CN101386831A - Recombinant bacterium of brucella abortus with immunity labeling and use thereof - Google Patents
Recombinant bacterium of brucella abortus with immunity labeling and use thereof Download PDFInfo
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- CN101386831A CN101386831A CNA2008102248186A CN200810224818A CN101386831A CN 101386831 A CN101386831 A CN 101386831A CN A2008102248186 A CNA2008102248186 A CN A2008102248186A CN 200810224818 A CN200810224818 A CN 200810224818A CN 101386831 A CN101386831 A CN 101386831A
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Abstract
The invention discloses a recombinant strain for Brucella abortus provided with an immune label. The recombinant strain for the Brucella melitensis provided with the immune label is a strain obtained by killing encoding genes of perosamine synthetase in the Brucella abortus. Compared with an original strain, the obtained strain has lower virulence, and can be used as a vaccine to immunize animals without killing the genes. By utilization of the strain to immunize a mouse, immunoreaction states different from those of the standard strain and the prior vaccine strain can be generated in the body of the mouse, and sicken animals can be identified from immune animal groups by immunological detection of serum of the animals. The modification of the brucelliasis vaccine and the research of a diagnosis and identification agent have important significance in developing a brucelliasis gene deletion marker vaccine and a matched diagnosis and identification agent and promoting the elimination and purification of brucelliasis of the animals all over the world.
Description
Technical field
The present invention relates to a kind of have immune labeled recombinant strain for Brucella abortus and application thereof.
Background technology
Brucellosis is a kind of transmissible disease of infecting both domestic animals and human, also is to be only second to foot and mouth disease in the large animal transmissible disease, has the disease of important politics, economic impact power, and is very severe in the popular situation of countries in the world.The strategy of global for a long time prevention and control brucellosis mainly is to quarantine-slaughter (developed country) or immunization (developing country), and the former slaughters animals such as ill ox, sheep, pig, costly, operational difficulty by quarantine; The latter is by immunization and assist the measure of slaughtering, though can stop the propagation of disease in fauna, but immune animal and clinical infected animal are difficult to differentiate, make infected animal in the medium-term and long-term existence of natural population, serious threat human and animal group's health hinders the elimination and the purification of brucellosis in the animal.
Summary of the invention
The purpose of this invention is to provide a kind of recombinant strain for Brucella abortus.
Recombinant strain for Brucella abortus provided by the invention is the reorganization bacterium that obtains after the encoding gene deactivation with the perosamine synthetic enzyme (perosamine synthetase) in Bacillus abortus (Brucella abortus) genome.
Described Bacillus abortus (Brucella abortus) can be existing bacterial strain, as wild strain (comprising type strain and virulent strain), vaccine strain.
Described perosamine synthetic enzyme (perosamine synthetase) can be the perosamine synthetic enzyme (perosamine synthetase) of any Bacillus abortus (Brucellaabortus), as is the perosamine synthetic enzyme shown in the sequence 1 (perosamine synthetase).
The encoding sequence of described perosamine synthetic enzyme (perosamine synthetase) is the nucleotide sequence shown in the sequence 2 in the sequence table.
Can be by the perosamine synthetic enzyme (perosamine synthetase) in the described Bacillus abortus of any existing ablation method deactivation (Brucella abortus), as this proteic encoding sequence, insertion external source or endogenous sequence, homologous recombination, RNA interference etc. in the missing gene group.
Described homologous recombination is that the dna fragmentation with DA14 imports in the described Bacillus abortus (Brucella abortus) and realizes; The dna fragmentation of described DA14 is followed successively by homology arm DA14-1 and homology arm DA14-2 to the downstream from the upstream; Described homology arm DA14-1 and homology arm DA14-2 can with the upstream sequence and the downstream sequence generation homologous recombination of perosamine synthetic enzyme (perosamine synthetase) encoding gene in the described Bacillus abortus genome, the encoding gene of the described perosamine synthetic enzyme of deactivation (perosamine synthetase).
The length of described homology arm DA14-1 and homology arm DA14-2 is 400-600bp.
