CN110484484A - One plant of brucella S2 vaccine strain ArsR gene deletion strains, its construction method and application - Google Patents
One plant of brucella S2 vaccine strain ArsR gene deletion strains, its construction method and application Download PDFInfo
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Abstract
The invention discloses one plant of brucella S2 vaccine strain ArsR gene deletion strains, the bacterial strain is the brucella S2 vaccine strain for knocking out ArsR gene, can be used for preparing brucella attenuated vaccine.The acquisition of brucella S2 vaccine strain ArsR gene deletion strains is to probe into brucella pathogenic mechanism and distinguish the characteristic of natural infection and artificial immunity to lay the foundation, to preventing and control cloth disease has great importance.
Description
Technical field
The present invention relates to biological fields, and in particular to one plant of brucella S2 vaccine strain ArsR gene deletion strains, its structure
Construction method and application.
Background technique
Brucella is also known as Brucella, is Grain-negative parasitic bacteria intracellular, Duo Cheng little club-shaped or rod-short.Bu Lu
Salmonella can infect people, domestic animal, wild animal and marine animal etc. as animal pathogen.Brucellosis is by cloth Lu Shi
Microbial bacillary Arbo infectious disease is still the weight for seriously threatening human health development and animal health cultivation so far
Big epidemic disease.Currently, brucellosis is still all the fashion in many places in the whole world, including the Middle East, Africa, Latin America, the Central Asia
Regional with Mediterranean basin etc., only a small number of developed countries have eliminated brucellosis by way of evolution, such as the U.S. and plus
It puts on airs.Brucellosis outburst in China's is concentrated mainly on the Northwests such as the Inner Mongol and Xinjiang.Further, since domestic animal across ground
The reasons such as area's transport, cause the spread scope of China's domestic animal brucellosis to be gradually expanded.
Brucella infects body, such as respiratory system, digestive system, skin and mucous membrane in several ways.People's infection
Brucella mainly passes through directly contact illness domestic animal and the edible animal product without stringent sterilization.People suffers from brucellosis master
The symptoms such as arthralgia and undulant fever are shown, serious patient leads to labor capacity is lost, burden on society is caused to aggravate.Mesh
Newly-increased number of patients is more than 500,000 people every year in the preceding whole world, and is difficult to cure completely by antibiotic, can also be pacified without vaccine
Complete effective prevention people's brucellosis.Brucellosis propagates rare report interpersonal, but from 1966 to
Have within 2015 45 report brucellosis in interpersonal propagation, main mode of transmission be placenta propagate, breast milk propagate,
Spread through sex intercourse with blood born etc..
Seek the hot subject that a kind of ideal anti-brucella disease vaccine is still scientist's research.However, to current
Until, the anti-brucella disease vaccine safely and effectively used for the mankind and dog is not developed yet.In ox, most successful epidemic disease
Seedling S19 is only used for calf, because Adult Bovine is inoculated with this vaccine and leads to male orchitis, and the persistent infection symptom that occurs together, even
It can lead to pregnant female Niu Fasheng miscarriage.Another widely used vaccine is mutation strain vaccine RB51, but this vaccine
The disadvantage is that not having higher Vaccine effectiveness.It is S2 vaccine strain that China prevents the widely used vaccine of domestic animal at present, still
There is also certain disadvantages for this vaccine, such as the miscarriage and indistinguishable natural infection and artificial immunity of the domestic animal that causes to be pregnant
Deng.
Summary of the invention
The purpose of the present invention is overcoming existing brucella vaccine defect, gene is passed through based on brucella S2 vaccine strain
The brucella that engineering technology, which obtains, to be had attenuation, distinguish natural infection and artificial immunity application value.
In order to achieve the above purpose, the present invention provides one plant of brucella S2 vaccine strain ArsR gene deletion strains,
Its construction method and application are based on brucella S2 vaccine strain, and using its genome as template, amplification ArsR gene upstream and downstream is homologous
Arm gene, while gentamicin resistance gene is expanded, construct recombinant vector PUC19-ArsR；By recombinant vector PUC19-ArsR electricity
Brucella S2 vaccine strain is converted, to replace ArsR gene, positive transformant obtained using gentamicin resistance gene
As brucella S2 vaccine strain ArsR gene-deleted strain.
To achieve the above object, the technical scheme adopted by the invention is as follows:
One plant of brucella S2 vaccine strain ArsR gene deletion strains, the bacterial strain are to knock outArsRThe brucella of gene
S2 vaccine strain.
