression of 1.2 recombination bacillus colis
1.2.1 express and identify at the beginning of recombinant protein
LB of e. coli bl21 (DE3) inoculation containing 50 μ g/ml Kan of the pET-39b-HbpA2 containing recombinant plasmid is cultivated
In base, 37 DEG C of shake 3~4h of culture, to OD values be 0.5~0.6 when, add IPTG to final concentration 1mmoL/mL, continue shake training
3~4h is supported, supernatant is removed in centrifugation, adds SDS-PAGE sample-loading buffers to boil 5min in precipitation, carries out SDS-PAGE.Gel is through examining
The bright blue dyeing rear decoloring of Maas, utilizes gel imaging system PHOTOGRAPHIC ANALYSIS.
1.2.2 the optimization of expression of recombinant proteins condition
1.2.2.1 the optimization of induction time
By recombinant bacterium with 1:100 ratio is inoculated in the LB fluid nutrient medium test tubes containing Kan, treats that culture to OD values are
When 0.5~0.6, IPTG to final concentration 1mmoL/mL is added, respectively in induced expression 0h, 1h, 2h, 3h, 4h, 5h, 6h are taken after 7h
1ml bacterium solutions, are handled sample, SDS-PAGE electrophoresis.
1.2.2.2IPTG the optimization of concentration
By recombinant bacterium with 1:100 are inoculated in the LB fluid nutrient medium test tubes containing 50 μ g/ml Kan (i.e. kalamycin) n respectively
In, when 37 DEG C of shaken cultivations to OD values are 0.5~0.6, IPTG is separately added into final concentration of：0mmol/L、0.2mmol/L、
0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L, 1.5mmol/L, the above-mentioned experiment of Fiber differentiation
The optimal induction time drawn, respectively takes bacterium solution 1mL, sample is post-processed, SDS-PAGE electrophoresis.
1.2.2.3 the optimization of inducing temperature
By recombinant bacterium with 1:100 are inoculated in the LB fluid nutrient mediums containing 50 μ g/ml Kan respectively, treat that culture to OD values are
When 0.5~0.6, add the optimal IPTG concentration that is drawn to final concentration of above-mentioned experiment of IPTG, respectively 25 DEG C, 30 DEG C, 37 DEG C,
The optimal induction time that 42 DEG C of above-mentioned experiments of Fiber differentiation are drawn, respectively takes bacterium solution 1mL, sample is post-processed, SDS-PAGE
Electrophoresis.
1.2.3 the great expression of recombinant protein
By recombinant bacterium with 1:100 are inoculated in the LB fluid nutrient mediums containing 50 μ g/ml Kan respectively, and culture to OD values is
When 0.5~0.6, in bacterium solution plus IPTG to final concentration of optimal induced expression concentration, then cultivated under optimum temperature to
Best Times.
1.3 isolate and purify
(1) the bacterium solution 12000g/min after induced expression is centrifuged into 5min, collects thalline, added and contain 1mg/mL lysozymes
Cellular lysate liquid 80mL, suspend precipitation.4 DEG C of reactions are placed in stay overnight.
(2) multigelation suspension 3 times between -20 DEG C and room temperature.
(3) rear suspension is dissolved to be placed on ice using the broken about 10min of the intensity of ultrasonic cell disruption instrument 40%.12000g/
Min centrifuges 10min, collects supernatant, and pH to 8.0 is adjusted with 1mmol/L NaOH, using 0.45 μm of membrane filtration sample, carries out
Subsequent purification.
(4) Ni ion fillers are packed into post overnight, after standby after filling compaction, i.e., nickel ion chelating affinity chromatography is filled out
Expect chromatographic column.
