CN105664148A - Genetic engineering subunit mixed vaccine as well as preparation method and application thereof - Google Patents
Genetic engineering subunit mixed vaccine as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a genetic engineering subunit mixed vaccine as well as a preparation method and an application thereof and belongs to the field of research of veterinary vaccines. Three recombinant proteins rApxIA, rApxIIA and rOMPD are expressed abundantly with a genetic engineering method and have better antigenicity. The rOMPD and rApxIA antibody levels of the mixed vaccine are higher than those during separate immunization of three proteins, the antibody level of rApxIIA and the antibody level of the mixed vaccine are remarkably higher than that of a control group, and the result proves that humoral immunity reaction induced by the recombined subunit mixed vaccine and rApxIA and rOMPD antibodies play an important role in cross immunization protection of mice. After inoculation, the genetic engineering subunit mixed vaccine is safe and harmless to an immune animal, is a novel vaccine with wide prospect, provides material reserve and technical support for control of porcine contagious pleuropneumonia in china and has great significance.
Description
Technical field
The invention belongs to live vaccine research field, relate to a kind of Actinobacillus pleuropneumoniae gene engineered subunit mixed vaccine and its preparation method and application, the invention particularly relates to a kind of Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mixed vaccine and its preparation method and application.
Background technology
Porcine contagious pleuropneumonia (Porcinecontagiouspleuropneumonia, PCP) it is oozed out the high degree in contact sexually transmitted disease for principal character by what Actinobacillus pleuropneumoniae (Actinobacilluspleuropneumoniae, APP) caused with pneumorrhagia, necrosis and cellulosic. APP serotype has 15 serotypes, and the popular serotype of each country also differs. In China, 1987, Harbin veterinary institute found clinical case first, by separating cause of disease and the cause of disease separated carries out serotype, Serologic test etc. demonstrating PCP in the existence of China and popular. China with serotype 1,3 and 7 maximum. Since two thousand, PCP occurs in that popular in China much places. The ground such as Shanghai, Zhejiang, Guangdong, Hubei, Hunan, Henan are particularly acute, it was reported that only pig farm, Suburb Areas of Hangzhou nearly 30,000 pig death because this disease results in.
PCP causes great economic loss to various countries' pig industry, PCP the pig that causes is dead, the reduction of slow, the feedstuff-meat ratio of the speed of growth and put into the increase for the treatment of fund, has carried out economic loss seriously to cultivation industrial belt. The various ages, sex pig to the equal susceptible of APP, the most susceptible of the pig at 6～10 monthly ages, when PCP acute popular time the incidence of infection and mortality rate more than 20%, the most acute popular time mortality rate then reach more than 80%. Clinical classical symptom is: body temperature more than 41.5 DEG C; Sick pig dyspnea is sitting position of dog gesture, and mouth and nose flow out blood sample exudate; Main pathology is changed to trachea color mucus; Diffusivity hemorrhagic pneumonia, fibrinous pleurisy.
APP can cause zoogenetic infection morbidity to be determined by virulence factor. APP has a lot of virulence factor, and research shows, the important virulence factor of APP is not only by the extracellular toxin (ApxI, ApxII, ApxIII) of APP secretion and outer membrane protein (OmpD) etc., is also important immunogenic factors simultaneously.ApxIA gene, apx II A gene are again the precursors of encoding toxin protein. D15/OmpD has been found to play an important role in the synthesis and assembling process of outer membrane protein.
At present, vaccination is prevention and a very important measure for the treatment of PCP. The pathogen of primary disease has 15 serotypes, and virulence factor is numerous, between different serotypes not or only have faint cross protection, thus develops difficult point and the key that the vaccine efficiently with cross-protection is this disease of prevention and control, is constantly subjected to the attention of people. The vaccine of various countries' research has a variety of: full bacterium inactivated vaccine, gene-deleted vaccine, bacterium shadow Seedling and subunit vaccine etc. At present, various countries prevention and control PCP clinically uses traditional APP whole cell inactivated vaccine. Owing to inactivated vaccine has type specificity, lack in APP yeast culture process to the extracellular toxin of cell exocrine; In the process of inactivation, some antigenic component is likely to be broken or loses. Weak poison deletion of vaccine and ghosts vaccine etc. can generation type cross-immunity, certain immune protective efficiency can be provided to attacking of different serotypes APP. While attenuated vaccine reduces along with virulence, its immunogenicity also declines to some extent, it sometimes appear that virulence returns strong phenomenon, is difficult to prediction and controls. Ghosts vaccine is it sometimes appear that crack incomplete situation. Comparatively speaking, it does not have the genetic engineering subunit vaccine of disadvantages mentioned above then becomes the focus of research.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, it is an object of the invention to provide a kind of Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mixed vaccine. The present invention uses engineered method great expression 3 kinds of recombiant protein rApx I A, rApxIIA and rOMPD, and prevention and the treatment of PCP are had good effect as the recombinant subunit vaccine of component by the RTX-toxins outer membrane protein (OmpD) of recombinant expressed APP.
Another object of the present invention is to provide above-mentioned Actinobacillus pleuropneumoniae Apx I A, Apx II preparation method of A and OMPD gene engineered subunit mixed vaccine.
It is still another object of the present invention to provide above-mentioned Actinobacillus pleuropneumoniae Apx I A, Apx II application of A and OMPD gene engineered subunit mixed vaccine.
