CN109294967A - The building and application of one plant of ExPEC yhaV-prlF gene-deleted strain - Google Patents
The building and application of one plant of ExPEC yhaV-prlF gene-deleted strain Download PDFInfo
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Abstract
The invention discloses one plant of ExPECyhaV‑prlFThe building and application of gene-deleted strain, belong to gene engineering technology field.The present invention constructs one plant using DNA homologous recombination technologyyhaV‑ prlFGene delection parenteral enteropathogenic E. Coli (Escherichia coli) ExPEC PPECC42-ΔyhaV‑ prlF, which is preserved in China typical culture collection center, deposit number are as follows: CCTCC NO:M 2018686.The bacterial strain is applied in the research of the formation novel drug target of the evaluation of bacterial biofilm Forming ability and control ExPEC bacterial biofilm.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to one plant of ExPECyhaV-prlFThe structure of gene-deleted strain
It builds and applies.
Background technique
Parenteral Enteropathogenic Escherichia coli (Extraintestinal PathogenicE. coli, ExPEC) and it is a kind of
Important pathogen, it is of increasing concern to animal husbandry (pig, fowl, cattle and sheep) and the harm of human health in recent years, multiple resistance to
The formation of pharmacological property and biofilm further increases the difficulty of its prevention and control and to sanitarian threat.ExPEC can cause people, dote on
The Extra intestinal infection of object and edible animal, such as neonatal meningitis, septicemia, pneumonia and mammitis.Furthermore ExPEC is also one
The important food source bacterium of kind, can cause food origin disease outbreak of epidemic by drinking-water, the food of pollution；ExPEC bacterial strain is not at present
Same country widely separates from retail chicken, beef, pork, restaurant, ready-to-eat food (including meat, fruits and vegetables)
It arrives, therefore speculates that meat products may be the important sources of people ExPEC infection.
Bacterial biofilm (Bacterial Biofilm) is that bacterial adhesion has gathered life or without the life surface of solids
A kind of group's sexual behaviour, be bacterium to adapt to environment and a kind of strategy that long-term surviving is taken.The formation of biofilm has
There is extensive harmfulness, once being formed biofilm, thallus is wrapped up by a large amount of extracellular matrix, forms a physical barriers, can
Enhance the resistance of the compounds such as bacterial antibiotic, disinfectant, cholate；It is thin that the phagocytosiss such as macrophage can be resisted in vivo
The immune clearance of host is escaped in the phagocytosis of born of the same parents；It is formed on body tissue surface, chronic infection and lasting sexuality can be caused
Dye；The propagation of food-borne microorganism, open date can be caused to shorten in the biofilm of the formation such as food processing equipment.
The presence of bacterial biofilm will seriously threaten the health of livestock and poultry and the mankind, the biofilm present in food production,
The important hidden danger for influencing public health security will be become.Therefore, control bacterial biofilm is formed in animal doctor, medicine, food
Equal fields all have particularly important meaning.
Toxin-antitoxin system (toxin-antitoxin, T-A) is widely present in bacterial genomes, in bacterium
It is played an important role in physiological activity: maintaining the stability of genome, promote the formation for withholding cell under antibiotic pressure, increase
The tolerance of strong phage-infect regulates and controls bacterial cell programmed death, plays a role in bacterium is caused a disease.Therefore T-A system
The new target drone for becoming multi drug resistant bacterial pathogens pathogenesis and Control Technology research, the height for causing domestic and international researcher are closed
Note.
The present invention is using ExPEC wild-type strain and building oxin-antitoxinyhaV-prlFGene deletion strains are evaluated
Effect of the YhaV-PrlF system in ExPEC biofilm is formed, it is raw to control ExPEC bacterium to can be used as new drug targets
The formation of object envelope.
Summary of the invention
It is an object of the present invention to construct one plant of ExPECyhaV-prlFGene-deleted strain ExPEC PPECC42-Δ yhaV-prlF, the classification naming of the bacterial strain be parenteral enteropathogenic E. Coli (Escherichia coli) ExPEC
PPECC42-ΔyhaV-prlF, on October 17th, 2018, it is deposited in China typical culture collection center, deposit number are as follows:
CCTCC NO:M 2018686, preservation address are as follows: the Chinese Wuhan Wuhan University.
Another object of the present invention is to provide one plant of ExPECyhaV-prlFGene-deleted strain ExPEC PPECC42-Δ yhaV-prlFConstruction method.
A further object of the present invention is to provide one plant of ExPECyhaV-prlFGene-deleted strain ExPEC PPECC42-Δ yhaV-prlFActive application is formed in ExPEC biofilm in evaluation YhaV-PrlF system, and to provide control ExPEC thin
The application of bacterium biofilm formed in novel drug target.
