CN108048383A - Brucella melitensis per gene deletion strains, its construction method and application - Google Patents
Brucella melitensis per gene deletion strains, its construction method and application Download PDFInfo
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Abstract
Invention provides one plant of 90 Δ per of brucella melitensis (Brucella melitensis, B.melitensis) M5, and the bacterium is built with the per gene coding regions of kalamycin resistance gene replacement brucella melitensis M5 90.The present invention is starting strain with sheep kind Bu Lushi bacterium M5 90, using its genome as template, by expanding per genes upstream and downstream homology arm, simultaneously per gene coding regions are replaced using kalamycin resistance gene, build pGEM Δ per homologous recombination plasmids, by 90 competent cells of plasmid electricity conversion sheep kind Bu Lushi bacterium M5, gained positive transformant is the 90 Δ per of sheep kind Bu Lushi bacterium M5 of per gene delections.The per plants of cell autophagies for brucella mediation of 90 Δs of M5 of the present invention have established important foundation with infection mechanism.
Description
【Technical field】
The present invention relates to biological technical fields, more particularly to one plant of brucella melitensis per gene deletion strains, further relate to
The construction method of the bacterial strain and its application.
【Background technology】
Brucellosis (Brucellosis) is that a kind of infecting both domestic animals and human infects as caused by brucella (Brucella)
Secondly disease, the disease main infection object sheep are pig and ox, cause huge economic loss to China's animal husbandry every year, while tight
Threaten the health of the mankind again.In recent years, which has the trend progressively gone up in the range of China.At present, the sick prevention master
Vaccine immunity is depended on, particularly in developing country, vaccine inoculation is to prevent the sick main means.Prevent the disease in China
One of vaccine be M5-90, can be prevented to a certain extent by the M5-90 vaccine strains of Harbin veterinary institute independent research
Sheep and goat brucellosis, but the vaccine is there is also many defects, as the vaccine strain be inoculated with virulence after animal it is also very strong,
Easily cause pregnant female miscarriage and natural immunity and vaccine immunity cannot be distinguished so that the application of the vaccine has very big office
Sex-limited, therefore, the further transformation of the vaccine is for successfully preventing brucellosis with very great meaning.
The study found that the virulence factor of brucella mainly include outer membrane protein (Outer membrane protein,
OMP) ingredient and lipopolysaccharides (Lipopolysaccharide, LPS) ingredient.Brucella lipopolysaccharides is mainly by lipid A
(LipidA), O- chains antigen and core polysaccharide composition, and the bone amine synzyme of crossing of per gene codes is then brucella lipopolysaccharides
The key enzyme of O- chains antigen synthesis.Brucella lipopolysaccharides is divided into two kinds of smooth type (Soomth, S) and rough type (Rough, R),
When per gene delections cause brucella lipopolysaccharides O- chains antigen that cannot synthesize so that the brucella melitensis M5- of smooth type
90 (Brucella melitensis, B.melitensis) have reformed into the B.melitensis M5-90 of rough type, and make cloth
The virulence of Shandong Salmonella occurs attenuation or disappears.
The synthesis crossed bone amine synzyme and participate in smooth type brucella lipopolysaccharides O side chains of per gene codes,
In B.abortus, the missing of the gene causes it that cannot synthesize LPS, so that smooth type brucella is changed into rough type, and
Make the virulence of brucella that attenuation occur or disappear.
【The content of the invention】
The purpose of the present invention is overcoming prior art defect, obtaining one plant by technique for gene engineering should with attenuated vaccine
With the sheep kind Bu Lushi bacterium of meaning.
To achieve these goals, thinking of the invention is using sheep kind Bu Lushi bacterium (B.melitensis) M5-90 to go out
Bacterium germination strain using its genome as template, by expanding per genes upstream and downstream homology arm, while utilizes kalamycin resistance gene
Per gene coding regions are replaced, build pGEM- Δ per homologous recombination plasmids, by plasmid electricity conversion sheep kind Bu Lushi bacterium M5-90
Competent cell, gained positive transformant are the sheep kind Bu Lushi bacterium M5-90- Δs per of per gene delections.
For this purpose, the present invention provides one plant of brucella melitensis (Brucella melitensis, B.melitensis) M5-
90- Δ p er, the bacterium are the per gene coding regions structures that brucella melitensis M5-90 is replaced with kalamycin resistance gene
It obtains.