The purpose of the present invention is achieved through the following technical solutions:
A kind of Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mixed vaccine, including recombiant protein rApx I A, rApxIIA and rOMPD;
Described recombiant protein rApx I A, the mass ratio of rApxIIA and rOMPD are (1～3): 1:(1～3);
Described recombiant protein rApx I A, the mass ratio of rApxIIA and rOMPD are preferably 1:1:1;
The aminoacid sequence of described recombiant protein rApx I A is such as shown in SEQIDNO:4.
The aminoacid sequence of described recombiant protein rApxIIA is such as shown in SEQIDNO:5.
The aminoacid sequence of described recombiant protein rOMPD is such as shown in SEQIDNO:6.
The preparation method of described Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mixed vaccine, comprises the steps:
(1) amplification Actinobacillus pleuropneumoniae apx I A, apx II A and OmpD gene, has following (a), (b) and the nucleotide sequence shown in (c):
Nucleotide sequence (apx I A gene) shown in (a) SEQIDNO:1;With
Nucleotide sequence (apx II A gene) shown in (b) SEQIDNO:2; With
Nucleotide sequence (OmpD gene) shown in (c) SEQIDNO:3;
(2) build the expression vector containing the gene described in (1) in steps, and proceed to escherichia coli respectively;
(3) abduction delivering obtains destination protein;
(4) purification of recombiant protein and detection;
(5) recombiant protein is mixed, obtain Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mixed vaccine.
The nucleotide sequence that primer sequence needed for extension increasing sequence gene is as follows:
Apx I A primer sequence:
F1 forward primer: 5 '-CGCGGATCCGCTGCAACCGGCTCATTA-3 ';
F2 downstream primer: 5 '-CCGCTCGAGTTAACCCGCATATACGATAGATG-3 ';
ApxIIA primer sequence:
F3 forward primer: 5 '-CGCGGATCCCCATTACTAACTCCAGGTGAA-3 ';
F4 forward primer: 5 '-CCGCTCGAGTTAAAAGGTGAGGTCTTTAAGA-3 ';
OmpD primer sequence:
F5 forward primer: 5 '-CGCGGATCCATGAAAAAATTCTTACTTTCTTCT-3 ';
F6 forward primer: 5 '-CCGCTCGAGTTAGAACGAGCTACCGATACT-3 ';
In above-mentioned primer sequence, CGC or CCG is protection base,GGATCCFor BamH I restriction enzyme site,CTCGAGFor Xho I restriction enzyme site.
The purpose fragment obtained is connected after double digestion with carrier pET-32a and PGEX-4T-1, proceeds to escherichia coli (E.coli) BL21 (DE3), extracts plasmid identified; Picking positive bacterium colony, obtains destination protein through IPTG abduction delivering; Collect induction thalline after ultrasonication, purification and will precipitate (inclusion body) be stored in the carbamide PBS of 8mol/L.
Recombiant protein rApx I A after described purification, concentration respectively 376, the 321 and 110 μ g/mL of rApxIIA and rOMPD.
The application in prevention porcine contagious pleuropneumonia of described Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mixed vaccine.
The 2382bp of 843bp and the OmpD containing the prediction 756bp fragment of discontinuous antigen dominant epitope out, apx II A that this research have chosen apx I A studies, and the present invention is obtained, and antigen immunogenicity is good.
The present invention, relative to prior art, has such advantages as and effect:
(1) utilize gene provided by the invention and method, it is possible to obtain higher expression product yield rate, and recombiant protein has good antigenicity. Therefore, utilize above recombiant protein as antigen, it is possible to set up APP immune diagnostic method and prepare effective subunit vaccine.
(2) mixed vaccine of the present invention is individually immune compared with three kinds of albumen; rOMPD, rApx I antibody horizontal when being above individually immunity of the antibody horizontal of A; antibody horizontal and the mixed vaccine antibody horizontal of rApx II A are significantly higher than matched group; the humoral immune reaction that recombinant subunit mixed vaccine is induced is described, rApxIA and rOMPD antibody plays effect of crucial importance in the Cross immunogenicity of mice. During with inclusion body immune mouse, the antibody horizontal that time during three kinds of albumen combined immunizations of rApxIA, rApxIIA and rOMPD compared with the individually immunity of three kinds of albumen, induction is higher. APP1,5,9,10 and 11 type is had protective effect by Apx I A, and other serotypes except APP serum 10 type are all had protective effect by Apx II A, and OMPD is outer membrane protein, and every kind of bacterium is had protective effect. So, subunit mixed vaccine of the present invention can for capturing owing to APP serotype is more and be that pig industry brings this difficult problem of heavy losses to specify a direction.
Accompanying drawing explanation
Fig. 1 is the electrophoretogram of genes of interest pcr amplification product; Wherein, Figure 1A is the amplification of apxIA and apxIIA gene, swimming lane M:DL2000DNAMarker; 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 lane M:DL10000DNAMarker; The amplification of swimming lane 1～8:OmpD gene; Swimming lane 9: negative control.
Fig. 2 is the double digestion qualification figure of genes of interest and plasmid; Wherein, the enzyme action of Fig. 2 A:apx I A and apx II A gene reclaims product, swimming lane M:DL1000DNAMarker; The double digestion of swimming lane 1:apx I A gene reclaims product; The double digestion of swimming lane 2:apx II A gene reclaims product; The double digestion of Fig. 2 B:OmpD gene reclaims product, swimming lane M:DL10000DNAMarker; The double digestion of swimming lane 1:OmpD gene reclaims product; Fig. 2 C: plasmid enzyme restriction reclaims product, swimming lane M:DL10000DNAMarker; Swimming lane 1: negative control; Swimming lane 2:PET-32a (+) enzyme action of plasmid reclaims product; The enzyme action of swimming lane 3:PGEX-4T-1 plasmid reclaims product.