To achieve the above object, the present invention adopts the following technical scheme:
Parenteral enteropathogenic E. Coli (ExPEC) is carried out using DNA homologous recombination technologyyhaV-prlFThe building of gene-deleted strain
With the measurement of biofilm Forming ability.
yhaV-prlFThe building of gene-deleted strain, comprises the following specific steps that:
1, the building of the clone of gene and homologous recombination plasmid
(1) extraction of genomic DNA: the genomic DNA of parent strain ExPEC PPECC42 is extracted using hot boiling method: choosing list
For bacterium colony to being added in the EP pipe of 50 μ L distilled waters, boiling water bath boils 5min, 10000rpm is centrifuged 3min, draws supernatant to newly
It is then genomic DNA in EP pipe, -20 DEG C save backup.
(2) design of primer
Utilize primer-design software Primer5.0, designyhaV-prlFThe primer of the upstream and downstream segment of DNA homolog arm, and lack
Identification primer is lost, primer sequence is shown in Table 1:
Table 1. yhaV-prlFGene-deleted strain constructs the primer sequence and PCR product size
(3) building of homologous recombination plasmid
1)yhaV-prlFGene upstream and downstream fragment amplification
According to the principle and method of over-lap PCR (overlapping-PCR), P1/P2(SEQ ID NO:1/SEQ ID NO is used:
2), P3/P4(SEQ ID NO:3/SEQ ID NO:4) it is two right respectively to primeryhaV-prlFThe fragment upstream of gene and downstream
Segment is expanded, P1/P2 amplificationyhaV-prlFUpstream region of gene clip size is 658 bp, P3/P4 amplificationyhaV-prlFBase
Because segments downstream is 715 bp.
Amplification system is as shown in table 2:
Table 2yhaV-prlFGene upstream and downstream fragment amplification system
PCR reaction condition: × 30cycles → 4 DEG C [98 DEG C of 10s → 56 DEG C 5s → 72 DEG C 1min].
2) over-lap PCR
The amplified production difference gel extraction of upstream and downstream segment is utilized into over-lap PCR pair by the product of recycling as templateyhaV-prlFGene upstream and downstream segment carries out over-lap PCR.With primer P1/P4(SEQ ID NO:1/SEQ ID NO:4) it is template
It is expanded, amplificationyhaV-prlFGene delection segment Δ needed for homologous recombinationyhaV-prlFSize be 1341
Bp, amplification system are as shown in table 3.
3 over-lap PCR amplification system of table
5 circulations are first expanded, each 1 μ L of primer P1/P4 is then added and carries out PCR amplification；
PCR reaction condition: [98 DEG C of 10s → 56 DEG C 5s → 72 DEG C 1min40sec] × cycles → 4 DEG C (5+28).
3) connection of digestion and digestion products
The Δ that above-mentioned over-lap PCR is obtainedyhaV-prlFGenetic fragment product carries out gel extraction, by recovery product and Suicide
CarrierpRE112Respectively carry out Xba I and Sac I double digestion, 37 DEG C of digestion 3h.Digestion products carry out gel extraction, by recycling
Target fragment and carrier segments are with the 16 DEG C of connections overnight of T4 DNA ligase.
Endonuclease reaction system is as shown in table 4:
4 endonuclease reaction system of table
Digestion products coupled reaction system is as shown in table 5:
5 digestion products coupled reaction system of table
4) connection product transformed competence colibacillus cell
10 μ L stay overnight connection product and are added in 100 μ L competent cell χ 7213, mix gently, ice bath 30min, stand after taking-up
That is 42 DEG C of water-bath heat shock 90sec, ice bath 90sec, adds the 600 μ L LB liquid training containing final concentration of 10 μ g/mlDAP immediately
Base is supported, is put into shaken cultivation 45min-60min in 37 DEG C of shaking tables, revolving speed≤200rpm carries out the recovery of bacterium solution.Take 200 μ L multiple
The bacterium solution of Soviet Union is coated on the L-A plate containing 25 μ g/ml chloramphenicol of final concentration and 10 μ g/mlDAP, and 37 DEG C of cultures may occur in which for 24 hours
Single colonie.Picking single colonie culture, extracts plasmid, and digestion is identified that correct plasmid send sequencing by digestion identification；Sequencing is correct,
Obtain homologous recombination plasmid pRE112- ΔyhaV-prlF。
2, engagement transfer withyhaV-prlFThe screening and identification of gene deletion strains