The construction method of the brucella S2 vaccine strain ArsR gene deletion strains, includes the following steps:
S1, design of primers
ArsR gene upstream and downstream primer, gentamicin gene and screening missing are designed using Primier Primier 5
ArsR identified for genes primer, primer sequence are as follows:
Upstream primer:
S-F:5 '-CTGCAG (Pst 1) CTGCAGCCATCTCGCTGTAGAAAGT-3 '
S-R:5 '-TCTAGA (Xba 1) TCTAGATTGCCGTCATACCCCCGT-3 '
Downstream primer:
X-F:5 '-GAGCTC (Sac 1) GAGCTCACCACATCCCATGAGGCC-3 '
X-R:5 '-GAATTC (EcoR 1) GAATTCCAAGAGCGGCGTTGATGT-3 '
Gentamicin gene primer:
G-F:5 '-TCTAGA (Xba 1) TTGACATAAGCCTGTTCGGTTCGTA-3 '
G-R:5 '-GAGCTC (Sac 1) TTAGGTGGCGGTACTTGGGTCGATA-3 '
Identify primer
F:GCCGCTATCTCGACCATAAT
R:GTGGCTTCATAGATACTGCG
S2, target gene PCR amplification and glue recycling
Using brucella S2 vaccine strain genome and PBBR1MCS5 carrier as template, carry out on PCR amplification ArsR respectively
Trip, downstream homology arm gene and gentamicin gene；
Reaction system is as follows: 5 × PS Buffer, 5 μ L；dNTP Mixture 2μL；F 0.5μL；R 0.5μL；1 μ of template
L；Prime star DNA polymerase 0.5μL；ddH2O 15.5μL；
PCR reaction condition is as follows:
95 DEG C of initial denaturation 10min；95 DEG C of denaturation 30s；58 DEG C of annealing 50s；72 DEG C of extension 1min, 30 circulations；72 DEG C are prolonged
Stretch 10min；13 DEG C of preservation 1h；
By PCR product through 1% agarose gel electrophoresis, 607bp, 825bp and 751bp size are cut respectively under bale cutting instrument
Purpose band blob of viscose, use Tiangeng biochemical technology company produce DNA purification and recovery kit recycle PCR product；
The acquisition of S3, cohesive terminus,cohesive termini target fragment
The upstream ArsR homology arm gene, the downstream ArsR homology arm gene and the gentamicin resistance gene that glue is recycled respectively
It is connect 2h at 16 DEG C with 19T (simple) carrier, linked system is as follows:
1 μ L of target gene；19T(simple)vector 4μL；Soluton1 5μL；
Product after connection is transformed into competent escherichia coli cell DH5 α, is applied to the A of preparatory 37 DEG C of preheatings+LB solid
Plate is smeared uniformly with spreader, after placing 37 DEG C of constant incubator 12h, takes out solid plate from 37 DEG C of constant incubators,
Choose the good bacterium colony of upgrowth situation with pipette tips, be inoculated in 50mL centrifuge tube, 20mL LB liquid medium is added, is placed in 37
12h is cultivated in DEG C constant-temperature table；Later, the plasmid produced using Tiangeng biochemical technology company is small to propose middle amount kit extraction recombination
Carrier；
The upstream recombinant vector 19T (the simple)-ArsR homology arm extracted, the downstream 19T (simple)-ArsR is homologous
Arm and 19T (simple)-G+Pst 1 and Xba 1, Sac 1 and EcoR 1 and 1 double digestion of Xba 1 and Sac, digestion are used respectively
Product carries out 1% agarose gel electrophoresis identification, and glue recycles the upstream ArsR homology arm gene, the downstream ArsR homology arm gene and celebrating
Big mycin gene；1 double digestion PUC19 carrier of EcoR 1 and Pst is used again, and digestion products carry out 1% agarose gel electrophoresis mirror
Fixed, glue recycles the PUC19 segment after double digestion；
The upstream 19T (simple)-ArsR homology arm digestion system:
Pst 1 1.5μL；Xba 1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
The downstream 19T (simple)-ArsR homology arm digestion system:
Sac 1 1.5μL；EcoR 1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
19T (simple)-G+ digestion system:
Sac 1 1.5μL；Xba 1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
PUC19 digestion system:
Pst 1 1.5μL；EcoR 1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
S4, homologous recombination vector PUC19G-ArsR building
The upstream ArsR homology arm gene, the downstream the ArsR homology arm gene, gentamicin resistance base that previous step glue is recycled
The PUC19 carrier of cause and double digestion 16 DEG C of connection 4h on connection instrument；Linked system is as follows:
3 μ L of the upstream ArsR homology arm gene；
3 μ L of the downstream ArsR homology arm gene；
3 μ L of gentamicin gene；
PUC19 1μL；
Solution 1 10μL；
Product after connection is transformed into competent escherichia coli cell DH5 α, is applied to the G+LB solid of preparatory 37 DEG C of preheatings
Plate is smeared uniformly with spreader, after placing 37 DEG C of constant incubator 12h, takes out solid plate from 37 DEG C of constant incubators,
Choose the good bacterium colony of upgrowth situation with pipette tips, be inoculated in 50mL centrifuge tube, 20mL LB liquid medium is added, is placed in 37
12h is cultivated in DEG C constant-temperature table；Later, the plasmid produced using Tiangeng biochemical technology company is small to propose middle amount kit extraction
PUC19G-ArsR recombinant vector；
