CN101575587B - Abortus Brucella vaccine recombinant strain and application thereof in preparing vaccine - Google Patents

Abstract

The invention discloses an abortus Brucella vaccine recombinant strain and an application thereof in preparing vaccine. The recombinant strain is a strain obtained by deactivating capsular polysaccharide synthesis protein gene and carboxyl uridine phosphate decarboxylase gene in the abortus Brucella strain S19. The obtained recombinant strain has small virulence and can be used as vaccines to immunize animals without needs of deactivation. The inoculated animal generates no lipopolysaccharide antibody and the virulent virus strain-infected animal can be distinguished from novel vaccine strain immunized animal by serological reaction. The recombinant strain is beneficial to improving the Brucella vaccine, researching diagnosis and identification reagents and developing the Brucella vaccine and matched diagnosis and identification reagents, and has extremely important significance in deracinating and purifying the global animal Brucella.

Description

A kind of Brucella abortus recombinant strain and the application in the preparation vaccine thereof

Technical field

The present invention relates to a kind of Brucella abortus recombinant strain and the application in the preparation vaccine 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 by quarantine is measure, costly, operational difficulty; 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 animal brucellosis.

Be used for seeing that according to now reaching making of China in the world rough type vaccine strain RB51 and vaccine strain S19 are attenuated live vaccines, but the virulence of S19 is strong, the inoculation dam easily causes miscarriage, utilizes serological method not infect with wild strain and distinguishes; And the virulence of RB51 relatively a little less than, though can carry out serological differential diagnosis, the immune efficacy of RB51 is controversial, does not also use in China.So the safety label vaccine strains is the technical bottlenecks of present countries in the world for the brucellosis prevention and control.

Summary of the invention

Purpose of the present invention provides a kind of Brucella abortus recombinant strain and the application in the preparation vaccine thereof.

Recombinant bacterial strain provided by the invention is the bacterial strain that the capsular polysaccharide synthetic proteins gene among Bacillus abortus (Brucella abortus) the bacterial strain S19 and the deactivation of carboxyuridine phosphate decarboxylase gene is obtained with being.

Described Bacillus abortus bacterial strain S19 is existing Bacillus abortus vaccine strain.

Described capsular polysaccharide synthetic proteins can be any capsular polysaccharide synthetic proteins that derives from Bacillus abortus (Brucella abortus), specifically can be the capsular polysaccharide synthetic proteins shown in the sequence 1 of sequence table.The encoding sequence of described capsular polysaccharide synthetic proteins specifically can be the nucleotide sequence shown in the sequence 2 of sequence table.

Described carboxyuridine phosphate decarboxylase can be any carboxyuridine phosphate decarboxylase that derives from Bacillus abortus (Brucella abortus), specifically can be the carboxyuridine phosphate decarboxylase shown in the sequence 3 of sequence table.The encoding sequence of described carboxyuridine phosphate decarboxylase specifically can be the nucleotide sequence shown in the sequence 4 of sequence table.

Can be by any existing method deactivation described capsular polysaccharide synthetic proteins gene and carboxyuridine phosphate decarboxylase gene, as this sequence deletion, external source or endogenous sequence insertion in the genome, homologous recombination, RNA interference etc.

The homologous recombination of deactivation capsular polysaccharide synthetic proteins gene can realize by dna fragmentation DA15 being imported Bacillus abortus bacterial strain S19; Described dna fragmentation DA15 is followed successively by homology arm DA15-1 and homology arm DA15-2 to the downstream from the upstream; Described homology arm DA15-1 and homology arm DA15-2 can with the upstream and downstream identical sequence generation homologous recombination of capsular polysaccharide synthetic proteins encoding sequence in the described strain gene group, the described capsular polysaccharide synthetic proteins of deactivation gene.

The length of described homology arm DA15-1 and homology arm DA15-2 is 400-600bp.

The genomic dna that described homology arm DA15-1 specifically can be with Bacillus abortus bacterial strain S19 is a template, carries out the dna fragmentation that pcr amplification obtains with following primer:

PWUO397 (upstream primer): 5 '-GGAATTCTCAATGTAACCAACTTCA-3 ' (sequence 5 of sequence table);

PWUO398 (downstream primer): 5 '-AAGCTTAGAGGTCATACCTTCCTG-3 ' (sequence 6 of sequence table).

The genomic dna that described homology arm DA15-2 specifically can be with Bacillus abortus bacterial strain S19 is a template, carries out the dna fragmentation that pcr amplification obtains with following primer:

PWUO399 (upstream primer): 5 '-CCTCTAAGCTTATCTGTTTGCTGATGCG-3 ' (sequence 7 of sequence table);

PWUO400 (downstream primer): 5 '-CGGGATCCGCGTTTCGGATAAGATG-3 ' (sequence 8 of sequence table).

The homologous recombination of deactivation carboxyuridine phosphate decarboxylase gene can realize by dna fragmentation DAT being imported Bacillus abortus bacterial strain S19; Described dna fragmentation DAT is followed successively by homology arm DAT-1 and homology arm DAT-2 to the downstream from the upstream; Described homology arm DAT-1 and homology arm DAT-2 can with the upstream and downstream identical sequence generation homologous recombination of carboxyuridine phosphate decarboxylase encoding sequence in the described strain gene group, the described carboxyuridine phosphate decarboxylase gene of deactivation.

The length of described homology arm DAT-1 and homology arm DAT-2 is 400-600bp.

