CN105664148B - A kind of gene engineered subunit polyvalent vaccine and its preparation method and application - Google Patents
A kind of gene engineered subunit polyvalent vaccine and its preparation method and application Download PDFInfo
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Abstract
The present invention discloses a kind of gene engineered subunit polyvalent vaccine and its preparation method and application, belongs to live vaccine research field.The present invention uses the method great expression of genetic engineering 3 kinds of recombinant protein rApx I A, rApxIIA and rOMPD, and recombinant protein has preferable antigenicity.Polyvalent vaccine of the invention is individually immunized compared with three kinds of albumen; the antibody level when antibody level of I A of rOMPD, rApx is above individually immune; the antibody level and polyvalent vaccine antibody level of II A of rApx is significantly higher than control group; illustrate that the humoral immune reaction of recombinant subunit polyvalent vaccine induction, rApxIA and rOMPD antibody play effect of crucial importance in the Cross immunogenicity of mouse.It is harmless to immune animal safety after inoculation, it is a kind of new generation vaccine with bright prospects, reserve supply and technical support will be provided to the prevention and control of China's porcine contagious pleuropneumonia, had great importance.
Description
Technical field
The invention belongs to live vaccine research fields, and it is sub- to be related to a kind of Actinobacillus pleuropneumoniae genetic engineering
Unit polyvalent vaccine and its preparation method and application, the invention particularly relates to a kind of Actinobacillus pleuropneumoniae Apx I
A, II A and OMPD gene engineered subunit polyvalent vaccine of Apx and its preparation method and application.
Background technique
Porcine contagious pleuropneumonia (Porcine contagious pleuropneumonia, PCP) is by pig transmissible
With empsyxis, necrosis and fibre caused by Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, APP)
Tie up the highly contagious disease that disposition exudation is main feature.APP serotype has 15 serotypes, each country's prevalence
Serotype is not also identical.In China, 1987, Harbin veterinary institute found clinical case for the first time, simultaneously by separation cause of disease
Serotype, Serologic test etc. are carried out to isolated cause of disease and demonstrate PCP in the presence and prevalence in China.China with serotype 1,
3 and 7 at most.Since two thousand, there is prevalence in many places in China in PCP.Shanghai, Zhejiang, Guangdong, Hubei, Hunan, river
The ground such as south are particularly acute, it was reported that only Suburb Areas of Hangzhou pig farm results in nearly 30,000 pig death because of the disease.
PCP is resulted in significant economic losses to various countries' pig breeding industry, the pig as caused by PCP is dead, it is the speed of growth it is slow,
The reduction of feedstuff-meat ratio and the increase for putting into treatment fund, bring serious economic loss to aquaculture.Various ages, gender
Pig is susceptible to APP, and the pig at 6~10 monthly ages is most susceptible, when PCP is acute popular the incidence of infection and the death rate 20% with
On, the death rate is then up to 80% or more when most acute popular.Clinical classical symptom are as follows: 41.5 DEG C of body temperature or more；Sick pig expiratory dyspnea
In sitting position of dog gesture, mouth and nose flow out blood sample exudate；Main pathological change is tracheae bloody mucus；Diffusivity hemorrhagic pneumonia, it is fine
Tie up disposition pleurisy.
APP can cause zoogenetic infection morbidity to be determined by virulence factor.APP has many virulence factors, grinds
Study carefully the exotoxin (ApxI, ApxII, ApxIII) for showing APP secretion and outer membrane protein (OmpD) etc. and is not only the important poison of APP
The power factor, while being also important immunogenic factors.ApxIA gene, II A gene of apx are the precursor of encoding toxin protein again.
D15/OmpD has been found to play an important role in the synthesis of outer membrane protein and assembling process.
Currently, vaccine inoculation is to prevent and treat a very important measure of PCP.The pathogen of this disease has 15
Serotype, virulence factor is numerous, and faint cross protection is had no or only between different serotypes, thus develops efficiently to have and intersect
The vaccine of protection is the difficult point and key of this disease of prevention and control, is constantly subjected to the attention of people.The vaccine of various countries' research has very
It is a variety of: full bacterium inactivated vaccine, gene-deleted vaccine, bacterium shadow seedling and subunit vaccine etc..Currently, various countries clinically prevention and control PCP
Use traditional APP whole cell inactivated vaccine.Since inactivated vaccine has type specificity, lack APP thallus incubation
The middle exotoxin to cell exocrine；Some antigenic components are likely to be broken or lose during inactivation.Weak poison deletion of vaccine
And ghosts vaccine etc. can produce type cross-immunity, can provide certain immune protective efficiency to the attack of different serotypes APP.
While attenuated vaccine is reduced with virulence, immunogenicity is also declined, it sometimes appear that virulence returns strong phenomenon, is difficult
Prediction and control.Ghosts vaccine is it sometimes appear that crack incomplete situation.In contrast, the not no gene work of disadvantages mentioned above
Journey subunit vaccine then becomes the hot spot of research.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a kind of porcine contagious pleuropneumonias
I II A and OMPD gene engineered subunit polyvalent vaccine of A, Apx of Actinobacillus Apx.The present invention is a large amount of with the method for genetic engineering
3 kinds of recombinant protein rApx I A, rApxIIA and rOMPD are expressed, the hemolytic exotoxin outer membrane protein (OmpD) of APP is recombinantly expressed
Recombinant subunit vaccine as component has preferable effect to the prevention and treatment of PCP.