Fig. 3 is recombiant plasmid pET-32a-apxIA, pET-32a-apxIIA and pGEX-OmpD bacterium colony PCR qualification figure; Wherein, Fig. 3 A:pET-32a-apxIA recombiant plasmid bacterium colony PCR identifies, swimming lane M:DL1000DNAMarker; Swimming lane 1～5: convert single bacterium colony PCR primer; Swimming lane 6: positive control; Swimming lane 7: negative control; Fig. 3 B:pET-32a-apxIIA recombiant plasmid bacterium colony PCR identifies, swimming lane M:DL1000DNAMarker; Swimming lane 1～4: convert single bacterium colony PCR primer; Swimming lane 5: positive control; Swimming lane 6: negative control; Fig. 3 C:pGEX-OmpD recombiant plasmid bacterium colony PCR identifies, swimming lane M:DL10000DNAMarker; Swimming lane 1～4: convert single bacterium colony PCR primer; Swimming lane 5: positive control; Swimming lane 6: negative control.
Fig. 4 is the double digestion qualification figure of recombiant plasmid; Wherein, swimming lane M1:DL2000DNAMarker; Swimming lane M2:DL10000DNAMarker; Swimming lane 1: the double digestion of recombiant plasmid pET-32a-apxIA; Swimming lane 2: the double digestion of recombiant plasmid pET-32a-apxIIA; Swimming lane 3: the double digestion of recombiant plasmid pGEX-OmpD.
Fig. 5 is recombiant protein SDS-PAGE detection figure; Wherein, the induction detection of Fig. 5 A:pET-32a-apx I A, swimming lane M:116kD albumen marker; The non-abduction delivering product of swimming lane 1:PET-32a; Swimming lane 2:PET-32a abduction delivering product; The non-abduction delivering product of swimming lane 3:pET32a-apx I A; Swimming lane 4:pET32a-apx I A abduction delivering product; The induction detection of Fig. 5 B:pET-32a-apxIIA, swimming lane M:116k albumen marker; The non-abduction delivering product of swimming lane 1:PET-32a; Swimming lane 2:PET-32a abduction delivering product; The non-abduction delivering product of swimming lane 3,5:pET-32a-apxIIA; Swimming lane 4:pET-32a-apxIIA abduction delivering product; The induction detection of Fig. 5 C:pGEX-OmpD, swimming lane M:116kD albumen marker; The non-abduction delivering product of swimming lane 1:PGEX-4T-1; Swimming lane 2,4, the non-abduction delivering product of 6:pGEX-OmpD; Swimming lane 3,5,7:pGEX-OMPD abduction delivering product.
Fig. 6 is the optimization figure of pET-32a-apxIA inductive condition; Wherein, the expression figure of Fig. 6 A:pET-32a-apxIA difference induced concentration, swimming lane M:116KD albumen marker;Swimming lane 1～5:IPTG concentration respectively 0,0.25,0.5,1.0,2.0mmol/L; Fig. 6 B:pET-32a-apxIA difference induction time expresses figure, and swimming lane M: standard protein Marker; Swimming lane 1～7: induce 1 respectively, 2,3,4,5,6,7h.
Fig. 7 is the optimization figure of pET-32a-apxIIA inductive condition; Wherein, the expression figure of Fig. 7 A:pET-32a-apxIIA difference induced concentration, swimming lane M:116KD albumen marker; Swimming lane 1～5:IPTG concentration respectively 0,0.25,0.5,1.0,2.0mmol/L; Fig. 7 B:pET-32a-apxIIA difference induction time expresses figure, and swimming lane M: standard protein Marker; Swimming lane 1～8: induce 0 respectively, 1,2,3,4,5,6,7h.
Fig. 8 is the optimization figure of pGEX-OmpD inductive condition; Wherein, the expression figure of Fig. 8 A:pGEX-OmpD difference induced concentration, swimming lane M:116KD albumen marker; Swimming lane 1～6:IPTG concentration respectively 0.5,1.0,1.5,2.0,2.5,3.0mmol/L; Fig. 8 B:pGEX-OMPD difference induction time expresses figure, and swimming lane M: standard protein Marker; Swimming lane 1～9: induce 0,1 respectively, 2,3,4,5,6,7,24h.
Fig. 9 is recombiant protein solubility detection figure; Wherein, Fig. 9 A:pET-32a-apx I A and pET-32a-apxIIA inclusion body detection figure, swimming lane M: standard protein Marker; Swimming lane 1:pET-32a-apx I A bacterial cell disruption supernatant; Swimming lane 2:pET-32a-apx I A bacterial cell disruption postprecipitation; Swimming lane 3:pET-32a-apxIIA bacterial cell disruption supernatant; Swimming lane 4:pET-32a-apxIIA bacterial cell disruption postprecipitation; Fig. 9 B:pGEX-OmpD inclusion body detection figure, swimming lane M: standard protein Marker; Swimming lane 1:pGEX-OmpD bacterial cell disruption supernatant; Swimming lane 2:pGEX-OmpD bacterial cell disruption postprecipitation.
Figure 10 is rApxIA recombinant protein purification condition optimizing figure; Wherein, swimming lane M:116kD protein standard; The imidazoles of swimming lane 1～3:30mmol/L combines, respectively with the albumen of the imidazoles eluting of 100,200,500mmol/L; The imidazoles of swimming lane 4～6:40mmol/L combines, respectively with the albumen of the imidazoles eluting of 100,200,500mmol/L; The imidazoles of swimming lane 7～9:50mmol/L combines, respectively with the albumen of the imidazoles eluting of 100,200,500mmol/L.