The genomic dna that described homology arm DA14-1 specifically can be with Bacillus abortus 2308 (Brucella abortus2308) type strain is a template, carries out the dna fragmentation that pcr amplification obtains with following primer:
PWUA393 (upstream primer): 5 '-GGGGTACCACGCTTAGGAACACTG-3 ';
PWUA394 (downstream primer): 5 '-CTGCAGTGGCTGGATCATAGGTA-3 '.
The genomic dna that described homology arm DA14-2 specifically can be with Bacillus abortus 2308 (Brucella abortus 2308) reference culture is a template, carries out the dna fragmentation that pcr amplification obtains with following primer:
PWUA395 (upstream primer): 5 '-AGCCACTGCAGGAGATTCAGGCGCTCCA-3 ';
PWUA396 (downstream primer): 5 '-CGGGATCCCACCTCGCCAAGTGTC-3 '.
The virulence that experiment showed, described reorganization bacterium is lower than starting strain, and has the serodiagnosis mark, and its immune protection effectiveness is suitable with the currently available vaccines, existing vaccines strain.Therefore, this reorganization bacterium can be used for developing the Bacillus abortus vaccine.
This brucella vaccine, its activeconstituents are above-mentioned recombinant strain for Brucella abortus.The present invention is by screening the functional gene in the brucella genome, the deactivation of finding perosamine synthetic enzyme (perosamine synthetase) encoding gene can make the virulence of Bacillus abortus significantly lower, and can change after the bacterial strain immunity antibody in the animal serum simultaneously and form.The present invention passes through the encoding gene deactivation with the perosamine synthetic enzyme (perosamine synthetase) of Bacillus abortus, obtained new bacterial strain, compare with starting strain, the virulence of this new bacterial strain is less, need not deactivation and just can be used as the vaccine immunity animal, and can make immunization animal and clinical infected animal differentiate out by serological technique.Use bacterial strain immune mouse of the present invention, can generation in the mouse body and reference culture, the existing different immune response state of vaccine strains, infected animal can be differentiated from immune animal colony by the immunology detection of animal serum.The present invention is to the improvement of brucella disease vaccine, the development of diagnosis identification reagent, and exploitation brucella genetically deficient marker vaccine and supporting differential diagnosis reagent promote the purification of global animal brucellosis and elimination to have crucial meaning.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Description of drawings
Fig. 1 is the PCR qualification result of DA14-1 and DA14-2
Fig. 2 is that the segmental PCR of DA14 connects the result
Fig. 3 is the PCR qualification result behind the recombinant plasmid pEX18AP-DA14 transformed into escherichia coli
Fig. 4 is the PCR qualification result of recombinant plasmid pEX18AP-DA14 through BamHI and KpnI double digestion after product
Fig. 5 is the structure schema of recombinant vectors pEX18AP-DA14
Fig. 6 is the PCR qualification result of reorganization bacterium BIDA14
Fig. 7 is the changes in weight trend of reorganization bacterium BIDA14 inoculation back mouse spleen
Fig. 8 is the bacterium quantitative change trend of carrying of reorganization bacterium BIDA14 inoculation back mouse spleen
Fig. 9 is that reorganization bacterium BIDA14 is in the intravital immune efficacy experimental result of mouse
Embodiment
Experimental technique described in the following embodiment if no special instructions, is ordinary method.
Bacterial strain and plasmid used in following examples are as follows:
Bacillus abortus reference culture 2308 (Brucella abortus 2308), Bacillus abortus vaccine strains S19 (available from China Veterinary Drugs Supervisory Inst. bacterial classification chamber); (construction process is referring to document: Tung T.Hoang, Gene 1998,212:77-86) for plasmid pEX18AP.
According to the perosamine synthetic enzyme in the brucella gene (perosamine synthetase) sequence (GenBank Accession Number:BAB1-0542), design two pairs of primers, the a pair of primer homology arm DA14-1 that is used to increase, another is used to the DA14-2 that increases to primer.Homology arm DA14-1 is connected the back and forms sequence D A14 with homology arm DA14-2, sequence D A14 is inserted the pEX18AP plasmid, obtains recombinant vectors pEX18AP-DA14.Recombinant vectors pEX18AP-DA14 and Bacillus abortus genome generation homologous recombination can obtain the Brucella abortus recombinant strain of deactivation perosamine synthetic enzyme (perosamine synthetase) encoding gene.