Another object of the present invention is to provide above-mentioned Actinobacillus pleuropneumoniae Apx I A, Apx II A and
The preparation method of OMPD gene engineered subunit polyvalent vaccine.
A further object of the present invention is to provide above-mentioned Actinobacillus pleuropneumoniae Apx I A, Apx II A and
The application of OMPD gene engineered subunit polyvalent vaccine.
The purpose of the invention is achieved by the following technical solution:
A kind of I II A and OMPD gene engineered subunit polyvalent vaccine of A, Apx of Actinobacillus pleuropneumoniae Apx,
Including recombinant protein rApx I A, rApxIIA and rOMPD；
The mass ratio of described recombinant protein rApx I A, rApxIIA and rOMPD are (1~3): 1:(1~3)；
The mass ratio of described recombinant protein rApx I A, rApxIIA and rOMPD are preferably 1:1:1；
The amino acid sequence of I A of recombinant protein rApx is as shown in SEQ ID NO:4.
The amino acid sequence of the recombinant protein rApxIIA is as shown in SEQ ID NO:5.
The amino acid sequence of the recombinant protein rOMPD is as shown in SEQ ID NO:6.
The Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mix epidemic disease
The preparation method of seedling, includes the following steps:
(1) I II A and OmpD gene of A, apx of amplification Actinobacillus pleuropneumoniae apx, has following (a), (b)
(c) nucleotide sequence shown in:
(a) nucleotide sequence shown in SEQ ID NO:1 (I A gene of apx)；With
(b) nucleotide sequence shown in SEQ ID NO:2 (II A gene of apx)；With
(c) nucleotide sequence shown in SEQ ID NO:3 (OmpD gene)；
(2) expression vector of the building containing gene described in step (1), and it is transferred to Escherichia coli respectively；
(3) inducing expression obtains destination protein；
(4) purifying and detection of recombinant protein；
(5) recombinant protein is mixed, obtains I II A and OMPD of A, Apx of Actinobacillus pleuropneumoniae Apx
Gene engineered subunit polyvalent vaccine.
The nucleotide sequence that primer sequence needed for extension increasing sequence gene is as follows:
I A primer sequence of apx:
F1 upstream primer: 5 '-CGCGGATCCGCTGCAACCGGCTCATTA-3′；
F2 downstream primer: 5 '-CCGCTCGAGTTAACCCGCATATACGATAGATG-3′；
ApxIIA primer sequence:
F3 upstream primer: 5 '-CGCGGATCCCCATTACTAACTCCAGGTGAA-3′；
F4 upstream primer: 5 '-CCGCTCGAGTTAAAAGGTGAGGTCTTTAAGA-3′；
OmpD primer sequence:
F5 upstream primer: 5 '-CGCGGATCCATGAAAAAATTCTTACTTTCTTCT-3′；
F6 upstream primer: 5 '-CCGCTCGAGTTAGAACGAGCTACCGATACT-3′；
CGC or CCG is protection base in above-mentioned primer sequence,GGATCCFor I restriction enzyme site of BamH,CTCGAGFor I enzyme of Xho
Enzyme site.
Obtained target fragment connects after double digestion with carrier pET-32a and PGEX-4T-1, is transferred to Escherichia coli
(E.coli) it is identified to extract plasmid by BL21 (DE3)；Picking positive bacterium colony obtains destination protein through IPTG inducing expression；It collects
The thallus of induction after ultrasonication, purifying and will precipitating (inclusion body) be stored in the urea PBS buffer solution of 8mol/L.
The concentration of described recombinant protein rApx I A, rApxIIA and the rOMPD after purification is respectively 376,321 and 110 μ
g/mL。
The Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mix epidemic disease
Application of the seedling in prevention porcine contagious pleuropneumonia.
This research has chosen the 756bp segment for containing the discontinuous antigen dominant epitope predicted of I A of apx, apx
The 2382bp of the 843bp and OmpD of II A are studied, and the present invention is obtained, and antigen immunogenicity is good.
The present invention compared with the existing technology, have following advantages and effects
(1) gene provided by the invention and method, available higher expression product yield rate are utilized, and recombinates egg
It is white that there is preferable antigenicity.Therefore, using the above recombinant protein as antigen, APP immune diagnostic method and system be can establish
Standby effective subunit vaccine.
(2) polyvalent vaccine of the invention is individually immunized compared with three kinds of albumen, and the antibody level of I A of rOMPD, rApx is above list
Antibody level when solely immune, the antibody level and polyvalent vaccine antibody level of II A of rApx are significantly higher than control group, illustrate to recombinate
The humoral immune reaction of subunit's polyvalent vaccine induction, rApxIA and rOMPD antibody play in the Cross immunogenicity of mouse
Effect of crucial importance.When mouse is immunized with inclusion body, compared with three kinds when tri- kinds of albumen combined immunizations of rApxIA, rApxIIA and rOMPD
Albumen induces higher antibody level when being individually immunized.I A of Apx has protective effect, II A of Apx to APP 1,5,9,10 and 11 types
There is protective effect to other serotypes in addition to 10 type of APP serum, OMPD is outer membrane protein, has protection to make every kind of bacterium
With.So subunit's polyvalent vaccine of the present invention can be that capture since APP serotype is more be that pig breeding industry brings heavy losses
This problem specifies a direction.
