CN103571867B - Brucella melitensis recombinant bacterial strain M5-Δ znuA and preparation method and application - Google Patents

Abstract

The present invention discloses a strain Brucella melitensis recombinant bacterial strain M5-Δ znuA and preparation method and application.The present invention passes through with Brucella melitensis M5 genomic dna for template, and pcr amplification obtains the upstream and downstream homology arm of znuA gene; The upstream and downstream homology arm of znuA gene is connected, obtains fragment Δ znuA; Fragment Δ znuA is cloned on carrier pRE112, obtains Suicide homologous recombination plasmid pRE-Δ znuA; This plasmid is imported in Brucella melitensis M5 cell, under the selective pressure of paraxin, obtain homologous recombination list recon, then homologous recombination list recon is coated on Selective agar medium, obtain Brucella melitensis recombinant bacterial strain M5-Δ znuA.This bacterial strain is M5 bacterial strain comparatively, has good growth characteristics and genetic stability, and virulence is little and immune effect suitable, and therefore, it is expected to become new generation vaccine and applies.

Description

Brucella melitensis recombinant bacterial strain M5-Δ znuA and preparation method and application

Technical field

The invention belongs to recombinant bacteria vaccines arts, particularly strain Brucella melitensis (Brucellamelitensis) recombinant bacterial strain M5-Δ znuA and preparation method and application.

Background technology

Brucellosis is the Arbo infectious disease caused by brucella, and this disease of prevention and control is apply vaccine to put prevention first always.Brucella vaccine kind is more, and traditional Attenuate vaccine low cost, the immunoprotection time length is long, immunoprotection efficiency is high, but there is toxicity comparatively great Yi reversion, cannot distinguish the shortcoming such as artificial immunization and natural infection; Recombinant protein vaccine and DNA vaccination have that security is high, preparation technology is simple, conveniently store advantages such as transporting, but also there is the limited shortcoming of immune effect difference, protectiveness; Though inactivated vaccine is safer in use, because the protection period is short, preparation cost is high and be difficult to widespread use.At present, attenuated strain for brucellosis prevention and control mainly contains external ox type No. 19 less-virulent strains (S19) and sheep type ReV.1 less-virulent strain, sheep type No. 5 (M5) less-virulent strains of domestic development and pig type No. 2 (S2) less-virulent strains, but the same existing defects of attenuated vaccine, after these smooth type brucella attenuated vaccine immunity animals, S-LPS antigen " O " chain specific antibody can be produced in animal body induction, be difficult to distinguish mutually with street strain Infected with Brucella thus disturb the diagnosis of conventional brucellosis.If the such vaccine of widespread use just to this sick epidemiology survey, detect epidemic disease source and produce very large obstacle, in addition, existing brucellar attenuated live vaccine all to animal generation side effect in various degree, more also can infect the mankind while causing animal slightly to miscarry.So build security high, without reversion, natural infection and artificial active immunity can be distinguished, to mark attenuated vaccine be the task of top priority for brucella that protective value is good.

In addition, brucellar existence and breed the multiple transition metal ion that must depend among environment or host.Especially zine ion is the necessary metal ion of brucella structure, is the cofactor of catalyzed reaction in thalline, and therefore, the zinc obtaining suitable degree is concerning extremely important bacterium.But because zinc ion content free in host is considerably less, the metabolism of bacterium self is ensured in order to absorb enough zinc, and breed in a large number in host, various bacterium as Bacillus brucellae evolve out several types albumen participate in picked-up and transhipment zine ion, the albumen maneuvering system of these transhipment zine ions is named as ZnuABC, this system comprises znuA, znuB, the multiple protein of znuC genes encoding, wherein ZnuA is a kind of periplasmic-solute binding proteins, still less to the study mechanism of this gene at present, only infer that this ZnuA albumen is relevant to Bacillus brucellae virulence factor.This research is intended to acquisition one strain and can lowers virulence, improve security, the efficient immunogenic brucellosis attenuated vaccine of vaccine can be maintained again, we will express the gene knockout of ZnuA albumen, to obtain a strain Brucella melitensis (Brucella melitensis) recombinant bacterial strain M5-Δ znuA and preparation method and application, lay the foundation for preparing safer effective novel gene engineered vaccine.

Summary of the invention

In order to overcome the shortcoming of above-mentioned existing vaccine with not enough, research brucella znuA genetically deficient is on the impact of vaccine strains.

Primary and foremost purpose of the present invention is the preparation method providing a strain Brucella melitensis recombinant bacterial strain M5-Δ znuA.

Another object of the present invention is to provide the Brucella melitensis recombinant bacterial strain M5-Δ obtained by above-mentioned preparation method znuA.

