CN102517232B - Resistance marker-free porcine actinobacillus pleuropneumoniae double-gene defective strain, construction method and application thereof - Google Patents
Resistance marker-free porcine actinobacillus pleuropneumoniae double-gene defective strain, construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a resistance marker-free porcine actinobacillus pleuropneumoniae (APP) serum type 7 double-gene defective strain, and belongs to the technical field of bacterial gene engineering. The recombinant strain APPdeltaclpPdeltaapx II C of APP is obtained by inactivating ClpP protease in the APP and a coded gene of a hemolysin activated factor Apx II C by adopting a directional homologous recombination technology, and expression of the ClpP protease and the Apx II C protein is destroyed. The obtained double-gene defective strain has lower toxicity compared with a parent strain, is safe to animals, provides an important basis for transformation of porcine contagious pleuropneumonia (PCP) vaccines and research of matched identification and diagnosis reagents, and has great significance for promoting elimination and purification of the global PCP.
Description
Technical field
The present invention relates to a kind of actinobacillus pleuropneumoniae ClpP proteolytic enzyme and the dual-gene disappearance strain of ApxIIC, its construction process and application that does not contain resistance marker, belong to the bacterial gene field of engineering technology.
Background technology
Porcine contagious pleuropneumonia (porcine contagious pleuropneumonia, PCP) be by actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, a kind of hyperinfection that APP) causes, the respiratory diseases of lethality.Should disease be widely current in countries in the world at present, cause enormous economic loss, become one of important transmissible disease of the modernized pig industry of internationally recognized harm.Along with the development of China's modern farming mass-producing, intensification, this disease be outburst trend, the positive rate on the pig farm that has has reached more than 70%, has seriously hindered the sound development of China's pig industry.
Antigenic different according to APP surface pod membrane (CPS) with lipopolysaccharides (LPS), APP can be divided into 15 serotypes, serum 1 type is divided into 1a and two hypotypes of 1b again, and serum 5 types are divided into 5a and two hypotypes of 5b again.Found and be separated to multiple serotypes such as serotype 1,2,3,4,5,7,8 and 15 at present in China, main popular serotype is 1,3,5 and 7 types.
Hemolysin is the topmost virulence factor of APP, also is topmost protective antigen.At present in 15 all serotypes of APP, find 4 kinds of Apx toxin altogether, be respectively Apx I, Apx II, Apx III and ApxIV.The serotype that APP is different produces different Apx toxin, and wherein serum 7 types contain Apx II and ApxIV.
The typical structure of Apx toxin operon contains complete CABD gene; Wherein, A genes encoding Apx toxin structure albumen does not have the toxin activity when beginning to synthesize, and C genes encoding Apx toxin activator is responsible for that structural protein are carried out acetylize and is activated, and forms the ApxC-ApxA complex body, and the toxin activity produces thereupon.The proteins encoded of B gene and D gene forms transmembrane channel, is responsible for the transhipment and the secretion of Apx toxin.Apx I and Apx III operon have complete CABD gene, and Apx II operon lacks B gene and D gene, and its product is responsible for transhipment by the BD gene encoding production of Apx I and is secreted into the extracellular.
Biofilm load (Biofilm) is long with the inorganic matter set of surfaces consor in vivo formed fixation of bacteria of the polysaccharide protein complex by bacterium and generation thereof group, and it is one of major reason of pathogenic bacteria persistent infection, generation resistance and immunologic escape.APP hides for a long time and usually cause persistent infection in the pig body, and many clinical separation strains of Huo Deing have multi-drug resistant in recent years, and can form biofilm load more at large, are considered to play a significant role in the APP pathogenic course.
The generation of biofilm load is a high complexity, by the dynamic process of several genes regulation and control, these genes relate to the sticking of bacterium, metabolism, quorum sensing (quorum sensing) and stress reaction (stress response) etc.Discovering pathogenic bacteria stress reaction and infectious diseases in recent years; this stress protein of ClpP proteolytic enzyme is a kind of crucial virulence factor; it is the serine protease that ATP relies on; regulating the adaptation (as heat-shocked, auxotrophy, oxidative stress etc.) of bacterium to the various pressure of environment; keep form and the virulence of bacterium, it is protected under various environment.Insertion sudden change or the disappearance of discovering this gene can reduce the ability to bear of bacterium to undesirable element usually, and thalli growth is destroyed, and viability descends, and influence biofilm load formation, cause virulence attenuation of.Though only explored the function of minority pathogenic bacteria ClpP proteolytic enzyme at present, in disclosing pathogenesis and vaccine research, demonstrated very important researching value and vaccine application prospect.