Detailed description of the invention
Fig. 1 is the electrophoretogram of target gene pcr amplification product；Wherein, Figure 1A is the amplification of apxIA and apxIIA gene,
Swimming lane M:DL 2000DNA Marker；The amplification of swimming lane 1~4:apxIA gene；The amplification of swimming lane 5~12:apxIIA gene；
The negative control of swimming lane 13:apxIA gene；The negative control of swimming lane 14:apxIIA gene；The amplification of Figure 1B: OmpD gene, swimming
Road M:DL 10000DNA Marker；The amplification of swimming lane 1~8:OmpD gene；Swimming lane 9: negative control.
Fig. 2 is the double digestion qualification figure of target gene and plasmid；Wherein, the digestion of II A gene of Fig. 2A: apx I A and apx is returned
Receive product, swimming lane M:DL 1000DNA Marker；The double digestion recovery product of I A gene of swimming lane 1:apx；II A base of swimming lane 2:apx
The double digestion recovery product of cause；The double digestion recovery product of Fig. 2 B:OmpD gene, swimming lane M:DL 10000DNA Marker；Swimming
The double digestion recovery product of road 1:OmpD gene；Fig. 2 C: plasmid enzyme restriction recovery product, swimming lane M:DL 10000DNA Marker；
Swimming lane 1: negative control；The digestion recovery product of swimming lane 2:PET-32a (+) plasmid；The digestion of swimming lane 3:PGEX-4T-1 plasmid is returned
Receive product.
Fig. 3 is recombinant plasmid pET-32a-apxIA, pET-32a-apxIIA and pGEX-OmpD bacterium colony PCR qualification figure；Its
In, Fig. 3 A:pET-32a-apxIA recombinant plasmid bacterium colony PCR identification, swimming lane M:DL 1000DNA Marker；Swimming lane 1~5: turn
Change single colonie PCR product；Swimming lane 6: positive control；Swimming lane 7: negative control；Fig. 3 B:pET-32a-apxIIA recombinant plasmid bacterium colony
PCR identification, swimming lane M:DL 1000DNA Marker；Swimming lane 1~4: conversion single colonie PCR product；Swimming lane 5: positive control；Swimming
Road 6: negative control；Fig. 3 C:pGEX-OmpD recombinant plasmid bacterium colony PCR identification, swimming lane M:DL 10000DNA Marker；Swimming lane 1
~4: conversion single colonie PCR product；Swimming lane 5: positive control；Swimming lane 6: negative control.
Fig. 4 is the double digestion qualification figure of recombinant plasmid；Wherein, swimming lane M1:DL 2000DNA Marker；Swimming lane M2:DL
10000DNA Marker；Swimming lane 1: the double digestion of recombinant plasmid pET-32a-apxIA；Swimming lane 2: recombinant plasmid pET-32a-
The double digestion of apxIIA；Swimming lane 3: the double digestion of recombinant plasmid pGEX-OmpD.
Fig. 5 is recombinant protein SDS-PAGE detection figure；Wherein, the induction detection of I A of Fig. 5 A:pET-32a-apx, swimming lane M:
116kD albumen marker；The non-inducing expression product of swimming lane 1:PET-32a；Swimming lane 2:PET-32a inducing expression product；Swimming lane 3:
The non-inducing expression product of I A of pET32a-apx；I A inducing expression product of swimming lane 4:pET32a-apx；Fig. 5 B:pET-32a-apxIIA
Induction detection, swimming lane M:116k albumen marker；The non-inducing expression product of swimming lane 1:PET-32a；Swimming lane 2:PET-32a induction
Expression product；The non-inducing expression product of swimming lane 3,5:pET-32a-apxIIA；Swimming lane 4:pET-32a-apxIIA inducing expression produces
Object；The induction of Fig. 5 C:pGEX-OmpD detects, swimming lane M:116kD albumen marker；The non-inducing expression of swimming lane 1:PGEX-4T-1 produces
Object；Swimming lane 2,4, the non-inducing expression product of 6:pGEX-OmpD；Swimming lane 3,5,7:pGEX-OMPD inducing expression product.
Fig. 6 is the optimization figure of pET-32a-apxIA inductive condition；Wherein, Fig. 6 A:pET-32a-apxIA not isogeneous induction is dense
The expression figure of degree, swimming lane M:116KD albumen marker；Swimming lane 1~5:IPTG concentration distinguishes 0,0.25,0.5,1.0,2.0mmol/
L；Fig. 6 B:pET-32a-apxIA difference induction time expression figure, swimming lane M: standard protein Marker；Swimming lane 1~7: it induces respectively
1、2、3、4、5、6、7h。
Fig. 7 is the optimization figure of pET-32a-apxIIA inductive condition；Wherein, Fig. 7 A:pET-32a-apxIIA not isogeneous induction
The expression figure of concentration, swimming lane M:116KD albumen marker；Swimming lane 1~5:IPTG concentration difference 0,0.25,0.5,1.0,
2.0mmol/L；Fig. 7 B:pET-32a-apxIIA difference induction time expression figure, swimming lane M: standard protein Marker；Swimming lane 1~
8: inducing 0,1,2,3,4,5,6,7h respectively.