The recombinant vector PUC19G-ArsR extracted is subjected to double digestion with Pst 1 and EcoR 1, digestion products carry out
The identification of 1% agarose gel electrophoresis；
The preparation of S5, brucella competence
S5.1, it the brucella after activation is diluted to suitable concentration is applied on TSA solid plate, be placed in 37 DEG C of constant temperature trainings
It supports in case and cultivates 72h；
S5.2, TSA solid plate is taken out from 37 DEG C of constant incubators, choose the good bacterium colony of upgrowth situation with pipette tips,
It is inoculated in 50mL centrifuge tube, 10mL TSB fluid nutrient medium is added, is placed in 37 DEG C of constant-temperature tables and cultivates about for 24 hours；
S5.3, the bacterium solution of previous step is inoculated in 50mL centrifuge tube according to 1:100, is placed in 37 DEG C of constant-temperature tables and cultivates
To OD600About 0.6；
S5.4,4 DEG C of the bacterium solution centrifugations by previous step, 5000r/min are centrifuged 10min, discard liquid, retain precipitating；
The sterile water 10mL that S5.5, addition are pre-chilled in advance, is resuspended precipitating, and 4 DEG C of centrifugation 10min of 5000r/min discard liquid
Body retains precipitating；
S5.6, step S5.5 is repeated；
The 10% glycerol 10mL that S5.7, addition are pre-chilled in advance, is resuspended precipitating, and 4 DEG C of centrifugation 10min of 5000r/min are abandoned
Fall liquid, retains precipitating；
S5.8, step S5.7 is repeated；
Precipitating is resuspended in the 10% glycerol 1mL that S5.9, addition are pre-chilled in advance, and 100 μ L packing, -80 DEG C save backup；
S6, electrotransformation
It takes 10 μ L of PUC19G-ArsR plasmid to be added in 100 μ L brucella competence, the electric shock of pre-cooling is moved into after mixing
In cup, 10min is placed on ice, is put into electroporation, voltage 1.8KV, time 6ms；37 DEG C of preheatings are added after having shocked by electricity immediately
TSB culture solution；180rpm/min culture in 37 DEG C of constant-temperature tables is placed in for 24 hours, to take out from 37 DEG C of constant-temperature tables, after centrifugation,
It is resuspended and is precipitated with about 200 μ L TSB, is applied on TSB gentamicin resistance plate later, cultivate 72h；
The screening of S7, muton
TSA solid plate is taken out from 37 DEG C of constant incubators, is chosen the good bacterium colony of upgrowth situation with pipette tips, is inoculated in
In 10mL centrifuge tube, 5mL TSB gentamicin resistance fluid nutrient medium is added, is placed in 37 DEG C of constant-temperature tables and cultivates about 48h；It
Afterwards, brucella genome is extracted, gene-deleted strain is screened from gene level, correct bacterial strain will be screened again by qRT-PCR
Identification.
Further, the product after connection is transformed into competent escherichia coli cell DH5 α by following steps:
(1) competent escherichia coli cell DH5 α is taken out from -80 DEG C of refrigerators, is placed in about 5min on ice, until competence
Cell melts completely；
(2) the competent escherichia coli cell DH5 α that 20 μ L have melted is added into the 1.5mL centrifuge tube being pre-chilled in advance,
Whole connection products are added, are mixed well, when operation not leave ice face；
(3) 1.5mL centrifuge tube is stood into 30min on ice after mixing, later 42 DEG C of thermostat water bath thermal shock 1min, later
2min on ice is placed again；
(4) from centrifuge tube is taken out on ice, LB culture solution 500L is added in superclean bench, is placed in 37 DEG C of constant temperature later
180r/min recovery 1h in shaking table；
(5) A of preparatory 37 DEG C of preheatings is taken+100L recovery product is added in LB solid plate, is smeared uniformly, is put with spreader
Set 37 DEG C of constant incubator 12h.
The present invention also provides the brucella ArsR gene deletion strains on preparation brucella attenuated vaccine
Application.
The present invention provides the brucella ArsR bases that one plant has attenuation, the characteristic for distinguishing natural infection and artificial immunity concurrently
Because of deletion mycopremna, have great importance to prevention and control cloth disease.
Detailed description of the invention
Fig. 1 is upstream homology arm, downstream homology arm and gentamicin gene PCR result；
In figure: M:maker；A: upstream homology arm；B: downstream homology arm；C: gentamicin.
Fig. 2 is 19T (simple)-upstream homology arm and downstream homology arm digestion result；
In figure: M:maker；A:19T (simple)-upstream homology arm digestion result；B:19T (simple)-downstream is homologous
Arm digestion result；C:19T (simple)-gentamicin digestion result；
Fig. 3 PUC19G-ArsR digestion result M:maker；A:PUC19G-ArsR.
Fig. 4 is Δ ArsR bacterial strain PCR the selection result；
In figure: M:DNA Marker；A: positive control；B and c: negative control；1-8: sample.