To have converted homologous recombination plasmid pRE112- ΔyhaV-prlF7213 bacterial strain of χ be donor bacterium, with parent strain ExPEC
PPECC42 is that recipient bacterium carries out engagement transfer.Homologous recombination plasmid pRE112- Δ is convertedyhaV-prlF7213 donor bacterium of χ
200rpm, 37 DEG C of overnight shaking cultures, with fresh LB liquid medium tune are respectively carried out with parent strain ExPEC PPECC42
Whole OD600Value is 0.7 ~ 0.9, respectively takes 100 μ L to mix, the nitrocellulose of sterilizing is affixed on containing final concentration of 10 μ g/mlDAP
L-A plate, 200 μ L mixed bacteria liquids are spread evenly across on filter membrane, after co-culturing 8 ~ 10h, with LB liquid medium by filter membrane
On bacterium wash twice, cleaning solution 6000rpm is centrifuged 5min, will the 200 μ L fresh LB liquid medium resuspension of washing precipitating bacterium,
It is spread evenly across the L-A plate of the 12.5 μ g/ml chloramphenicol containing final concentration, 37 DEG C of 12 ~ 14h of culture, picking single bacterium is fallen within containing dense eventually
It spends in the LB liquid medium of 25 μ g/ml chloramphenicol, 37 DEG C, 200 rpm shaken cultivation 8h, streak inoculation is in chloramphenicol final concentration
To screen chlorampenicol resistant, the joint element of sucrose sensitivity on the L-A plate of 12.5 μ g/ml and sucrose final concentration 50mg/ml.
By the above-mentioned chlorampenicol resistant screened, the joint element of sucrose sensitivity is seeded in no NaCl, the LB liquid of non-resistant
In culture medium, 37 DEG C, 10 times after 200 17 ~ 18h of rpm shaken cultivation, 100 times, 1000 times of dilutions, by the bacterium of each dilution gradient
Liquid takes the 100 μ L coating 50mg/ml of final concentration containing sucrose respectively and non-resistant is without on the L-A plate of NaCl, 37 DEG C of culture 12-14h,
Picking single colonie is inoculated with the L-A plate containing chloramphenicol final concentration of 12.5 μ g/ml and sucrose final concentration 50mg/ml, and screening chlorine is mould
Plain sensitive, the bacterium colony of sucrose resistance uses primer P5/P6(SEQ ID NO:5/SEQ ID NO:6) carry out PCR identification, product
Send sequencing.Sequencing is correct, obtains one plant of ExPEC gene-deleted strain, i.e. ExPEC PPECC42-ΔyhaV-prlFBacterial strain.
PCR identification reaction system such as table 6:
6 PCR identification reaction system of table
PCR reaction condition: × 30cycles → 4 DEG C [98 DEG C of 10s → 56 DEG C 5s → 72 DEG C 1min40sec].
3, ExPEC wild strain andyhaV-prlFThe biofilm Forming ability of gene deletion strains measures
It is modified and is carried out according to the method in bibliography (Stepanovic et al 2000), by parent strain ExPEC
PPECC42 andyhaV-prlFGene deletion strains, i.e. ExPEC PPECC42-ΔyhaV-prlFBacterial strain is inoculated in LB training respectively
Base is supported, 37 DEG C, 200rpm shaken cultivation is stayed overnight, and overnight culture is carried out 1:100 dilution with fresh M9 culture medium respectively, is diluted
Good bacterium solution is separately added into 96 porocyte culture plates, and every 100 μ L of hole, every plant of bacterium sets 8 repetitions respectively.With aseptic culture medium
Make blank control, 28 DEG C or 37 DEG C stationary culture 5 days, culture is gently sucked out, three times with aseptic distillation water washing micropore, is dried in the air
Dry, in 96 orifice plates, every hole adds the violet staining 15min of the 1wt% of 125 μ L, gently washes out dye liquor with flowing water, gently gets rid of and dry, then
The 150 μ L of acetic acid of 33vol% is added in every hole, and oscillation dissolution 10min is placed under microplate reader and measures OD630Value, with statistical analysis
The data obtained.
The present invention has the advantages that one plant of ExPEC of the inventionyhaV-prlFGene-deleted strain, i.e. ExPEC
PPECC42-ΔyhaV-prlFBacterial strain is in parent strain ExPEC PPECC42yhaV-prlFThe bacterial strain of gene delection, should
Bacterial strain is applied to the evaluation of bacterial biofilm Forming ability and control ExPEC bacterial biofilm forms grinding for novel drug target
In studying carefully, it is of great significance to the research of bacterial toxin-antitoxin systematic research and bacterium pathogenesis and Control Technology.