Fig. 8 is the optimization figure of pGEX-OmpD inductive condition；Wherein, the expression of Fig. 8 A:pGEX-OmpD difference induced concentration
Figure, swimming lane M:116KD albumen marker；Swimming lane 1~6:IPTG concentration distinguishes 0.5,1.0,1.5,2.0,2.5,3.0mmol/L；
Fig. 8 B:pGEX-OMPD difference induction time expression figure, swimming lane M: standard protein Marker；Swimming lane 1~9: induce 0,1 respectively, 2,
3、4、5、6、7、24h。
Fig. 9 is recombinant protein solubility detection figure；Wherein, Fig. 9 A:pET-32a-apx I A and pET-32a-apxIIA forgives
Physical examination mapping, swimming lane M: standard protein Marker；I A bacterial cell disruption supernatant of swimming lane 1:pET-32a-apx；Swimming lane 2:pET-32a-
It is precipitated after I A bacterial cell disruption of apx；Swimming lane 3:pET-32a-apxIIA bacterial cell disruption supernatant；Swimming lane 4:pET-32a-apxIIA thallus
It is precipitated after broken；Fig. 9 B:pGEX-OmpD inclusion body detection figure, swimming lane M: standard protein Marker；Swimming lane 1:pGEX-OmpD bacterium
Body is crushed supernatant；It is precipitated after swimming lane 2:pGEX-OmpD bacterial cell disruption.
Figure 10 is rApxIA recombinant protein purification condition optimizing figure；Wherein, swimming lane M:116kD protein standard；Swimming lane 1~3:
The imidazoles of 30mmol/L combines, the albumen eluted respectively with 100,200,500mmol/L imidazoles；4~6:40mmol/L of swimming lane
Imidazoles combine, respectively with 100,200,500mmol/L imidazoles elution albumen；The imidazoles knot of 7~9:50mmol/L of swimming lane
It closes, the albumen eluted respectively with 100,200,500mmol/L imidazoles.
Figure 11 is rApxIIA recombinant protein purification condition optimizing figure；Wherein, swimming lane M:116kD protein standard；Swimming lane 1~
The imidazoles of 3:30mmol/L combines, the albumen eluted respectively with 100,200,500mmol/L imidazoles；4~6:40mmol/ of swimming lane
The imidazoles of L combines, the albumen eluted respectively with 100,200,500mmol/L imidazoles；The imidazoles knot of 7~9:50mmol/L of swimming lane
It closes, the albumen eluted respectively with 100,200,500mmol/L imidazoles.
Figure 12 is the detection figure of rOMPD recombinant protein purification；Wherein, swimming lane M:116kD protein standard；Swimming lane 1: purifying
ROMPD recombinant protein；Swimming lane 2: unpurified rOMPD albumen.
Figure 13 is WB detection figure after recombinant protein purification；Wherein, Figure 13 A: the WB inspection of purifying I A and rApxIIA albumen of rApx
It surveys, swimming lane M: the albumen marker of pre-dyed；Swimming lane 1: the PET-32a of induction；Swimming lane 2: the I A albumen of rApx of purifying；Swimming lane 3: pure
The rApxIIA albumen of change；Figure 13 B: the WB detection of purifying rOMPD albumen, swimming lane M: the albumen marker of pre-dyed；Swimming lane 1: purifying
ROMPD albumen.
Figure 14 is I A specific antibody indirect ELISA measurement result figure of rApx in serum after immune mouse.
Figure 15 is II A specific antibody indirect ELISA measurement result figure of rApx in serum after immune mouse.
Figure 16 is rOMPD specific antibody indirect ELISA measurement result figure in serum after immune mouse.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Method in the following example is unless otherwise instructed conventional method.
Percentage composition in the following example is unless otherwise instructed mass percentage.
Embodiment 1, APP Apx I A, Apx II A and OMPD recombinant protein preparation
1. main material
1.1 strain APP are China Veterinery Drug Inspection Office's product, number Actinobacillus
pleuropneumoniae CVCC259。
1.2 plasmids, carrier and competent cell
Escherichia coli (E.coli) BL21 (DE3), Escherichia coli (E.coli) DH5 α competent cell are public purchased from Takara
Department, expression vector PET-32a (+), PGEX-4T-1 are purchased from Novagen company.
2. method
2.1 using PCR methods to I II A and OMPD genes of SEQ ID NO:1 of A, Apx of APP Apx, SEQ ID NO:2, SEQ
Sequence is extended in ID NO:3, and amplification length is respectively 753bp, 843bp, 2382bp, as a result as shown in Figure 1.Above and below used
Trip primers F 1, F2, F3, F4, F5, F6 are primer mentioned above, contain BamH I, I restriction enzyme site of Xho.PCR reaction system:
10 μ L of 2 × KOD FX Buffer25 μ L, 2mM dNTPs, each 1 μ L of 1 μ L, KOD FX of upstream and downstream primer, the app gene of extraction
Group DNA 2 μ L, ddH210 μ L of O, totally 50 μ L.
Reaction condition is following (a), (b), (c) PCR program:
(a) I A gene of apx: 94 DEG C of initial denaturation 2min；94 DEG C of thermal denaturation 15s, 50.5 DEG C of 30s, 68 DEG C of 1min, totally 30 are followed
Ring；68 DEG C of 7min extend eventually.
(b) apxIIA gene: 94 DEG C of initial denaturation 2min；94 DEG C of thermal denaturation 15s, 50.5 DEG C of 30s, 68 DEG C of 1min, totally 30
Circulation；68 DEG C of 7min extend eventually.