(5) the ultrapure water elution alcohol by 2ml/min of flow velocity, to chromatography column equilibration.Again with sample-loading buffer with lml/
Min flow velocity balance chromatographic column, about 30min.Then with 0.5ml/min flow velocity loadings, it is complete on sample after it is flat with sample-loading buffer
The chromatographic column that weighs extremely is balanced.In order with containing 30mmol/L, 50mmol/L, 80mmol/L, 100mmol/L, 150mmol/L,
The elution buffer of 200mmol/L, 250mmol/L imidazoles, albumen is eluted with 1ml/min flow velocity, is started when there is rising peak
Collect eluent to lower decresting to terminate, the imidazoles elution buffer that a concentration is changed after ready to balance continues to elute.Collect with containing
The eluent that the elution buffer of 100mmol/L imidazoles is purified by flash, the as solution containing recombinant protein HbpA2 of the present invention ,-
80 DEG C are stored for future use, and purity of protein is collected in SDS-PAGE analyses.
2nd, determine
2.1Western blotting
After HbpA2 albumen after purification is carried out into SDS-PAGE electrophoresis through 12% gel, Western is carried out
blotting.It is comprised the following steps that：
(1) by the gel piece after SDS-PAGE electrophoresis through appropriate shearing, while shearing six and gel filter of the same size
Paper and a NC film；It is soaked into transfering buffering liquid and places 1h；
(2) transfer box, the filter paper that transferred buffer solution was soaked, gel and NC films, by " filter paper -- gel -- NC are opened
Film -- filter paper " is stacked on transfer box negative pole, and is gently rolled across with a clean small test tube, to eliminate bubble, shuts transfer
Box simultaneously inserts transfer cell；
(3) switch on power, 25V transfers 30min；
(4) after transfer terminates, NC films are taken out, appropriate Block buffer closing 1h or so is added；Confining liquid is outwelled, TBST is washed
Film 3 times, 10min/ times；
NC films are put into sealing small plastic bag, 1 is added：The rabbit-anti CVCC3361 primary antibodies of 1000 dilutions, in 37 DEG C of effects
Lh, abandons primary antibody, film is washed with TBST 3 times, 10min/ times；
(5) NC films are put into sealing small plastic bag, add 1：The goat anti-rabbit igg secondary antibody of 1000 dilutions, in 37 DEG C of effects
1h；TBST washes film 3 times, 10min/ times；
(6) appropriate nitrite ion is added, develop the color 20min~30min, treats that purpose band is high-visible, film is washed with deionized water
Color development stopping, blotting paper blots moisture on film, and lucifuge is dried.
(7) gel images processing system photographic analysis.
3rd, measurement result
As shown in figure 1, using bacterial strain CVCC3361 genomic DNAs template, being expanded through PCR, there is 1 and expected fragment
(159bp) band of the same size (Fig. 1).Sequencing result shows, the HbpA2 bases announced in the fragment and Gen-Bank amplified
Because the homology of sequence is 99.9%.The recombinant expression plasmid pET-39b-HbpA2 of structure is through XhoI and ScaI double digestions visible 2
Band, size is consistent with expected stripe size.Illustrate that HbpA2 genes have been successfully plugged on pET39b carriers.
As shown in figs. 2 to 4, during induced expression, IPTG optium concentrations be 1.5mmol/L under, most preferably 30 DEG C of inducing temperature,
Most induction time most preferably 5h.
As shown in Fig. 5~6, after Escherichia coli induced expression of the present invention containing recombinant plasmid pET-39b-HbpA2,
There is 1 obvious thicker band, its size and expected fusion protein theoretical molecular size at 70kd in SDS-PAGE analyses
Unanimously, illustrate to obtain recombinant protein HbpA2, after being isolated and purified according to the inventive method, obtain sterling albumen.
Recombinant protein-HbpA2 amino acid sequence (SEQ ID NO:2) it is：
As shown in fig. 7, after recombinant protein HbpA2 of the present invention fully reacts with immune serum, having in the position of recombinant protein
Specific chromogenic band, it was confirmed that the albumen has good reactionogenicity.
Experimental result illustrates that the present invention is by way of genetic engineering, and recombination expression has obtained the recombinant protein of sterling
HbpA2。
The preparation of the subunit vaccine of the present invention of embodiment 2
The μ g of sterling recombinant protein HbpA2 50 prepared by Example 1, are made 135 μ lHbpA2 protein solutions, plus 115 μ l
Physiological saline, with complete Freund's adjuvant according to volume ratio 1:1 emulsification, is produced.