At present full bacterium inactivated vaccine of using and subunit vaccine can alleviate the clinical symptom that homology serotype infected pigs causes and reduce mortality ratio; but can not stop pulmonary lesion and chronic infection; infection to allos serotype can not provide good cross protection, and natural infection or experimental infection can be induced the protection to any allos serotype.Therefore, perhaps the development of attenuated vaccine efficiently is the insufficient a kind of feasible method that solves current pleuropneumonia vaccine.
In view of above-mentioned background, be structured in China popular APP serum 7 type ClpP proteolytic enzyme and the dual-gene disappearance strain of Apx II C, study its hereditary property, growth characteristics, virulence, to the biological characteristicses such as pathogenic and immunogenicity of animal, for the control of porcine contagious pleuropneumonia, the drug research and the vaccine research of other respiratory infectious disease are established important basis.
Summary of the invention
One of purpose of the present invention is to obtain a kind of actinobacillus pleuropneumoniae serum 7 type clpP and dual-gene disappearance strain of apx IIC that does not contain resistance marker.
A kind of actinobacillus pleuropneumoniae serum 7 type clpP and dual-gene disappearance strain of apx II C that does not contain resistance marker provided by the invention is the disappearance strain that obtains after the encoding gene deactivation with the ClpP proteolytic enzyme of actinobacillus pleuropneumoniae and hemolysin incitant Apx II C.
The dual-gene disappearance strain of a kind of actinobacillus pleuropneumoniae of the present invention, it is characterized in that described gene-deleted strain APP Δ clpP Δ apx II C, classification called after actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, APP), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC No.5444, preservation date are on November 3rd, 2011.
Two of purpose of the present invention is to provide a kind of method that makes up the dual-gene disappearance strain of described actinobacillus pleuropneumoniae, it is characterized in that lacking strain and realizes through homologous recombination by dna fragmentation Δ clpP and Δ apx II C are imported described actinobacillus pleuropneumoniae serum 7 type bacterial strains;
Wherein, described dna fragmentation Δ clpP is followed successively by homology arm ClpPS and following homology arm ClpPX from upstream to downstream: described go up homology arm ClpPS and following homology arm ClpPX can with the encoding gene of the upstream sequence of ClpP protease-encoding gene the described actinobacillus pleuropneumoniae and downstream sequence generation homologous recombination, the described ClpP proteolytic enzyme of deactivation, obtain the clpP single-gene and lack strain APP Δ clpP;
Wherein, described dna fragmentation Δ apxIIC is followed successively by homology arm apx II CS and following homology arm apx II CX to the downstream from the upstream, described go up homology arm apx II CS and following homology arm apxII CX can with the upstream sequence of Apx II C protein coding gene in the described actinobacillus pleuropneumoniae and downstream sequence generation homologous recombination, the proteic encoding gene of the described Apx II of deactivation C, obtain clpP and the dual-gene disappearance strain of apx II C APP Δ clpP Δ apx II C.
In specific embodiments of the invention, the encoding gene of described ClpP proteolytic enzyme is shown in SEQ ID NO.1, and the proteic encoding gene of described Apx II C is shown in SEQ ID NO.3.
In specific embodiments of the invention, the primer sequence of homology arm ClpPS is as follows in the amplification:
clpSF：5’-CG
(EcoR I)GGGGCGTTACTGGATGC-3’
clpSR：5’-
CCATCGCTTC 
GGTTTGC-3’
The primer sequence of homology arm ClpPX is as follows under the amplification:
clpXF：5’-
TCCAAAGGCG 
AATACGGTC-3’
clpXR：5’-CG
(BamH I)TTCTCTGCTTTAAGTGTCGGC-3’
The primer sequence of homology arm apx II CS is as follows in the amplification:
II CSF：5’-CG
(EcoR I)ATGACAACACCAATGATTGATTTAC-3’
II CSR：5’-
AATCCCCGAA 
CTCCCATTC-3’
The primer sequence of homology arm apx II CX is as follows under the amplification:
II CXF：5’-
GGATGATGCT 
CATCTCTATTG-3’
II CXR：5’-CG
(BamH I)GTTGTAATAAGTCCCGTAACACCAG-3’
Described actinobacillus pleuropneumoniae serum 7 type bacterial strains are the 7 type CVCC265 strains of APP serum.
