CN110305889A - A kind of building and its application of tubercle bacillus Pup deletion mutant bacterial strain - Google Patents
A kind of building and its application of tubercle bacillus Pup deletion mutant bacterial strain Download PDFInfo
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Abstract
The invention discloses a kind of construction method of tubercle bacillus Pup deletion mutant bacterial strain and its applications, based on fusion DNA vaccine technology and carrier T clone technology, the displaced type deletion mutation carrier T-Pup-N-K-Pup-C of gene is constructed and identified for the first time, the Pup deletion mutant carrier of recombination is made to enter mycobacterium tuberculosis competent cell by electrotransformation technology, using homology of chromosome recombination and multiplex screening, identification, the tubercle bacillus Pup deletion mutant bacterial strain of resistant gene containing kanamycin is obtained for the first time.The tubercle bacillus Pup deletion mutant bacterial strain that the present invention constructs, the Pup gene in MTB Pup- proteasomal system is knocked out, the drug resistance of clinical drug-resistant tubercle bacillus separation strains can be reduced, its Antituberculous Drugs is set to restore sensibility, help is provided to explore pathogenic mechanism and the control propagation lungy of MTB, provides new direction to find antituberculotic target.
Description
A kind of building and its application of tubercle bacillus Pup deletion mutant bacterial strain.
Technical field
The invention belongs to recombinations, deletion mutant strain construction technical field, are related to tubercle bacillus Pup gene and lack
Lose the building and its application of mutant strain.
Background technique
Tuberculosis (Tuberculosis, TB) is by tuberculosis branch core bacillus (Mycobacterium
Tuberculosis, MTB) infect caused chronic infectious disease.There are about 1/3 people to infect MTB in the whole world, and TB is a kind of serious
The pandemic infection disease and single pathogenic infection for endangering human health lead to the highest infectious diseases of the death rate.In recent years
Carry out the appearance due to drug resistance MTB, in addition the rising of MTB and AIDS virus double infection epidemic situation, so that global tuberculosis prevention and treatment
Situation becomes very severe, all the time, resistance mechanism and pathogenesis about Mtb be all emphasis in tuberculosis research and
Hot spot.The resistance mechanism in relation to tubercle bacillus and pathogenesis mainly pass through inactivation or the specific gene of missing at present,
Tubercle bacillus mutant strain various change is analyzed, to parse the function of target gene.
A brand-new prokaryotes proteolytic pathway is presented in face of us i.e. MTB ubiquitin-like sample albumen in recent years
(Pup) the tubercle bacillus Pup- proteasomal system formed with MTB proteasome, the system mainly mediate and regulate and control Mtb albumen
The degradation selectivity of matter, to Mtb the intracorporal growth of host, breeding, toxicity, it is pathogenic, in terms of play it is important
Regulating and controlling effect.Wherein Pup is a kind of unordered albumen, there is unstable secondary structure, can mark a variety of destination proteins, and mediate
Labeled albumen enters proteasome degradation, and the Pup of tubercle bacillus is in by a kind of C- terminal region that 64 amino acid form
The pairs of protein of spiral helicine internal structure confusion.Its end N- still keeps disordered state, the end C- after Pup labelled protein
S21-K61 forms helical structure, can be with the end the N- spiral knot for the confactor Mpa for mediating tubercle bacillus Pup- proteasomal system
Structure domain noncovalent interaction, is transmitted into proteasome by Mpa and degrades.Therefore Pup may have there are two types of function, it is not only to allow
Substrate becomes the marker of proteasome identification, and makes unordered label on target protein band, is conducive to the degradation of target protein.
Gene Knockout is one of common method in gene functional research, by particular technology make organism some
Gene is accurately inactivated or is lacked, and is compared with wild-type organisms phenotype, and the function of the gene is recognized according to its variation.