The preparation of the subunit vaccine of the present invention of embodiment 3
The μ g of sterling recombinant protein HbpA2 50 prepared by Example 1, are made 135 μ lHbpA2 protein solutions, plus 115 μ l
Physiological saline, with incomplete Freund's adjuvant according to volume ratio 1:1 emulsification, is produced.
The preparation of the subunit vaccine of the present invention of embodiment 4
The μ g of sterling recombinant protein HbpA2 50 prepared by Example 1, are made 135 μ lHbpA2 protein solutions, plus 115 μ l
Physiological saline, with incomplete Freund's adjuvant according to volume ratio 1:1 emulsification, adds appropriate thimerosal as preservative, produces.
Illustrate beneficial effects of the present invention with the mode of experimental example below：
The protective effect in vivo of the subunit vaccine of the present invention of experimental example 1 and subunit vaccine
First, experimental method
The measure of 1.1 haemophilus parasuis CVCC3361 median lethal doses (LD50)
1.1.1LD0 with LD100 measure
10 mouse are one group, every mouse peritoneal injection living bacterial liquid 1 × 109CFU (LD0 estimated in preliminary experiment), 7 days
Dead mouse situation is counted afterwards, if dead two or more than two, is attacked toxic agent amount and is reduced 0.1 × 109CFU, if all survivals,
Then attack toxic agent amount increase by 0.1 × 109CFU, untill there was only a death in one group of mouse, then the low dosage adjacent with this is
For LD0.LD100 is tried to achieve with method.
1.1.2 experimental group attacks the determination of toxic agent amount
Mouse stochastic averagina is divided into 7 groups, every group of mouse is 10, wherein 7 groups are control group, 1-6 groups are test group.
R values are obtained by following equation,Wherein G is group number, and Dm/Dn is the ratio between LD100 and LD0.Then test group is attacked
Toxic agent amount is respectively D1=Dn=LD0, D2=D1 × r, D3=D2 × r, D4=D3 × r, D5=D4 × r, D6=D5 × r.Press
Attacked according to above-mentioned dosage after poison, the death condition of observed and recorded mouse.
1.1.3LD50 calculating
LD50 is calculated by Bliss-LD50 softwares, each group is inputted in a program and attacks toxic agent amount, number of animals, death toll, no
With input control group data, clicking " calculating " can show CVCC3361 to parameters such as the LD50 of mouse.
The protest test of 1.2 mouse
40 mouse are randomly divided into 4 groups, every group 10.The HbpA2+ of 50 micrograms purifying is immunized in l group dorsal scs multiple spot
(first immunisation is Freund's complete adjuvant to adjuvant, is immunized for the second time as incomplete Freund's adjuvant, preparation method：Take 135 μ lHbpA2
Protein solution, plus 115 μ l physiological saline, with adjuvant 1:1 emulsification)；2nd group of immune haemophilus parasuis CVCC3361 inactivated vaccine+
Adjuvant is used as positive controls；3rd group is used as negative control group with physiological saline；4th group is used as negative control group with adjuvant.It is first
14th day booster immunization 1 time after exempting from, immunizing dose and mode are exempted from head.Two exempt from after the 14th day all mouse bloodthirsty bar of secondary pig
Bacterium CVCC3361 attacks poison by abdominal cavity.Attack before poison, gather mice serum, detect HbpA2 specific antibody titres.Attack every after poison
It was observed and records clinical manifestation and the death condition of mouse, until the 5th day.Mouse is implemented after 5d and is euthanized, and to all
Dead mouse carries out the separation identification of bacterium, to confirm as the infection of haemophilus parasuis.
The detection of 1.3 specific antibodies
For detecting that the serum of HbpA2 specific antibodies is picked up from before attacking poison.Mouse docking blood sampling, the serum adopted in-
20 DEG C save backup.The detection indirect elisa method of antibody, specific method is as follows：HbpA2 after purification, with pH 9.6 carbonic acid
Salt is diluted, and 96 hole elisa plates add 100 μ L to be incubated 1h in 37 DEG C per hole, and 4 DEG C of coatings are stayed overnight.Next day, washed after 3 times, used with PBST
5% skim milk closes 1.5h in 37 DEG C.Confining liquid is abandoned, is washed with PBST 3 times.By test serum from l：100 to l：12500 dilutions
Add 100 μ L, 37 DEG C of incubation 1h per hole afterwards.Wash after 3 times, goat anti-mouse igg is diluted with PBST, 100 μ L, 37 DEG C of effects are added per hole
30min.Wash 3 times, add 100 μ LTMB solution to react 15min in room temperature lucifuge per hole, plus 50 μ L 2.0mol/L sulfuric acid solutions are terminated
Reaction, with ELIASA at 450nm reading.The hole that value is more than 0.2 is considered as with the presence of HbpA2 specific antibodies.