Still a further object of the present invention is to provide above-mentioned Brucella melitensis recombinant bacterial strain M5-Δ znuA for the preparation of the application prevented and/or treated in the vaccine of brucellosis.

Object of the present invention is realized by following proposal:

The preparation method of one strain Brucella melitensis recombinant bacterial strain M5-Δ znuA, comprises the following steps:

(1) structure of Suicide homologous recombination plasmid

1. be that template carries out PCR with Brucella melitensis (Brucella melitensis) M5 genomic dna, when the primer of use is for P1 and P2, obtain the upstream homology arm of znuA gene; When the primer used is for P3 and P4, obtain the downstream homology arm of znuA gene;

P1:5 '-GAT gGTACCcGTCCTCGTTTGCTTGTGC-3 ' (horizontal line part is KpnI restriction enzyme site);

P2：5′-TCGCTGAAAGACTGCCTGT-3′；

P3：5′-GCAGTCTTTCAGCGAAGCCAGAAAGGCAGAAGC-3′；

P4:5 '-AGA gAGCTCcAATGTCCCCTTGGTCCC-3 ' (horizontal line part is Sac1 restriction enzyme site);

2. utilize the method for Overlap PCR, with step, 1. the upstream homology arm of the znuA gene of middle preparation and the downstream homology arm of znuA gene are for template, P1 and P4 is primer pair, obtains the object fragment Δ znuA for homologous recombination;

3. double digestion is carried out, purifying to suicide plasmid carrier pRE112 with for the object fragment Δ znuA of homologous recombination respectively with KpnI and SacI; The fragment Δ znuA that purifying reclaims is connected with carrier segments pRE112, obtains Suicide homologous recombination plasmid pRE-Δ znuA;

(2) structure of Brucella melitensis recombinant bacterial strain and screening

1. Suicide homologous recombination plasmid pRE-Δ znuA is imported in the competent cell of Brucella melitensis M5; Then coat on Triptic soya yeast extract agar substratum (TSA-YE) flat board containing paraxin, under the selective pressure of paraxin, obtain homologous recombination list recon; PCR is identified correct homologous recombination list recon is cultivated in Triptic soya yeast extract broth culture (TSB-YE), lax plasmid resistance;

2. then the homologous recombination list recon after lax plasmid resistance is coated on TSA-YE-Sucrose Selective agar medium and cultivate, utilize the susceptibility of sacB gene pairs sucrose in Suicide homologous recombination plasmid pRE-Δ znuA, suicide plasmid is eliminated, primer P1 and P4 is utilized to carry out PCR screening to the bacterium colony that TSA-YE-Sucrose Selective agar medium grows, obtain homologous recombination double exchange, namely obtain Brucella melitensis recombinant bacterial strain M5-Δ znuA;

Step (1) 1. described in the condition optimization of PCR be 94 DEG C of 2min denaturations; 98 DEG C of 10s, 56 DEG C of 30s, 68 DEG C of 1.5min, 30 circulations; 68 DEG C of 5 ~ 10min extend eventually;

Step (1) 2. described in the condition optimization of Overlap PCR be 94 DEG C of 2min denaturations; 98 DEG C of 10s, 56 DEG C of 30s, 68 DEG C of 3min, 30 circulations; 68 DEG C of 5 ~ 10min extend eventually;

Step (2) 1. described in the mode of importing import preferably by electric transform mode;

The condition optimization that described electricity transforms is 1mm pole cup, 1.8kv, 400 Ω;

Step (2) 1. described in the final concentration of paraxin in described Triptic soya yeast extract agar substratum (TSA-YE) be preferably 5 μ g/mL;

Step (2) 1. described in the condition optimization of cultivation be 37 DEG C, about 18h cultivated by 200r/min shaking table;

Step (2) 1. described in PCR be accredited as and identified by primer P1 and P4;

Step (2) 1. described in PCR qualification condition optimization as follows: 94 DEG C of 2min denaturations; 98 DEG C of 10s, 56 DEG C of 30s, 68 DEG C of 3min, 30 circulations; 68 DEG C of 5 ~ 10min;

Step (2) 2. described in TSA-YE-Sucrose Selective agar medium composed as follows: Triptic soya yeast extract agar substratum (TSA-YE)+final concentration is the sucrose of mass volume ratio (g/mL) 7%;

Described TSA-YE-Sucrose Selective agar medium prepares preferably by following steps: by after Triptic soya yeast extract agar substratum (TSA-YE) sterilizing for preparing, the sucrose that concentration degerming is after filtration mass volume ratio (g/mL) 50% is added when being cooled to 60 ~ 70 DEG C, its final concentration is made to be mass volume ratio (g/mL) 7%, be distributed into after mixing in sterilizing plate, solidify rear 4 DEG C of preservations.