And Pup albumen and the specific drug resistance regulatory mechanism of MTB are unclear.Based on this, it is proposed that being constructed using gene Knockout
A kind of tubercle bacillus Pup deletion mutant bacterial strain, the bacterial strain pass through the Pup base in Pup- proteasomal system in missing MTB
Cause is mainly used for studying tubercle bacillus ubiquitin-like sample proteasomal system GAP-associated protein GAP Pup to mycobacterium tuberculosis in host
Growth, drug resistance, permeability adjusting, acid resistance, pathogenic etc. effect, and it is new for intervening and treating tuberculosis
The R and D of medicine.
Summary of the invention
A kind of construction method for being designed to provide tubercle bacillus Pup deletion mutant bacterial strain of invention and its application,
The urgency of emergence and antituberculotic research and development for clinical resistance mycobacterium tuberculosis, the present invention pass through knockout
Pup gene in MTB Pup- proteasomal system constructs tubercle bacillus Pup deletion mutant bacterial strain, and further examines
The variation of such drug tolerance of strain is surveyed, it will new approach is provided to the research of mycobacterium tuberculosis pathogenic mechanism, to be
The infection for controlling propagation and inhibition tuberculosis mycomycete lungy provides help, while providing to find antituberculotic target
New direction.
The purpose of the present invention one be to provide it is a kind of building, identification and purifying mycobacterium tuberculosis Pup gene substitution type lack
The method for losing mutational vector, using SOE-PCR technology and carrier T clone technology building deletion mutation segment and further
Deletion mutation carrier is constructed, will identify that successful MTB Pup gene substitution type deletion mutation carrier is named as T- through gene sequencing
Pup-N-K-Pup-C, and simultaneously plasmid purification is extracted using miniplasmids extracts kit, purified with DNA gel electroresis appraisal, simultaneously
With the concentration and purity of ultraviolet specrophotometer measurement plasmid.
The purpose of the present invention two is to provide a kind of building, screening and identification tubercle bacillus Pup deletion mutant bacterial strains
Method makes T-Pup-N-K-Pup-C enter mycobacterium tuberculosis competent cell, passes through chromosome using electrotransformation technology
Homologous recombination, multiplex screening and identification obtain the tubercle bacillus Pup deletion mutant bacterial strain of resistant gene containing kanamycin.
Its technical solution is as follows:
The building of Mycobacterium tuberculosis Pup gene substitution type deletion mutation carrier and identification method, comprising the following steps:
The extracting of step 1, mycobacterium tuberculosis international standard velogen strain (H37Rv bacterial strain) genomic DNA
With improvement Roche solid medium be inoculated with tubercle bacillus H37Rv bacterial strain, 37 DEG C stationary culture 3-4 week, picking mycoderm extract
Genomic DNA shows DNA without degradation, no RNA and protein residue through the detection of 1% agarose gel electrophoresis.
Step 2, PCR primer design and synthesis
H37Rv bacterial strain Pup gene order and Pup gene upstream and downstream homology arm sequence are downloaded from GenBank, uses primer
5. 0 software designed, designed primer of premier, primer are synthesized by Shanghai Sheng Gong bio-engineering corporation.
The PCR amplification and purifying of step 3, Pup gene and kalamycin resistance gene upstream and downstream homology arm gene
Identify that successful H37Rv bacterial strain complete genome DNA as reaction template, is expanded with primer Pup-N-F and Pup-N-R to extract
The N-terminal homology arm of Pup gene takes PCR amplification with the C-terminal homology arm of primer Pup-C-F and Pup-C-R amplification Pup gene respectively
5 μ L of product is identified with the agarose gel electrophoresis of 1.5% concentration, will identify that successfully this segment is named as Pup-N, Pup-C.With dilute
PUC19K plasmid gene group DNA after releasing is PCR reaction template, anti-with Kan- F, Kan- R primer amplification kanamycins respectively
Property gene, take 5 μ L of pcr amplification product to identify clip size through the agarose gel electrophoresis that concentration is 1.5%, and be named as
K；Using Ago-Gel QIAquick Gel Extraction Kit (centrifugal column type) is to Pup gene upstream and downstream homology arm sequence fragment and to block that mould
Plain resistance gene fragment is recycled and is purified.