The construction method of brucella melitensis M5-90- Δs per comprises the following steps：
(1) according to the gene order of brucella melitensis M5-90 and the gene order of pEGFP-N1 carriers and p GEM-
7Zf (+) carrier feature, the corresponding primer needed for design construction per gene-deleted strains；
(2) genome of brucella melitensis M5-90 bacterium is extracted and in -20 DEG C of preservations；
(3) genome obtained using step (2) is template, the left homology arm per-C ends of amplification per genes, per-N ends, with
PEGFP-N1 plasmids are template, expand kalamycin resistance gene Kana；
(4) pcr amplification product with purification step (3) is separately recovered, obtains per-C ends, per-N ends and K ana genes；
(5) per-C ends, per-N ends and the Kana genes obtained step (4) respectively with pMD20-T carriers 4 DEG C be connected,
Respectively obtain recombinant plasmid；
(6) connection product that step (5) obtains converts bacillus coli DH 5 alpha competent cell respectively, is confirmed by sequencing
To recombinant plasmid pMD20-per-C, pMD20-per-N and pMD20-Kana；
(7) Sma I and Sac I are utilized respectively, double digestion is carried out to recombinant plasmid pMD20-per-C, utilize Sma I and Sac
I carries out double digestion to suicide vector pGEM-7Zf (+), using Xho I and Sma I respectively to recombinating plasmid pGEM-C and pMD20-
Kana carries out double digestion, and plastic recovery kit purifying target fragment obtains cohesive end, with T4 ligases by 4 DEG C of cohesive end
Connection overnight；Connection product is converted into DH5 α competent cells, screening positive clone uses Apa I and Xho I double after extracting plasmid
Digestion is identified, identifies that correct plasmid is named as pGEM-C-K-N, gained plasmid is sequenced；
(8) line of brucella melitensis M5-90 bacterium is incubated on the TSA solid mediums of non-resistant, picking after culture
Single bacterium colony is inoculated in non-resistant TSB fluid nutrient mediums, 37 DEG C, 220rpm culture 48h after according to volume ratio 1:1000 are inoculated in
In 10mL non-resistants TSB；When OD values are 0.389, supernatant is abandoned after centrifugal treating, thalline is placed in precooling 30min on ice, then
4000rpm centrifuges 10min, abandons supernatant；Thalline is resuspended with 10 weight % glycerine water solutions of 5mL again, 8000rpm is centrifuged after mixing
2min abandons supernatant；It repeats that step 3 time is resuspended；Thalline is resuspended with 10 weight % glycerine water solutions 2.5mL of precooling, after mixing again
Packing, obtains brucella melitensis M5-90 competent cells, -70 DEG C save backup；
(9) by the brucella melitensis M5-90 competent cells of homologous recombination plasmid pGEM-C-K-N step of converting (8),
Confirm per gene delections, obtain brucella melitensis M5-90- Δs per.
The present invention also provides above-mentioned brucella melitensis (Brucella melitensis, B.melitensis) M5-90-
Applications of the Δ per in brucella attenuated vaccine is prepared.
The present invention also provides above-mentioned brucella melitensis (Brucella melitensis, B.melitensis) M5-90-
Applications of the Δ per in infection cell autophagy level is reduced.
【Description of the drawings】
The left and right homology arm of Fig. 1 per genes and the PCR product of Kana genes
A：M：D2000DNAMarker；1：The left homology arm of per genes；
B：M：D2000DNAMarker；1：The right homology arm of per genes；
C：M：D2000DNAMarker；1：Kana genes；
The digestion qualification figure of Fig. 2 homologous recombinations plasmid pGEM-Δ per；
A:M1, λ DNA/Hind III DNAMarker.1, pGEM- Δ per homologous recombination plasmid .2, pGEM- Δs per are same
Source recombinant plasmid product .M2, D2000DNAMarker. after Sma I and Sac I digestions
B:M1, D2000DNAMarker.1, pGEM- Δ per homologous recombinations plasmid product after Xho I and Sma I digestions
.2, pGEM- Δs per homologous recombination plasmids M2, λ DNA/Hind III DNAMarker.
C:M1, D2000DNAMarker.1, pGEM- Δ per homologous recombinations plasmid product after Apa I and Xho I digestions
.2, pGEM- Δs per homologous recombination plasmids .M2, λ DNA/Hind III DNAMarker.