The genomic dna that described homology arm DAT-1 specifically can be with Bacillus abortus bacterial strain S19 is a template, carries out the dna fragmentation that pcr amplification obtains with following primer:

PWUO659 (upstream primer): 5 '-GGAATTCAAGTCTTCTCACCCACATCC-3 ' (sequence 9 of sequence table);

PWUO660 (downstream primer): 5 '-AAGCTTCGTCATCGTGCAATTCTG-3 ' (sequence 10 of sequence table).

The genomic dna that described homology arm DAT-2 specifically can be with Bacillus abortus bacterial strain S19 is a template, carries out the dna fragmentation that pcr amplification obtains with following primer:

PWUO661 (upstream primer): 5 '-TGACGAAGCTTCGCTCGATTGGAAGAAGT-3 ' (sequence 11 of sequence table);

PWUO662 (downstream primer): 5 '-CGGGATTCCCGTTCGCCTGTAAGGA-3 ' (sequence 12 of sequence table).

The virulence that experiment showed, this reorganization bacterium is lower than the maternal bacterial strain that sets out, and immune protection effectiveness is suitable with existing vaccine strain, and has clinical quarantine, diagnosis with immune labeled.Therefore, this reorganization bacterium can be used for preparing brucella vaccine.

Described recombinant bacterial strain can be applicable to prepare the Bacillus abortus disease vaccine.

The present invention also protects a kind of Bacillus abortus disease vaccine, and its activity becomes described recombinant bacterial strain.

The present invention finds, the rough type Bacillus abortus vaccine of deactivation capsular polysaccharide synthetic proteins gene and carboxyuridine phosphate decarboxylase gene is stablized mutant strain and is compared with original maternal bacterial strain-international standard vaccine strain S19, because of the lipopolysaccharides dyssynthesis, its virulence reduces relatively, but immanoprotection action is similar to original strain; Of paramount importance is that this bacterial strain is a rough type, can distinguish vaccine immunity animal and clinical wild toxic bacterial strain infection animal simply by serological technique in reality quarantine practice.The present invention can be applicable to the transformation of brucella disease vaccine, the development of diagnosis identification reagent, is applied to develop brucella disease vaccine and supporting differential diagnosis reagent, and elimination and the purification that promotes global animal brucellosis had crucial meaning.

Description of drawings

Fig. 1 is the pcr amplification electrophorogram of DA15-1 and DA15-2.

Fig. 2 is the pcr amplification electrophorogram of DA15.

Fig. 3 is that pEX18AP-DA15 transformed into escherichia coli DH5 α is after the evaluation figure of pcr amplification.

Fig. 4 is that BamHI and the EcoRI double digestion of pEX18AP-DA15 identified figure.

Fig. 5 is the structure schema of recombinant vectors pEX18AP-DA15.

Fig. 6 is the pcr amplification electrophorogram of DAT-1 and DAT-2.

Fig. 7 is the pcr amplification electrophorogram of DAT.

Fig. 8 is that pEX18AP-DAT transformed into escherichia coli DH5 α is after the evaluation figure of pcr amplification.

Fig. 9 is that BamHI and the EcoRI double digestion of pEX18AP-DAT identified figure.

Figure 10 is the structure schema of recombinant vectors pEX18AP-DAT.

Figure 11 identifies figure for the PCR of reorganization bacterium RB15.

Figure 12 identifies figure for the PCR of reorganization bacterium RB15T.

Figure 13 measures change curve in time for reorganization bacterium RB15T inoculation back mouse spleen carries bacterium.

Figure 14 is the preliminary immunoprotection test-results of reorganization bacterium RB15T inoculation mouse.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.

Bacterial strain and plasmid used in following examples are as follows:

Bacillus abortus (Brucella abortus) bacterial strain 2308 (reference culture), Bacillus abortus (Brucella abortus) bacterial strain S19 (vaccine strains) are all available from China Veterinary Drugs Supervisory Inst. bacterial classification chamber; Bacillus abortus (Brucella abortus) bacterial strain RB51 (rough type vaccine strains) is from the isolated strains of U.S.'s business men industry Brucella abortus rough type vaccine; (construction process is referring to document: Tung T.Hoang, Gene 1998,212:77-86) for the pEX18AP plasmid.

The structure of embodiment 1, recombinant bacterial strain

Capsular polysaccharide synthetic proteins gene (GenBank AccessionNumber:BAbS19I04980) according in the Bacillus abortus genome designs two pairs of primers, a pair of primer homology arm DA15-1 that is used to increase, and another is used to the DA15-2 that increases to primer.Homology arm DA15-1 is connected the back and forms sequence D A15 with homology arm DA15-2, sequence D A15 is inserted the pEX18AP plasmid, obtains recombinant vectors pEX18AP-DA15.According to two pairs of primers of the carboxyuridine phosphate decarboxylase gene in the Bacillus abortus genome (GenBank Accession Number:BAbS19I19940) design, a pair of primer homology arm DAT-1 that is used to increase, another is used to the DAT-2 that increases to primer.Homology arm DAT-1 is connected the back and forms sequence D AT with homology arm DAT-2, sequence D AT is inserted the pEX18AP plasmid, obtains recombinant vectors pEX18AP-DAT.The recombinant bacterial strain that homologous recombination can obtain deactivation capsular polysaccharide synthetic proteins gene and carboxyuridine phosphate decarboxylase gene takes place in recombinant vectors pEX18AP-DA15 and pEX18AP-DAT and Bacillus abortus bacterial strain S19.