Fig. 5 is Δ ArsR bacterial strain PCR qualification result；
In figure: M:DNA Marker；A: upstream homology arm and gentamicin gene；B: downstream homology arm and gentamicin base
Cause；C: gentamicin gene；D:ArsR.
Fig. 6 is Δ ArsR bacterial strain qRT-PCR qualification result.
Fig. 7 is ne ar variation.
Fig. 8 is growth curve of bacteria.Fig. 9 is that stress survive in vitro.
Figure 10 is endogenous multiplication.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
The present invention provides one plant of brucella S2 vaccine strain ArsR gene deletion strains, are constructed by following steps:
S1, design of primers
ArsR gene upstream and downstream primer, gentamicin gene and screening missing are designed using Primier Primier 5
ArsR identified for genes primer, primer sequence are as follows:
Upstream primer:
S-F:5 '-CTGCAG (Pst 1) CTGCAGCCATCTCGCTGTAGAAAGT-3 '
S-R:5 '-TCTAGA (Xba 1) TCTAGATTGCCGTCATACCCCCGT-3 '
Downstream primer:
X-F:5 '-GAGCTC (Sac 1) GAGCTCACCACATCCCATGAGGCC-3 '
X-R:5 '-GAATTC (EcoR 1) GAATTCCAAGAGCGGCGTTGATGT-3 '
Gentamicin gene primer:
G-F:5 '-TCTAGA (Xba 1) TTGACATAAGCCTGTTCGGTTCGTA-3 '
G-R:5 '-GAGCTC (Sac 1) TTAGGTGGCGGTACTTGGGTCGATA-3 '
Identify primer
F:GCCGCTATCTCGACCATAAT
R:GTGGCTTCATAGATACTGCG
S2, target gene PCR amplification and glue recycling
Using brucella S2 vaccine strain genome and PBBR1MCS5 carrier as template, carry out on PCR amplification ArsR respectively
Trip, downstream homology arm gene and gentamicin gene；
Reaction system is as follows: 5 × PS Buffer, 5 μ L；dNTP Mixture 2μL；F 0.5μL；R 0.5μL；1 μ of template
L；Prime star DNA polymerase 0.5μL；ddH2O 15.5μL；
PCR reaction condition is as follows:
95 DEG C of initial denaturation 10min；95 DEG C of denaturation 30s；58 DEG C of annealing 50s；72 DEG C of extension 1min, 30 circulations；72 DEG C are prolonged
Stretch 10min；13 DEG C of preservation 1h；
By PCR product through 1% agarose gel electrophoresis, 607bp, 825bp and 751bp size are cut respectively under bale cutting instrument
Purpose band blob of viscose, use Tiangeng biochemical technology company produce DNA purification and recovery kit recycle PCR product；
The acquisition of S3, cohesive terminus,cohesive termini target fragment
Respectively by glue recycling the upstream ArsR homology arm gene, the downstream ArsR homology arm gene and gentamicin gene with
For 19T (simple) carrier in 16 DEG C of connection 2h, linked system is as follows:
1 μ L of target gene；19T(simple)vector 4μL；Soluton1 5μL；
Product after connection is transformed into competent escherichia coli cell DH5 α, specifically, (1) takes out from -80 DEG C of refrigerators
Competent escherichia coli cell DH5 α is placed in about 5min on ice, until competent cell melts completely；(2) to being pre-chilled in advance
1.5mL centrifuge tube in the competent escherichia coli cell DH5 α that has melted of 20 μ L is added, add whole connection products, fill
It point mixes, when operation not leave ice face；(3) 1.5mL centrifuge tube is stood into 30min on ice after mixing, later 42 DEG C of constant temperature
Water-bath thermal shock 1min, places 2min on ice again later；(4) from centrifuge tube is taken out on ice, LB is added in superclean bench
500 μ L of culture solution is placed in 180r/min recovery 1h in 37 DEG C of constant-temperature tables later；(5) take the A+LB of preparatory 37 DEG C of preheatings solid
100L recovery product is added in body plate, is smeared uniformly with spreader, places 37 DEG C of constant incubator 12h.