Three of purpose of the present invention is to provide the application of the dual-gene disappearance strain of described actinobacillus pleuropneumoniae in preparation prevention porcine contagious pleuropneumonia vaccine.
Four of purpose of the present invention is to provide a kind of porcine contagious pleuropneumonia vaccine, and its activeconstituents is the dual-gene disappearance strain of actinobacillus pleuropneumoniae of the present invention.
The present invention is achieved through the following technical solutions:
Resistance marker actinobacillus pleuropneumoniae serum 7 type clpP and the dual-gene deletion mutantion bacterial strain of apxIIC of not containing of the present invention, be derived from that China Veterinary Drugs Supervisory Inst. buys actinobacillus pleuropneumoniae CVCC265 strain, with the clpP genetically deficient on its genome 491bp, deactivation clpP gene, ClpP proteolytic enzyme is not expressed, obtain single-gene deletion mutantion bacterial strain; Again with the apx II C genetically deficient on its genome 270bp, deactivation apx II C gene is not expressed Apx II C albumen, obtains dual-gene deletion mutantion bacterial strain.
Basic construction method of the present invention is:
1. at first (be called for short APP-7 with actinobacillus pleuropneumoniae serum 7 types, down together) the CVCC265 strain is a parent material, from the genome of CVCC265, amplify the last homology arm ClpPS and the following homology arm ClpPX of clpP gene with the method for PCR, adopt the upper and lower homology arm of overlapping extension PCR method splicing clpP gene, amplification Δ clpP gene fragment, this fragment deletion the sequence fragment of 491bp of clpP gene.Then this Δ clpP sequence clone through transforming to carrier pUC18, finally become homologous recombination vector (suicide plasmid) pUC Δ clpP.The method that adopts electricity to transform is transformed into suicide plasmid pUC Δ clpP in the APP CVCC265 strain, since homologous recombination vector (suicide plasmid) can not be in the APP bacterium self-replicating, therefore changing the intracellular supercoiled suicide plasmid of recipient bacterium over to just can be linearized.Because of containing the homologous sequence of recipient bacterium gene on the suicide plasmid, it just can insert in the karyomit(e) that is subjected to thalline CVCC265, follows linearizing plasmid and the clpP gene is replaced, and the generation of homologous recombination incident is changed in just usually said single cross.Have only and replace suicide plasmid linearized on the CVCC265 karyomit(e) and could exist along with THE REPLICATION OF CHROMOSOME.Recipient bacterium is coated on the TSA flat board that contains selection markers, filters out single recon easily.Because it is that whole carrier sequence (containing the screening of medicaments resistant gene) is all replaced on the karyomit(e) that single cross changes, therefore, we must further screen goal gene and be incorporated into and delete double exchange of carrier sequence on the karyomit(e) simultaneously again.The single recon of the homologous recombination that obtains after liquid culture, is coated on the nonresistant TSA flat board, and picking list bacterium colony carries out PCR screening positive homologous recombination double exchange, i.e. clpP single-gene disappearance strain APP Δ clpP.
2. then, from the genome of CVCC265, amplify the last homology arm apx II CS and the following homology arm apx II CX of apx II C gene with the method for PCR, adopt the upper and lower homology arm of overlapping extension PCR method splicing apx II C gene, amplification Δ apx II C gene fragment, this fragment deletion the sequence fragment of 270bp of apx II C gene.Then this Δ apx II C sequence clone through transforming to carrier pUC18, finally become homologous recombination vector (suicide plasmid) pUC Δ apx II C, again according to 1 method, lacking strain APP Δ clpP with the clpP single-gene that obtains is parent material, further disappearance apx II C gene obtains clpP and the dual-gene disappearance strain of apx II C APP Δ clpP Δ apx II C.With APP Δ clpP Δ apx II C inoculation animal, according to clinical manifestation and the survival condition analysis of animal, APP Δ clpP Δ apx II C virulence is weaker than its parent plant CVCC265.
Major advantage of the present invention is:
1, the used material of the present invention is the 7 type CVCC265 strains of actinobacillus pleuropneumoniae serum, and available from China Veterinary Drugs Supervisory Inst., APP serum 7 type bacterial strains are that present China is popular and seriously cause the advantage serotype of pig morbidity.Therefore, the vaccine that is foundation development with the clpP and the dual-gene deletion mutantion bacterial strain of apx II C of this strain construction can have very strong specific aim from now on, and wide market application prospect is arranged.