(c) OmpD gene: 94 DEG C of initial denaturation 2min；94 DEG C of thermal denaturation 15s, 53 DEG C of 30s, 68 DEG C of 3min, totally 30 recycle；
68 DEG C of 7min extend eventually.
2.2 expression recombination APP Apx I A, Apx II A and OMPD bacterial strain building, comprising the following steps:
2.2.1 through 1% agarose gel electrophoresis Preliminary Identification, pcr amplification product purify and recycling, PET-32a (+) and
After the preparation of PGEX-4T-1 carrier, since restriction enzyme site is contained at the both ends of PCR product, by PCR product and PET-32a (+),
PGEX-4T-1 after BamH I and the identification of I double digestion of Xho with recycling, as a result as shown in Figure 2.
2.2.2 target gene is connect with expression vector.I A, apxIIA and PET-32a carrier of target fragment apx, OmpD
With the connection of PGEX-4T-1 carrier, connection mixed liquor is gently blown and beaten into mixing, 16 DEG C of water-baths are stayed overnight.
Reaction system: 2 × Ligation solution, I 5 μ L, PCR recovery product 2.5 μ L, PET-32a or PGEX-4T-
10.5 μ L, ddH22 μ L of O, totally 10 μ L.
2.2.3 connection product is converted to bacillus coli DH 5 alpha competent cell, is coated on LB plate.
The screening of 2.3 recombinant expression plasmids and identification, comprising the following steps:
2.3.1 the single colonie on picking LB plate carries out bacterium colony PCR verifying, reaction system: ddH2O 17.85 μ L, Taq
2.5 2.5 μ L of μ L, 10 × buffer of 0.15 μ L of enzyme, upstream and downstream primer each 1 μ L, dNTPs, totally 25 μ L.
Reaction condition is following (a), (b), (c) PCR program:
(a) A:94 DEG C of initial denaturation 2min of pET-32a-apx I；94 DEG C of thermal denaturation 15s, 50.5 DEG C of 30s, 68 DEG C of 1min, totally 30
A circulation；68 DEG C of 7min extend eventually.
(b) pET-32a-apxIIA:94 DEG C of initial denaturation 2min；94 DEG C of thermal denaturation 15s, 50.5 DEG C of 30s, 68 DEG C of 1min, altogether
30 circulations；68 DEG C of 7min extend eventually.
(c) pGEX-OmpD:94 DEG C of initial denaturation 2min；94 DEG C of thermal change 15s, 53 DEG C of 30s, 68 DEG C of 3min, totally 30 recycle；
68 DEG C of 7min extend eventually.
After PCR, 5 μ L amplified productions electrophoresis 25min under 110V voltage on 1% Ago-Gel is taken respectively, benefit
It is scanned with Labworks image acquisition and analysis software, Preliminary Identification, as a result as shown in Figure 3.
2.3.2 the double digestion identification of recombinant expression plasmid.It will be accredited as the bacterium colony of positive colony, shakes bacterium, extracts bacterium solution
Plasmid.With restriction enzyme, under 37 DEG C of constant temperatures, the recombinant clone plasmid that double digestion 3h is extracted, digestion products are with 1%
Agarose gel electrophoresis detection, as a result as shown in Figure 4.
Double enzyme digestion reaction system: I 1 I 1 μ L, 10 × K buffer of μ L, Xho of BamH 2 μ L, recombinant plasmid 4 μ L, ddH2O 12
μ L, totally 20 μ L.
2.3.3 recombinant strains are obtained
Positive recombinant expression plasmid will be accredited as to convert to Escherichia coli (E.coli) BL21 (DE3), obtain recombination table
Up to bacterial strain.
2.4 recombination APP Apx I A, Apx II A and OMPD albumen inducing expression condition optimizing and purifying, including following step
It is rapid:
2.4.1 recombinant protein inducing expression
2.4.1.1 positive single colonie will be accredited as to be inoculated in containing final concentration of 0.1mg/mL Amp (ampicillin)
5mL LB liquid medium, 37 DEG C of constant-temperature tables are incubated overnight first order seed with 200r/min.
2.4.1.2, first order seed is added to the LB liquid to 5mL containing final concentration of 0.1mg/mL Amp in the ratio of 1:50
In culture medium, 37 DEG C, 220r/min cultivates bacterium solution OD600 to 0.6~0.8.
2.4.1.3 IPTG to final concentration of 1.0mmol/L is added into the bacterium solution that needs induce, 37 DEG C, 200r/min shakes
Swing culture about 3h, inducing expression.
2.4.1.4 thallus 1mL is collected, is centrifuged 1min in 12000r/min.With the 100 sterile ddH of μ L2Thallus is resuspended in O, is added
Isometric 2 × SDS sample-loading buffer, boiling water bath 10min or so after mixing, 12000r/min are centrifuged 10min, supernatant are taken to carry out
SDS-PAGE electrophoresis, as a result as shown in Figure 5.
2.4.2 inducing expression condition optimizing
In order to obtain more expressing quantities, the inducing expression condition of albumen is optimized:
2.4.2.1IPTG concentration optimization: the use of IPTG inducer concentrations is 0,0.25,0.5,1.0,1.5,2,2.5,
3mmol/L is induced respectively.
2.4.2.2 induction time optimizes: select the optimal concentration of IPTG, induction time is set to 0,1,2,3,4,5,6,
7h, as a result as shown in Fig. 6,7,8: I A and pET-32a-apxIIA IPTG induced concentration of pET-32a-apx be determined as 1mmol/L,
Induction time is 3h；PGEX-OmpD IPTG induced concentration is determined as 2.5mmol/L, induction time 3h.