Competent escherichia coli cell DH5 α is applied to the A of preparatory 37 DEG C of preheatings+LB solid plate is smeared equal with spreader
It is even, after placing 37 DEG C of constant incubator 12h, solid plate is taken out from 37 DEG C of constant incubators, chooses upgrowth situation with pipette tips
Good bacterium colony is inoculated in 50mL centrifuge tube, and 20mL LB liquid medium is added, is placed in 37 DEG C of constant-temperature tables and cultivates
12h；Later, the plasmid produced using Tiangeng biochemical technology company is small to mention middle amount kit extraction recombinant vector；
The upstream recombinant vector 19T (the simple)-ArsR homology arm extracted, the downstream 19T (simple)-ArsR is homologous
Arm and 19T (simple)-G+Pst 1 and Xba 1, Sac 1 and EcoR 1 and 1 double digestion of Xba 1 and Sac, digestion are used respectively
Product carries out 1% agarose gel electrophoresis identification, and glue recycles the upstream ArsR homology arm gene, the downstream ArsR homology arm gene and celebrating
Big mycin gene；1 double digestion PUC19 carrier of EcoR 1 and Pst is used again, and digestion products carry out 1% agarose gel electrophoresis mirror
Fixed, glue recycles the PUC19 segment after double digestion；
The upstream 19T (simple)-ArsR homology arm digestion system:
Pst1 1.5μL；Xba1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
The downstream 19T (simple)-ArsR homology arm digestion system:
Sac1 1.5μL；EcoR1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
19T(simple)-G+Digestion system:
Sac1 1.5μL；Xba1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
PUC19 digestion system:
Pst1 1.5μL；EcoR1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
S4, homologous recombination vector PUC19G-ArsR building
The upstream ArsR homology arm gene that previous step glue is recycled, the downstream ArsR homology arm gene, gentamicin gene and
The PUC19 carrier of double digestion 16 DEG C of connection 4h on connection instrument；Linked system is as follows:
3 μ L of the upstream ArsR homology arm gene；
3 μ L of the downstream ArsR homology arm gene；
3 μ L of gentamicin gene；
PUC19 1μL；
Solution 1 10μL；
Product after connection is transformed into competent escherichia coli cell DH5 α, specifically,
(1) competent escherichia coli cell DH5 α is taken out from -80 DEG C of refrigerators, is placed in about 5min on ice, until competence
Cell melts completely；(2) competent escherichia coli cell that 20 μ L have melted is added into the 1.5mL centrifuge tube being pre-chilled in advance
DH5 α adds whole connection products, mixes well, and when operation not leave ice face；(3) by 1.5mL centrifuge tube after mixing
30min is stood on ice, and 42 DEG C of thermostat water bath thermal shock 1min, place 2min on ice again later later；(4) from take out on ice from
500 μ L of LB culture solution is added in superclean bench, is placed in 180r/min recovery 1h in 37 DEG C of constant-temperature tables later for heart pipe；
(5) the A+LB solid plate for taking preparatory 37 DEG C of preheatings, is added 100 μ L recovery products, is smeared uniformly with spreader, place 37 DEG C of perseverances
Warm incubator 12h.
Competent escherichia coli cell DH5 α is applied to the G of preparatory 37 DEG C of preheatings+LB solid plate is smeared equal with spreader
It is even, after placing 37 DEG C of constant incubator 12h, solid plate is taken out from 37 DEG C of constant incubators, chooses upgrowth situation with pipette tips
Good bacterium colony is inoculated in 50mL centrifuge tube, and 20mL LB liquid medium is added, is placed in 37 DEG C of constant-temperature tables and cultivates
12h；Later, the plasmid produced using Tiangeng biochemical technology company is small to mention middle amount kit extraction PUC19G-ArsR recombinant vector；
The recombinant vector PUC19G-ArsR extracted is subjected to double digestion with Pst1 and EcoR1, digestion products carry out 1%
Agarose gel electrophoresis identification；
The preparation of S5, brucella competence
S5.1, it the brucella after activation is diluted to suitable concentration is applied on TSA solid plate, be placed in 37 DEG C of constant temperature trainings
It supports in case and cultivates 72h；
S5.2, TSA solid plate is taken out from 37 DEG C of constant incubators, choose the good bacterium colony of upgrowth situation with pipette tips,
It is inoculated in 50mL centrifuge tube, 10mL TSB fluid nutrient medium is added, is placed in 37 DEG C of constant-temperature tables and cultivates about for 24 hours；
S5.3, the bacterium solution of previous step is inoculated in 50mL centrifuge tube according to 1:100, is placed in 37 DEG C of constant-temperature tables and cultivates
To OD600About 0.6；
S5.4,4 DEG C of the bacterium solution centrifugations by previous step, 5 000r/min are centrifuged 10min, discard liquid, retain precipitating；
The sterile water 10mL that S5.5, addition are pre-chilled in advance, is resuspended precipitating, and 54 DEG C of 000r/min centrifugation 10min discard liquid
Body retains precipitating；
S5.6, step S5.5 is repeated；
The 10% glycerol 10mL that S5.7, addition are pre-chilled in advance, is resuspended precipitating, and 54 DEG C of 000r/min centrifugation 10min are abandoned
Fall liquid, retains precipitating；
S5.8, step S5.7 is repeated；
Precipitating is resuspended in the 10% glycerol 1mL that S5.9, addition are pre-chilled in advance, and 100 μ L packing, -80 DEG C save backup；
S6, electrotransformation
It takes 10 μ L of PUC19G-ArsR plasmid to be added in 100 μ L brucella competence, the electric shock of pre-cooling is moved into after mixing
In cup, 10min is placed on ice, is put into electroporation, voltage 1.8KV, time 6ms；37 DEG C of preheatings are added after having shocked by electricity immediately
TSB culture solution；180rpm/min culture in 37 DEG C of constant-temperature tables is placed in for 24 hours, to take out from 37 DEG C of constant-temperature tables, after centrifugation,
It is resuspended and is precipitated with about 200 μ L TSB, is applied on TSB gentamicin resistance plate later, cultivate 72h；
The screening of S7, muton
TSA solid plate is taken out from 37 DEG C of constant incubators, is chosen the good bacterium colony of upgrowth situation with pipette tips, is inoculated in
In 10mL centrifuge tube, 5mL TSB gentamicin resistance fluid nutrient medium is added, is placed in 37 DEG C of constant-temperature tables and cultivates about 48h；It
Afterwards, brucella genome is extracted, gene-deleted strain is screened from gene level, correct bacterial strain will be screened again by qRT-PCR
Identification.