Step (2) 2. described in the condition optimization of cultivation be 37 DEG C and cultivate 48 ~ 96h;

One strain Brucella melitensis recombinant bacterial strain M5-Δ znuA, is obtained by above-mentioned preparation method.

Above-mentioned Brucella melitensis recombinant bacterial strain M5-Δ znuA is for the preparation of the application prevented and/or treated in the vaccine of brucellosis.

The present invention, relative to prior art, has following advantage and beneficial effect:

(1) Brucella melitensis recombinant bacterial strain M5-Δ znuA provided by the invention has lower virulence compared with parent plant M5, after inoculation BALB/c mouse, spleen swelling degree is little, ponderal index is low, Inflammatory response is weak, spleen bacterium quantity is few and it is fast to clear out of external speed.Meanwhile, this bacterial strain maintains good immunogenicity, and the antibody horizontal of anti-BLS and L7/L12 of generation is suitable with parent plant M5.

(2) experiment proves that Brucella melitensis recombinant bacterial strain M5-Δ znuA provided by the invention has good genetic stability, its growth characteristics and immune protective effect equal no significant difference compared with Brucella melitensis parent vaccine strain M5.

(3) Brucella melitensis recombinant bacterial strain M5-Δ znuA provided by the invention lays the foundation for preparing safer effective novel gene engineered vaccine.

Accompanying drawing explanation

Fig. 1 is the agarose gel electrophoresis figure of the gene fragment Δ znuA that the upstream and downstream homology arm gene fragment of znuA gene and upstream and downstream homology arm gene fragment Overlap PCR thereof obtain, wherein: swimming lane M is DNA molecular quality standard, swimming lane 1 is the upstream homology arm gene fragment of znuA, swimming lane 2 is the downstream homology arm gene fragment of znuA, and swimming lane 3 is that the upstream and downstream homology arm gene fragment of znuA gene is connected the gene fragment Δ znuA obtained.

Fig. 2 is the agarose gel electrophoresis figure of pRE-Δ znuA digestion verification result, wherein: swimming lane M is DNA molecular quality standard, swimming lane 1 is the pRE-Δ znuA plasmid cut without enzyme, and swimming lane 2 is the fragment obtained after using KpnI and SacI double digestion pRE-Δ znuA plasmid.

Fig. 3 is the agarose gel electrophoresis figure of Brucella melitensis gene-deleted strain M5-Δ znuA homologous recombination list recon screening, wherein: swimming lane M is DNA molecular quality standard, swimming lane 1 is pRE-Δ znuA positive control, swimming lane 2 is M5 positive control, and swimming lane 3 ~ 6 is M5-Δ znuA strain homologous recombination list recon; Swimming lane 7 is blank.

Fig. 4 is the agarose gel electrophoresis figure of Brucella melitensis gene-deleted strain M5-Δ znuA homologous recombination double exchange screening, wherein: swimming lane M is DNA molecular quality standard, swimming lane 1 is pRE-Δ znuA positive control, swimming lane 2 is M5 positive control, swimming lane 3 ~ 20 is homologous recombination double exchange, and swimming lane 21 is blank.

Fig. 5 is the result figure of Brucella melitensis recombinant bacterial strain M5-Δ znuA genetic stability, wherein: swimming lane M is DNA molecular quality standard, swimming lane 1 is pRE-Δ znuA positive control, swimming lane 2 is M5 positive control, swimming lane 3 ~ 12 is respectively M5-Δ znuA the 2nd, 4,6,8,10,12,14,16,18,20 generation bacterium colony, and swimming lane 13 is blank.

Fig. 6 is the measurement result figure of Brucella melitensis parent plant M5 and recombinant bacterial strain M5-Δ znuA growth curve.

Fig. 7 is the result figure on mouse spleen weight impact after Brucella melitensis parent plant M5 and recombinant bacterial strain M5-Δ znuA immune mouse.

Fig. 8 is to the result figure that mouse spleen bacterium quantity detects after Brucella melitensis parent plant M5 and recombinant bacterial strain M5-Δ znuA immune mouse.

Fig. 9 is the ELISA detected result figure of Brucella melitensis parent plant M5 and recombinant bacterial strain M5-Δ znuA immune mouse mice serum specific antibody after 8 weeks.

Embodiment

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

Material involved in embodiment:

1. KOD FX DNA Polymerase high-fidelity enzyme is purchased from TOYOBO company; Restriction enzyme Kpn I, Sac I, T4DNA ligase enzyme, DNA Marker etc. are purchased from TaKaRa company; Glue reclaims test kit, mini-scale plasmid extraction test kit is Omega Products; QIAamp DNA extraction kit is QIAGEN Products; Pancreas peptone soybean broth (TSB), agar powder are purchased from Huankai Microbes Tech Co., Ltd., Guangdong; Yeast extract (Yeast Extract is called for short YE) is purchased from OXOID company.