One, the structure of recombinant vectors pEX18AP-DA14
The building process synoptic diagram is seen Fig. 5.
1, the genomic extraction of brucella
Get the Bacillus abortus reference culture 2308 of 300 μ l deactivations, add 1ml TNE (every liter contains 1.21gTris, 5.84g NaCl, 0.37g EDTA) mixing, 10000r/min is centrifugal, stays precipitation to abandon supernatant.Add the precipitation after the resuspended washing of 135 μ l TNE afterwards.Contain the TNE of 2%Triton X-100, mixing to wherein adding 135 μ l again.Add the freshly prepared N,O-Diacetylmuramidases of 30 μ l (5mg/ml), flick the test tube mixing.30min is hatched in 37 ℃ of water-baths, adds 15 μ l Proteinase Ks (20mg/ml) afterwards, the vortex mixing.2h is hatched in 65 ℃ of water-baths at least.
Add RNAse (making RNAse concentration reach 10 μ g/ml), after agarose gel electrophoresis is identified that the genomic dna that extracts is standby in 4 ℃ of preservations.
2, pcr amplification upstream fragment DA14-1 (477bp) and downstream fragment DA14-2 (574bp)
The segmental primer in amplification upstream is as follows:
PWUA393 (upstream primer): 5 '-GGGGTACCACGCTTAGGAACACTG-3 ';
PWUA394 (downstream primer): 5 '-CTGCAGTGGCTGGATCATAGGTA-3 '.
Add the KpnI restriction enzyme site in the upstream primer, add the PstI restriction enzyme site in the downstream primer.
The segmental primer in amplification downstream is as follows:
PWUA395 (upstream primer): 5 '-AGCCACTGCAGGAGATTCAGGCGCTCCA-3 ';
PWUA396 (downstream primer): 5 '-CGGGATCCCACCTCGCCAAGTGTC-3 '
Add the PstI restriction enzyme site in the upstream primer, add the BamHI restriction enzyme site in the downstream primer.
The genomic dna that obtains with step 1 is a template, uses above-mentioned two pairs of primers to carrying out touchdown PCR.The actual conditions of touchdown PCR reaction is: 94 ℃ of pre-sex change 3min of elder generation; 94 ℃ of sex change 45s then; 2 circulations of 1 ℃ of operation whenever fall in annealing temperature from 65 ℃ to 50 ℃, each circulation is extended 1min for 72 ℃; Last 72 ℃ are extended 10min.Amplified production carries out 1% agarose gel electrophoresis to be identified, sees Fig. 1.Wherein, the pcr amplification product of 1:DA14-1, the pcr amplification product of 2:DA14-2, M:DNA molecular weight standard.The result shows, has successfully amplified the purpose fragment.
3, the segmental acquisition of DA14
By pcr amplification upstream fragment DA14-1 is connected with downstream fragment DA14-2, obtains fragment and be the DA14 fragment.The used primer of pcr amplification is as follows:
PWUA393 (upstream primer): 5 '-GGGGTACCACGCTTAGGAACACTG-3 ';
PWUA396 (downstream primer): 5 '-CGGGATCCCACCTCGCCAAGTGTC-3 '
The pcr amplification system:
DA14-1 fragment 1ul
DA14-2 fragment 1ul
dNTP(2.5mM) 1ul
Taq enzyme 2ul
10XTaq?buffer 5ul
pWUA393(20pmol/μl) 1ul
pWUA396(20pmol/μl) 1ul
Distilled water complements to 50ul 38ul
Amplified production carries out electrophoresis detection, sees Fig. 2.The result shows, has successfully obtained the DA14 fragment that DA14-1 is connected with DA14-2.