Scanning electron microscopic observation ne ar
1. sampling: cell culture at after suspension be centrifuged after remove supernatant.
2. rinsing: the PBS buffer solution pH 6.8 of 0.1M embathes 4 times, each 10min, each dosage about 2ml.
It is fixed after 3.: fixed after only animal tissue needs: 1-2 hours fixed with 4 DEG C of 1% osmic acid of 0.7ml or so
4. dehydration: 10%, 30%, 50%, 70%, 80%, 90% ethanol solution is each primary, each 15-20min；
100% 3 each 30min of ethyl alcohol.5. displacement among: acetone displacement is primary, time 10-20min.6.CO2It is dry
7. viscous platform: sample after dry is distinguished front and back sides, is adhered on conducting resinl.
8. metal spraying-loading observation.
As a result: by scanning electron microscopic observation, compared with wild-type strain, Δ ArsR bacterial strain is in single ne ar and carefully
There is no significant difference in terms of bacterium outer membrane.
The extracellular growth curve detection of bacterium
From picking them separately the good B.suis S2 of growth conditions and Δ ArsR bacterium colony on TSA plate in TSB culture solution,
It is placed in constant-temperature table, 37 DEG C, 180r/min, shaken cultivation to OD600≈1.0；Then in 1: 100 ratio respectively by bacterium solution
It is inoculated in fresh TSB, shaken cultivation, draws 500 μ L bacterium solutions every 4h, above-mentioned bacterium solution is placed in boiling water bath 10min and is gone out
It is living, after with enzyme-linked immunosorbent assay instrument detect bacterium solution OD value, until 72h.
As a result: showing Δ ArsR bacterial strain and wild-type strain by growth curve detection, proliferation having the same is lived in vitro
Power.
External stress tests detection bacterium survival rate
OD will be grown to600The bacterium solution doubling dilution of ≈ 1.0, is coated with TSA plate, and bacterium colony counts；Choose appropriate dilutions multiple
Bacterium solution 1mL centrifugation, use D (-)-Sorbitol solution of 0.5M respectively, the TSB culture solution of 50 μ g/mL polymyxin Bs contains
0.5mM H2O2TSB culture solution, the TSB culture solution of the TSB culture solution of pH 5.0 and 42 DEG C is resuspended, and gradient dilution is coated with TSA
Plate, bacterium colony count.Simultaneously using three bacterial strain untreated fish groups as control, test is in triplicate.
As a result: showing Δ ArsR bacterial strain to osmotic pressure, polymyxin B, H by external stress test2O2, acid and heat
Sensibility increases.
Proliferation variation in 264.7 macrophage of RAW
RAW 264.7 is pressed 2 × 105Cells/well is inoculated in 24 orifice plates for being put into dedicated cell climbing sheet.Cultivate 12h
Afterwards, by B.suis S2 bacterial strain and Δ ArsR bacterial strain according to 100: 1 infection multiplicity (multiplicity of infection,
MOI it) is added in 24 orifice plates.24 orifice plates are put into incubator and cultivate 4h, discard culture solution in hole later, PBS is washed 3 times, added
Enter containing 50 μ g/mL ammonia benzyl mycin culture solutions.24 orifice plates being put into after cultivating 1h in incubator, discards culture solution, PBS is washed 3 times,
The culture solution containing 25 μ g/mL ammonia benzyl mycins is added.Using this time point as 0h, 0h, 6h, 12h, thin with 48h for 24 hours is taken respectively
Born of the same parents, each time point do 3 repetitions, and every hole is added the PBS of the X-100 containing 0.5%Triton, is put into constant incubator and acts on
10min lytic cell.Doubling dilution cell pyrolysis liquid applies TSA plate, chooses Colony Forming Unit (Colony Forming
Units, CFU) plate between 30-300, carry out CFU counting.Test is repeated 3 times.
As a result: Δ ArsR bacterial strain and wild-type strain existence energy intracellular having the same are found by detection endogenous multiplication
Power.Virulence does not increase after showing ArsR gene delection.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make a variety of changes or modify within the scope of the claims, this not shadow
Ring substantive content of the invention.In the absence of conflict, the feature in embodiments herein and embodiment can any phase
Mutually combination.