One, the structure of recombinant vectors pEX18AP-DA15

The building process synoptic diagram is seen Fig. 5.

1, the genomic extraction of brucella

Get the Bacillus abortus bacterial strain S19 of 15 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, the upstream fragment DA15-1 (579bp) of pcr amplification capsular polysaccharide synthetic proteins gene and downstream fragment DA15-2 (505bp)

The segmental primer in amplification upstream is as follows:

PWUO397 (upstream primer): 5 '-GGAATTCTCAATGTAACCAACTTCA-3 ';

PWUO398 (downstream primer): 5 '-AAGCTTAGAGGTCATACCTTCCTG-3 '.

Add the EcoRI restriction enzyme site in the upstream primer, add the HindIII restriction enzyme site in the downstream primer.

The segmental primer in amplification downstream is as follows:

PWUO399 (upstream primer): 5 '-CCTCTAAGCTTATCTGTTTGCTGATGCG-3 ';

PWUO400 (downstream primer): 5 '-CGGGATCCGCGTTTCGGATAAGATG-3 '.

Add the HindIII 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 primers to carrying out touchdown PCR, and parameter is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 45s, 2 circulations of 1 ℃ of operation whenever fall in annealing temperature from 65 ℃ to 50 ℃, and each circulation is extended 1min for 72 ℃; Last 72 ℃ are extended 10min eventually.

Amplified production carries out 1% agarose gel electrophoresis to be identified, sees Fig. 1.Among Fig. 1, the pcr amplification product of 1:DA15-1; The pcr amplification product of 2:DA15-2; The 3:DNA molecular weight standard.The result shows, has successfully amplified the purpose fragment.

3, the segmental acquisition of DA15

By pcr amplification upstream fragment DA15-1 is connected with downstream fragment DA15-2, obtains fragment and be the DA15 fragment.The used primer of pcr amplification is as follows:

PWUO397 (upstream primer): 5 '-GGAATTCTCAATGTAACCAACTTCA-3 ';

PWUO400 (downstream primer): 5 '-CGGGATCCGCGTTTCGGATAAGATG-3 '.

The pcr amplification system:

DA15-1 fragment 1ul

DA15-2 fragment 1ul

dNTP(2.5mM) 1ul

Taq enzyme 2ul

10XTaq?buffer 5ul

pWUM397(20pmol/μl) 1ul

pWUM400(20pmol/l) 1ul

Distilled water complements to 50ul 38ul

Amplified production carries out electrophoresis detection, sees Fig. 2.Among Fig. 2, the 1:PCR product; The 2:DNA molecular weight standard.The result shows, has successfully obtained the DAT fragment that DA15-1 is connected with DA15-2.

4, the structure of pEX18AP-DA15 plasmid

The pEX18AP plasmid that extraction contains AMP resistance, SacB gene and lac-Z gene and contains multiple clone site.To be connected with the DA15 fragment of the step 3 of identical double digestion through the pEX18AP plasmid behind BamHI and the EcoRI double digestion.With the recombinant plasmid called after pEX18AP-DA15 that obtains.

Be transformed into bacillus coli DH 5 alpha with pEX18AP-DA15, 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:

PWUO397 (upstream primer): 5 '-GGAATTCTCAATGTAACCAACTTCA-3 ';

PWUO400 (downstream primer): 5 '-CGGGATCCGCGTTTCGGATAAGATG-3 '.

The results are shown in Figure 3.Among Fig. 3, the 1:DNA molecular weight standard; 2:pEX18AP-DA15 transforms the PCR product of positive strain.

Extract plasmid then and carry out BamHI and the evaluation of EcoRI double digestion.What enzyme was cut evaluation the results are shown in Figure 4.Among Fig. 4, the 1:DNA molecular weight standard; 2: the double digestion product of recombinant plasmid in the positive strain.

With the bacterial strain propagation that contains recombinant plasmid that obtains, be kept at-80 ℃ standby.

Two, the structure of recombinant vectors pEX18AP-DAT

The building process synoptic diagram is seen Figure 10.

1, the genomic extraction of brucella

Get the Bacillus abortus bacterial strain S19 of 15 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, the upstream fragment DAT-1 (403bp) of pcr amplification carboxyuridine phosphate decarboxylase gene and downstream fragment DAT-2 (408bp)

The segmental primer in amplification upstream is as follows:

PWUO659 (upstream primer): 5 '-GGAATTCAAGTCTTCTCACCCACATCC-3 ';

PWUO660 (downstream primer): 5 '-AAGCTTCGTCATCGTGCAATTCTG-3 '.

Add the EcoRI restriction enzyme site in the upstream primer, add the HindIII restriction enzyme site in the downstream primer.

The segmental primer in amplification downstream is as follows:

PWUO661 (upstream primer): 5 '-TGACGAAGCTTCGCTCGATTGGAAGAAGT-3 ';

PWUO662 (downstream primer): 5 '-CGGGATTCCCGTTCGCCTGTAAGGA-3 '.

Add the HindIII 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 primers to carrying out touchdown PCR, and parameter is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 45s, 2 circulations of 1 ℃ of operation whenever fall in annealing temperature from 65 ℃ to 50 ℃, and each circulation is extended 1min for 72 ℃; Last 72 ℃ are extended 10min eventually.

Amplified production carries out 1% agarose gel electrophoresis to be identified, sees Fig. 6.Among Fig. 1, the 1:DNA molecular weight standard; The pcr amplification product of 2:DAT-1; The pcr amplification product of 3:DAT-2.The result shows, has successfully amplified the purpose fragment.