2.4.2.3 recombinant protein soluble analysis
Under the optimal inductive condition of 2.4.2.2, recombinant bacterium 100mL is cultivated.Recombinant bacterium in 4 DEG C of 12 000r/min from
Heart 10min collects precipitating, is washed 2~3 times with PBS, and precipitating is resuspended with PBS.Under condition of ice bath, Trixon114 ultrasound is added
Wave cracks bacterium, and cracking to bacterium solution becomes limpid, and 12 000r/min are centrifuged 20min, collects supernatant precipitating respectively.Pass through SDS-
PAGE detects the solubility of albumen, as a result as shown in Figure 9: the I miscellaneous egg of A and pET-32a-apxIIA albumen supernatant of pET-32a-apx
White more, precipitating contains more destination protein, and band is single, shows that expressing albumen is mainly inclusion body；PGEX-OmpD albumen is exhausted
Major part illustrates that the albumen of expression also mainly exists in the form of inclusion body in broken precipitating.
2.4.3 recombinant protein great expression and purifying
2.4.3.1 Trixon114 ultrasonication is added after collecting thallus in recombinant protein great expression, inducing amount 2L, and
(inclusion body) will be precipitated to be dissolved with the urea PBS buffer solution of 8mol/L, 4 DEG C overnight.Precipitating is abandoned in centrifugation after dissolution, stays supernatant.It will
The above-mentioned urea PBS buffer solution supernatant containing 8mol/L is in bag filter, respectively with the urea PBS buffer solution of 6,4,2,0mol/L
Gradient dialysis, each dialysis time are 4h or so, finally make its final concentration urea 0mol/L, are finally carried out with 0.45 μm of filter column
Filtering, filtrate deposit in -20 DEG C.
2.4.3.2 endotoxic removal
In clean centrifuge tube, PMXB liquid will be added by just pure albumen；The weight ratio of protein and PMXB is
1000:5~15.At room temperature, it is slowly mixed together 20~35min in vertical blenders, isometric organic solvent is then added
Ergol mix two minutes, in desk centrifuge 10000~13000r/min be centrifuged 3~4min, after centrifugation two-phase it
Between form thin film, this film is the compound that PMXB and endotoxin are formed.It is (untouchable carefully to draw protein-contg water phase
Film) and move in another clean tubule, it is centrifuged after adding isometric organic solvent Ergol mixing.It so repeats to wash
It washs 3~4 times, detect the concentration of albumen with BCA protein quantification kit and detects remaining endotoxin content with reagents.
2.4.3.3 recombinant protein purification condition optimizing includes following (a) and (b):
(a) purifying of I A, rApxIIA recombinant protein of rApx
Selection 30mM, 40mM, 50mM imidazole buffer balances purification column respectively；After having adsorbed pretreatment sample, the miaow of 5mL
Azoles buffer rinses purification column, and 1~2mL/min of flow velocity collects eluent；Using 5mL100mM, 200mM, 500mM imidazole buffer
Liquid carries out gradient elution to destination protein, and 1~2mL/min of flow velocity collects eluent.It is examined with SDS-PAGE to samples are collected
It surveys, optimal combination buffer and elution buffer is determined, as a result as shown in Figure 10,11: I A, rApxIIA recombinant protein of rApx
When combination buffer is 50mmol/L, the protein content that eluent elutes when being 200mmol/L is most.
(b) purifying of rOMPD recombinant protein
OMPD albumen is carried out cutting glue purification specific steps are as follows: preparing according to the method for preparing PAGE gel
Unlike glass plate when configuring separation gel, the gap of 1~2cm is reserved thereon, and is not inserted into comb.By processed albumen
It is added into gap, runs glue to the preceding band of bromophenol blue to gel sub by the identical mode of SDS-PAGE.It takes out gel and is put in Sheng
There is ddH25min is gently washed in the plate of O, discards ddH20.25mol/L KCl solution is added in O, until there is the bright band of a white to go out
It is existing.It is cut, grinds and be put in EP pipe, add PBS in 4 DEG C of overnight soluble proteins.4 DEG C of centrifugations will be taken out by rear EP pipe overnight,
8000r/min is centrifuged 10min, takes supernatant in new EP pipe, this is albumen after purification, as a result as shown in figure 12.
2.4.3.4 purification of recombinant proteins SDS-PAGE
Take 100 μ L that 2 × SDS-PAGE sample-loading buffer, 100 μ L is added in the above-mentioned sample of recombinant protein after purification respectively,
10min is boiled in boiling water, carries out SDS-PAGE, and observation electrophoresis result is analyzed.
2.4.3.5 purification of recombinant proteins Western-blotting is detected
By above-mentioned resulting APP recombinant protein rApx I A, rApx II A and rOMPD carry out SDS-PAGE electrophoresis, constant current
200mA, electricity turn 60min for the protein delivery on gel to nitrocellulose filter (NC film), wash 3 times by PBST buffer
Afterwards, with 5% skimmed milk power, 37 DEG C of shaking table 60r/min close 2h, are washed 3 times with PBST buffer, with 37 DEG C of primary antibody (rabbit anteserum)
Water-bath acts on 1h, then is washed 3 times with PBST buffer, (uses diluted ELIAS secondary antibody HRP (the horseradish mistake of PBST buffer with secondary antibody
Oxide enzyme) label goat anti-rabbit igg) (1:5000 dilution) 37 DEG C of water-baths act on 1h, shown after the washing of BST buffer 3 times
Show, as a result as shown in figure 13: respectively having a clear band at about 46kD, 50kD and 110KD, illustrate that the recombinant protein of expression has
Good antigenicity.