The synthesis and identification of step 4, deletion mutation box Pup-N-K-Pup-C
Pup-N, K, Pup-C of purifying are mixed to the template reacted as SOE-PCR according to 1:2:1, any primer is not added and carries out
The SOE-PCR amplification of the first run one circulation, because fusion segment will be used for TA clone, so 2xTaq PCR should be selected
MasterMix is used for the PCR amplification of this wheel, and after reaction, 4 μ L of Pup-N-F is added in the first round in the reaction product,
4 μ L of Pup-C-R carries out the pcr amplification reaction of the second wheel.Take 5 μ L PCR reaction products in the fine jade of 1.5% concentration after reaction
Sepharose electroresis appraisal will identify that successfully this segment is named as Pup-N-K-Pup-C.
The building and identification of step 5, Pup deletion mutant carrier T-Pup-N-K-Pup-C
Deletion mutation box Pup-N-K-Pup-C, T-Vector pMD 19, efficient connection liquid Solution I after purification is mixed
It is combined as experimental group；T-Vector pMD 19, efficiently connection liquid Solution I mixing is used as negative control group 1；Deionization
Water, efficiently connection liquid Solution I mixing is used as negative control group 2.After 3 groups carry out TA connection respectively, by each group connection product
It is transferred to E. coli DH5 α competence, is coated on the dual anti-solid medium culture of containing kanamycin and ammonia benzyl mycin LB, it is secondary
The single positive transformant of daily aseptic inoculating ring picking each group is inoculated in the containing kanamycin and dual anti-liquid of ammonia benzyl mycin LB
Culture medium, 37 DEG C of shaking table concussions are incubated overnight.It takes bacterium solution to detect as reaction template row bacterium solution PCR, and send Hua Da gene sequencing,
It will identify that successfully this carrier is named as T-Pup-N-K-Pup-C.
The building of tubercle bacillus Pup deletion mutant bacterial strain and screening technique, comprising the following steps:
The extraction and purification of step 1, deletion mutation carrier T-Pup-N-K-Pup-C
Bacterium solution containing deletion mutation carrier T-Pup-N-K-Pup-C is taken into 10 μ l, is coated on kanamycins and ammonia benzyl mycin
The dual anti-solid medium culture of LB, the single positive transformant of picking are inoculated in the dual anti-liquid training of LB of kanamycins and ammonia benzyl mycin
Base is supported, 37 DEG C of shaking table concussions are incubated overnight, and are extracted simultaneously plasmid purification using miniplasmids extracts kit, are reflected with DNA gel electrophoresis
Determine, while measuring the concentration and purity of plasmid with ultraviolet specrophotometer.
The preparation of step 2, tubercle bacillus bacterial strain competent cell
Divide the MTB bacterial strain of the appropriate logarithmic growth phase of picking to be placed in sterile mycobacterium tuberculosis mill tube with aseptic inoculation ring, is vortexed
Mixer, which shakes, sufficiently smashes mycoderm；Bacterium solution is moved in sterile centrifugation tube, stands 30min on ice, supernatant is abandoned in 4 DEG C of centrifugations；Nothing
Thallus is resuspended in bacterium deionized water, stands 5 min on ice, and 4 DEG C of centrifugations are abandoned supernatant, are repeated 3 times；10% pre- cold glycerol is added to be resuspended
Thallus, with standard Maxwell opacity tube than turbid rear plus 10% pre- cold glycerol, making into bacterial population is about 1.0 × 108The bacteria suspension of a/ml.
Step 3, deletion mutation carrier T-Pup-N-K-Pup-C electrotransformation to its deletion mutant strain of MTB strain construction
The freshly prepared MTB competent cell of 10 μ l deletion mutation carrier T-Pup-N-K-Pup-C and 100 μ l is added sterile
2mlEP pipe mixes, while the MTB competent cell for taking 110 μ l that any plasmid is not added stands 30min as blank control on ice
Afterwards, it is transferred to respectively in the electric shock cup of pre-cooling, setting electricity turns voltage, will contain deletion mutation carrier and blank pair after electrotransformation
According to 10 min of electrotransformation product ice bath, be all coated in the modified Russell medium containing kanamycins, 37 DEG C cultivate 4 weeks
After observe result.