3, the segmental acquisition of DAT

By pcr amplification upstream fragment DAT-1 is connected with downstream fragment DAT-2, obtains fragment and be the DAT fragment.The used primer of pcr amplification is as follows:

PWUO659 (upstream primer): 5 '-GGAATTCAAGTCTTCTCACCCACATCC-3 ';

PWUO662 (downstream primer): 5 '-CGGGATTCCCGTTCGCCTGTAAGGA-3 '.

The pcr amplification system:

DAT-1 fragment 1ul

DAT-2 fragment 1ul

dNTP(2.5mM) 1ul

Taq enzyme 2ul

10XTaq?buffer 5ul

Pwum659(20pmol/μl) 1ul

pWUM662(20pmol/μl) 1ul

Distilled water complements to 50ul 38ul

Amplified production carries out electrophoresis detection, sees Fig. 7.Among Fig. 7, the 1:PCR product; The 2:DNA molecular weight standard.The result shows, has successfully obtained the DAT fragment that DAT-1 is connected with DAT-2.

4, the structure of pEX18AP-DAT plasmid

The pEX18AP plasmid that extraction contains AMP resistance, SacB gene and lac-Z gene and contains multiple clone site.To be connected with the DAT fragment of the step 3 of identical double digestion through the pEX18AP plasmid behind BamHI and the EcoRI double digestion.With the recombinant plasmid called after pEX18AP-DAT that obtains.

Be transformed into bacillus coli DH 5 alpha with pEX18AP-DAT, 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:

PWUO659 (upstream primer): 5 '-GGAATTCAAGTCTTCTCACCCACATCC-3 ';

PWUO662 (downstream primer): 5 '-CGGGATTCCCGTTCGCCTGTAAGGA-3 '.

The results are shown in Figure 8.Among Fig. 8,1:pEX18AP-DAT transforms the PCR product of positive strain; The 2:DNA molecular weight standard.

Extract plasmid then and carry out BamHI and the evaluation of EcoRI double digestion.What enzyme was cut evaluation the results are shown in Figure 9.Among Fig. 9,1: the double digestion product of recombinant plasmid in the positive strain; The 2:DNA molecular weight standard.

With the bacterial strain propagation that contains recombinant plasmid that obtains, be kept at-80 ℃ standby.

Three, the acquisition of the acquisition of recombinant bacterial strain

1, the acquisition of the acquisition RB15 of recombinant bacterial strain

Inoculation 50ul Bacillus abortus bacterial strain S19 cultivates after 24 hours for 37 ℃ in the centrifuge tube that contains the 20mlTSB substratum, 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 recombinant plasmid pEX18AP-DA15 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 (penbritin that contains 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, positive bacteria is dropped into performing PCR identify.

The primer that PCR identifies is as follows:

PWUO397 (upstream primer): 5 '-GGAATTCTCAATGTAACCAACTTCA-3 ';

PWUO400 (downstream primer): 5 '-CGGGATCCGCGTTTCGGATAAGATG-3 '.

The PCR qualification result of recombinant bacterial strain RB15 is seen Figure 11.Among Figure 11, the 1:DNA molecular weight standard; 2: recombinant bacterial strain RB15; 3: Bacillus abortus bacterial strain S19.

The result shows, obtained the Bacillus abortus bacterial strain of deactivation capsular polysaccharide synthetic proteins by double exchange, with its called after recombinant bacterial strain RB15.

2, the acquisition of recombinant bacterial strain RB15T

According to the program of step 1, be female parent with the recombinant bacterial strain RB15 that obtains, by electric transformation technology recombinant plasmid pEX18AP-DAT is imported, the screening positive bacteria is dropped into performing PCR and is identified.

The primer that PCR identifies is as follows:

PWUO659 (upstream primer): 5 '-GGAATTCAAGTCTTCTCACCCACATCC-3 ';

PWUO662 (downstream primer): 5 '-CGGGATTCCCGTTCGCCTGTAAGGA-3 '.

The PCR qualification result of recombinant bacterial strain RB15T is seen Figure 12.Among Figure 12, the 1:DNA molecular weight standard; 2: Bacillus abortus bacterial strain S19; 3: recombinant bacterial strain RB15T.

The result shows, has obtained the Bacillus abortus bacterial strain by double exchange deactivation capsular polysaccharide synthetic proteins gene and carboxyuridine phosphate decarboxylase gene, with its called after recombinant bacterial strain RB15T.

The application of embodiment 2, recombinant bacterial strain RB15T

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 four groups at random, 30 every group.Concrete vaccination regimen is as follows:

First group (test group): the recombinant bacterial strain RB15T of inoculation embodiment 1 preparation, every mouse peritoneal dosage of inoculation 0.1ml includes 10 ⁷Bacterium.

Second group (control group 1): inoculation Bacillus abortus bacterial strain 2308, every mouse peritoneal dosage of inoculation 0.1ml includes 10 ⁷Bacterium.

The 3rd group (control group 2): inoculation Bacillus abortus bacterial strain S19, every mouse peritoneal dosage of inoculation 0.1ml includes 10 ⁷Bacterium.

The 4th group (control group 3): inoculation Bacillus abortus rough type vaccine strains RB51, every mouse peritoneal dosage of inoculation 0.1ml includes 10 ⁷Bacterium.

The 1st, 3,4,6 and 10 weeks from every group of mouse, selected 5 respectively at the inoculation back, carry out spleen and carry bacterium amount and serology detection evaluation.