2.4.3.6 the measurement of purifying protein concentration
It utilizesBCA Protein Assay Kit, step obtains recombinant protein rApx after purification as requested
The concentration of I A, rApxIIA and rOMPD is respectively 376,321 and 110 μ g/mL.
2 Actinobacillus pleuropneumoniae Apx of embodiment I A, Apx II A and OMPD gene engineered subunit mix epidemic disease
The analysis of seedling immune efficacy
1. main material
1.1 animals: 4~6 week old female Balb/c mouse are purchased from Guangzhou south Animal Science Co., Ltd, medical university
1.2 vaccines: complete using Freund for the mass mixings object such as gained albumen after purification and three kinds of albumen in embodiment 1
Full adjuvant and incomplete Freund's adjuvant emulsified protein, are purchased from SIGMA company.
2. method
2.1 take the Balb/c mouse of 60 4~6 week old or so, random point 6 groups, rApx I A, rApxIIA, rOMPD and
I A+rApxIIA+rOMPD albumen of rApx is respectively one group, sets physiological saline separately as blank control group and porcine contagious pleuropneumonia three
Valence inactivated vaccine (being purchased from Wuhan predecessor company, section) is used as positive control.Mouse is immunized in each group in a manner of hypodermic, and albumen is exempted from
Epidemic disease amount is 100 μ g/, and being immunized for inactivated vaccine is 0.2mL/.Booster immunization after initial immunity 14d and 28d, it is immune for the first time to make
With Freund's complete adjuvant emulsified protein, second and third time is using incomplete Freund's adjuvant emulsified protein, immunizing dose and mode and just
It is secondary be immunized it is identical.
2.2 indirect ELISA methods detect serum specific antibody
Adopt positive serum and negative serum (the Agricultural University Of South China animal doctor of serum of second of protein immunization mouse after two weeks
The preparation of microorganism teaching and research room, institute) square matrix titration is carried out, determine the best antigen coat concentration of rApxIA, rApx II A, rOMPD
With the optimum dilution degree of primary antibody.With the specific antibody in indirect ELISA method detection immune mouse serum.Concrete operations are as follows:
Elisa plate, every 100 μ L of hole, ambient temperature overnight coating are coated with the optimum dilution degree of rApxIA, rApx II A and rOMPD of purifying；
PBST is washed liquid 3 times, and every hole adds confining liquid (5%BSA) 100 μ L, 37 DEG C of closing 2h；It is washed liquid 3 times with PBST again, with an anti-binding
(being detected serum with PBST buffer doubling dilution), every 100 μ L of hole, wherein the negative serum with BALB/c mouse compares；Again
It is washed liquid 3 times with PBST, with two anti-bindings (with the sheep anti mouse of the diluted ELIAS secondary antibody horseradish peroxidase-labeled of 0.5%BSA
IgG), every hole adds 100 μ L, 37 DEG C of incubation 1h；It is washed liquid 3 times with PBST again, every hole adds substrate solution (phosphate-lemon acid buffering
Liquid 100mL, o-phenylenediamine 40mg, 30%H2O2100 μ L), the 15min that develops the color in room temperature magazine is set, after developing the color, 50 μ are added in every hole
L terminate liquid (2mol/LH2SO4) terminate reaction；Use the OD value of microplate reader measurement 450nm.
According to square matrix titration as a result, antigen coat concentration and primary antibody dilution when P/N value maximum are optimum dilution degree.
Wherein the optimum dilution degree of purifying protein rApxIA, rApx II A and rOMPD is respectively 1:320,1:320 and 1:10.Primary antibody is most
Good dilution is 1:100.The sheep anti-mouse igg dilution of HRP label is 1:5000.Immune mouse is detected according to this reaction condition
The dynamic change of II A and rOMPD specific antibody of serum rApxIA, rApx.
As a result as shown in Figure 14,15,16: rApxIA group and three kinds of albumen mixing groups first immunisation after two weeks, just have bright
Aobvious antibody generates, and antibody level is higher than tervalence inactivated vaccine group and control group.Twice after booster immunization, rApxIA group and three
The trend that rises rapidly is presented in kind albumen mixing group antibody, and antibody height still maintains original trend.Three times after booster immunization,
Three groups of antibody level reduces, and antibody level is compared: I A group > tri- kind albumen mixing group of rApx > tervalence inactivated vaccine group > blank
Group.However, the I A antibody level of rApx of tervalence inactivated vaccine group is generated without apparent antibody after being immunized three times；For the first time
It is immune after two weeks, the antibody level of rApxIIA group and three kinds of albumen mixing groups is suitable, hence it is evident that higher than tervalence inactivated vaccine group and right
According to group.Twice after booster immunization, the trend that rises rapidly, antibody level is presented in II A group of rApx and three kinds of albumen mixing group antibody
It compares: II A group > of rApx, tri- kinds of albumen mixing group > tervalence inactivated vaccine group > blank groups.Three times after booster immunization, II A of rApx
Group reduces and identical with the trend of antibody height afterwards immune twice with the antibody level of three kinds of albumen mixing group antibody, and trivalent is gone out
The II A antibody level of rApx and control group of live vaccine group almost maintain same level.However, tervalence inactivated vaccine group rApx II
A antibody level its antibody level and antibody level of PBS control group after being immunized three times are not much different；Three kinds of albumen mixing groups and
The rOMPD antibody level of rOMPD protein groups successively reduces and is apparently higher than tervalence inactivated vaccine group and control group.First immunisation two
Zhou Hou, rOMPD protein groups and three kinds of albumen mixing groups have apparent antibody to generate and are on close level, and are higher than tervalence inactivated vaccine
Group and control group.Twice after booster immunization, rOMPD protein groups, three kinds of albumen mixing groups and tervalence inactivated vaccine group antibody are in
Existing ascendant trend, antibody level are compared: three kinds of albumen mixing group > rOMPD protein groups > tervalence inactivated vaccine group > control groups.