Triptic soya yeast extract broth culture (TSB-YE) prepares as follows: pancreas peptone soybean broth 30g, YE2g, be dissolved in 1000mL tri-distilled water, heating mixes, 121 DEG C of autoclaving 15min, and cooling is placed on 4 DEG C of preservations.

Triptic soya yeast extract agar substratum (TSA-YE) prepares as follows: pancreas peptone soybean broth (TSB) 30g, YE2g, be dissolved in 1000mL tri-distilled water, adding agar powder to final concentration is 1.5%(w/v), after heated and stirred is even, 121 DEG C of autoclaving 15min, to be cooledly to be distributed in sterilizing plate to about 60 DEG C, to solidify rear 4 DEG C of preservations.

Triptic soya yeast extract agar substratum (TSA-YE) containing paraxin prepares as follows: by after Triptic soya yeast extract agar substratum (TSA-YE) sterilizing for preparing, paraxin (final concentration is 5 μ g/mL) is added when being cooled to 40 ~ 50 DEG C, be distributed into after mixing in sterilizing plate, solidify rear 4 DEG C of preservations.

TSA-YE-Sucrose Selective agar medium prepares as follows: by after Triptic soya yeast extract agar substratum (TSA-YE) sterilizing for preparing, the sucrose that concentration degerming is after filtration mass volume ratio (g/mL) 50% is added when being cooled to 60 ~ 70 DEG C, the final concentration of sucrose is made to be mass volume ratio (g/mL) 7%, be distributed into after mixing in sterilizing plate, solidify rear 4 DEG C of preservations.

2. Brucella melitensis (Brucella melitensis) M5 strain is purchased from Xinjiang Tiankang Raise Livestock Biotechnology Co., Ltd;

3. following biomaterial is all open at document " structure of brucella bp26 gene-deleted strain; Chinese veterinary science; 2011; 41(03): 280-286 ": bacillus coli DH 5 alpha λ pir(genotype is supE44 Δ lacU169 (φ 80lacZ Δ M15) hsdR17recA1endA1gyrA96thi-relA1 λ pi) and pRE112 suicide plasmid (cat, sacB, oriT _rP4, oriV _r6K, MCS).

4. following methods is all open in patent application " Brucella abortus recombinant bacterial strain S19-Δ bp26-BL and preparation method thereof and application, 201310153715.6 ": the preparation method of BL fusion rotein.

Embodiment 1

(1) structure of Suicide homologous recombination plasmid

1. the extraction of bacterium genomic dna: illustrate according to QIAamp DNA extraction agent box and extract Brucella melitensis M5 pnca gene group DNA.

2. the Design and synthesis of primer: with reference to the gene order of Brucella melitensis (CP001489.1) in Genbank, experimental Demand Design primer, as shown in table 1.Wherein primer P1 and P2 is for the upstream homology arm of the znuA gene that increases, primer P3 and P4 for the downstream homology arm of the znuA gene that increases, and introduces the restriction enzyme site of KpnI and SacI respectively at the 5 ' end of primer P1 and P4.Above-mentioned primer synthesizes by Shanghai Li Fei Bioisystech Co., Ltd.

Table 1 is for the PCR primer sequence of the different genes fragment that increases

Primer	Sequence 5 ' → 3 '
		P1	5′-GATGGTACCCGTCCTCGTTTGCTTGTGC-3′
P2	5′-TCGCTGAAAGACTGCCTGT-3′
		P3	5′-GCAGTCTTTCAGCGAAGCCAGAAAGGCAGAAGC-3′
P4	5′-AGAGAGCTCCAATGTCCCCTTGGTCCC-3′

3. the pcr amplification of different genes fragment: with the genomic dna of Brucella melitensis M5 strain for template, the upstream and downstream homology arm fragment of the znuA gene that increases respectively with corresponding primer (P1/P2, P3/P4); Utilize the method for Overlap PCR, with the upstream and downstream homology arm of znuA gene for template, increase with primer P1 and P4, obtain the fragment Δ znuA of the disappearance znuA gene that arm before and after is connected.The PCR reaction system of the upstream and downstream homology arm of amplification znuA gene is: 2 × L Buffer12.5 μ L, dNTPs5 μ L, P1(P3) and P2(P4) each 1 μ L, KOD-FX enzyme 0.5 μ L, DNA profiling 1.0 μ L, adds ddH ₂o makes cumulative volume reach 25 μ L; Response procedures is: 94 DEG C of 2min; 98 DEG C of 10s, 56 DEG C of 30s, 68 DEG C of 1.5min, 30 circulations, 68 DEG C of 5 ~ 10min.The Overlap PCR reaction system of amplification Δ znuA fragment is each 1.0 μ L of 2 × LBuffer12.5 μ L, dNTPs5 μ L, P1 and P4, and KOD-FX enzyme 1.5 μ L, znuA upstream region of gene homology arm 1.0 μ L and znuA downstream of gene homology arm 1.0 μ L, add ddH ₂o makes cumulative volume reach 25 μ L: response procedures is: 94 DEG C of 2min; 98 DEG C of 10s, 56 DEG C of 30s, 68 DEG C of 3min, 30 circulations, 68 DEG C of 5 ~ 10min.