4, the structure of pEX18AP-DA14 plasmid
The pEX18AP plasmid that extraction contains AMP resistant gene, SacB gene and contains multiple clone site.To be connected with the DA14 fragment that step 3 obtains through the pEX18AP plasmid behind BamHI and the KpnI double digestion.With the recombinant plasmid called after pEX18AP-DA14 that obtains.
Be transformed into DH5 α intestinal bacteria with pEX18AP-DA14, carry out blue hickie screening then.Single bacterium colony of picking positive strain shakes bacterium and cultivates, and advanced performing PCR identifies that primers designed is as follows:
PWUA393 (upstream primer): 5 '-GGGGTACCACGCTTAGGAACACTG-3 ';
PWUA396 (downstream primer): 5 '-CGGGATCCCACCTCGCCAAGTGTC-3 '
The results are shown in Figure 3.Among Fig. 3, the M:DNA molecular weight standard; 1: the PCR product of positive strain.
Extract plasmid then and carry out BamHI and the evaluation of KpnI double digestion.What enzyme was cut evaluation the results are shown in Figure 4.Among Fig. 4, the M:DNA molecular weight standard; 1: the PCR result of recombinant plasmid pEX18AP-DA14 double digestion product.
Contain recombinant plasmid bacterial strain propagation with what obtain, be kept at-80 ℃ standby.
Two, the cloth Lu Shi recombinant bacterial strain of deactivation perosamine synthetic enzyme (perosamine synthetase) encoding gene obtains
Inoculation Bacillus abortus reference culture 230850ul in the centrifuge tube that contains the 25mlTSB liquid nutrient medium cultivates after 24 hours for 37 ℃, and centrifugal thalline also suspends and changes in the 1.5ml centrifuge tube of precooling continuation centrifuge washing 3-4 time over to the tri-distilled water of precooling.Then, add an amount of cold tri-distilled water suspension bacteria liquid, get the pole cup that 87ul bacterium liquid and 3ul plasmid (pEX18AP-DA14) add 1mm, pole cup is put into the BIORAD electroporation apparatus, adjust instrument to the Agr program, shock by electricity by the Pulse button.The inquiry shock parameters is 2.22KV.After the electric shock, in pole cup, add 1ml SOC nutrient solution, change the 1.5ml centrifuge tube over to, leave standstill 10min under the room temperature, place 37 ℃ of shaking table recovery 12-18h.The bacterium liquid that transforms after recovering is coated TSB+Amp (100ug/ul).Grow single bacterium colony after 5-8 days.Picking list bacterium colony with after 10-100 times of this bacterium liquid dilution, is got 100ul and is coated the TSA plate culture medium that contains 5% sucrose again to 37 ℃ of propagation of TSB liquid nutrient medium (antibiotic-free) 48h.After 3-5 days, grow single bacterium colony, this bacterium colony is carried out PCR to be identified, positive strain is the brucellar reorganization bacterium of deactivation perosamine synthetic enzyme (perosamine synthetase) encoding gene, and its called after is had immune labeled recombinant strain for Brucella abortus BIDA14.
PCR identifies that the primer of usefulness is as follows:
PWUA393 (upstream primer): 5 '-GGGGTACCACGCTTAGGAACACTG-3 ';
PWUA396 (downstream primer): 5 '-CGGGATCCCACCTCGCCAAGTGTC-3 '.
The PCR that has immune labeled recombinant strain for Brucella abortus BIDA14 identifies and sees Fig. 6.Among Fig. 6, the 1:DNA molecular weight standard; 2: the PCR product of Bacillus abortus reference culture 2308; 3: the PCR product of reorganization bacterium BIDA14.
The result shows, has obtained the brucella by double exchange deactivation perosamine synthetic enzyme (perosamine synthetase) encoding gene.
The evaluation of the recombinant bacterium of brucella of embodiment 2, deactivation perosamine synthetic enzyme (perosamine synthetase)
The female mouse of experimental animal: 4-6 week SPF level Balb/C in age, available from Beijing Vital River Experimental Animals Technology Co., Ltd., license licensed licenser licence is numbered SCXK (capital) 2007-0001.