Compared with the control group, three kinds of recombinant proteins produce higher antibody level after mouse is immunized, and are higher than control group.
When the mixing of three kinds of albumen, rOMPD albumen, I A of rApx antibody level of antibody level when being above individually immune illustrate that recombination is sub-
The humoral immune reaction of subunit vaccine induction, rApxIA and rOMPD antibody play extremely heavy in the Cross immunogenicity of mouse
It acts on.
There is not phenomena such as red and swollen, fever in immune animal injection site, and also without there is inoculation adverse reaction, appetite is just
Often, the state of mind is good.
As a result the I II A and OMPD genetic engineering of A, Apx of Actinobacillus pleuropneumoniae Apx for prompting us to prepare
Subunit's polyvalent vaccine has good immunogenicity, high-caliber protection antibody can be generated after immune mouse, after inoculation
It is harmless to immune animal safety, it is a kind of new generation vaccine with bright prospects, China's porcine contagious pleuropneumonia will be prevented
Control provides reserve supply and technical support, has great importance.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (9)
1. a kind of Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mix epidemic disease
Seedling, it is characterised in that including recombinant protein rApx I A, rApxIIA and rOMPD；
The amino acid sequence of I A of recombinant protein rApx is as shown in SEQ ID NO:4；
The amino acid sequence of the recombinant protein rApxIIA is as shown in SEQ ID NO:5；
The amino acid sequence of the recombinant protein rOMPD is as shown in SEQ ID NO:6.
2. I II A and OMPD gene work of A, Apx of Actinobacillus pleuropneumoniae Apx according to claim 1
Journey subunit polyvalent vaccine, it is characterised in that:
The mass ratio of described recombinant protein rApx I A, rApxIIA and rOMPD are (1~3): 1:(1~3).
3. I II A and OMPD gene work of A, Apx of Actinobacillus pleuropneumoniae Apx according to claim 1
Journey subunit polyvalent vaccine, it is characterised in that:
The mass ratio of described recombinant protein rApx I A, rApxIIA and rOMPD are 1:1:1.
4. I II A and OMPD of A, Apx of the described in any item Actinobacillus pleuropneumoniae Apx of claims 1 to 3
The preparation method of gene engineered subunit polyvalent vaccine, it is characterised in that include the following steps:
(1) I II A and OmpD gene of A, apx of amplification Actinobacillus pleuropneumoniae apx, has following (a), (b)
(c) nucleotide sequence shown in:
(a) nucleotide sequence shown in SEQ ID NO:1 (I A gene of apx)；With
(b) nucleotide sequence shown in SEQ ID NO:2 (II A gene of apx)；With
(c) nucleotide sequence shown in SEQ ID NO:3 (OmpD gene)；
(2) expression vector of the building containing gene described in step (1), and it is transferred to Escherichia coli respectively；
(3) inducing expression obtains destination protein；
(4) purifying and detection of recombinant protein；
(5) recombinant protein is mixed, obtains I II A and OMPD of A, Apx of Actinobacillus pleuropneumoniae Apx
Gene engineered subunit polyvalent vaccine.
5. I II A and OMPD gene work of A, Apx of Actinobacillus pleuropneumoniae Apx according to claim 4
The preparation method of journey subunit polyvalent vaccine, it is characterised in that:
The nucleotide sequence that primer sequence needed for extension increasing sequence gene is as follows:
I A primer sequence of apx:
F1 upstream primer: as shown in SEQ ID NO:7；
F2 downstream primer: as shown in SEQ ID NO:8.
6. I II A and OMPD gene work of A, Apx of Actinobacillus pleuropneumoniae Apx according to claim 4
The preparation method of journey subunit polyvalent vaccine, it is characterised in that:
The nucleotide sequence that primer sequence needed for extension increasing sequence gene is as follows:
ApxIIA primer sequence:
F3 upstream primer: as shown in SEQ ID NO:9；
F4 upstream primer: as shown in SEQ ID NO:10.
7. I II A and OMPD gene work of A, Apx of Actinobacillus pleuropneumoniae Apx according to claim 4
The preparation method of journey subunit polyvalent vaccine, it is characterised in that:
The nucleotide sequence that primer sequence needed for extension increasing sequence gene is as follows:
OmpD primer sequence:
F5 upstream primer: as shown in SEQ ID NO:11；
F6 upstream primer: as shown in SEQ ID NO:12.
8. I II A and OMPD gene work of A, Apx of Actinobacillus pleuropneumoniae Apx according to claim 4
The preparation method of journey subunit polyvalent vaccine, it is characterised in that:
The concentration of described recombinant protein rApx I A, rApxIIA and the rOMPD after purification is respectively 376,321 and 110 μ g/
mL。
9. I II A and OMPD of A, Apx of the described in any item Actinobacillus pleuropneumoniae Apx of claims 1 to 3
Application of the gene engineered subunit polyvalent vaccine in the drug of preparation prevention porcine contagious pleuropneumonia.
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