The result of the per genes of Fig. 3 PCR identification gene-deleted strain M5-90- Δs per；
M:D2000DNAMarker.1, M5-90 vaccine strain per gene PCR amplified production .2, M5-90- Δ per gene-deleted strains
Per gene PCR amplified productions
The genetic stability detection figure of Fig. 4 gene-deleted strain M5-90- Δs per；
M:In 15 generation of D2000DNA Marker.1-15, M5-90- Δ per gene-deleted strains continuous passage, expands Kana genes respectively；
Fig. 5 is the qualification figure of the LC3-II and LC3-I of control type M5-90 and gene-deleted strain M5-90- Δs per.
【Specific embodiment】
Following embodiment is used to explain technical scheme without limitation.
In the present invention, unless otherwise specified, for representing that " % " of concentration is mass percent, "：" it is quality
Than " part " is mass parts.
The present invention relates to following culture medium or glue：
TSA solid mediums：Amount of preparation 1L：It weighs 43g BrucellaAgar dry powder to be dissolved in 1L deionized waters, fully
Stirring and dissolving, 121 DEG C of high pressure 15min.According to 1:1000 ratio adds in antibiotic kanamycins (Kanamycin), uniformly falls
Enter in the culture dish that sterilizes, 4 DEG C of preservations.
TSB culture mediums：Amount of preparation 1L：It weighs 28g Brucella Broth dry powder to be dissolved in 1L deionized waters, fully stir
Mix dissolving, 121 DEG C of high pressure 15min.
SOC culture mediums：Amount of preparation 100mL：1mol/L glucose solutions are prepared first, i.e., 18g glucose are dissolved in 90mL
In deionized water, 100mL fully is settled to after dissolving, with 0.22 μm of filter filtration sterilization；It is added in into 100mL SOB culture mediums
The 1mol/L glucose solution 2mL of degerming are uniformly mixed, 4 DEG C of preservations.
Complete medium：I.e. containing penicillin (100U/mL), streptomysin (100 μ g/mL), 10% hyclone (FBS)
Doby gram improves her grignard complete medium (Dulbecco's Modified Eagle's Medium, DMEM).
12% separation gel：1.6mL deionized waters, 30% acrylamide solution 2mL, 1.5moL/L Tris (pH 8.8)
50 μ L of 1.3mL, 10%SDS, 10% ammonium persulfate, 50 μ L, TEMED 2 μ L.Encapsulating is at glass plate 1.0cm after abundant mixing,
The closing of 3mL deionized waters is added in, 45min is stored at room temperature, outwells deionized water.
5% concentration glue：Deionized water 2.1mL, 30% acrylamide solution 0.5mL, 380 μ of 1moL/L Tris (pH 6.8)
L, 30 μ L of 10%SDS, 10% ammonium persulfate, 30 μ L, TEMED 3 μ L.Encapsulating after abundant mixing, is inserted into comb, pays attention to avoiding producing
Anger bubble, if any bubble, can be broken with syringe needle, be stored at room temperature 1.5h, gently extract comb.
The structure of 1 brucella melitensis M5-90- Δs per of embodiment
Brucella melitensis M5-90 plants of the gene order (accession number according to disclosed in GenBank：CP001851) and
Gene order (the accession number of pEGFP-N1 carriers：U55762) and pGEM-7Zf (+) carrier feature, and per genes are in cloth Shandong
It is located at 6434-7537bp positions, the corresponding primer needed for design construction per gene-deleted strains on Salmonella M5-90 genomes：
1 primer of table and sequence
The sheep kind Bu Lushi bacterium M5-90 strains that ultra low temperature freezer preserves is taken to be coated on the TSA (Triptic soyas of non-resistant
Agar) culture medium flat plate, 37 DEG C of culture 48h, picking single bacterium falls within 5mL TSB (pancreas peptone soybean broth) culture medium, 37 DEG C
1mL bacterium solutions, simultaneously -20 DEG C of preservations of extraction genome are taken after culture 48h；
Using gained genome as template, using per-C-F, per-C-R as the left homology arm per-C ends of primer amplification per genes,
Using per-N-F, per-N-R as the right homology arm per-N ends of primer amplification per genes；Using pEGFP-N1 plasmids as template, with Kanar-F、Kana r- R is primer amplification kalamycin resistance gene Kana；
Above-mentioned pcr amplification product per-C ends, per-N ends and Kana are separately recovered and purify that (method is precious referring to Takara