Figure 11 is rApxIIA recombinant protein purification condition optimizing figure; Wherein, swimming lane M:116kD protein standard; The imidazoles of swimming lane 1～3:30mmol/L combines, respectively with the albumen of the imidazoles eluting of 100,200,500mmol/L; The imidazoles of swimming lane 4～6:40mmol/L combines, respectively with the albumen of the imidazoles eluting of 100,200,500mmol/L; The imidazoles of swimming lane 7～9:50mmol/L combines, respectively with the albumen of the imidazoles eluting of 100,200,500mmol/L.
Figure 12 is the detection figure of rOMPD recombinant protein purification; Wherein, swimming lane M:116kD protein standard; Swimming lane 1: the rOMPD recombiant protein of purification; Swimming lane 2: unpurified rOMPD albumen.
Figure 13 is WB detection figure after recombinant protein purification; Wherein, the WB detection of Figure 13 A: purification rApx I A and rApxIIA albumen, swimming lane M: the albumen marker of pre-dyed; Swimming lane 1: the PET-32a of induction; Swimming lane 2: the rApx I A albumen of purification; Swimming lane 3: the rApxIIA albumen of purification; Figure 13 B: the WB detection of purification rOMPD albumen, swimming lane M: the albumen marker of pre-dyed; Swimming lane 1: the rOMPD albumen of purification.
Figure 14 is rApx I A specific antibody indirect ELISA measurement result figure in serum after immune mouse.
Figure 15 is rApx II A specific antibody indirect ELISA measurement result figure in serum after immune mouse.
Figure 16 is rOMPD specific antibody indirect ELISA measurement result figure in serum after immune mouse.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Method in the following example, if no special instructions, is conventional method.
Percentage composition in the following example, if no special instructions, is weight/mass percentage composition.
Embodiment 1, APPApx I A, Apx II the recombiant protein of A and OMPD prepare
1. main material
1.1 strain APP are China Veterinery Drug Inspection Office's product, are numbered ActinobacilluspleuropneumoniaeCVCC259.
1.2 plasmids, carrier and competent cell
Escherichia coli (E.coli) BL21 (DE3), escherichia coli (E.coli) DH5 α competent cell purchased from Takara company, expression vector PET-32a (+), PGEX-4T-1 is purchased from Novagen company.
2. method
2.1 utilize PCR method that sequence in APPApx I A, Apx II A and OMPD genes of SEQ IDNO:1, SEQIDNO:2, SEQIDNO:3 is extended, and amplification length is 753bp, 843bp, 2382bp respectively, and result is as shown in Figure 1. Upstream and downstream primers F 1 used, F2, F3, F4, F5, F6 are primer mentioned above, all contain BamH I, Xho I restriction enzyme site. PCR reaction system: 2 × KODFXBuffer25 μ L, 2mMdNTPs10 μ L, upstream and downstream primer each 1 μ L, KODFX1 μ L, app gene group DNA2 μ L, the ddH of extraction2O10 μ L, totally 50 μ L.
Reaction condition is following (a), (b), (c) PCR program:
(a) apx I A gene: 94 DEG C of denaturation 2min; 94 DEG C of thermal denaturation 15s, 50.5 DEG C of 30s, 68 DEG C of 1min, totally 30 circulations; 68 DEG C of 7min extend eventually.
(b) apxIIA gene: 94 DEG C of denaturation 2min; 94 DEG C of thermal denaturation 15s, 50.5 DEG C of 30s, 68 DEG C of 1min, totally 30 circulations; 68 DEG C of 7min extend eventually.
(c) OmpD gene: 94 DEG C of denaturation 2min; 94 DEG C of thermal denaturation 15s, 53 DEG C of 30s, 68 DEG C of 3min, totally 30 circulations; 68 DEG C of 7min extend eventually.
2.2 express restructuring APPApx I A, Apx II structure of A and OMPD bacterial strain, comprise the following steps:
2.2.1 through 1% agarose gel electrophoresis Preliminary Identification, pcr amplification product purification and recovery, PET-32a (+) and the preparation of PGEX-4T-1 carrier after, owing to restriction enzyme site is contained at the two ends of PCR primer, by PCR primer and PET-32a (+), PGEX-4T-1 reclaim after identifying with BamH I and Xho I double digestion, result is as shown in Figure 2.
2.2.2 genes of interest is connected with expression vector. Purpose fragment apx I A, apxIIA and PET-32a carrier, the connection of OmpD and PGEX-4T-1 carrier, connection mixed liquor is blown and beaten mixing gently, 16 DEG C of water-baths are overnight.
Reaction system: 2 × Ligationsolution I 5 μ L, PCR reclaim product 2.5 μ L, PET-32a or PGEX-4T-10.5 μ L, ddH2O2 μ L, totally 10 μ L.
2.2.3 will connect product to convert to bacillus coli DH 5 alpha competent cell, coat on LB flat board.
2.3 recombinant expression plasmid screening and qualifications, comprise the following steps:
2.3.1 the single bacterium colony on picking LB flat board, carries out bacterium colony PCR checking, reaction system: ddH2O17.85 μ L, Taq enzyme 0.15 μ L, each 1 μ L, dNTPs2.5 μ L, 10 × buffer2.5 μ L, the totally 25 μ L of upstream and downstream primer.
Reaction condition is following (a), (b), (c) PCR program:
A:94 DEG C of denaturation 2min of (a) pET-32a-apx I; 94 DEG C of thermal denaturation 15s, 50.5 DEG C of 30s, 68 DEG C of 1min, totally 30 circulations; 68 DEG C of 7min extend eventually.