One, virulence is identified
Mouse is divided into three groups at random, 30 every group.Concrete vaccination regimen is as follows:
First group (test group): inoculate the immune labeled recombinant strain for Brucella abortus BIDA14 that has of embodiment 1 preparation, every mouse peritoneal dosage of inoculation 0.1ml includes 10
7Bacterium.
Second group (control group 1): inoculation Bacillus abortus reference culture 2308, every mouse peritoneal dosage of inoculation 0.1ml includes 10
7Bacterium.
The 3rd group (control group 2): inoculation Bacillus abortus vaccine strains S19, every mouse peritoneal dosage of inoculation 0.1ml includes 10
7Bacterium.
The 1st, 2,3,4,5 and 10 weeks from every group of mouse, selected 5 respectively at inoculation back, carry out spleen size, spleen and carry bacterium amount and serology and detect and estimate.
Spleen weight is seen Fig. 7 over time.Wherein, wild strain represents to inoculate the experimental result of Bacillus abortus reference culture 2308, S19 represents to inoculate the experimental result of Bacillus abortus vaccine strains S19, and BIDA14 represents to inoculate the experimental result that has immune labeled recombinant strain for Brucella abortus BIDA14 of embodiment 1 preparation.The result shows, have immune labeled recombinant strain for Brucella abortus BIDA14 inoculation after, the spleen of mouse is compared early stage enlargement with control group 1 with 2, but the later stage descend rapidly, suitable with inoculation Bacillus abortus vaccine strains S19 (control group 2).Spleen carries the bacterium amount sees Fig. 8 over time.The result shows, after having immune labeled recombinant strain for Brucella abortus BIDA14 inoculation, the spleen of mouse carries bacterium amount to be compared in early days with inoculation Bacillus abortus vaccine strains S19 (control group 2) and slightly raises, but the later stage significantly descend, even descend also soon than vaccine group.
The serology detected result shows, (China Veterinery Drug Inspection Office makes the mice serum of inoculation Bacillus abortus reference culture 2308 by the red plate agglutination test antigen of brucella tiger, when lot number 200801) detecting, all tangible agglutination reaction can appear in 2 minutes, (China Veterinery Drug Inspection Office makes by tube agglutination test antigen, lot number 200603) detected antibody titer reaches more than the 1:100, and that inoculation has the detected result of mice serum of immune labeled recombinant strain for Brucella abortus strain BIDA14 is all negative.
Two, vaccine potency evaluation
Above-mentioned healthy mice is divided into three groups at random, 5 every group.Three groups of mouse are injected respectively and have immune labeled recombinant strain for Brucella abortus strain BIDA14, Bacillus abortus vaccine strains S19 and PBS, and are specific as follows:
First group (test group): inoculate the immune labeled recombinant strain for Brucella abortus strain BIDA14 that has of embodiment 1 preparation, every mouse peritoneal dosage of inoculation 0.1ml includes 10
7Bacterium.
Second group (vaccine control group): inoculation Bacillus abortus vaccine strains S19, every mouse peritoneal dosage of inoculation 0.1ml includes 10
7Bacterium.
The 3rd group (blank group): inoculation 0.01M pH7.2 phosphate buffered saline buffer (PBS), every mouse peritoneal dosage of inoculation 0.1ml.
Above-mentioned three groups of mouse inoculations are attacked malicious protection test with Bacillus abortus reference culture 2308 after 8 weeks, attack poison back and cut open mouse and blood sampling extremely two weeks, get spleen and weigh and calculate spleen and carry the bacterium quantitative changeization, estimate immune efficacy.Three repetition are established in experiment, on average spleen carry the bacterium quantitative changeization the result as shown in Figure 9.Among the figure, the experimental result of blank group is not represented in the inoculation contrast, and S19 represents the experimental result of vaccine control group, and BIDA14 represents to inoculate the experimental result that has immune labeled recombinant strain for Brucella abortus strain BIDA14 of embodiment 1 preparation.Mouse body inside fire attack poison experimental result shows that the immune protection effectiveness that has immune labeled recombinant strain for Brucella abortus BIDA14 of perosamine synthetic enzyme (perosamine synthetase) encoding gene disappearance obviously is better than vaccine strain S19.Explanation is compared with Bacillus abortus vaccine strains S19, the virulence that has immune labeled recombinant strain for Brucella abortus BIDA14 of perosamine synthetic enzyme (perosamine synthetase) encoding gene disappearance is less, need not deactivation and just can be used as the vaccine immunity animal, and can make immunization animal and clinical infected animal differentiate out by serological technique.