Sequence table
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Claims (4)
1. one plant of brucella S2 vaccine strain ArsR gene deletion strains, it is characterised in that: the bacterial strain is to knock outArsRGene
Brucella S2 vaccine strain.
2. the construction method of brucella S2 vaccine strain ArsR gene deletion strains as described in claim 1, it is characterised in that:
Include the following steps:
S1, design of primers
ArsR gene upstream and downstream primer, gentamicin gene and screening missing ArsR base are designed using Primier Primier 5
Because identifying that primer, primer sequence are as follows:
Upstream primer:
S-F:5 '-CTGCAG (Pst 1) CTGCAGCCATCTCGCTGTAGAAAGT-3 '
S-R:5 '-TCTAGA (Xba 1) TCTAGATTGCCGTCATACCCCCGT-3 '
Downstream primer:
X-F:5 '-GAGCTC (Sac 1) GAGCTCACCACATCCCATGAGGCC-3 '
X-R:5 '-GAATTC (EcoR 1) GAATTCCAAGAGCGGCGTTGATGT-3 '
Gentamicin gene primer:
G-F:5 '-TCTAGA (Xba 1) TTGACATAAGCCTGTTCGGTTCGTA-3 '
G-R:5 '-GAGCTC (Sac 1) TTAGGTGGCGGTACTTGGGTCGATA-3 '
Identify primer
F:GCCGCTATCTCGACCATAAT
R:GTGGCTTCATAGATACTGCG
S2, target gene PCR amplification and glue recycling
Using brucella S2 vaccine strain genome and PBBR1MCS5 carrier as template, respectively carry out the upstream PCR amplification ArsR, under
Swim homology arm gene and gentamicin resistance gene；
Reaction system is as follows: 5 × PS Buffer, 5 μ L；dNTP Mixture 2μL；F 0.5μL；R 0.5μL；1 μ L of template；
Prime star DNA polymerase 0.5μL；ddH2O 15.5μL；
PCR reaction condition is as follows:
95 DEG C of initial denaturation 10min；95 DEG C of denaturation 30s；58 DEG C of annealing 50s；72 DEG C of extension 1min, 30 circulations；72 DEG C of extensions
10min；13 DEG C of preservation 1h；
By PCR product through 1% agarose gel electrophoresis, the mesh of 607bp, 825bp and 751bp size is cut respectively under bale cutting instrument
Band blob of viscose, use Tiangeng biochemical technology company produce DNA purification and recovery kit recycle PCR product；
The acquisition of S3, cohesive terminus,cohesive termini target fragment
Respectively by glue recycling the upstream ArsR homology arm gene, the downstream ArsR homology arm gene and gentamicin resistance gene with
For 19T (simple) carrier in 16 DEG C of connection 2h, linked system is as follows:
1 μ L of target gene；19T vector 4μL；Soluton 1 5μL；
Product after connection is transformed into competent escherichia coli cell DH5 α, is applied to the A of preparatory 37 DEG C of preheatings+LB solid plate,
It is smeared uniformly with spreader, after placing 37 DEG C of constant incubator 12h, takes out solid plate from 37 DEG C of constant incubators, use rifle
Choicest goes out to grow bacterium colony in order, is inoculated in 50mL centrifuge tube, 20mL LB liquid medium is added, is placed in 37 DEG C of perseverances
12h is cultivated in warm shaking table；Later, the plasmid produced using Tiangeng biochemical technology company is small to propose middle amount kit extraction recombination load
Body；
By the upstream recombinant vector 19T (the simple)-ArsR homology arm extracted, 19T (simple)-ArsR downstream homology arm and
19T(simple)-G+Pst 1 and Xba 1, Sac 1 and EcoR 1 and 1 double digestion of Xba 1 and Sac, digestion products are used respectively
1% agarose gel electrophoresis identification is carried out, it is big mould that glue recycles the upstream ArsR homology arm gene, the downstream ArsR homology arm gene and celebrating
Plain gene；1 double digestion PUC19 carrier of EcoR 1 and Pst is used again, digestion products carry out 1% agarose gel electrophoresis identification,
Glue recycles the PUC19 segment after double digestion；
The upstream 19T (simple)-ArsR homology arm digestion system:
Pst 1 1.5μL；Xba 1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
The downstream 19T (simple)-ArsR homology arm digestion system:
Sac 1 1.5μL；EcoR 1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
19T (simple)-G+ digestion system:
Sac 1 1.5μL；Xba 1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
PUC19 digestion system:
Pst 1 1.5μL；EcoR 1 1.5μL；10×M Buffer 5μL；30 μ L of plasmid；TB 12μL；
S4, homologous recombination vector PUC19G-ArsR building
The upstream ArsR homology arm gene that previous step glue is recycled, the downstream ArsR homology arm gene, gentamicin resistance gene and
The PUC19 carrier of double digestion 16 DEG C of connection 4h on connection instrument；Linked system is as follows:
3 μ L of the upstream ArsR homology arm gene；
3 μ L of the downstream ArsR homology arm gene；
3 μ L of gentamicin gene；
PUC19 1μL；
Solution 1 10μL；
Product after connection is transformed into competent escherichia coli cell DH5 α, is applied to the G of preparatory 37 DEG C of preheatings+LB solid plate,