The DNA recovery purifyings kit specification of bioengineering (Dalian) Co., Ltd sale)；
Recycle obtain per-C ends, per-N ends and Kana genes, respectively with pMD20-T carriers 4 DEG C be connected overnight, then
Connection product pMD20-per-C, pMD20-per-N and pMD20-Kana are converted into bacillus coli DH 5 alpha competent cell respectively,
Obtain respective converted product；
Three plants of single bacterium colonies of picking are distinguished from the tablet of converted product to identify for bacterium colony PCR, identify correctly restructuring matter
Grain pMD20-per-C, pMD20-Kana and pMD20-per-N send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced；
For correct recombinant plasmid is sequenced, wherein：Double digestion is carried out to pMD20-per-C with Sma I and Sac I, is used
Sma I and Sac I carry out double digestion to suicide vector pGEM-7Zf (+), and gained digestion products are attached to obtain pGEM-C；So
Double digestion is carried out to pGEM-C with Xho I and Sma I afterwards, double digestion, gained are carried out to pMD20-Kana with Xho I and Sma I
Digestion products are attached to obtain pGEM-C-K；Finally, double digestion is carried out to pGEM-C-K with Apa I and Xho I, with Apa I
Double digestion is carried out to pMD20-per-N with Xho I, gained digestion products are attached to obtain pGEM-C-K-N.Pass through glue respectively
QIAquick Gel Extraction Kit purifies target fragment, obtains cohesive end, is connected cohesive end for 4 DEG C overnight with T4 ligases；Connection is produced
Object converts DH5 α competent cells, and screening positive clone identifies that identification is just with Apa I and Xho I double digestions after extracting plasmid
True plasmid is named as pGEM-C-K-N (i.e. homologous recombination suicide plasmid pGEM- Δ per), and is sent raw work bioengineering
(Shanghai) limited company is sequenced；
Sheep kind Bu Lushi bacterium M5-90 line is incubated on the TSA solid mediums of non-resistant, is chosen after 37 DEG C of culture 72h
Single bacterium colony is taken to be inoculated in 5mL non-resistant TSB fluid nutrient mediums, according to volume ratio 1 after 37 DEG C of 220rpm cultures 48h:1000 connect
Kind is in 10mL non-resistants TSB；When OD values are 0.389,4000rpm centrifugation 10min abandon supernatant, thalline are placed in pre- on ice
Cold 30min, then 4000rpm centrifugations 10min, abandons supernatant；Thalline is resuspended with 10% glycerine water solutions of 5mL, gently mixing,
8000rpm centrifuges 2min, abandons supernatant；Repeat previous step step 3 time；Bacterium is resuspended with 10% glycerine water solution 2.5mL of precooling
Body, gently mixing, 100 μ L often pipe packing obtain M5-90 competent cells, -70 DEG C save backup；
Homologous recombination plasmid pGEM-C-K-N is converted into M5-90 competence using electric conversion instrument (voltage 2.1KV, 80 μ s)
Cell obtains bacterial strain to be detected；
The genome of bacterial strain to be detected is extracted after conversion, whether is lacked using per-F, per-R primer PCR identification per genes
It loses successfully, utilizes Kanar-F、Kana r- R identifies that Kana genes whether there is in the dual crossing positive colony for primer PCR；
PCR is identified that correct per gene-deleted strains M5-90- Δs per carries out genetic stability detection：Single bacterium colony is taken to connect
Kind is arrived on the TSA culture medium flat plates containing kanamycins (50 μ g/mL), and vaccinization reached for 15 generations, and genome is extracted by generation, sharp
Use Kanar-F、Kana r- R primers identify that Kana genes can stablize hereditary (as shown in Figure 4) by PCR, confirm as per bases
Because of the sheep kind Bu Lushi bacterium M5-90- Δs per of missing.
2 infecting mouse mononuclear macrophage RAW264.7 of embodiment
(1) Example 1 obtains sheep kind Bu Lushi bacterium M5-90- Δ per, using wild type brucella melitensis as control,
Infecting mouse mononuclear macrophage RAW264.7 (buying from Chinese Academy of Sciences's cell bank) respectively.