2, actinobacillus pleuropneumoniae serum 7 type clpP of the present invention and the dual-gene deletion mutantion bacterial strain of apx II C do not contain any resistance marker, meet China's vaccine Biosafety requirement fully.
Description of drawings
The structure schema of Fig. 1 actinobacillus pleuropneumoniae reorganization suicide plasmid pUC Δ clpP;
The structure schema of Fig. 2 actinobacillus pleuropneumoniae reorganization suicide plasmid pUC Δ apxIIC;
The pcr amplification result of homology arm ClpPS and following homology arm ClpPX on Fig. 3;
1:ClpPS among the figure (1200bp); 2:ClpPX (1249bp); M:DL2000DNAMarker
The PCR splicing result of Fig. 4 Δ clpP gene;
Among the figure 1: Δ clpP gene (2449bp); M:DL15000DNA Marker
The recombinate PCR qualification result of suicide plasmid pUC Δ clpP of Fig. 5;
1-3 among the figure: Δ clpP; M:DL15000DNA Marker
The PCR qualification result of strain is changed in Fig. 6 APP Δ clpP single cross;
1-3 among the figure: strain is changed in single cross; M:DL2000DNA Marker
The PCR qualification result of Fig. 7 APP Δ clpP disappearance strain;
M:DL2000DNA Marker among the figure; No. 8 clpP single-gene disappearance strains for screening
The real face result of genetic stability of Fig. 8 APP Δ clpP disappearance strain;
1-10 among the figure: 1-10 is for the disappearance strain; M:DL2000DNA Marker
The pcr amplification result of homology arm apx II CS and following homology arm apx II Cx on Fig. 9;
1:apx II CS (1412bp) among the figure; 2:apx II CX (1443bp); M:DL5000DNA Marker
The PCR splicing result of Figure 10 Δ apx II C gene;
Among the figure 1: Δ apxIIC gene (2855bp); M:DL5000DNA Marker
The recombinate PCR qualification result of suicide plasmid pUC Δ apx II C of Figure 11;
1-5 among the figure: Δ apx II C; M:DL5000DNA Marker
The PCR qualification result of strain is changed in Figure 12 APP Δ clpP Δ apx II C single cross;
1-5 among the figure: strain is changed in single cross; M:DL2000DNA Marker
The PCR qualification result of the dual-gene disappearance strain of Figure 13 APP Δ clpP Δ apx II C;
M:DL2000DNA Marker among the figure; No. 1 and No. 5 clpP and the dual-gene disappearance strains of apxIIC for screening
The genetic stability experimental result of the dual-gene disappearance strain of Figure 14 APP Δ clpP Δ apx II C.
1-10 among the figure: 1-10 is for the disappearance strain; M:DL2000DNA Marker
Embodiment
Further describe the present invention below in conjunction with specific examples, advantage of the present invention and characteristics will be more clear along with description.But these examples only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Related material in the embodiment of the invention:
Bacterial classification: the 7 type CVCC265 strains of APP serum are bought from China Veterinary Drugs Supervisory Inst.
Carrier and reagent: pUC18 plasmid, Ex Taq archaeal dna polymerase 2U, T4 dna ligase are available from TaKaRa company.
The structure of embodiment 1APP serum 7 type clpP single-gene deletion mutantion strains
1.1 the structure of reorganization suicide vector pUC Δ clpP
1.1.1ClpP the design of primers of the upper and lower homology arm of proteinase gene and pcr amplification
According to two pairs of primers of the APP-7 type AP76 strain sequence (is the gene order of CP001091.1 with reference to the GenBank accession number) reported design increase respectively the last homology arm ClpPS and the following homology arm ClpPX of clpP gene, the amplified fragments size is respectively 1200bp and 1249bp, last homology arm upstream primer 5 ' end design EcoR I restriction enzyme site, following homology arm downstream primer 5 ' end design BamH I restriction enzyme site.Above-mentioned primer is synthetic by the big genome company of Beijing China.