Spleen carries the bacterium amount sees Figure 13 over time.Among Figure 13, WT represents the experimental result of inoculating strain 2308, and S19 represents the experimental result of inoculating strain S19, and RB15T represents to inoculate the experimental result of recombinant bacterial strain RB15T, and RB51 represents the experimental result of inoculating strain RB51.The result shows: after the recombinant bacterial strain RB15T inoculation, the spleen of mouse carries the bacterium amount and prolongs reduction gradually in time, and removing speed in vivo proves that far away faster than S19 and RB51 this bacterial strain may be much shorter than vaccine strains S19 in the intravital time length of mouse.

The serology detected result shows, (China Veterinery Drug Inspection Office makes the mice serum of inoculation Bacillus abortus reference culture and Bacillus abortus vaccine strains S19 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 1: 100, and the detected result of the mice serum of recombinant bacterial strain RB15T is negative, and the detected result of inoculation recombinant bacterial strain RB6 and RB51 mice serum is negative.

Two, vaccine potency evaluation

Above-mentioned healthy mice is divided into four groups at random, 15 every group.Four groups of mouse are injected recombinant bacterial strain RB15T, Bacillus abortus vaccine strains S19, Bacillus abortus bacterial strain RB51 and PBS respectively, and are specific as follows:

First group (test group): the recombinant bacterial strain RB15T of inoculation embodiment 1 preparation, every mouse peritoneal dosage of inoculation 0.1ml includes 10 ⁷Bacterium.

Second group (vaccine control group): inoculation Bacillus abortus bacterial strain S19, every mouse peritoneal dosage of inoculation 0.1ml includes 10 ⁷Bacterium.

The 3rd group (rough strains of bacteria control group): inoculation Bacillus abortus rough type vaccine strains RB51, every mouse peritoneal dosage of inoculation 0.1ml includes 10 ⁷Bacterium.

The 4th group (blank group): inoculation 0.01M pH7.2 phosphate buffered saline buffer (PBS), every mouse peritoneal dosage of inoculation 0.1ml.

After the above-mentioned bacterial strains inoculation, every group of mouse is divided into three groups (5 of each groups), respectively at inoculation the 12nd week of back, 16 weeks and 20 weeks attacking poison with Bacillus abortus bacterial strain 2308 (Brucella abortus), dosage is 10 ⁶CFU/ml.Take a blood sample after two weeks and cut open extremely and respectively organize mouse, get spleen and weigh and calculate spleen and carry the bacterium quantitative changeization, estimate immune efficacy.Test is provided with three repetitions, results averaged.

The average of spleen carried bacterium quantitative change result as shown in figure 14.Among Figure 14, PBS represents the experimental result of blank group, and S19 represents the experimental result of vaccine control group, and RB15T represents the experimental result of test group, and RB51 represents the experimental result of rough strains of bacteria control group.Mouse body inside fire attack poison is the result show; the immune protection effectiveness of this recombinant bacterial strain RB15T is better than bacterial strain RB51; similar with maternal vaccine strains S19, and this recombinant bacterial strain RB15T has the immunodiagnosis mark, and inoculation animal and clinical infection animal can be differentiated by serological reaction.

Sequence table

＜110〉China Agricultural University

＜120〉a kind of Brucella abortus recombinant strain and the application in the preparation vaccine thereof

<130>CGGNARY92323

<160>12

<210>1

<211>622

<212>PRT

＜213〉Bacillus abortus (Brucella abortus)