Detected by the agarose gel electrophoresis of mass volume ratio (g/mL) 1%, result as shown in Figure 1, the size of the upstream homology arm of znuA gene is 1149bp, the size of the downstream homology arm of znuA gene is 1098bp, the be connected size of the gene fragment Δ znuA obtained of the upstream and downstream homology arm gene fragment of znuA gene is 2247bp, conforms to theoretical value.

4. the structure of Suicide homologous recombination plasmid: respectively double digestion is carried out to suicide plasmid carrier pRE112 and Δ znuA with KpnI and SacI, wherein, plasmid pRE112 double digestion reaction system is: 10 × LBuffer2.0 μ L, the each 2.0 μ L of KpnI and SacI, pRE112(concentration≤1 μ g/ μ L) 2.0 μ L, add ddH ₂o makes cumulative volume reach 20 μ L; DNA fragmentation Δ znuA double digestion reaction system is: 10 × LBuffer2.0 μ L, KpnI and SacI each 2.0 μ L, Δ znuA6.0 μ L, adds ddH ₂o makes cumulative volume reach 20 μ L.Fragment Δ znuA enzyme being cut the recovery of rear purifying is connected with carrier segments pRE112 T4DNA ligase enzyme (reaction system and the operation of reaction conditions by specification), and product conversion bacillus coli DH 5 alpha λ pir will be connected, primer P1 and P4 is used through PCR() identify that correct positive colony carries out plasmid extraction, KpnI and SacI double digestion is verified, result as shown in Figure 2, the fragment that enzyme cuts rear acquisition is identical with object clip size, carry out gene sequencing, the sequence of the sequence obtained and Δ znuA is completely the same, shows to obtain Suicide homologous recombination plasmid pRE-Δ znuA.

(2) structure of Brucella melitensis recombinant bacterial strain M5-Δ znuA and screening

1. the preparation of Brucella melitensis M5 competent cell: single bacterium colony of inoculation M5 strain, in 100mLTSB-YE substratum, 37 DEG C, 200r/min shaking table is cultivated, is cultured to OD600 and is about 0.6, cooled on ice 30min.The centrifugal 10min of 4000r/min, discards substratum, washs 3 times with the aqueous glycerin solution of volume percent 10%.Finally the thalline of acquisition is resuspended in the aqueous glycerin solution of 2.5mL volume percent 10%, namely obtains Brucella melitensis M5 competent cell.Be sub-packed in the Eppendorf tube of precooling by 100 μ L/ pipes, put-80 DEG C of Refrigerator stores for subsequent use.

2. transformed by electricity, Bio-rad electric shock cup 1mm, 1.8kv, recombination suicide vector pRE-Δ znuA imports in Brucella melitensis M5 competent cell by 400 Ω, bacterium liquid after being turned by electricity is coated on the TSA-YE flat board containing paraxin (5 μ g/mL), cultivate more than 48h for 37 DEG C, under the selective pressure of paraxin, obtain pRE-Δ znuA and be integrated into homologous recombination list recon on Brucella melitensis M5 genome.Utilize primer P1 and P4 to carry out PCR screening to bacterium colony, PCR response procedures is: 94 DEG C of 2min denaturations; 98 DEG C of 10s, 56 DEG C of 30s, 68 DEG C of 4min, 30 circulations; 68 DEG C of 5 ~ 10min.PCR qualification result as shown in Figure 3, has band at 3180bp and 2247bp place respectively, proves to obtain homologous recombination list recon.

3. bacterium colony PCR is identified correct homologous recombination list recon in TSB-YE 37 DEG C, 18h cultivated by 200r/min shaking table, after lax plasmid resistance, suitable dilution spread is in TSA-YE-Sucrose Selective agar medium, cultivate more than 48h for 37 DEG C, utilize the susceptibility of sacB gene pairs sucrose in plasmid pRE-Δ znuA, obtain homologous recombination double exchange.Utilize primer P1 and P4 to carry out PCR screening to bacterium colony, PCR response procedures is: 94 DEG C of 2min denaturations; 98 DEG C of 10s, 56 DEG C of 30s, 68 DEG C of 4min, 30 circulations; 68 DEG C of 5 ~ 10min.There are two kinds of situations: one is that pRE-Δ znuA is again from karyomit(e) disappearance (reverse mutation) at homologous recombination double exchange of TSA-YE-Sucrose grow on plates; Two is that Δ znuA gene integration is to genome, and lose in parent znuA gene swapping to carrier, PCR qualification result is done as shown in Figure 4 to homologous recombination double exchange, band is 2247bp(and swimming lane 5,11,12,13) homologous recombination double exchange son be the Brucella melitensis recombinant bacterial strain that will screen, by its called after Brucella melitensis recombinant bacterial strain M5-Δ znuA.