Detailed description of the invention
Fig. 1yhaV-prlFGene upstream and downstream fragment amplification PCR product electrophoresis result.Swimming lane M:DNA Marker；Swimming lane
1: primer P3/P4 amplificationyhaV-prlFDownstream of gene segment, 715bp；Swimming lane 2: primer P1/P2 amplificationyhaV-prlFBase
Because of fragment upstream, 658bp.
Fig. 2yhaV-prlFGene upstream and downstream segment overlapping PCR products electrophoresis result.Swimming lane M:DNA Marker；Swimming lane
1: primer P1/P4 amplificationyhaV-prlFGene delection overlapping PCR products segment, 1341bp.
Fig. 3 identifies that primer P5/P6 expands deletion mycopremna ExPEC PPECC42-ΔyhaV-prlFAnd parent strain
ExPEC PPECC42'syhaV-prlFGene electrophoresis result.Swimming lane M:DNA Marker；Swimming lane 1: with parent strain ExPEC
PPECC42 genome is template, and amplification gained purpose band is 987bp；Swimming lane 2-5: being with ExPEC PPECC42-Δ yhaV-prlFStrain gene group is template, and amplification gained purpose band is 327bp；Swimming lane 6: using water as the negative control of template.
Fig. 4 is in M9 culture medium, measured by way of crystal violet dyeing ExPEC PPECC42-ΔyhaV-prlFWith parent strain
The biofilm formational situation of ExPEC PPECC42.ΔYhaV-PrlF: ExPEC PPECC42-ΔyhaV-prlF ；WT: parent
This bacterial strain EXPEC PPECC42；NC: the blank control of aseptic culture medium；*:P < 0.01。
Specific embodiment
The present invention is described further with reference to embodiments
Embodiment 1
Parenteral enteropathogenic E. Coli (ExPEC) is carried out using DNA homologous recombination technologyyhaV-prlFGene-deleted strain ExPEC
PPECC42-ΔyhaV-prlFBuilding and biofilm Forming ability measurement.
yhaV-prlFGene-deleted strain ExPEC PPECC42-ΔyhaV-prlFBuilding, comprise the following specific steps that:
1, the building of the clone of gene and homologous recombination plasmid
(1) extraction of genomic DNA: the genomic DNA of parent strain ExPEC PPECC42 is extracted using hot boiling method: choosing list
For bacterium colony to being added in the EP pipe of 50 μ L distilled waters, boiling water bath boils 5min, 10000rpm is centrifuged 3min, draws supernatant to newly
It is then genomic DNA in EP pipe, -20 DEG C save backup.
(2) design of primer
Utilize primer-design software Primer5.0, designyhaV-prlFThe primer of the upstream and downstream segment of gene, and missing mirror
Determine primer, primer sequence is shown in Table 7:
Table 7.yhaV-prlFGene-deleted strain constructs the primer sequence and PCR product size
3) building of homologous recombination plasmid
1)yhaV-prlFGene upstream and downstream fragment amplification
According to the principle and method of over-lap PCR (overlapping-PCR), using P1/P2, P3/P4 two is right respectively to primeryhaV-prlFThe fragment upstream and segments downstream of gene are expanded, P1/P2 amplificationyhaV-prlFUpstream region of gene clip size
For 658 bp, P3/P4 amplificationyhaV-prlFDownstream of gene segment is 715 bp.
Amplification system is as shown in table 8:
Table 8yhaV-prlFGene upstream and downstream fragment amplification system
PCR reaction condition: × 30cycles → 4 DEG C [98 DEG C of 10s → 56 DEG C 5s → 72 DEG C 1min].
2) over-lap PCR
The amplified production difference gel extraction of upstream and downstream segment is utilized into over-lap PCR pair by the product of recycling as templateyhaV-prlFGene upstream and downstream segment carries out over-lap PCR.It is expanded using primer P1/P4 as template, amplificationyhaV-prlF
Gene delection segment Δ needed for homologous recombinationyhaV-prlFSize be 1341 bp, amplification system is as shown in table 9.
9 over-lap PCR amplification system of table
5 circulations are first expanded, each 1 μ L of primer P1/P4 is then added and carries out PCR amplification；
PCR reaction condition: [98 DEG C of 10s → 56 DEG C 5s → 72 DEG C 1min40sec] × cycles → 4 DEG C (5+28).
3) connection of digestion and digestion products
The Δ that above-mentioned over-lap PCR is obtainedyhaV-prlFGenetic fragment product carries out gel extraction, by recovery product and Suicide
CarrierpRE112Respectively carry out Xba I and Sac I double digestion, 37 DEG C of digestion 3h.Digestion products carry out gel extraction, will recycle
Target fragment and carrier segments with the connection overnight of 16 DEG C of T4 DNA ligase.