<400>1

Met?Ser?Leu?Lys?Tyr?Pro?Trp?Gln?Arg?Leu?Ala?Leu?Leu?Pro?Arg?Ile

1 5 10 15

Ser?Lys?Gln?Ile?Ile?Leu?Val?Leu?Ser?Asp?Cys?Leu?Leu?Leu?Leu?Ala

20 25 30

Ser?Ala?Tyr?Leu?Ala?Phe?Val?Val?Arg?Phe?Gly?Phe?Val?Phe?Val?Pro

35 40 45

Asn?Leu?Ala?Gln?Leu?Phe?Leu?Ile?Leu?Ile?Ala?Pro?Leu?Leu?Ala?Ile

50 55 60

Pro?Val?Phe?Ile?Arg?Phe?Gly?Leu?Tyr?Arg?Ala?Ile?Ile?Arg?Tyr?Leu

65 70 75 80

Ala?Glu?Arg?Ala?Ile?Trp?Ser?Ile?Phe?Gln?Ala?Thr?Ala?Val?Ala?Ala

85 90 95

Leu?Phe?Trp?Val?Ala?Leu?Val?Phe?Leu?Met?Glu?Leu?Tyr?Gly?Ser?Thr

100 105 110

Gly?Leu?Pro?Arg?Ser?Val?Pro?Leu?Leu?Tyr?Trp?Leu?Leu?Ser?Thr?Val

115 120 125

Phe?Ile?Ser?Ala?Ser?Arg?Phe?Gly?Ala?Lys?Trp?Leu?Leu?Arg?Thr?Ala

130 135 140

Glu?His?Asp?Lys?Arg?Tyr?Thr?Ser?Ser?Ala?Leu?Ile?Ile?Gly?Ile?Gly

145 150 155 160

Glu?Pro?Ala?Arg?Gln?Leu?Ala?Thr?Ala?Leu?Arg?Ser?His?Ser?Asp?Thr

165 170 175

Leu?Val?Val?Gly?Phe?Ile?Asp?Pro?Ala?Gly?Gln?Leu?Ala?Gly?Met?Asp

180 185 190

Ile?Ile?Gly?Leu?Arg?Val?Tyr?Arg?Thr?Glu?Glu?Ile?Pro?Ser?Leu?Ile

195 200 205

Glu?Asn?Tyr?Gly?Ile?Lys?Gln?Val?Val?Val?Ser?Glu?Pro?Ala?Leu?Glu

210 215 220

Gln?Lys?Glu?Arg?Gln?Glu?Phe?Ala?Arg?Leu?Leu?Gly?Arg?Leu?Pro?Val

225 230 235 240

Asn?Thr?Arg?Ile?Leu?Pro?Pro?Ile?Ala?Asp?Leu?Thr?Ala?Gly?Lys?Tyr

245 250 255

Leu?Val?Ser?Ala?Leu?Arg?Asn?Val?Glu?Ile?Asp?Asp?Leu?Leu?Gly?Arg

260 265 270

Ser?Pro?Val?Pro?Pro?Asp?Thr?Thr?Leu?Leu?Arg?Glu?Val?Val?Glu?Gly

275 280 285

Arg?Arg?Ile?Met?Ile?Thr?Gly?Ala?Gly?Gly?Ser?Ile?Gly?Ser?Gln?Leu

290 295 300

Cys?Leu?Thr?Ile?Ala?Gln?Trp?Asn?Pro?Ala?Ala?Ile?Val?Leu?Phe?Glu

305 310 315 320

Ser?Ser?Glu?Phe?Ala?Leu?Tyr?Gln?Ile?Asp?Arg?Gln?Leu?Arg?Gln?Phe

325 330 335

Ala?Ser?Cys?Thr?Val?Val?Pro?Val?Leu?Gly?Ser?Val?Arg?Asp?Arg?Ala

340 345 350

Cys?Val?Glu?Lys?Pro?Ile?Arg?Asp?His?Ser?Ile?Asp?Thr?Val?Tyr?His

355 360 365

Cys?Ala?Ala?Tyr?Lys?His?Val?Pro?Leu?Val?Glu?Arg?Asn?Pro?Leu?Val

370 375 380

Gly?Ile?Phe?Asn?Asn?Val?Phe?Gly?Thr?Leu?Glu?Val?Ala?Glu?Ala?Ala

385 390 395 400

Leu?Asn?Thr?Asp?Val?Glu?Arg?Met?Val?Leu?Ile?Ser?Ser?Asp?Lys?Ala

405 410 415

Val?Arg?Pro?Thr?Asn?Val?Met?Gly?Ala?Thr?Lys?Arg?Trp?Ala?Glu?Leu

420 425 430

Val?Ile?Tyr?Tyr?Tyr?Gly?Arg?Leu?Ala?Glu?Gln?Ala?Gly?Lys?Lys?Lys

435 440 445

Ala?Phe?Tyr?Ser?Val?Arg?Phe?Gly?Asn?Val?Leu?Gly?Ser?Asn?Gly?Ser

450 455 460

Val?Val?Pro?Leu?Phe?Arg?Glu?Gln?Ile?Ala?Asn?Gly?Gly?Pro?Val?Thr

465 470 475 480

Leu?Thr?His?Glu?Asp?Met?Thr?Arg?Tyr?Phe?Met?Ser?Ile?Lys?Glu?Ala

485 490 495

Ala?Glu?Leu?Ile?Val?Gln?Ser?Gly?Ala?Ile?Ala?Gln?Ser?Gly?Asp?Thr

500 505 510

Val?Leu?Leu?Glu?Met?Gly?Glu?Pro?Val?Lys?Ile?Arg?Asp?Leu?Ala?Glu

515 520 525

Asn?Met?Ile?Leu?Leu?Ala?Gly?Leu?Thr?Val?Arg?Asn?Glu?Glu?Asn?Pro

530 535 540

Gln?Gly?Asp?Ile?Ala?Ile?Glu?Val?Thr?Gly?Ile?Arg?Glu?Gly?Glu?Lys

545 550 555 560

Met?Tyr?Glu?Glu?Leu?Phe?Tyr?Asp?Pro?Ser?Leu?Ala?Gln?Arg?Thr?Arg

565 570 575

His?Pro?Lys?Ile?Met?Arg?Ala?Pro?Gln?Gly?Ser?Lys?Ala?Val?Val?Asp

580 585 590

Ile?Gln?Glu?Ser?Leu?Ala?Val?Leu?Arg?Thr?Ala?Met?Glu?Lys?Glu?Asp

595 600 605

Ile?Glu?Met?Val?Arg?Lys?Val?Leu?Phe?Glu?Val?Ile?Ala?Ser

610 615 620

<210>2

<211>1869

<212>DNA

＜213〉Bacillus abortus (Brucella abortus)