2nd, experimental result
1st, mouse Serum Antibody Detection
As a result it is as shown in table 1：
The mice serum antibody test result of table 1
The antibody titer of HbpA2 immune groups apparently higher than negative control group and positive controls, illustrate expression HbpA2 and
Its subunit vaccine prepared has good immunogenicity.
2nd, protest test
To evaluate the protective efficacy that HbpA2 is immune, this research is using mouse as animal model, after 2 times are immune, with
6x109CFU (5 × LD50) virulent strains CVCC3361 attacks poison, observes and record the clinical symptoms and death condition of mouse, experiment
As a result as shown in table 2 and Fig. 8：
The protest test result of table 2
Negative control group mouse is all dead in 1 day after poison is attacked it can be seen from such as table 2 and Fig. 8, is found after dissection
Multi viscera lesion, lesion internal organs, which can be separated to, attacks poison bacterial strain；HbpA2 immune groups are attacked after poison dead 3 in 2 days, and remaining is small
Mouse, which was observed to 5 days, still to survive.Positive controls mouse is attacked after poison dead 2 in 2 days.
As a result illustrate, HbpA2 immunity energies of the present invention significantly protect the attack of mouse resistance haemophilus parasuis virulent strain,
Preferably, protective rate is up to 70% to subunit vaccine immune protective that it is prepared, and protecting effect is close to full bacterium inactivated vaccine.
To sum up, the present invention successfully constructs genetic engineering bacterium by the way of genetic engineering, and expression is recombinated
Albumen HbpA2.Recombinant protein HbpA2 has good immunogenicity and protection originality, is exempted from its subunit vaccine prepared
The HbpA2 specific antibody levels produced after epidemic disease are high, can significantly protect mouse to resist the strong toadstool of haemophilus parasuis Serotype 5
The attack of strain, it is the protective antigens of haemophilus parasuis to illustrate recombinant protein HbpA2, can be prepared as vaccine, clinic should
With having good prospects.
Claims (10)
1. amino acid sequence such as SEQ ID NO：Restructuring HbpA2 albumen shown in 2 is preparing the medicine of prevention Haemophilus parasuis
Purposes in thing；The albumen is by SEQ ID NO：It is nucleotide sequence coded shown in 1.
2. purposes according to claim 1, it is characterised in that：The medicine includes the restructuring HbpA2 for causing immune effective dose
Albumen.
3. a kind of subunit vaccine, it is characterised in that：It is by amino acid sequence such as SEQ ID NO：Restructuring HbpA2 shown in 2
Albumen and pharmaceutically acceptable auxiliary material or carrier are prepared from.
4. vaccine according to claim 3, it is characterised in that：The vaccine includes the amino acid sequence for causing immune effective dose
Such as SEQ ID NO：Restructuring HbpA2 albumen and pharmaceutically acceptable auxiliary material or carrier shown in 2；The albumen is by SEQ
ID NO：It is nucleotide sequence coded shown in 1.
5. vaccine according to claim 4, it is characterised in that：Described cause immune effective dose is to be not less than 0.1 μ g/ml.
6. vaccine according to claim 4, it is characterised in that：Described auxiliary material or carrier are immunologic adjuvant and/or anti-
Rotten agent.
7. vaccine according to claim 6, it is characterised in that：The adjuvant is complete Freund's adjuvant or incomplete Freund
Agent.
8. vaccine according to claim 6, it is characterised in that：The preservative is thimerosal.
9. purposes of the vaccine in the medicine for preparing prevention Haemophilus parasuis described in claim 3-8 any one.
10. purposes according to claim 9, it is characterised in that：The medicine of described prevention Haemophilus parasuis is logical
The mode for crossing injection is administered.
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