(b) pET-32a-apxIIA:94 DEG C of denaturation 2min; 94 DEG C of thermal denaturation 15s, 50.5 DEG C of 30s, 68 DEG C of 1min, totally 30 circulations; 68 DEG C of 7min extend eventually.
(c) pGEX-OmpD:94 DEG C of denaturation 2min; 94 DEG C of heat become 15s, 53 DEG C of 30s, 68 DEG C of 3min, totally 30 circulations; 68 DEG C of 7min extend eventually.
After PCR terminates, take respectively 5 μ L amplified productions on 1% agarose gel under 110V voltage electrophoresis 25min, utilize Labworks image acquisition and analysis software to scan, Preliminary Identification, result is as shown in Figure 3.
2.3.2 the double digestion of recombinant expression plasmid is identified. The bacterium colony of positive colony will be accredited as, shake bacterium, extract the plasmid of bacterium solution. With restricted enzyme, under 37 DEG C of constant temperatures, the recombinant clone plasmid that double digestion 3h extracts, digestion products detects with 1% agarose gel electrophoresis, and result is as shown in Figure 4.
Double digestion reaction system: BamH I 1 μ L, Xho I 1 μ L, 10 × Kbuffer2 μ L, recombiant plasmid 4 μ L, ddH2O12 μ L, totally 20 μ L.
2.3.3 recombinant strains is obtained
The recombinant expression plasmid being accredited as the positive is converted to escherichia coli (E.coli) BL21 (DE3), it is thus achieved that recombinant strains.
2.4 restructuring APPApx I A, Apx II A and OMPD albumen abduction delivering condition optimizing and purification, comprise the following steps:
2.4.1 recombiant protein abduction delivering
2.4.1.1 being accredited as positive single colony inoculation in the 5mLLB fluid medium containing final concentration of 0.1mg/mLAmp (ampicillin), 37 DEG C of constant-temperature tables are with 200r/min incubated overnight first order seed.
2.4.1.2 being added to the 5mL LB fluid medium containing final concentration of 0.1mg/mLAmp in the ratio of 1:50 by first order seed, 37 DEG C, 220r/min cultivates bacterium solution OD600 to 0.6～0.8.
2.4.1.3 in the bacterium solution need induction, IPTG to final concentration of 1.0mmol/L is added, 37 DEG C, the about 3h of 200r/min shaken cultivation, abduction delivering.
2.4.1.4 thalline 1mL is collected, in the centrifugal 1min of 12000r/min. With the 100 aseptic ddH of μ L2The resuspended thalline of O, adds isopyknic 2 × SDS sample-loading buffer, and after mixing, the centrifugal 10min of boiling water bath about 10min, 12000r/min, takes supernatant and carry out SDS-PAGE electrophoresis, and result is as shown in Figure 5.
2.4.2 abduction delivering condition optimizing
In order to obtain more expressing quantity, the abduction delivering condition of albumen is optimized:
2.4.2.1IPTG concentration optimization: to use IPTG inducer concentrations be 0,0.25,0.5,1.0,1.5,2,2.5,3mmol/L induces respectively.
2.4.2.2 induction time optimization: select the optimal concentration of IPTG, induction time is set to 0,1,2,3,4,5,6,7h, shown in result such as Fig. 6,7,8: pET-32a-apx I A and pET-32a-apxIIAIPTG induced concentration is defined as 1mmol/L, induction time is 3h; PGEX-OmpDIPTG induced concentration is defined as 2.5mmol/L, induction time is 3h.
2.4.2.3 recombiant protein soluble analysis
Under the best inductive condition of 2.4.2.2, cultivate recombinant bacterium 100mL. Recombinant bacterium, in the centrifugal 10min of 4 DEG C of 12000r/min, is collected precipitation, is washed 2～3 times with PBS, and precipitation PBS is resuspended. Under condition of ice bath, adding Trixon114 ultrasonic treatment antibacterial, crack and become limpid to bacterium solution, 12000r/min is centrifuged 20min, collects upper cleer and peaceful precipitation respectively.Detected the solubility of albumen by SDS-PAGE, result as shown in Figure 9: pET-32a-apx I A and pET-32a-apxIIA albumen supernatant foreign protein is more, and precipitation is containing more destination protein, and band is single, it was shown that expressing protein is mainly inclusion body; PGEX-OmpD albumen overwhelming majority precipitation after crushing, illustrates that the albumen expressed also mainly exists with the form of inclusion body.
2.4.3 recombiant protein great expression and purification
2.4.3.1 recombiant protein great expression, inducing amount is 2L, adds Trixon114 ultrasonication, and precipitation (inclusion body) dissolved with the carbamide PBS of 8mol/L after collecting thalline, and 4 DEG C overnight. It is centrifuged after dissolving and abandons precipitation, stay supernatant. The above-mentioned carbamide PBS supernatant containing 8mol/L is contained in bag filter, respectively with 6,4,2, the carbamide PBS gradient dialysis of 0mol/L, each dialysis time is about 4h, finally making its final concentration carbamide is 0mol/L, finally being filtered with 0.45 μm of filter post, filtrate deposits in-20 DEG C.
2.4.3.2 endotoxic removal
By through just pure albumen in clean centrifuge tube, add PMXB liquid; The weight ratio of protein and PMXB is 1000:5～15. At room temperature, 20～35min it is slowly mixed together in vertical blenders, it is subsequently added isopyknic organic solvent benzyl benzoate to mix two minutes, centrifugal 3～the 4min of 10000～13000r/min in desk centrifuge, after centrifugal biphase between form thin film, this thin film is the PMXB complex formed with endotoxin. The protein-contg aqueous phase of careful absorption (untouchable thin film) also moves in another clean tubule, centrifugal after adding the mixing of equal-volume organic solvent benzyl benzoate. Such repeated washing 3～4 times, the concentration detecting albumen with BCA protein quantification test kit the endotoxin content remained with tachypleus amebocyte lysate detection.