<400>2

atgagtttaa?aatatccttg?gcaaagattg?gcgcttctgc?cgcgcatcag?caaacagatc 60

attctggtgt?tgagcgattg?cctgttgctg?cttgcaagcg?cctatctagc?ttttgtcgtc 120

cgtttcggtt?tcgtcttcgt?acccaatctg?gcgcagcttt?tcctgatcct?gattgcgccg 180

cttctggcca?ttccggtctt?catccgtttt?gggctttacc?gcgccatcat?tcgttatctg 240

gccgagcgcg?ccatctggtc?gatcttccag?gccaccgcag?tcgccgcctt?gttctgggtg 300

gcgctggtgt?tcctgatgga?gctttacggc?agcaccggcc?tgccgcgttc?tgttccgctt 360

ctttattggc?tcctgagtac?ggttttcatt?tccgcaagcc?gcttcggcgc?aaaatggctg 420

ctacgcactg?ccgaacacga?caagcggtat?accagttcag?ccctgataat?cggcattggc 480

gaaccggcgc?gtcagcttgc?gacggccctt?cgcagccata?gcgatacgtt?ggtggtgggt 540

tttatcgatc?ctgccgggca?actggccgga?atggacatta?ttggcctgcg?tgtctatcgc 600

acggaagaaa?ttccaagcct?gatcgaaaat?tacggtatca?agcaggttgt?cgtttcagaa 660

cccgcgctgg?agcagaagga?gcgccaggag?tttgcgcgcc?ttttggggcg?cctgccggtc 720

aatacgcgca?ttcttccgcc?cattgctgat?ctcacggccg?ggaaatatct?cgttagtgcg 780

ctgcgcaatg?tcgagatcga?tgatcttttg?gggcgttcgc?cggttccgcc?tgatacaacg 840

cttctgcgtg?aggtcgtgga?ggggcggcgc?atcatgataa?ccggcgcggg?cggttccatc 900

ggcagccagc?tttgcctcac?gattgcgcaa?tggaacccgg?ctgcgattgt?tctctttgaa 960

tcgagtgagt?ttgcattata?ccagatcgac?aggcagcttc?gccagtttgc?ctcttgcacg 1020

gtggtgccgg?tccttggctc?cgtgcgtgac?cgcgcctgcg?tggaaaaacc?gatccgcgat 1080

cattccatcg?atacggttta?tcactgtgcg?gcttacaagc?atgtgccgct?ggtggagagg 1140

aacccgctgg?tcggcatttt?caacaatgta?ttcggcacgc?tggaggtggc?cgaggcagcg 1200

ttgaacacgg?atgtggaacg?catggtgctg?atctcttccg?acaaggccgt?gcgccccacc 1260

aatgtcatgg?gcgcgaccaa?gcggtgggcg?gaactggtta?tctattatta?cggacggctg 1320

gctgagcagg?ccggcaagaa?aaaagctttc?tattccgtcc?gttttggcaa?tgtgcttggc 1380

tccaacggtt?cggtagtgcc?gcttttccgc?gagcagattg?caaatggcgg?ccccgttacg 1440

ctgacgcatg?aagacatgac?gcgctatttc?atgtccatca?aggaggcggc?ggaactgatc 1500

gtgcagtccg?gcgcgattgc?ccaatccggt?gataccgttc?ttttggaaat?gggtgagccg 1560

gtgaaaatcc?gcgatctggc?cgaaaacatg?atcctgctcg?cgggactcac?cgtgcgcaac 1620

gaggaaaacc?cgcagggcga?tatcgccatt?gaggtgacgg?gtattcgcga?aggcgagaag 1680

atgtatgaag?agcttttcta?cgatccttcg?ctcgcccagc?gcacccgtca?tccgaaaatc 1740

atgcgtgcgc?cccagggcag?caaggccgtg?gtagatattc?aggaaagcct?ggcggtactg 1800

cggaccgcca?tggaaaagga?ggatatcgaa?atggtccgca?aggtgctttt?cgaggtcata 1860

gcttcctga 1869

<210>3

<211>238

<212>PRT

＜213〉Bacillus abortus (Brucella abortus)

<400>3

Met?Thr?Thr?Glu?Leu?His?Asp?Asp?Ala?Ser?Gly?Arg?Leu?Ile?Val?Gly

1 5 10 15

Leu?Asp?Val?Pro?Thr?Ile?Ala?Glu?Ala?Glu?Lys?Val?Val?Glu?Glu?Leu

20 25 30

Gly?Asn?Ala?Val?Ser?Phe?Tyr?Lys?Ile?Gly?Tyr?Gln?Leu?Val?Phe?Ala

35 40 45

Gly?Gly?Leu?Asp?Phe?Ala?Lys?Ser?Leu?Val?Ala?Ala?Arg?Lys?Lys?Val

50 55 60

Phe?Leu?Asp?Met?Lys?Leu?Leu?Asp?Ile?Asp?Asn?Thr?Ile?Ala?Lys?Gly

65 70 75 80

Val?Glu?Asn?Val?Ala?Lys?Met?Gly?Val?Ser?Met?Leu?Thr?Leu?His?Ala

85 90 95

Tyr?Pro?Lys?Ala?Met?Arg?Ala?Ala?Val?Glu?Ala?Ala?Arg?Gly?Ser?Asp

100 105 110

Leu?Cys?Leu?Leu?Gly?Val?Thr?Val?Leu?Thr?Ser?Met?Asp?Asn?Ala?Asp

115 120 125

Leu?Arg?Glu?Ala?Gly?Tyr?Phe?Asp?Asn?Ala?Glu?Thr?Leu?Val?Leu?Lys

130 135 140

Arg?Ala?Arg?Gln?Ala?His?Glu?Ala?Gly?Met?Gly?Gly?Ile?Val?Ala?Ser

145 150 155 160

Ala?Val?Glu?Ala?Gln?Ala?Ile?Arg?Gln?Ala?Val?Arg?Pro?Asp?Met?Ala

165 170 175

Ile?Val?Thr?Pro?Gly?Ile?Arg?Pro?Ala?Gly?Ser?Glu?Lys?Gly?Asp?Gln

180 185 190

Lys?Arg?Val?Met?Thr?Pro?Ala?Asp?Ala?Leu?Arg?Ala?Gly?Ala?Ser?His

195 200 205

Leu?Ile?Val?Ala?Arg?Pro?Ile?Val?Gly?Ala?Pro?Asp?Arg?Lys?Ala?Ala

210 215 220

Ala?Leu?Ala?Ile?Leu?Lys?Glu?Met?Arg?Ser?Ile?Gly?Arg?Ser

225 230 235

<210>4

<211>717

<212>DNA

＜213〉Bacillus abortus (Brucella abortus)