Step 4, the screening of M. tuberculosis strains deletion mutation strain, identification
T-Pup-N-K-Pup-C electricity is gone in MTB bacterial strain using electrotransformation technology, and is sieved using kanamycins as antibiotic
Choosing label preliminary screening positive colony extracts positive colony genomic DNA as reaction template, under being with Pup-N-F, Kan-R
Primer is swum, carries out bacterium colony PCR identification by downstream primer of Kan-F, Pup-C-R, detects specific splice segment Pup-N-K and K-
Pup-C, to identify whether deletion mutant strain constructs success.
The preparation of tubercle bacillus competent cell of the present invention and the knot in tubercle bacillus Pup deletion mutant bacterial strain
Core bacillus is suitable for clinical various antibody-resistant bacterium.
Beneficial effects of the present invention:
The present invention is based on SOE-PCR technology, carrier T clone technology and electrotransformation technologies to construct resistance containing kanamycin for the first time
The tubercle bacillus Pup deletion mutant bacterial strain of gene, we further investigations have shown that, tubercle bacillus Pup- protease system
Pup deletion mutant can reduce the drug resistance of clinical drug-resistant tubercle bacillus separation strains in system, restore its Antituberculous Drugs
Sensibility provides help to explore pathogenic mechanism and the control propagation lungy of MTB, while to find antituberculotic target
Mark provides new direction.
Detailed description of the invention
Fig. 1: the electrophoretic analysis of tubercle bacillus H37Rv strain gene group DNA；
1,2: tubercle bacillus H37Rv strain gene group DNA；
Fig. 2: target gene N-terminal, C-terminal homology arm PCR amplification；
M:DNA molecular mass standard I；1:Pup gene N-terminal homology arm；2:Pup gene C end homology arm；
The amplification of Fig. 3: Kana resistance gene fragment；
M:DNA molecular mass standard II；1: blocking that resistant gene PCR product；
Fig. 4: SOE-PCR synthesis deletion mutation box；
M:DNA molecular mass standard III；1:Pup-N-K-Pup-C deletion mutation box；
Fig. 5: the positive colony bacterium colony of the Pup-N-K-Pup-C of box containing deletion mutation；
Fig. 6: the bacterium colony PCR identification of deletion mutation carrier；
M:DNA molecular mass standard III；1:Pup-N-K-Pup-C segment；
Fig. 7: Pup gene deletion mutants bacterium colony PCR identification；
M:DNA molecular mass mark III；1:INH-MTB Pup genetic fragment (primer Pup-F, Pup-R)；2:Pup gene delection is prominent
Mutant Pup genetic fragment (primer Pup-F, Pup-R)；3:Pup gene deletion mutants specificity splice segment Pup-N-K(draws
Object Pup-N-F, Kan-R)；4:Pup gene deletion mutants specificity splice segment Pup-N-K(primer Kan-F, Pup-C-R)；
Specific embodiment
Technical solution of the present invention is described in more detail with reference to the accompanying drawings and detailed description.