Endonuclease reaction system is as shown in table 10:
10 endonuclease reaction system of table
Digestion products coupled reaction system is as shown in table 11:
11 digestion products coupled reaction system of table
4) connection product transformed competence colibacillus cell
10 μ L stay overnight connection product and are added in 100 μ L competent cell χ 7213, mix gently, ice bath 30min, stand after taking-up
That is 42 DEG C of water-bath heat shock 90sec, ice bath 90sec, adds the 600 μ L LB Liquid Cultures of final concentration of 10 μ g/mlDAP immediately
Base, is put into shaken cultivation 45min in 37 DEG C of shaking tables, and revolving speed 200rpm carries out the recovery of bacterium solution.The bacterium solution for taking 200 μ L to recover applies
It is distributed on the L-A plate containing 25 μ g/ml chloramphenicol of final concentration and 10 μ g/mlDAP, 37 DEG C of cultures may occur in which single colonie for 24 hours.Picking
Single colonie culture, extracts plasmid, and digestion is identified that correct plasmid send Takara company to be sequenced by digestion identification；Sequencing is correct, obtains
Obtain homologous recombination plasmid pRE112- ΔyhaV-prlF。
2, engagement transfer withyhaV-prlFThe screening and identification of gene deletion strains
To have converted homologous recombination plasmid pRE112- ΔyhaV-prlF7213 bacterium of χ be donor bacterium, with parent strain ExPEC
PPECC42 is that recipient bacterium carries out engagement transfer.Homologous recombination plasmid pRE112- Δ is convertedyhaV-prlF7213 donor bacterium of χ
200rpm, 37 DEG C of overnight shaking cultures, with fresh LB liquid medium tune are respectively carried out with parent strain ExPEC PPECC42
Whole OD600Value is 0.8, respectively takes 100 μ L to mix, the nitrocellulose of sterilizing is affixed on the L-A containing final concentration of 10 μ g/mlDAP
200 μ L mixed bacteria liquids are spread evenly across on filter membrane by plate, after co-culturing 8-10h, with LB liquid medium by the bacterium on filter membrane
It washes twice, cleaning solution 6000rpm is centrifuged 5min, and the washing precipitating bacterium fresh LB liquid medium of 200 μ L is resuspended, is uniformly applied
It is distributed in the L-A plate of the 12.5 μ g/ml chloramphenicol containing final concentration, 37 DEG C of culture 13h, picking single bacterium is fallen within containing 25 μ g/ml of final concentration
In the LB liquid medium of chloramphenicol, 37 DEG C, 200 rpm shaken cultivation 8h, streak inoculation is in the final concentration of 12.5 μ g/ of chloramphenicol
On the L-A plate of ml and sucrose final concentration 50mg/ml, chlorampenicol resistant, the joint element of sucrose sensitivity are screened.
By the above-mentioned chlorampenicol resistant screened, the joint element of sucrose sensitivity is seeded in no NaCl, the LB liquid of non-resistant
In culture medium, 37 DEG C, 10 times after 200 rpm shaken cultivation 18h, 100 times, 1000 times of dilutions, by the bacterium solution of each dilution gradient
Take that 100 μ L are coated on the 50mg/ml of final concentration containing sucrose and non-resistant is without on the L-A plate of NaCl respectively, 37 DEG C of culture 13h choose
Single colonie is taken to be inoculated in the L-A plate containing chloramphenicol final concentration of 12.5 μ g/ml and sucrose final concentration 50mg/ml, screening chlorine is mould
Plain sensitive, the bacterium colony of sucrose resistance carries out PCR identification using primer P5/P6, and product send Nanjing Jin Sirui biotechnology limited public affairs
Sequencing.Sequencing is correct, obtains one plant of ExPEC gene-deleted strain, i.e. ExPEC PPECC42-ΔyhaV-prlFBacterial strain.
PCR identification reaction system such as table 12:
12 PCR identification reaction system of table
PCR reaction condition: × 30cycles → 4 DEG C [98 DEG C of 10s → 56 DEG C 5s → 72 DEG C 1min40sec].