<400>4

atgacgacag?aattgcacga?tgacgcatcc?gggcgcctga?tcgtgggcct?tgacgtgccg 60

acaattgccg?aggcggaaaa?ggtggtcgaa?gaactgggca?atgctgtttc?cttctacaag 120

atcggctatc?agcttgtttt?tgccggtggg?cttgatttcg?ccaaaagcct?tgtggcggcg 180

cgcaaaaaag?tcttcctcga?catgaagctg?ctcgatatcg?acaacacgat?tgccaaaggc 240

gtggaaaatg?tcgcgaagat?gggtgtttcc?atgctcacgc?ttcatgccta?tccgaaggca 300

atgcgtgcgg?cggtggaagc?cgccagaggg?tcggacctat?gccttttggg?cgtgacggtc 360

ctgacctcca?tggacaatgc?cgatcttcgg?gaagccggtt?atttcgacaa?tgcggaaacg 420

ctggtgctga?aacgtgcccg?tcaggcgcat?gaggccggta?tgggcggcat?tgtcgcctcg 480

gcggtggagg?cgcaggccat?tcgccaggcg?gttcgccccg?atatggccat?cgtcacaccg 540

ggcatccgcc?cggcagggag?cgagaagggc?gaccagaagc?gcgtgatgac?gccagccgat 600

gccttgaggg?ccggtgcgag?ccatctcatc?gtggcgcgcc?cgatcgtggg?cgcaccggat 660

cgcaaggcgg?cagcccttgc?catcctgaag?gaaatgcgct?cgattggaag?aagttag 717

<210>5

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>5

ggaattctca?atgtaaccaa?cttca 25

<210>6

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>6

aagcttagag?gtcatacctt?cctg 24

<210>7

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>7

cctctaagct?tatctgtttg?ctgatgcg 28

<210>8

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>8

cgggatccgc?gtttcggata?agatg 25

<210>9

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>9

ggaattcaag?tcttctcacc?cacatcc 27

<210>10

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>10

aagcttcgtc?atcgtgcaat?tctg 24

<210>11

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>11

tgacgaagct?tcgctcgatt?ggaagaagt 29

<210>12

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>12

cgggattccc?gttcgcctgt?aagga 25

Claims (6)

1. Brucella abortus recombinant strain strain is the bacterial strain that the capsular polysaccharide synthetic proteins gene among Bacillus abortus (Brucella abortus) the bacterial strain S19 and the deactivation of carboxyuridine phosphate decarboxylase gene are obtained;

Described capsular polysaccharide synthetic proteins is shown in the sequence 1 of sequence table; Described carboxyuridine phosphate decarboxylase is shown in the sequence 3 of sequence table.

2. recombinant bacterial strain as claimed in claim 1 is characterized in that: the encoding sequence of described capsular polysaccharide synthetic proteins is shown in the sequence 2 of sequence table; The encoding sequence of described carboxyuridine phosphate decarboxylase is shown in the sequence 4 of sequence table.

3. recombinant bacterial strain as claimed in claim 1 or 2 is characterized in that: described deactivation realizes by homologous recombination.

4. recombinant bacterial strain as claimed in claim 3 is characterized in that:

The homologous recombination of the described capsular polysaccharide synthetic proteins of described deactivation gene imports described Bacillus abortus bacterial strain S19 with dna fragmentation DA15 and realizes; Described dna fragmentation DA15 comprises homology arm DA15-1 and homology arm DA15-2 successively to the downstream from the upstream; Described homology arm DA15-1 is that the genomic dna with Bacillus abortus bacterial strain S19 is a template, and the primer of forming with sequence 5 and sequence 6 is to carrying out the dna fragmentation that pcr amplification obtains; Described homology arm DA15-2 is that the genomic dna with Bacillus abortus bacterial strain S19 is a template, and the primer of forming with sequence 7 and sequence 8 is to carrying out the dna fragmentation that pcr amplification obtains;

The homologous recombination of the described carboxyuridine phosphate decarboxylase gene of described deactivation imports described Bacillus abortus bacterial strain S19 with dna fragmentation DAT and realizes; Described dna fragmentation DAT comprises homology arm DAT-1 and homology arm DAT-2 successively to the downstream from the upstream; Described homology arm DAT-1 is that the genomic dna with Bacillus abortus bacterial strain S19 is a template, and the primer of forming with sequence 9 and sequence 10 is to carrying out the dna fragmentation that pcr amplification obtains; Described homology arm DAT-2 is that the genomic dna with Bacillus abortus bacterial strain S19 is a template, and the primer of forming with sequence 11 and sequence 12 is to carrying out the dna fragmentation that pcr amplification obtains.

5. the application of arbitrary described recombinant bacterial strain in preparation Bacillus abortus disease vaccine in the claim 1 to 4.

6. Bacillus abortus disease vaccine, its activeconstituents is arbitrary described Brucella abortus recombinant strain strain in the claim 1 to 4.

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