<160>2
<210>1
<211>252
<212>PRT
＜213〉artificial sequence
<400>1
Met?Ile?Gln?Pro?Ser?Ile?Thr?Leu?Ser?Asn?Val?His?Leu?His?Tyr?Ala
1 5 10 15
Ala?Ser?Ala?Phe?Lys?Glu?Arg?Ser?Val?Lys?Thr?Leu?Val?Asn?Ala?Leu
20 25 30
Leu?Ser?Met?Arg?Arg?Ser?Ala?Gly?Ala?Asn?Ile?Glu?Asp?Ile?His?Ala
35 40 45
Leu?Lys?Gly?Ile?Ser?Val?Asp?Ile?Ala?Arg?Gly?Glu?Arg?Val?Ala?Leu
50 55 60
Ile?Gly?His?Asn?Gly?Ala?Gly?Lys?Ser?Thr?Phe?Leu?Lys?Thr?Ile?Ala
65 70 75 80
Gly?Leu?Tyr?Pro?Ile?Ser?Ser?Gly?Thr?Leu?Lys?Val?Thr?Gly?Thr?Val
85 90 95
Arg?Ser?Leu?Phe?Asp?Ile?Gly?Leu?Gly?Phe?Glu?Pro?Asp?Ala?Thr?Gly
100 105 110
Arg?Glu?Asn?Ile?Leu?Tyr?Arg?Gly?Leu?Leu?Leu?Gly?Leu?Thr?Pro?Arg
115 120 125
Phe?Met?Arg?Glu?Ile?Glu?Asp?GluIleIle?Glu?Phe?Ala?Asp?Leu?Gly
130 135 140
Asp?Phe?Ile?Asp?Tyr?Pro?Ile?Lys?Thr?Tyr?Ser?Ala?Gly?Met?Gln?Val
145 150 155 160
Arg?Leu?Ala?Phe?Ala?Ile?Ser?Thr?Ala?Val?Ala?Gly?AspIle?Leu?Leu
165 170 175
Leu?Asp?Glu?Val?Ile?Gly?Ala?Gly?Asp?Ala?Ala?Phe?Met?Thr?Lys?Ala
180 185 190
Lys?Ala?Arg?Ile?Met?Asn?Met?Val?Glu?Lys?Ala?Glu?Ile?Met?Val?Leu
195 200 205
Ala?Ser?His?Asp?Leu?Ala?Asn?Val?Arg?Gln?Leu?Cys?Thr?Arg?Ala?Leu
210 215 220
Val?Phe?Lys?Ala?Gly?Thr?Ile?Ala?Phe?Asp?Gly?Arg?Val?Glu?Asp?Ala
225 230 235 240
Ile?Ser?Phe?Tyr?Asn?Ser?Gly?Met?Gly?Ala?Ile?Ala
245 250
<210>2
<211>759
<212>DNA
＜213〉artificial sequence
<400>2
atgatccagc?catcgattac?cctgtcaaat?gttcatctgc?actacgctgc?atcagcgttc 60
aaagaacgct?cagtcaaaac?tctagtaaac?gccttattga?gtatgcggcg?cagcgcagga 120
gcaaacattg?aagacatcca?tgccctaaag?ggtatttctg?tagatatagc?gcggggcgaa 180
cgcgttgccc?tgataggtca?caacggggct?ggcaaaagta?cgttcttgaa?aactatagcc 240
ggtctctacc?ctatatcatc?tgggacatta?aaagtgacgg?gtaccgtaag?atccctgttc 300
gatattggtc?ttgggtttga?gcctgatgca?actggccgtg?agaatattct?ttaccgtggg 360
ttgcttctcg?gactaacgcc?acgtttcatg?cgagagatcg?aggatgagat?catcgagttc 420
gcggatctcg?gcgattttat?cgattatcca?atcaaaactt?attctgccgg?catgcaagtt 480
cggctcgcct?tcgcgatttc?gacagcagtc?gccggcgaca?tactccttct?agacgaagtt 540
ataggtgcag?gtgatgcggc?attcatgact?aaggcgaagg?cccgcataat?gaatatggtc 600
gagaaggctg?agataatggt?tctagcaagc?catgaccttg?cgaacgtccg?tcagctttgc 660
acacgagcat?tggttttcaa?agccggcaca?attgcatttg?atggcagggt?agaagacgcg 720
atttccttct?ataactcggg?aatgggagct?atagcatga 759
Claims (7)
1, recombinant strain for Brucella abortus is the bacterial strain that the encoding gene deactivation with the perosamine synthetic enzyme (perosamine synthetase) in the Bacillus abortus (Brucella abortus) obtains.