2 ExPEC wild strain of embodiment andyhaV-prlFThe biofilm Forming ability of gene deletion strains measures
It is modified and is carried out according to the method in bibliography (Stepanovic et al 2000), by parent strain ExPEC
PPECC42 andyhaV-prlFGene deletion strains, i.e. ExPEC PPECC42-ΔyhaV-prlFBacterial strain is inoculated in LB training respectively
Base is supported, 37 DEG C, 200rpm shaken cultivation is stayed overnight, and overnight culture is carried out 1:100 dilution with fresh M9 culture medium respectively, is diluted
Good bacterium solution is separately added into 96 porocyte culture plates, and every 100 μ L of hole, every plant of bacterium sets 8 repetitions respectively.With aseptic culture medium
Making blank control, culture is gently sucked out 28 DEG C of 5 days of stationary culture, three times with aseptic distillation water washing micropore, dry,
In 96 orifice plates, every hole adds the violet staining 15min of the 1wt% of 125 μ L, gently washes out dye liquor with flowing water, gently gets rid of and dry, then often
33% 150 μ L of acetic acid is added in hole, and oscillation dissolution 10min is placed under microplate reader and measures OD630Value carries out non-matching through Excel
Student’s t- test. is examined, under the conditions of 28 DEG C, after culture 5 days,yhaV-prlFGene deletion strains, i.e. ExPEC
PPECC42-ΔyhaV-prlF(ΔYhaV-PrlF) bacterial strain is with parent strain ExPEC PPECC42(WT) compared with, biofilm
Forming ability be remarkably decreased (P < 0.01).
3 result of embodiment
The building of gene-deleted strain is carried out according to the principle of homologous recombination and suicide plasmid pRE112.It expands firstyhaV-prlFBase
The upstream and downstream segment of cause, size are respectively 658 bp, 715 bp, amplification such as Fig. 1.The upstream and downstream segment of amplification is pure
Gene delection segment Δ needed for method amplification homologous recombination after change using over-lap PCRyhaV-prlF, size 1341
Bp, amplification such as Fig. 2.By the segment recycling and suicide vector pRE112 digestion simultaneously of overlapping PCR products, purify back
Digestion products are received, are transferred to after connectionχ7213, choose single colonie and expand culture extracting plasmid progress digestion identification, by digestion band position
Setting correct plasmid 2 confirms that sequence deletion is correct by sequencing.
It after engagement transfer, is screened twice, the correct single colonie of phenotype is inoculated in LB culture medium, after expanding culture, is adopted
PCR identification is carried out with primer P5/P6, PCR product is shown in Fig. 3 through electroresis appraisal, as the result is shownyhaV-prlFGene deletion strains,
That is ExPEC PPECC42-ΔyhaV-prlFThe pcr amplification product size of bacterial strain is 327 bp, control group parent strain ExPEC
The pcr amplification product size of PPECC42 is that 987bp is small；It is consistent with expected results, tentative confirmationyhaV-prlFGene delection bacterium
Strain constructs successfully, and sequencing after PCR product segment recovery purifying is confirmed that deletion sequence is correct.
To explore effect of the YhaV-PrlF in ExPEC biofilm is formed, using crystal violet staining assay to parent strain
ExPEC PPECC42 andyhaV-prlFGene deletion strains ExPEC PPECC42-ΔyhaV-prlFBiofilm formed energy
Power is measured and compares.It is cultivated 5 days under the conditions of 28 DEG C of M9 culture medium as the result is shown, parent strain ExPEC PPECC42's
Biofilm Forming ability is very strong, forms bacterial strain for strong biofilm, andyhaV-prlFGene deletion strains ExPEC
PPECC42-ΔyhaV-prlFBiofilm Forming ability be remarkably decreased (P < 0.01), as a result see Fig. 4, therefore YhaV-
PrlF can provide the control new potential drug target that ExPEC biofilm is formed.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
The building and application of<120>one plants of ExPEC yhaV-prlF gene-deleted strains
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<170> PatentIn version 3.3
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aggaggatgt cctctttgcg tgagagcgag cattagc 37
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tgctcgctct cacgcaaaga ggacatcctc ct 32
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Claims (8)
1. one plant of ExPECyhaV-prlFGene-deleted strain ExPEC PPECC42-ΔyhaV-prlF, it is characterised in that: it is described
ExPEC PPECC42-ΔyhaV-prlFBacterial strain, that is, parenteral enteropathogenic E. Coli (Escherichia coli) ExPEC
PPECC42-ΔyhaV-prlFIt is parent strain ExPEC PPECC42 missingyhaV-prlFObtained by gene, in October, 2018
It is deposited in China typical culture collection center, deposit number within 17th are as follows: CCTCC NO:M 2018686.