2.4.3.3 recombinant protein purification condition optimizing, comprises following (a) and (b):
The purification of (a) rApx I A, rApxIIA recombiant protein
Select 30mM, 40mM, 50mM imidazole buffer balance purification column respectively; After having adsorbed pretreatment sample, the imidazole buffer of 5mL rinses purification column, and flow velocity 1～2mL/min collects eluent; Adopt 5mL100mM, 200mM, 500mM imidazole buffer that destination protein carries out gradient elution, flow velocity 1～2mL/min, collect eluent. With SDS-PAGE to collecting samples detection, determine binding buffer liquid and the elution buffer of the best, result such as Figure 10, shown in 11: rApx I A, rApxIIA recombiant protein is when binding buffer liquid is 50mmol/L, and when eluent is 200mmol/L, the protein content of eluting is maximum.
The purification of (b) rOMPD recombiant protein
OMPD albumen is cut glue purification concrete operation step as follows: prepare glass plate the difference is that when configuring separation gel, it reserving the gap of 1～2cm, and is not inserted into comb according to the method for preparation PAGE gel. The albumen processed is added to space, runs glue by the mode that SDS-PAGE is identical and bring to gel sub to bromophenol blue. Taking-up gel is put in and fills ddH2The plate of O washs 5min gently, discards ddH2O, adds 0.25mol/LKCl solution, occurs to the bright band having a white. Cut, grind and be put in EP pipe, added PBS in 4 DEG C of overnight soluble proteins. To overnight rear EP pipe taking-up 4 DEG C be centrifuged, 8000r/min is centrifuged 10min, takes supernatant in new EP pipe, and this is albumen after purification, and result is as shown in figure 12.
2.4.3.4 purification of recombinant proteins SDS-PAGE
Respectively recombiant protein sample after above-mentioned purification is taken 100 μ L and adds 2 × SDS-PAGE sample-loading buffer 100 μ L, boiling water boils 10min, carry out SDS-PAGE, observe electrophoresis result and be analyzed.
2.4.3.5 purification of recombinant proteins Western-blotting detection
By APP recombiant protein rApx I A of above-mentioned gained, rApx II A and rOMPD carries out SDS-PAGE electrophoresis, constant current 200mA, electricity turns 60min by the protein delivery on gel to nitrocellulose filter (NC film), after PBST buffer solution 3 times, use 5% defatted milk powder, 37 DEG C of shaking table 60r/min close 2h, with PBST buffer solution 3 times, with 37 DEG C of water-bath effect 1h of primary antibodie (rabbit anteserum), use PBST buffer solution 3 times again, anti-with two (goat anti-rabbit igg with ELIAS secondary antibody HRP (horseradish peroxidase) labelling of PBST buffer dilution) (1:5000 dilution) 37 DEG C of water-bath effect 1h, display after BST buffer solution 3 times, result is as shown in figure 13: at about 46kD, 50kD and 110KD place is respectively arranged with a clear band, illustrate that the recombiant protein expressed has good antigenicity.
2.4.3.6 the mensuration of purifying protein concentration
UtilizeBCAProteinAssayKit, step draws concentration respectively 376, the 321 and 110 μ g/mL of recombiant protein rApx I A after purification, rApxIIA and rOMPD as requested.
Embodiment 2 Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mixed vaccine immune efficacy analyze
1. main material
1.1 animals: 4～6 female Balb/c mices of week old, purchased from south, Guangzhou Animal Science company limited of medical university
1.2 vaccines: for mass mixing things such as the albumen after gained purification in embodiment 1 and three kinds of albumen, use Freund's complete adjuvant and incomplete Freund's adjuvant emulsified protein, all purchased from SIGMA company.
2. method
2.1 take the Balb/c mice about 60 4～6 week old, random point 6 groups, rApx I A, rApxIIA, rOMPD and rApx I A+rApxIIA+rOMPD albumen be respectively one group, separately set normal saline as blank group and porcine contagious pleuropneumonia inactivated trivalent vaccine (purchased from Wuhan predecessor company of section) as positive control. Respectively organizing all with hypodermic mode immune mouse, protein immunization amount is 100 μ g/, and the immunity of inactivated vaccine is 0.2mL/. Booster immunization after initial immunity 14d and 28d, first time immunity uses Freund's complete adjuvant emulsified protein, and second and third time uses incomplete Freund's adjuvant emulsified protein, and immunizing dose and mode are identical with initial immunity.
2.2 indirect ELISA method detection serum specific antibodies
Adopt the second time positive serum of protein immunization mice serum after two weeks and negative serum (prepared by microorganism teaching and research room of College of Veterinary Medicine, South China Agricultural University) carry out square formation titration, it is determined that rApxIA, rApx II A, rOMPD the antigen coated concentration of the best and the optimum dilution degree of primary antibodie. The specific antibody in immune mouse serum is detected with indirect ELISA method. Concrete operations are as follows: with purification rApxIA, rApx II the optimum dilution degree of A and rOMPD be coated elisa plate, every hole 100 μ L, ambient temperature overnight is coated; PBST washs liquid 3 times, and every hole adds confining liquid (5%BSA) 100 μ L, closes 2h for 37 DEG C; Liquid is washed 3 times again with PBST, and an anti-binding (with the PBST tested serum of buffer doubling dilution), every hole 100 μ L, wherein compare with the negative serum of BALB/c mouse;Washing liquid 3 times with PBST again, with two anti-bindings (sheep anti-mouse igg by the ELIAS secondary antibody horseradish peroxidase-labeled of 0.5%BSA dilution), every hole adds 100 μ L, hatches 1h for 37 DEG C; Washing liquid 3 times with PBST again, every hole adds substrate solution (phosphate-citrate buffer 100mL, o-phenylenediamine 40mg, 30%H2O2100 μ L), put colour developing 15min in room temperature magazine, after developing the color, every hole adds 50 μ L stop buffer (2mol/LH2SO4) terminate reaction; Microplate reader is used to measure the OD value of 450nm.