The primer sequence of homology arm is as follows in the amplification:
clpSF：5’-CG
(EcoR I)GGGGCGTTACTGGATGC-3’
clpSR：5’-
CCATCGCTTC 
GGTTTGC-3’
The primer sequence of homology arm is as follows under the amplification:
clpXF：5’-
TCCAAAGGCG 
AATACGGTC-3’
clpXR：5’-CG
(BamH I)TTCTCTGCTTTAAGTGTCGGC-3’
With APP serum 7 type CVCC265 pnca gene group DNA is the template upper and lower homology arm of clpP gene that increases respectively: the pcr amplification reaction system is 50 μ L, template DNA 10ng wherein, each 1 μ M of upstream and downstream primer, dNTP 200 μ M, 10 * Taq buffer, 10 μ L, Ex Taq archaeal dna polymerase 2U (TaKaRa).The PCR response procedures is: 95 ℃ of pre-sex change 5min, 94 ℃ of 60s, 55 ℃ of 60s, 72 ℃ of 90s, 30 circulations, 72 ℃ of 10min.The PCR product reclaims test kit with glue and reclaims after gel electrophoresis.
1.1.2 overlapping extension PCR method amplification Δ clpP gene fragment
PCR recovery product with upper and lower homology arm is a template, adopt the upper and lower homology arm of overlapping extension PCR method splicing clpP gene, amplification Δ clpP gene fragment, the base of primer clpSR 5 ' end underscore part and the base reverse complemental of the oblique bolded section of primer clpXF, the base of primer clpXF 5 ' end underscore part and the base reverse complemental of the oblique bolded section of primer clpSR.The pcr amplification reaction system is 50 μ L, template DNA 10ng wherein, each 1 μ M of upstream and downstream primer, dNTP 200 μ M, 10 * Taq buffer, 10 μ L, Ex Taq archaeal dna polymerase 2U (TaKaRa).The PCR response procedures is: 95 ℃ of pre-sex change 5min, 94 ℃ of 60s, 55 ℃ of 90s, 72 ℃ of 150s, 30 circulations, 72 ℃ of 10min.The PCR product reclaims test kit with glue and reclaims after gel electrophoresis, carry out enzyme with EcoR I and BamH I then and cut, and reclaims test kit with glue and reclaims.
1.1.3 the structure of reorganization suicide plasmid pUC Δ clpP
With the Δ clpP gene fragment of purifying with T4DNA ligase enzyme (TaKaRa) be connected with the pUC18 carrier of BamH I double digestion digestion through EcoR I, 16 ℃ connect 24h, heat shock is transformed into the bacillus coli DH 5 alpha competent cell, being accredited as the male clone through bacterium colony PCR increases bacterium and utilizes alkaline lysis to extract plasmid in a small amount, cut the positive plasmid called after pUC Δ clpP of evaluation through enzyme, to clone's the Δ clpP gene evaluation of checking order.
1.2APP the structure of serum 7 type clpP genetically deficient mutant strains
The reorganization suicide plasmid pUC Δ clpP electricity conversion that makes up is entered APP serum 7 type CVCC265, at the positive bacterium colony of 10ug/mLAmp resistant panel top sieve menu exchange strain, and at the upper and lower homology arm indoor design of clpP primer, carrying out PCR identifies, wild strain can increase and obtain the 858bp fragment, the disappearance strain acquisition 367bp fragment that can increase, single cross are changed strain can increase simultaneously acquisition 858bp and 367bp fragment.Above-mentioned primer is synthetic by the big genome company of Beijing China.
Primer sequence is as follows:
clpJDF：5’-CGTGGTGTCGCTTGAAACTC-3’
clpJDR：5’-AATTAGACCGTATTCCATCGC-3’
Single cross is changed the positive single bacterium colony of strain after the TSB that does not contain the Amp resistance (horse serum of former times acid and 10% is examined in the fast cry of certain animals two of 1% nicotinoyl amine gland) cultivates propagation, coat nonresistant TSA flat board, picking list bacterium colony, carrying out PCR identifies, wild strain can increase and obtain the 858bp fragment, the disappearance strain only can be increased and be obtained 367bp fragment, this positive colony called after APP Δ clpP.
The clpP genetically deficient mutant strain APP Δ clpP of the present invention preparation TSB substratum continuous passage 10 times, is identified with PCR,, show that mutant strain of the present invention can genetic stability if every monobasic mutant strain can both amplify the 367bp fragment; If amplify the 858bp fragment, show that mutant strain of the present invention can not genetic stability.The result is as shown in Figure 8: the clpP genetically deficient mutant strain of the present invention's preparation can genetic stability.