2, reorganization bacterium as claimed in claim 1 is characterized in that: the protein that described perosamine synthetic enzyme (perosaminesynthetase) is made up of the aminoacid sequence shown in the sequence in the sequence table 1.
3, reorganization bacterium as claimed in claim 1 or 2, it is characterized in that: described deactivation realizes by homologous recombination.
4, reorganization bacterium as claimed in claim 3 is characterized in that: described homologous recombination imports described Bacillus abortus (Brucella abortus) with dna fragmentation DA14 and realizes; Described dna fragmentation DA14 is followed successively by homology arm DA14-1 and homology arm DA14-2 to the downstream from the upstream; Described homology arm DA14-1 and homology arm DA14-2 can with the upstream sequence and the downstream sequence generation homologous recombination of perosamine synthetic enzyme (perosamine synthetase) encoding gene in the described Bacillus abortus genome, the encoding gene of the described perosamine synthetic enzyme of deactivation (perosaminesynthetase).
5, reorganization bacterium as claimed in claim 4, it is characterized in that: the length of described homology arm DA14-1 and homology arm DA14-2 is 400-600bp.
6, the application of arbitrary described reorganization bacterium in the preparation brucella vaccine in the claim 1 to 5.
7, a kind of brucella vaccine, its activeconstituents are arbitrary described recombinant strain for Brucella abortus in the claim 1 to 5.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629151B (en) * | 2009-08-07 | 2011-08-03 | 中国农业大学 | Brucella abortus immune marking recombinant strain and application thereof in preparation of vaccine |
| US20120183576A1 (en) * | 2009-09-21 | 2012-07-19 | Jean-Pierre Gorvel | Modified gram-negative bacteria for use as vaccines |
| CN109142757A (en) * | 2018-11-02 | 2019-01-04 | 中国农业科学院兰州兽医研究所 | A kind of brucella indirect ELISA testing kit |
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2008
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629151B (en) * | 2009-08-07 | 2011-08-03 | 中国农业大学 | Brucella abortus immune marking recombinant strain and application thereof in preparation of vaccine |
| US20120183576A1 (en) * | 2009-09-21 | 2012-07-19 | Jean-Pierre Gorvel | Modified gram-negative bacteria for use as vaccines |
| CN102665739A (en) * | 2009-09-21 | 2012-09-12 | 国家医疗保健研究所 | Modified gram-negative bacteria for use as vaccines |
| US8778655B2 (en) * | 2009-09-21 | 2014-07-15 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Modified gram-negative bacteria for use as vaccines |
| US9034630B2 (en) | 2009-09-21 | 2015-05-19 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Modified gram-negative bacteria for use as vaccines |
| CN109142757A (en) * | 2018-11-02 | 2019-01-04 | 中国农业科学院兰州兽医研究所 | A kind of brucella indirect ELISA testing kit |
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| CN101386831B (en) | 2010-12-01 |
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