(3) the genetic stability checking of Brucella melitensis recombinant bacterial strain M5-Δ znuA

Obtained Brucella melitensis recombinant bacterial strain M5-Δ znuA was reached for 20 generations continuously on TSA-YE flat board, with primer P1/P4, pcr amplification is carried out to the bacterium colony on TSA-YE flat board, in every two generations, carry out a bacterium colony PCR and order-checking qualification, the genetic stability of checking recombinant bacterium, result as shown in Figure 5, all increase and obtain the band of 2247bp, show that M5-Δ znuA has good genetic stability.

(4) growth curve of Brucella melitensis recombinant bacterial strain M5-Δ znuA measures

By M5 and M5-Δ znuA respectively at the flat lining out of TSA-YE, after growing single bacterium colony, by single colony inoculation in fresh TSB-YE nutrient solution, 37 DEG C, 200r/min shakes bacterium and is about 20h, logarithmic phase is reached to bacterium, collect thalline, bacterial concentration is adjusted to same numerical value (OD600=0.6), be inoculated in 200mL TSB-YE nutrient solution according to the ratio of volume ratio 1:100,37 DEG C, 200r/min cultivation, take out a certain amount of bacterium liquid every 4h, measures OD value and also do 10 times of serial dilutions, be applied on TSA-YE flat board, each extent of dilution 3 flat boards.After bacterium colony grows, count and add up, drawing growth curve as shown in Figure 6, through the growth characteristics of statistical analysis two kinds of bacterial strains there are no significant difference.

(5) Brucella melitensis recombinant bacterial strain M5-Δ znuA virulence determination test

By the female BAl BIc in 36 5 ~ 6 week ages/c mouse (Guangdong Medical Lab Animal Center), be divided into 3 groups by often organizing 12.By 1 × 10 ⁸the dosage of inoculation intraperitoneal inoculation M5 and M5-Δ znuA respectively of CFU/ mouse, simultaneously using the PBS solution of 10mM, pH7.4 as blank group.BALB/c mouse neck is broken execution in after inoculation the 2nd, 4,6,8 week, often organize 3, eyeball venous blood collection, centrifuging and taking serum.Aseptic spleen of getting is weighed and by spleen homogenate, is made 10 times of serial dilutions after homogenate, and get 100 μ L and be coated with TSA-YE flat board, 37 DEG C of cultivations, laggard row data statistic analysis appears in bacterium colony.

The result display of Fig. 7, from spleen weight, mouse immune is after 2 weeks, and the spleen swelling degree caused by M5-Δ znuA is obviously weaker than parent plant M5, and the two has significant difference; Latter 4,6,8 weeks of immunity, the spleen swelling degree caused by M5-Δ znuA is slightly weak compared with parent plant M5, shows that the inflammatory reaction that M5-Δ znuA strain causes when infecting mouse is less than parent plant M5.The result display of Fig. 8, from spleen colony number, latter 2,4,6,8 weeks of immunity, M5-Δ znuA bacterial strain bacterium quantity of (in spleen) in Mice Body is few compared with parent plant M5, there is significant difference, and after immunity 6 weeks, there is decline sharply than parent plant M5 bacterium quantity of (in spleen) in Mice Body in M5-Δ znuA bacterial strain, infers that recombinant bacterial strain can substantially be cleared out of external by mouse in the process of relative Long-term Infection.Above result shows that the Inflammatory response that B.melitensis recombinant bacterial strain M5-Δ znuA causes compared with parent plant M5 is weak, and virulence is little, infers that znuA gene is brucellar virulence correlation factor.