2. one plant of ExPEC as described in claim 1yhaV-prlFGene-deleted strain ExPEC PPECC42-ΔyhaV-prlF
Construction method, which comprises the following steps:
(1) building of the clone of gene and homologous recombination plasmid: extract parent strain ExPEC PPECC42's using hot boiling method
Genomic DNA；Utilize primer-design software Primer5.0, designyhaV-prlFThe primer of the upstream and downstream segment of gene；AmplificationyhaV-prlFThe upstream and downstream segment of gene；Utilize over-lap PCR pairyhaV-prlFGene upstream and downstream segment carries out over-lap PCR, obtains
?yhaV-prlFGene delection segment needed for homologous recombinationΔyhaV-prlF；What over-lap PCR obtainedΔyhaV-prlFBase
Because segment carries out gel extraction, recovery product and suicide vector pRE112 are respectively subjected to Xba I and Sac I double digestion, enzyme
It cuts product and carries out gel extraction, the target fragment of recycling is connected with carrier segments with T4 DNA ligase；Connection product conversion
Competent cell, transformant extract plasmid, and digestion is identified and is sequenced, and obtain homologous recombination plasmid pRE112-ΔyhaV-prlF；
(2) engagement transfer withyhaV-prlFThe screening and identification of gene deletion strains: to have converted homologous recombination plasmid
pRE112-ΔyhaV-prlF7213 bacterial strain of χ be donor bacterium, engaged using parent strain ExPEC PPECC42 as recipient bacterium
After transfer, on the L-A plate containing chloramphenicol and sucrose, chlorampenicol resistant, the joint element of sucrose sensitivity are screened；Screening obtains chlorine
Chloramphenicol resistance, the joint element of sucrose sensitivity are seeded in no NaCl, in the LB liquid medium of non-resistant, dilute after shaken cultivation；
Diluted bacterium solution is coated on containing sucrose and non-resistant on the L-A plate of NaCl without cultivating, picking single colonie is inoculated in mould containing chlorine
On the L-A plate of element and sucrose, Chloramphenicol-sensitive is screened, the bacterium colony of sucrose resistance carries out PCR identification, and product send sequencing.
3. one plant of ExPEC according to claim 2 yhaV-prlFGene-deleted strain ExPEC PPECC42-ΔyhaV- prlFConstruction method, which is characterized in that in step (1), for expandingyhaV-prlFThe primer of upstream region of gene segment is SEQ
ID NO:1 and SEQ ID NO:2；For expandingyhaV-prlFThe primer of downstream of gene segment is SEQ ID NO:3 and SEQ ID
NO:4；Primer for over-lap PCR is SEQ ID NO:1 and SEQ ID NO:4.
4. one plant of ExPEC according to claim 2yhaV-prlFGene-deleted strain ExPEC PPECC42-ΔyhaV- prlFConstruction method, which is characterized in that it is SEQ ID NO:5 and SEQ ID NO that the primer that PCR is identified is carried out in step (2):
6。
5. one plant of ExPEC according to claim 2 yhaV-prlFGene-deleted strain ExPEC PPECC42-ΔyhaV- prlFConstruction method, which is characterized in that the building of the clone of step (1) gene and homologous recombination plasmid, including following specific
Step:
(1) extraction of genomic DNA: the genomic DNA of parent strain ExPEC PPECC42 is extracted using hot boiling method: choosing list
For bacterium colony to being added in the EP pipe of 50 μ L distilled waters, boiling water bath boils 5min, 10000rpm is centrifuged 3min, draws supernatant to newly
It is then genomic DNA in EP pipe, -20 DEG C save backup；
(2) design of primer:
Utilize primer-design software Primer5.0, designyhaV-prlFThe primer of the upstream and downstream segment of gene, and missing mirror
Determine primer；
(3) building of homologous recombination plasmid:
1)yhaV-prlFGene upstream and downstream fragment amplification
According to the principle and method of over-lap PCR (overlapping-PCR), using SEQ ID NO:1, SEQ ID NO:2 and
SEQ ID NO:3, SEQ ID NO:4 two are right respectively to primeryhaV-prlFThe fragment upstream and segments downstream of gene are expanded
Increase, SEQ ID NO:1, SEQ ID NO:2 amplificationyhaV-prlFUpstream region of gene clip size is 658 bp；SEQ ID NO:
3, SEQ ID NO:4 is expandedyhaV-prlFDownstream of gene segment is 715bp；
2) over-lap PCR
The amplified production difference gel extraction of upstream and downstream segment is utilized into over-lap PCR pair by the product of recycling as templateyhaV-prlFGene upstream and downstream segment carries out over-lap PCR；It is expanded, is expanded with primer SEQ ID NO:1, SEQ ID NO:4
'syhaV-prlFGene delection segment needed for homologous recombinationΔyhaV-prlFSize be 1341 bp；
3) connection of digestion and digestion products