RAW264.7 cells are in 37 DEG C, 5%CO2, carry out cellar culture in 10% hyclone, by 5 × 105Density is inoculated with
Into six porocyte culture plates, after bed board cell, the culture of strain is carried out first, then collects wild type sheep kind Bu Lushi respectively
In centrifuge tube, 5000rpm centrifugation 2min abandon supernatant, receive per plants of bacterium solution 1.5mL of bacterium M5-90 and the M5-90- Δs of embodiment 1
Collect thalline, be so repeated 3 times；It being resuspended and precipitated with 1mL physiological saline, mixing, 5000rpm centrifugation 2min abandon supernatant and collect thalline,
So it is repeated 3 times.It is carried out using No. 4 pipe standards pipes in the opacity tube of Maxwell than turbid, adjustment cell concentration is surveyed to No. 4 standard pipes
Determine OD values.It is 10 according to MOI:1 is infected and (respectively takes 100 μ L bacterium solutions mixing into 3mL complete mediums, replace fresh training
Base is supported, is placed in 37 DEG C, 5%CO2Incubator continues to cultivate 4h).
Per plants of M5-90 plants of wild type brucella melitensis and M5-90- Δs infect RAW264.7 cell 4h, i.e. bed board respectively
After cell during 48h, cell culture medium is sopped up, PBS cleaning cellular layer 3 times, adds in 0.02%EDTA and 0.25% pancreatin digests
2min adds in 1mL complete mediums and terminates digestion, blow and beat cell repeatedly, collects cell.
(2) extraction of total protein：The cell of collection step (1) centrifuges 3min in 8000rpm, collects cell, PBS cleaning 2
It is secondary；200 μ L IP cell pyrolysis liquids are added in (according to 1:1000 ratios add in protease inhibitors), it is placed in and blows and beats repeatedly on ice, split
Solve 15min；Then 12000rpm centrifuges 20min, collects supernatant as sample, -70 DEG C save backup.
BCA methods measure the concentration of albumen：First, the making of standard curve is carried out, standard items are diluted to according to equal proportion
In 96 hole elisa Plates, total volume be 20 μ L, each 3 repeating holes of ratio setting；Foregoing each sample is added into 2 μ L to 96 hole enzyme marks
In plate, 18 μ L deionized waters are added in, each sample sets 3 repeating holes；200 μ LBCA reagents are added in per hole, 37 DEG C are protected from light standing
30min；A570 absorbances are detected with microplate reader；Standard curve is drawn according to the A570 absorbances of the standard items of equal proportion,
The concentration of sample is calculated further according to the A570 absorbances of sample.
The testing result of microplate reader is：M5-90 plants of infection RAW264.7 group A570 absorbances of wild type brucella melitensis
It is worth that infect RAW264.7 group A570 absorbances respectively for per plants of 1.876, M5-90- Δs be 2.106, which can
For illustrating the protein concentration of each group sample.
BCA methods measure cellular protein concentration after, add in isometric 2 × Loading Buffer sample-loading buffers (according to
1:4 ratios add in DTT), 10min is boiled in 100 DEG C of water-baths makes albumen be denatured completely, stands 3min, 3000rpm centrifugations on ice
5min collects supernatant, discards precipitation, and supernatant carries out SDS-PAGE.
Total protein is subjected to loading according to 60 μ g, with 2 × Loading Buffer sample-loading buffer polishing samples to totality
Product carries out 5% concentration gel electrophoresis for 25 μ L, 75V burning voltages, and 90V burning voltages carry out 12% separation gel electrophoresis；Electrophoresis is completed
After carry out transferring film, pvdf membrane is activated into 10s in methyl alcohol, is then transferred in deionized water and impregnates 10min, transfers to ice
10min is impregnated in TBST, transferring film is carried out for anode black negative pole according to red, one layer of sponge and filter is placed in black module
Then paper puts glue, then puts pvdf membrane, finally put one layer of filter paper and sponge again, and 3h, transferring film are transferred on ice with 200mA stabling currents
After by pvdf membrane impregnate deionized water in 5min；It is closed after the completion of transferring film, the skimmed milk power that 5% is prepared with TBST is
Confining liquid puts pvdf membrane in confining liquid, washes film after room temperature shaker closing 2h, washes film 5 times with TBST, each 5min；
The incubation that film carries out primary antibody after finishing is washed, is 1 with confining liquid dilution primary antibody rabbit-anti mouse MAP LC3 β:1000, room temperature
After being incubated 2h on shaking table, TBST washes film 5 times, each 5min；Secondary antibody mountain sheep anti mouse dilution ratio is 1:5000, it incubates on room temperature shaker
2h is educated, TBST washes film 5 times, each 5min, utilizes SuperSignal West Pico Chemiluminescent
The super developing solutions of Substrate carry out chemiluminescence colour developing, and the CCD time for exposure is 20s, by this film with stripping buffer at 50 DEG C
It is boiled in water-bath and stands 30min, TBST washes film 5 times, each 5min；GAPDH with confining liquid dilution primary antibody goat source is 1:
5000,2h is incubated on room temperature shaker, TBST washes film 5 times, each 5min, and secondary antibody rabbit-anti goat dilution ratio is 1:5000, room temperature
2h is incubated on shaking table, TBST washes film 5 times, each 5min, utilizes SuperSignal West Pico Chemiluminescent
The super developing solutions of Substrate carry out chemiluminescence colour developing, and the CCD time for exposure is 20s.