(6) Brucella melitensis recombinant bacterial strain M5-Δ znuA specific antibody level detection experiment

By the female BAl BIc in 36 5 ~ 6 week ages/c mouse (Guangdong Medical Lab Animal Center), be divided into 3 groups by often organizing 12.By 5 × 10 ⁵the dosage of inoculation intraperitoneal inoculation M5 and M5-Δ znuA respectively of CFU/ mouse, simultaneously using the PBS solution of 10mM, pH7.4 as blank group.BALB/c mouse neck was broken execution in after inoculation the 2nd, 4,6,8 week, often organize 3, eyeball venous blood collection, centrifuging and taking serum is as sample to be checked, the BL fusion rotein prepared with this laboratory is as antigen coated elisa plate, detect the level of the specific antibody of anti-BL in serum, and carry out statistical analysis, step is as follows:

1. the preparation of BL fusion rotein: 1. prepare by patent application 201310153715.6 embodiment 1 step (6), specific as follows: respectively double digestion to be carried out to pET32a expression vector and the correct Δ bp26-BL of gene sequencing with KpnI and SacI, the reaction system of double digestion is: 10 × L Buffer2.0 μ L, the each 2.0 μ L of KpnI and SacI, pET32a(concentration≤1 μ g/ μ L) 2.0 μ L, Δ bp26-BL6.0 μ L, adds ddH ₂o makes cumulative volume reach 20 μ L.The fragment Δ bp26-BL reclaimed by purifying is connected with carrier segments pET32a T4DNA ligase enzyme (reaction system and the operation of reaction conditions by specification), and connection product conversion is entered to express in bacterium e. coli bl21 (DE3) (purchased from Novagen company, production code member: 69449-3).The mono-colony inoculation of picking BL-pET-Δ bp26-BL in containing in the LB liquid nutrient medium of penbritin (final concentration is 100 μ g/mL), 37 DEG C, after 200r/min incubated overnight.Get above-mentioned cultivation bacterium by volume 1:50 ratio be inoculated in fresh in the LB nutrient solution of penbritin (final concentration is 100 μ g/mL), 37 DEG C, 200r/min cultivates 3h, adding IPTG(final concentration is 1.5mmol/L) induction time 4h, collection thalline.After ultrasonication, through HisTrap FF purification of recombinant proteins, obtain the BL fusion rotein that purifying is good.

2. BL-iELISA method detects M5-Δ znuA specific antibody level: using the BL fusion rotein after purifying as envelope antigen, with coating buffer (pH=9.6,0.05M carbonate buffer solution: Na ₂cO ₃1.59g, NaHCO ₃2.93g, adding distil water is to 1L) being diluted to 100 μ g/mL, every hole adds 100 μ L, 4 DEG C are spent the night, and add Tween-20 with lavation buffer solution PBST(at pH=7.4,0.15mol/L phosphate buffered saline buffer, the final concentration of Tween-20 is volume percent 0.05%, specifically composed as follows: KH ₂pO ₄0.2g, Na ₂hPO ₄12H ₂o2.9g, NaCl8g, KCl0.2g, Tween-200.5mL, distilled water is settled to 1L) washing 3 times, each 3min, pats dry.Then every hole adds the skim-milk 200 μ L of mass volume ratio (g/mL) 0.03%, and 37 DEG C of closed 3h, washing methods is the same.With serum after 8 weeks of M5, M5-Δ znuA bacterial strain immune mouse of above-mentioned collection as serum to be checked, 20 μ L/ holes add enzyme plate and erect row first hole, again by the first perpendicular row μ L/ hole, hole 180, all the other 100 μ L/ holes add two anti-diluents, carry out transverse direction 10 times of doubling dilutions, 37 DEG C of effect 1h, washing methods is the same; Every hole adds the sheep anti mouse HRP-IgG(1:5000 of horseradish peroxidase-labeled) 100 μ L, 37 DEG C of effects 1h, PBST wash 5 times, and each 3min, pats dry; Every hole adds the substrate nitrite ion TMB(substrate buffer solution of 100 μ L: Na ₂hPO ₄14.6g, citric acid 9.33g, 0.75%H ₂o ₂6.4mL, pH=5.4, distilled water is settled to 1L; TMB uses liquid: TMB200g, dehydrated alcohol 100mL; Get substrate buffer solution 950 μ L and TMB during use and use liquid 50 μ L mixing, be nitrite ion), 37 DEG C of darkroom effect 10min, then add stop buffer (2mol/L sulfuric acid) 50 μ L termination reaction, in 2min, measure OD450nm value with enzyme mark detector.

As shown in Figure 9, immunity the 6th week, serum proportion reached 1:80 to result, and the specific antibody amount that recombinant bacterial strain M5-Δ znuA produces compared with parent plant M5 is suitable, and both difference is not obvious.Show that the disappearance of znuA gene fails to affect the immunogenicity of M5 recombinant vaccine strain.Thus can infer, new generation vaccine strain M5-Δ znuA compared with parent plant M5 virulence reduce, have better security while, immune protective effect still can be kept not subtract.

Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (10)

1. a strain Brucella melitensis recombinant bacterial strain M5- Δ znuApreparation method, it is characterized in that comprising the following steps:

(1) structure of Suicide homologous recombination plasmid

1. with Brucella melitensis ( brucella melitensis) M5 genomic dna is that template carries out PCR, when the primer of use is for P1 and P2, obtain znuAthe upstream homology arm of gene; When the primer used is for P3 and P4, obtain znuAthe downstream homology arm of gene;

P1：5′-GATGGTACCCGTCCTCGTTTGCTTGTGC-3′；

P2：5′-TCGCTGAAAGACTGCCTGT-3′；

P3：5′-GCAGTCTTTCAGCGAAGCCAGAAAGGCAGAAGC-3′；

P4：5′-AGAGAGCTCCAATGTCCCCTTGGTCCC-3′；

2. utilize the method for Overlap PCR, with step 1. in preparation znuAthe upstream homology arm of gene and znuAthe downstream homology arm of gene is template, P1 and P4 is primer pair, obtains the object fragment Δ for homologous recombination znuA;

3. KpnI and SacI is used respectively to suicide plasmid carrier pRE112 and the object fragment Δ for homologous recombination znuAcarry out double digestion, purifying; By the fragment Δ that purifying reclaims znuAbe connected with carrier segments pRE112, obtain Suicide homologous recombination plasmid pRE-Δ znuA;

(2) structure of Brucella melitensis recombinant bacterial strain and screening

1. by Suicide homologous recombination plasmid pRE-Δ znuAimport in the competent cell of Brucella melitensis M5; Then coat on the TSA-YE flat board containing paraxin, under the selective pressure of paraxin, obtain homologous recombination list recon; PCR is identified correct homologous recombination list recon is cultivated in TSB-YE substratum, lax plasmid resistance;

2. then the homologous recombination list recon after lax plasmid resistance is coated on TSA-YE-Sucrose Selective agar medium and cultivate, utilize Suicide homologous recombination plasmid pRE-Δ znuAin sac1 B gene, to the susceptibility of sucrose, is eliminated suicide plasmid, utilizes primer P1 and P4 to carry out PCR screening to the bacterium colony that TSA-YE-Sucrose Selective agar medium grows, and obtains homologous recombination double exchange, namely obtains Brucella melitensis recombinant bacterial strain M5-Δ znuA;

Described TSA-YE-Sucrose Selective agar medium composed as follows: Triptic soya yeast extract agar substratum+final concentration is the sucrose of mass volume ratio 7%.

2. Brucella melitensis recombinant bacterial strain M5-according to claim 1 Δ znuApreparation method, it is characterized in that: step (1) 1. described in the condition of PCR be 94 DEG C of 2min; 98 DEG C of 10s, 56 DEG C of 30s, 68 DEG C of 1.5min, 30 circulations; 68 DEG C of 5 ~ 10min extend eventually.

3. Brucella melitensis recombinant bacterial strain M5-according to claim 1 Δ znuApreparation method, it is characterized in that: step (1) 2. described in the condition of Overlap PCR be 94 DEG C of 2min denaturations; 98 DEG C of 10s, 56 DEG C of 30s, 68 DEG C of 3min, 30 circulations; 68 DEG C of 5 ~ 10min extend eventually.

4. Brucella melitensis recombinant bacterial strain M5-according to claim 1 Δ znuApreparation method, it is characterized in that: step (2) 1. described in the mode of importing for be imported by electric transform mode.

5. Brucella melitensis recombinant bacterial strain M5-according to claim 4 Δ znuApreparation method, it is characterized in that: the condition that described electricity transforms is 1mm pole cup, 1.8kv, 400 Ω.

6. Brucella melitensis recombinant bacterial strain M5-according to claim 1 Δ znuApreparation method, it is characterized in that: step (2) 1. described in the final concentration of paraxin in described TSA-YE substratum be 5 μ g/mL.

7. Brucella melitensis recombinant bacterial strain M5-according to claim 1 Δ znuApreparation method, it is characterized in that: step (2) 1. described in the condition of cultivation be 37 DEG C, 36 ~ 48h cultivated by 200r/min shaking table;

Step (2) 2. described in the condition of cultivation be 37 DEG C and cultivate 48 ~ 96h.

8. Brucella melitensis recombinant bacterial strain M5-according to claim 1 Δ znuApreparation method, it is characterized in that: step (2) 1. described in PCR be accredited as and identified by primer P1 and P4;

The condition of described PCR qualification is as follows: 94 DEG C of 2min denaturations; 98 DEG C of 10s, 56 DEG C of 30s, 68 DEG C of 3min, 30 circulations; 68 DEG C of 5 ~ 10min.

9. a strain Brucella melitensis recombinant bacterial strain M5-Δ znuA, it is characterized in that: obtained by the preparation method described in any one of claim 1 ~ 8.

10. Brucella melitensis recombinant bacterial strain M5-according to claim 9 Δ znuAfor the preparation of the application prevented and/or treated in the vaccine of brucellosis.