The Δ that above-mentioned over-lap PCR is obtainedyhaV-prlFGenetic fragment carries out gel extraction, by recovery product and suicide vector
PRE112 respectively carries out Xba I and Sac I double digestion, 37 DEG C of digestion 3h；Digestion products carry out gel extraction, by the purpose of recycling
Segment and carrier segments are with the 16 DEG C of connections overnight of T4 DNA ligase；
4) connection product transformed competence colibacillus cell
10 μ L stay overnight connection product and are added in 100 μ L competent cell χ 7213, mix gently, ice bath 30min, stand after taking-up
That is 42 DEG C of water-bath heat shock 90sec, immediately ice bath 90sec add 2,6- diamino heptan of the 600 μ L containing final concentration of 10 μ g/ml
The LB liquid medium of diacid (DAP), is put into shaken cultivation 45min ~ 60min in 37 DEG C of shaking tables, and revolving speed≤200 rpm carries out
The recovery of bacterium solution；It is flat that the bacterium solution for taking 200 μ L to recover is coated on the L-A containing final concentration of 25 μ g/ml chloramphenicol and 10 μ g/ml DAP
On plate, there is transformant for 24 hours in 37 DEG C of cultures；Picking transformant culture, extracts plasmid, and digestion identification is identified digestion correct
Plasmid send sequencing, and sequencing is correct, obtains homologous recombination plasmid pRE112-ΔyhaV-prlF。
6. one plant of ExPEC according to claim 2yhaV-prlFGene-deleted strain ExPEC PPECC42-ΔyhaV- prlFConstruction method, which is characterized in that step (2) engagement transfer withyhaV-prlFThe screening and identification of gene deletion strains,
It comprises the following specific steps that:
(1) to have converted homologous recombination plasmid pRE112-ΔyhaV-prlF7213 bacterial strain of χ be donor bacterium, with parent strain
ExPEC PPECC42 is that recipient bacterium carries out engagement transfer；Homologous recombination plasmid pRE112- Δ is convertedyhaV-prlFχ 7213
Donor bacterium and ExPEC PPECC42 wild strain PPECC42 respectively carry out 200rpm, 37 DEG C of overnight shaking cultures, with fresh
LB liquid medium adjusts OD600Value is 0.7 ~ 0.9, and 100 μ L is respectively taken to mix；The nitrocellulose of sterilizing is affixed on containing dense eventually
Degree is that 200 μ L mixed bacteria liquids are spread evenly across on filter membrane on the L-A plate of 10 μ g/ml DAP, after co-culturing 8 ~ 10h, uses LB
Fluid nutrient medium washes twice the bacterium on filter membrane, and cleaning solution 6000rpm is centrifuged 5min, and washing precipitating bacterium is fresh with 200 μ L
LB liquid medium is resuspended, and is spread evenly across on the L-A plate containing final concentration of 12.5 μ g/ml chloramphenicol, and 37 DEG C of cultures 12 ~
14h, picking single bacterium are fallen in the LB liquid medium containing final concentration of 25 μ g/ml chloramphenicol, and 37 DEG C, 200 rpm shaken cultivations
8h, on the L-A plate of 12.5 μ g/ml final concentration of containing chloramphenicol and sucrose final concentration 50mg/ml, screening chlorine is mould for streak inoculation
Plain resistance, the joint element of sucrose sensitivity；
(2) by the above-mentioned chlorampenicol resistant screened, the joint element of sucrose sensitivity is seeded in no NaCl, the LB liquid training of non-resistant
It supports in base, 37 DEG C, 10 times after 200 17 ~ 18h of rpm shaken cultivation, 100 times, 1000 times of dilutions；By the bacterium solution of each dilution gradient
The 100 μ L coating 50mg/ml of final concentration containing sucrose is taken respectively and non-resistant is without on the L-A plate of NaCl, and 37 DEG C of 12 ~ 14h of culture choose
It takes on L-A plate of the single colonie inoculation containing chloramphenicol final concentration of 12.5 μ g/ml and sucrose final concentration 50mg/ml, screening chlorine is mould
Plain sensitive, the bacterium colony of sucrose resistance carries out PCR identification using primer SEQ ID NO:5, SEQ ID NO:6, and product send sequencing,
Sequencing is correct, obtains one plant of ExPECyhaV-prlFGene-deleted strain, i.e. ExPEC PPECC42-ΔyhaV-prlFBacterial strain.
7. one plant of ExPEC as described in claim 1yhaV-prlFGene-deleted strain ExPEC PPECC42-ΔyhaV-prlF
Active application is formed in ExPEC biofilm in evaluation YhaV-PrlF system.
8. one plant of ExPEC as described in claim 1 yhaV-prlFGene-deleted strain ExPEC PPECC42-ΔyhaV-prlF
The application of control ExPEC bacterial biofilm formed in novel drug target is being provided.
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| CN102164950A (en) * | 2008-08-20 | 2011-08-24 | 新泽西内科与牙科大学 | Novel toxin-antitoxin system |
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