The results are shown in Figure 5.
LC3 (Light Chain 3) is identified that the subunit of microtubule associated protein a 1A and 1B are (former to be claimed originally originally
For MAP1LC3), it is subsequently found similar to Yeast protein APG8/aut7/cvt5 and occurs that there is key effect for autophagy.Three
The LC3 hypotypes (LC3A, LC3B and LC3C) of a mankind can be translated during cell autophagy after processing and modification ---
First expose LC3 c-terminuses in post synthesis immediately by the cutting of ATG4 (there is protein endoenzyme enzymatic activity) and play the 120th
Glycine residue generates a relic LC3-I for being positioned at endochylema.Then, LC3-I includes ATG7 and Atg3 deciles by being related to
The processing of the esterified coupling system of ubiquitin sample of son makes LC3 be covalently attached with phosphatidyl-ethanolamine (PtdEth, PE) and is inserted into certainly
Bite in the duplicature of body, it is this be connected with PE after the LC3 segments that are formed be also referred to as LC3-II.LC3 be coupled on autophagosome with
And LC3-I is converted into LC3-II, when autophagy is formed, endochylema type LC3 is digested under the action of enzyme, after removing a bit of polypeptide
LC3-I is formd, free PE can specifically bind the LC3-II i.e. autophagosome that LC3-I forms membranous type, therefore, available
LC3-II/I ratio sizes carry out the height of reacting cells autophagy level.
Fig. 5 is shown, the sheep kind of the per gene coding regions of brucella melitensis M5-90 is replaced with kalamycin resistance gene
Ratio of the LC3-II/I ratios of brucella M5-90- Δs per significantly less than control brucella melitensis M5-90, therefore judge
Brucella melitensis M5-90- Δs per has the characteristic for making the horizontal reduction of RAW264.7 cell autophagies.
In conclusion utilize per plants of difference infecting mouse monokaryons of M5-90 plants of wild type sheep kind Bu Lushi bacterium and M5-90- Δs
Macrophage.Find that the RAW264.7 cell autophagy levels of per plants of M5-90- Δs significantly reduce after 4h, this will be further research
The cell autophagy of brucella mediation establishes important foundation with infection mechanism.
Sequence table
<120>Brucella melitensis per gene deletion strains, its construction method and application
<130> 17233.1
<160> 8
<170> SIPOSequenceListing 1.0
<210> 2
<211> 30
<212> DNA
<213>Primer Per-C-F (artificial sequence)
<400> 2
gcgccccggg tgagacgatt tcgtatgata 30
<210> 3
<211> 27
<212> DNA
<213>Primer Per-C-R (artificial sequence)
<400> 3
cgcggagctc cccgttgtga cctatca 27
<210> 4
<211> 30
<212> DNA
<213>Primer Kana r-F (artificial sequence)
<400> 4
gcgcctcgag atgattgaac aagatggatt 30
<210> 3
<211> 28
<212> DNA
<213>Primer Kana r-R (artificial sequence)
<400> 3
cgcgcccggg tcagaagaac tcgtcaag 28
<210> 5
<211> 28
<212> DNA
<213>Primer Per-N-F (artificial sequence)
<400> 5
gcgcgggccc aagcgtcgca cctcgcaa 28
<210> 7
<211> 33
<212> DNA
<213>Primer Per-N-R (artificial sequence)
<400> 7
cgcgctcgag gtatggatca ttccttaggg gcg 33
<210> 7
<211> 27
<212> DNA
<213>Primer Per-F (artificial sequence)
<400> 7
atggatatac cagtttactc tccctat 27
<210> 8
<211> 24
<212> DNA
<213>Primer Per-R (artificial sequence)
<400> 8
ctaaatgtgg ttggaatgat taga 24
Claims (5)
1. one plant of brucella melitensis (Brucella melitensis, B.melitensis) M5-90- Δs per, the bacterium are
It is built with the per gene coding regions of kalamycin resistance gene replacement brucella melitensis M5-90.