Result according to square formation titration, antigen coated concentration and primary antibodie dilution factor when P/N value is maximum are optimum dilution degree. Wherein optimum dilution degree respectively 1:320,1:320 and the 1:10 of purifying protein rApxIA, rApx II A and rOMPD. The optimum dilution degree of primary antibodie is 1:100. The sheep anti-mouse igg dilution factor of HRP labelling is 1:5000. Immune serum rApxIA, rApx II dynamic change of A and rOMPD specific antibody is detected according to this reaction condition.
Shown in result such as Figure 14,15,16: rApxIA group and three kinds of albumen mixing groups, after first immunisation two weeks, just have obvious antibody to produce, and antibody horizontal is higher than tervalence inactivated vaccine group and matched group. After twice booster immunization, rApxIA group and three kinds of albumen mixing group antibody all present rapid ascendant trend, and antibody height remains in that original trend. After three booster immunizations, the antibody horizontal of three groups all reduces, and antibody horizontal is compared: rApx I A group > three kinds of albumen mixing groups > tervalence inactivated vaccine group > blank group. But, the rApx I A antibody horizontal of tervalence inactivated vaccine group does not all have obvious antibody to produce after three immunity; After first immunisation two weeks, the antibody horizontal of rApxIIA group and three kinds of albumen mixing groups is suitable, hence it is evident that higher than tervalence inactivated vaccine group and matched group. After twice booster immunization, rApx II A group and three kinds of albumen mixing group antibody all present rapid ascendant trend, and antibody horizontal is compared: tri-kinds of albumen mixing group > tervalence inactivated vaccine group > blank groups of rApx II A group >. After three booster immunizations, rApx II A group all reduces with the antibody horizontal of three kinds of albumen mixing group antibody and identical with the trend of antibody height after twice immunity, and the rApx II A antibody horizontal of tervalence inactivated vaccine group and matched group almost maintain same level. But, tervalence inactivated vaccine group rApx II A antibody horizontal antibody horizontal of its antibody horizontal and PBS control group after three immunity is more or less the same; The rOMPD antibody horizontal of three kinds of albumen mixing groups and rOMPD protein groups reduces and successively apparently higher than tervalence inactivated vaccine group and matched group. After first immunisation two weeks, rOMPD protein groups and three kinds of albumen mixing groups all have obvious antibody produce and be on close level, higher than tervalence inactivated vaccine group and matched group. After twice booster immunization, rOMPD protein groups, three kinds of albumen mixing groups and tervalence inactivated vaccine group antibody all present ascendant trend, and antibody horizontal is compared: three kinds of albumen mixing group > rOMPD protein groups > tervalence inactivated vaccine group > matched groups.
Compared with matched group, all create higher antibody horizontal after three kinds of recombiant protein immune mouses, higher than matched group. During three kinds of albumen mixing; rOMPD albumen, rApx I A antibody horizontal antibody horizontal when being above individually immunity the humoral immune reaction of recombinant subunit vaccine induction is described, rApxIA and rOMPD antibody plays effect of crucial importance in the Cross immunogenicity of mice.
There is not the phenomenon such as redness, heating in immune animal injection site, and also without there is inoculation untoward reaction, appetite is normal, and the mental status is good.
Result point out Actinobacillus pleuropneumoniae Apx I A, Apx that we prepare II A and OMPD gene engineered subunit mixed vaccine there is good immunogenicity; high-caliber protection antibody can be produced after immune mouse; after inoculation immune animal is safe and harmless; it it is a kind of new generation vaccine with bright prospects; by the prevention and control of China's porcine contagious pleuropneumonia being provided reserve supply and technical support, have great importance.
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention are also not restricted to the described embodiments; the change made under other any spirit without departing from the present invention and principle, modification, replacement, combination, simplification; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. an Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mixed vaccine, it is characterised in that include recombiant protein rApx I A, rApxIIA and rOMPD;
The aminoacid sequence of described recombiant protein rApx I A is such as shown in SEQIDNO:4;
The aminoacid sequence of described recombiant protein rApxIIA is such as shown in SEQIDNO:5;
The aminoacid sequence of described recombiant protein rOMPD is such as shown in SEQIDNO:6.
2. Actinobacillus pleuropneumoniae Apx I A, Apx according to claim 1 II A and OMPD gene engineered subunit mixed vaccine, it is characterised in that:
Described recombiant protein rApx I A, the mass ratio of rApxIIA and rOMPD are (1～3): 1:(1～3).
3. Actinobacillus pleuropneumoniae Apx I A, Apx according to claim 1 II A and OMPD gene engineered subunit mixed vaccine, it is characterised in that:
Described recombiant protein rApx I A, the mass ratio of rApxIIA and rOMPD are 1:1:1.