It is smeared uniformly with spreader, after placing 37 DEG C of constant incubator 12h, takes out solid plate from 37 DEG C of constant incubators, use rifle
Choicest goes out to grow bacterium colony in order, is inoculated in 50mL centrifuge tube, 20mL LB liquid medium is added, is placed in 37 DEG C of perseverances
12h is cultivated in warm shaking table；Later, the plasmid produced using Tiangeng biochemical technology company is small to mention middle amount kit extraction PUC19G-
ArsR recombinant vector；
The recombinant vector PUC19G-ArsR extracted is subjected to double digestion with Pst 1 and EcoR 1, digestion products carry out 1% fine jade
Sepharose electroresis appraisal；
The preparation of S5, brucella competence
S5.1, it the brucella after activation is diluted to suitable concentration is applied on TSA solid plate, be placed in 37 DEG C of constant incubators
Middle culture 72h；
S5.2, TSA solid plate is taken out from 37 DEG C of constant incubators, choose the good bacterium colony of upgrowth situation with pipette tips, be inoculated with
In 50mL centrifuge tube, 10mL TSB fluid nutrient medium is added, is placed in 37 DEG C of constant-temperature tables and cultivates about for 24 hours；
S5.3, the bacterium solution of previous step is inoculated in 50mL centrifuge tube according to 1:100, be placed in 37 DEG C of constant-temperature tables cultivate to
OD600About 0.6；
S5.4,4 DEG C of the bacterium solution centrifugations by previous step, 5 000r/min are centrifuged 10min, discard liquid, retain precipitating；
The sterile water 10mL that S5.5, addition are pre-chilled in advance, is resuspended precipitating, and 54 DEG C of 000r/min centrifugation 10min discard liquid,
Retain precipitating；
S5.6, step S5.5 is repeated；
The 10% glycerol 10mL that S5.7, addition are pre-chilled in advance, is resuspended precipitating, and 54 DEG C of 000r/min centrifugation 10min discard liquid
Body retains precipitating；
S5.8, step S5.7 is repeated；
Precipitating is resuspended in the 10% glycerol 1mL that S5.9, addition are pre-chilled in advance, and 100 μ L packing, -80 DEG C save backup；
S6, electrotransformation
It takes 10 μ L of PUC19G-ArsR plasmid to be added in 100 μ L brucella competence, the electric shock cup of pre-cooling is moved into after mixing
In, 10min is placed on ice, is put into electroporation, voltage 1.8KV, time 6ms；37 DEG C of preheatings are added after having shocked by electricity immediately
TSB culture solution；It is placed in 180rpm/min culture in 37 DEG C of constant-temperature tables for 24 hours, to take out from 37 DEG C of constant-temperature tables, after centrifugation, use
Precipitating is resuspended in about 200 μ L TSB, is applied on TSB gentamicin resistance plate later, cultivates 72h；
The screening of S7, muton
TSA solid plate is taken out from 37 DEG C of constant incubators, is chosen the good bacterium colony of upgrowth situation with pipette tips, is inoculated in 10mL
In centrifuge tube, 5mL TSB gentamicin resistance fluid nutrient medium is added, is placed in 37 DEG C of constant-temperature tables and cultivates about 48h；Later,
Brucella genome is extracted, gene-deleted strain is screened from gene level, correct bacterial strain will be screened and reflected again by qRT-PCR
It is fixed.
3. the application of brucella ArsR gene deletion strains as claimed in claim 2, it is characterised in that: pass through following steps
Product after connection is transformed into competent escherichia coli cell DH5 α:
(1) competent escherichia coli cell DH5 α is taken out from -80 DEG C of refrigerators, is placed in about 5min on ice, until competent cell
Melt completely；
(2) it is added the competent escherichia coli cell DH5 α that 20 μ L have melted into the 1.5mL centrifuge tube being pre-chilled in advance, then plus
Enter whole connection products, mix well, when operation not leave ice face；
(3) 1.5mL centrifuge tube is stood into 30min on ice after mixing, later 42 DEG C of thermostat water bath thermal shock 1min, later again
Place 2min on ice；
(4) from centrifuge tube is taken out on ice, LB culture solution 500L is added in superclean bench, is placed in 37 DEG C of constant-temperature tables later
Middle 180r/min recovery 1h；
(5) the A+LB solid plate for taking preparatory 37 DEG C of preheatings, is added 100L recovery product, is smeared uniformly with spreader, places 37
DEG C constant incubator 12h.
4. the application of brucella ArsR gene deletion strains as described in claim 1, it is characterised in that: it can be used for preparing
Brucella attenuated vaccine.
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