2. the construction method of brucella melitensis M5-90- Δs per, the described method comprises the following steps：
(1) according to the gene order of brucella melitensis M5-90 and the gene order of pEGFP-N1 carriers and pGEM-7Zf (+)
Carrier feature, the corresponding primer needed for design construction per gene-deleted strains；
(2) genome of brucella melitensis M5-90 bacterium is extracted and in -20 DEG C of preservations；
(3) genome obtained using step (2) is template, the left homology arm per-C ends of amplification per genes, per-N ends, with
PEGFP-N1 plasmids are template, expand kalamycin resistance gene Kana；
(4) pcr amplification product with purification step (3) is separately recovered, obtains per-C ends, per-N ends and Kana genes
(5) per-C ends, per-N ends and the Kana genes obtained step (4) respectively with pMD20-T carriers 4 DEG C be connected, respectively
Obtain recombinant plasmid；
(6) connection product that step (5) obtains converts bacillus coli DH 5 alpha competent cell respectively, and weight is obtained by the way that confirmation is sequenced
Group plasmid pMD20-per-C, pMD20-per-N and pMD20-Kana；
(7) Sma I and Sac I are utilized respectively, double digestion is carried out to recombinant plasmid pMD20-per-C, utilize Sma I and Sac I couple
Suicide vector pGEM-7Zf (+) carries out double digestion, using Xho I and Sma I respectively to recombinating plasmid pGEM-C and pMD20-
Kana carries out double digestion, and plastic recovery kit purifying target fragment obtains cohesive end, with T4 ligases by 4 DEG C of cohesive end
Connection overnight；Connection product is converted into DH5 α competent cells, screening positive clone uses Apa I and Xho I double after extracting plasmid
Digestion is identified, identifies that correct plasmid is named as pGEM-C-K-N, gained plasmid is sequenced
(8) line of brucella melitensis M5-90 bacterium is incubated on the TSA solid mediums of non-resistant, picking single bacterium after culture
Fall to be inoculated in non-resistant TSB fluid nutrient mediums, 37 DEG C, 220rpm culture 48h after according to volume ratio 1:1000 are inoculated in 10mL
In non-resistant TSB；When OD values are 0.389, supernatant is abandoned after centrifugal treating, thalline is placed in precooling 30min on ice, then
4000rpm centrifuges 10min, abandons supernatant；Thalline is resuspended with 10 weight % glycerine water solutions of 5mL again, 8000rpm is centrifuged after mixing
2min abandons supernatant；It repeats that step 3 time is resuspended；Thalline is resuspended with 10 weight % glycerine water solutions 2.5mL of precooling, after mixing again
Packing, obtains brucella melitensis M5-90 competent cells, -70 DEG C save backup；
(9) by the brucella melitensis M5-90 competent cells of homologous recombination plasmid pGEM-C-K-N step of converting (8), confirm
Per gene delections obtain brucella melitensis M5-90- Δs per.
3. brucella melitensis (Brucella melitensis, B.melitensis) M5-90- Δs per of claim 2 exists
Prepare the application in brucella attenuated vaccine.
4. brucella melitensis (Brucella melitensis, B.melitensis) M5-90- Δs per of claim 2 exists
Reduce the application in infection cell autophagy level.
5.per gene delections are reducing application of the brucella melitensis to RAW264.7 cell autophagies level.
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| US20120183576A1 (en) * | 2009-09-21 | 2012-07-19 | Jean-Pierre Gorvel | Modified gram-negative bacteria for use as vaccines |
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2017
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| US20120183576A1 (en) * | 2009-09-21 | 2012-07-19 | Jean-Pierre Gorvel | Modified gram-negative bacteria for use as vaccines |
| CN107267432A (en) * | 2016-04-07 | 2017-10-20 | 中国人民解放军军事医学科学院生物工程研究所 | Brucella 104M vaccine strains knock out recombinant bacterium and the application of Per genes |
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