4. Actinobacillus pleuropneumoniae Apx I A, Apx described in any one of claims 1 to 3 II preparation method of A and OMPD gene engineered subunit mixed vaccine, it is characterised in that comprise the steps:
(1) amplification Actinobacillus pleuropneumoniae apx I A, apx II A and OmpD gene, has following (a), (b) and the nucleotide sequence shown in (c):
Nucleotide sequence (apx I A gene) shown in (a) SEQIDNO:1; With
Nucleotide sequence (apx II A gene) shown in (b) SEQIDNO:2; With
Nucleotide sequence (OmpD gene) shown in (c) SEQIDNO:3;
(2) build the expression vector containing the gene described in (1) in steps, and proceed to escherichia coli respectively;
(3) abduction delivering obtains destination protein;
(4) purification of recombiant protein and detection;
(5) recombiant protein is mixed, obtain Actinobacillus pleuropneumoniae Apx I A, Apx II A and OMPD gene engineered subunit mixed vaccine.
5. Actinobacillus pleuropneumoniae Apx I A, Apx according to claim 4 II preparation method of A and OMPD gene engineered subunit mixed vaccine, it is characterised in that:
The nucleotide sequence that primer sequence needed for extension increasing sequence gene is as follows:
Apx I A primer sequence:
F1 forward primer: as shown in SEQIDNO:7;
F2 downstream primer: as shown in SEQIDNO:8.
6. Actinobacillus pleuropneumoniae Apx I A, Apx according to claim 4 II preparation method of A and OMPD gene engineered subunit mixed 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 forward primer: as shown in SEQIDNO:9;
F4 forward primer: as shown in SEQIDNO:10.
7. Actinobacillus pleuropneumoniae Apx I A, Apx according to claim 4 II preparation method of A and OMPD gene engineered subunit mixed 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 forward primer: as shown in SEQIDNO:11;
F6 forward primer: as shown in SEQIDNO:12.
8. Actinobacillus pleuropneumoniae Apx I A, Apx according to claim 4 II preparation method of A and OMPD gene engineered subunit mixed vaccine, it is characterised in that:
Recombiant protein rApx I A after described purification, concentration respectively 376, the 321 and 110 μ g/mL of rApxIIA and rOMPD.
9. Actinobacillus pleuropneumoniae ApxIA, Apx described in any one of claims 1 to 3 II A and OMPD gene engineered subunit mixed vaccine application in prevention porcine contagious pleuropneumonia.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110038124A (en) * | 2019-05-13 | 2019-07-23 | 天康生物股份有限公司 | Swine fever-porcine contagious pleuropneumonia bigeminy subunit vaccine and its preparation method and application |
| CN112704732A (en) * | 2020-12-24 | 2021-04-27 | 华中农业大学 | Porcine infectious actinobacillus pleuropneumoniae subunit vaccine |
| CN115960184A (en) * | 2022-09-28 | 2023-04-14 | 华中农业大学 | Mannheimia haemolytica A6 serotype leukotoxin antigen protein, antibody and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1713921A (en) * | 2002-11-20 | 2005-12-28 | 西班牙海博乐生物大药厂 | Live attenuated vaccine against porcine pleuropneumonia |
-
2016
- 2016-01-25 CN CN201610053070.2A patent/CN105664148B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1713921A (en) * | 2002-11-20 | 2005-12-28 | 西班牙海博乐生物大药厂 | Live attenuated vaccine against porcine pleuropneumonia |
Non-Patent Citations (3)
| Title |
|---|
| YOSHIHISA HAGA等: "Protective Efficacy of an Affinity-Purified Hemolysin Vaccine against Experimental Swine Pleuropneumonia", 《THE JOURNAL OF VETERINARY MEDICAL SCIENCE》 * |
| 廖永洪: "猪胸膜肺炎放线杆菌免疫蛋白质组学及亚单位疫苗的研究", 《中国博士学位论文全文数据库》 * |
| 邵美丽: "猪传染性胸膜肺炎重组亚单位疫苗的研究", 《中国优秀博硕士学位论文全文数据库(博士) 农业科技辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110038124A (en) * | 2019-05-13 | 2019-07-23 | 天康生物股份有限公司 | Swine fever-porcine contagious pleuropneumonia bigeminy subunit vaccine and its preparation method and application |
| CN110038124B (en) * | 2019-05-13 | 2023-04-14 | 天康生物股份有限公司 | Swine fever-porcine infectious pleuropneumonia bigeminal subunit vaccine and preparation method and application thereof |
| CN112704732A (en) * | 2020-12-24 | 2021-04-27 | 华中农业大学 | Porcine infectious actinobacillus pleuropneumoniae subunit vaccine |
| CN112704732B (en) * | 2020-12-24 | 2022-02-25 | 华中农业大学 | Porcine infectious actinobacillus pleuropneumoniae subunit vaccine |
| CN115960184A (en) * | 2022-09-28 | 2023-04-14 | 华中农业大学 | Mannheimia haemolytica A6 serotype leukotoxin antigen protein, antibody and application thereof |
| CN115960184B (en) * | 2022-09-28 | 2023-12-12 | 华中农业大学 | Leucobacillus mannii A6 serotype leukotoxin antigen protein, antibody and application thereof |
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Inventor after: Zhao Mingqiu Inventor after: Wang Lijun Inventor after: Chen Jinding Inventor after: Liu Cuicui Inventor after: Sun Xiu Inventor after: Wu Yunyan Inventor after: Zhu Junling Inventor after: Wu Zuxiong Inventor before: Chen Jinding Inventor before: Wang Lijun Inventor before: Zhao Mingqiu Inventor before: Liu Cuicui Inventor before: Sun Xiu Inventor before: Wu Yunyan Inventor before: Zhu Junling Inventor before: Wu Zuxiong |