The structure of embodiment 2APP serum 7 type clpP and the dual-gene deletion mutantion strain of apxIIC
2.1 the structure of reorganization suicide vector pUC Δ apxIIC
2.1.1apx the design of primers and the pcr amplification of the upper and lower homology arm of II C gene
According to two pairs of primers of the APP-7 type AP76 strain sequence (is the gene order of CP001091.1 with reference to the GenBank accession number) reported design increase respectively the last homology arm apx II CS and the following homology arm apx II Cx of apx II C gene, the amplified fragments size is respectively 1412bp and 1443bp, last homology arm upstream primer 5 ' end design EcoR I restriction enzyme site, following homology arm downstream primer 5 ' end design BamH I restriction enzyme site.Above-mentioned primer is synthetic by the big genome company of Beijing China.
The primer sequence of homology arm is as follows in the amplification:
II CSF：5’-CG
(EcoR I)ATGACAACACCAATGATTGATTTAC-3’
II CSR：5’-
AATCCCCGAA 
CTCCCATTC-3’
The primer sequence of homology arm is as follows under the amplification:
II CXF：5’-
GGATGATGCT 
CATCTCTATTG-3’
II CXR：5’-CG
(BamH I)GTTGTAATAAGTCCCGTAACACCAG-3’
With APP serum 7 type CVCC265 pnca gene group DNA is the template upper and lower homology arm of apxIIC gene that increases respectively: the pcr amplification reaction system is 50 μ L, template DNA 10ng wherein, each 1 μ M of upstream and downstream primer, dNTP 200 μ M, 10 * Taq buffer, 10 μ L, Ex Taq archaeal dna polymerase 2U (TaKaRa).The PCR response procedures is: 95 ℃ of pre-sex change 5min, 94 ℃ of 60s, 55 ℃ of 60s, 72 ℃ of 90s, 30 circulations, 72 ℃ of 10min.The PCR product reclaims test kit with glue and reclaims after gel electrophoresis.
2.1.2 overlapping extension PCR method amplification Δ apxIIC gene fragment
PCR recovery product with upper and lower homology arm is a template, adopt the upper and lower homology arm of overlapping extension PCR method splicing apx II C gene, amplification Δ apx II C gene fragment, the base of primer I I CSR 5 ' end underscore part and the base reverse complemental of the oblique bolded section of primer I I CXF, the base of primer I I CXF 5 ' end underscore part and the base reverse complemental of the oblique bolded section of primer I I C SR.The pcr amplification reaction system is 50 μ L, template DNA 10ng wherein, each 1 μ M of upstream and downstream primer, dNTP 200 μ M, 10 * Taq buffer, 10 μ L, Ex Taq archaeal dna polymerase 2U (TaKaRa).The PCR response procedures is: 95 ℃ of pre-sex change 5min, 94 ℃ of 60s, 55 ℃ of 90s, 72 ℃ of 150s, 30 circulations, 72 ℃ of 10min.The PCR product reclaims test kit with glue and reclaims after gel electrophoresis, carry out enzyme with EcoR I and BamH I then and cut, and reclaims test kit with glue and reclaims.
2.1.3 the structure of reorganization suicide plasmid pUC Δ apx II C
With the Δ apx II C gene fragment of purifying with T4DNA ligase enzyme (TaKaRa) be connected with the pUC18 carrier of BamH I double digestion digestion through EcoR I, 16 ℃ connect 24h, heat shock is transformed into the bacillus coli DH 5 alpha competent cell, being accredited as the male clone through bacterium colony PCR increases bacterium and utilizes alkaline lysis to extract plasmid in a small amount, cut the positive plasmid called after pUC Δ apx II C of evaluation through enzyme, to clone's the Δ apx II C gene evaluation of checking order.
2.2APP the structure of serum 7 type clpP and the dual-gene deletion mutantion strain of apx II C
The reorganization suicide plasmid pUC Δ apx II C electricity conversion that makes up is entered the APP Δ clpP that builds among the embodiment 1, at the positive bacterium colony of 10ug/mL Amp resistant panel top sieve menu exchange strain, and at the upper and lower homology arm indoor design of apx II C primer, carrying out PCR identifies, the strain of clpP single-gene disappearance can be increased and be obtained the 564bp fragment, the dual-gene disappearance strain of clpP and apxII C can be increased and be obtained the 294bp fragment, and single cross is changed strain and can be increased simultaneously and obtain 564bp and 294bp fragment.Above-mentioned primer is synthetic by the big genome company of Beijing China.
Primer sequence is as follows:
II CJDF：5’-GAAGAGCCATTACCCAACAAC-3’
II CJDR：5’-ATACAATAGAGATGAATCCCCG-3’
Single cross is changed the positive single bacterium colony of strain after the TSB that does not contain the Amp resistance (horse serum of former times acid and 10% is examined in the fast cry of certain animals two of 1% nicotinoyl amine gland) cultivates propagation, coat nonresistant TSA flat board, picking list bacterium colony, carrying out PCR identifies, the strain of clpP single-gene disappearance can be increased and be obtained the 564bp fragment, the dual-gene disappearance strain of clpP and apx II C only can be increased and be obtained 294bp fragment, this positive colony called after APP Δ clpP Δ apx II C.
With the clpP of the present invention preparation and the dual-gene deletion mutantion strains A of apx II C PP Δ clpP Δ apx II C TSB substratum continuous passage 10 times, identify with PCR, if every monobasic mutant strain can both amplify the 294bp fragment, show that mutant strain of the present invention can genetic stability; If amplify the 564bp fragment, show that mutant strain of the present invention can not genetic stability.The result is as shown in figure 14: the clpP and the dual-gene deletion mutantion bacterial strain of apx II C of the present invention's preparation can genetic stabilities.
The virulence of embodiment 3APP Δ clpP Δ apx II C mutant strain is identified and the immunoprotection experiment
The female mouse of experimental animal: 4-6 week SPF level Balb/C in age is available from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.
3.1 virulence is identified
Mouse is divided into two groups at random, 10 every group.Concrete vaccination regimen is as follows:
First group (test group): the APP Δ clpP Δ apx II C of inoculation embodiment 2 preparation, dilute and be listed concentration (CFU) in the table 1, every mouse peritoneal dosage of inoculation 0.1ml.
Second group (control group): inoculation actinobacillus pleuropneumoniae CVCC265, dilute and be listed concentration (CFU) in the table 1, every mouse peritoneal dosage of inoculation 0.1ml.
The mouse survival condition sees Table 1.
Table 1
3.2 immunoprotection experiment
Mouse is divided into three groups at random, 10 every group.Concrete vaccination regimen is as follows:
First group (test group 1): the APP Δ clpP Δ apx II C of inoculation embodiment 2 preparations, every mouse peritoneal dosage of inoculation 0.1ml includes 1 * 10
7Bacterium.
Second group (test group 2): the actinobacillus pleuropneumoniae CVCC265 of inoculation deactivation, every mouse peritoneal dosage of inoculation 0.1ml counts it and includes 1 * 10 before the deactivation
7Bacterium.
The 3rd group (control group): inoculation TSB liquid nutrient medium, every mouse peritoneal dosage of inoculation 0.1ml.
After 4 weeks of immunity, each group is used APP serum 7 type CVCC265 bacterium (viable bacteria contents 2 * 10 respectively
7CFU) mouse is attacked poison, the mouse survival condition sees Table 2.
Table 2
| Test strain | Mouse survival number | Protection ratio |
| |
10/10 | 100 |
| CVCC265 | ||
| 2/10 | 20％ | |
| TSB | 0/10 | 0 |
Claims (5)
1. the application of the dual-gene disappearance strain of actinobacillus pleuropneumoniae in preparation prevention porcine contagious pleuropneumonia vaccine that does not contain resistance marker, it is characterized in that: this bacterial strain has lacked ClpP proteolytic enzyme and the proteic encoding gene of Apx II C.
2. application as claimed in claim 1 is characterized in that: the aminoacid sequence of described ClpP proteolytic enzyme is shown in SEQ ID NO.2, and the proteic aminoacid sequence of Apx II C is shown in SEQ ID NO.4.
3. application as claimed in claim 1 is characterized in that: described gene-deleted strain is APP △ clpP △ apx II C, is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its preserving number is CGMCC No.5444.
4. dual-gene disappearance strain of actinobacillus pleuropneumoniae that does not contain resistance marker, it is characterized in that described gene-deleted strain is APP △ clpP △ apx II C, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, its preserving number is CGMCC No.5444.
5. porcine contagious pleuropneumonia vaccine, its activeconstituents is the dual-gene disappearance strain of the described actinobacillus pleuropneumoniae of claim 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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