The building of 1 Mycobacterium tuberculosis Pup gene substitution type deletion mutation carrier of embodiment and identification method
1. materials and methods
1.1 main agents materials
1.1.1 main material: mycobacterium tuberculosis international standard velogen strain (H37Rv) is purchased from Chinese pharmaceutical biological product calibrating
Institute is saved by this laboratory；Mycobacterium tuberculosis medicaments insensitive bacterial strain (Drug-sensitive-MTB), merely resistance to isoniazid
Mycobacterium tuberculosis clinical separation strain (the RFP- of mycobacterium tuberculosis clinical separation strain (INH-MTB), simple resistance to rifampin
MTB), the tuberculosis branch bar of the mycobacterium tuberculosis clinical separation strain (SM-MTB) of simple resistance to streptomycin, simple resistance to ethambutol
Bacterium clinical separation strain (EB-MTB), multi-drug resistant mycobacterium tuberculosis clinical separation strain (MDR-MTB) strain are acquired by this laboratory
Sample, identification and preservation；E.coli Competent Cells DH5 α and T-Vector pMD 19 (Simple) is purchased from treasured
Bioengineering Co., Ltd (Dalian TaKaRa company)；
TE buffer, PCR primer are purchased from Shanghai bioengineering Co., Ltd；50 × TAE buffer, kanamycins, ammonia benzyl
Penicillin is purchased from Beijing Suo Laibao Science and Technology Ltd；SDS(lauryl sodium sulfate), Tris, chloroform, RNase A,
Resazurin sodium salt is purchased from U.S. Sigma company；2×Pfu Master Mix,2×Taq Master Mix,
DP209 DNA gel QIAquick Gel Extraction Kit (centrifugal column type), Marker I, Marker II, Marker III, TIANpure
Midi Plasmid Kit is purchased from TIANGEN Biotech (Beijing) Co., Ltd.；Tween-80 is purchased from Tianjin chemical reagent factory；Electricity
Cup is hit purchased from Eppendorf company, Germany.
1.1.2 key instrument equipment: by Shihezi of Xinjiang's University Medical College " Xinjiang place and national high fall ill " education
Key lab, portion provides
The adjustable single track pipettor of Research is purchased from Eppendorf company, Germany；MLS3750 type autoclave is purchased from Japan
Sanyo；Generic centrifuge originates from Changsha Xiang Yi centrifuge Instrument Ltd.；Electroporation is public purchased from Germany Eppendorf
Department；SCS-24 shaking table is purchased from ShangHai City centrifugal Machine Institute；DYY-4 type electrophoresis apparatus is purchased from Beijing Liu Yichang；Bio-Rad ladder
Spend PCR instrument, Bio-Rad gel imager is purchased from Bio Rad Laboratories；170S type CO2 incubator is public purchased from Britain Galaxy
Department；2-16K high speed freezing centrifuge is purchased from Sigma company, Germany；Millipore ultrapure water equipment derives from the U.S.
Millipore company；SYNYO low temperature refrigerator is purchased from SYNYO company, Japan；1300 type second level of Thermo Scientific is raw
Object safety cabinet, 2000 type ultraviolet specrophotometer of Nanodrop are purchased from U.S. Thermo Fisher Scientific company.
1.2 experimental method
1.2.1 the culture of mycobacterium tuberculosis international standard velogen strain (H37Rv bacterial strain)
H37Rv bacterial strain is purchased from Chinese drug biological products assay institute, is passed on and is saved by this laboratory cultures.H37Rv bacterial strain is answered
It revives and is not inoculated in improvement Roche solid medium, cultivated 3-4 weeks in 37 DEG C of constant incubators of water isolation type, observed within every 7 days
Once, bacterium colony is that faint yellow bacterium colony is grown in particle or cauliflower-like, and, contaminating strains bad to upgrowth situation carry out timely clear
It removes.The above experimental implementation is both needed to be operated in two stage biological safety cabinet, and strictly observes the base of tuberculosis laboratory operation
This regulation.
1.2.2 the preparation of mycobacterium tuberculosis international standard velogen strain (H37Rv bacterial strain) genomic DNA
With it is disposable sterilized connect collarium picking one and expire the good bacterium colony of ring growth conditions set in 2mL sterile EP tube, first by 300 μ
L 1 × TE buffer solution is added in EP pipe, and is put into 80 DEG C of thermostat water baths and inactivates 30-40min；It is taken from water-bath
EP is managed out, is put on vortex oscillator and is shaken 20min or be moderately ground into bacteria suspension with 1000 μ L pipette tips, then in bacteria suspension
In sequentially add 500 μ L, 125g/L lysozyme soln of 10%SDS solution, 80 μ L, 25% sucrose solution, 50 μ L and 20g/L Proteinase K
20 μ L of solution is put into 37 DEG C of thermostat water bath water-bath 5h after playing uniformly；By 3,000rpm, 4 DEG C of bacteria suspension after water-bath, centrifugation
10min；It moves in supernatant Yu Yixin sterile EP tube, isometric 700 μ L of phenol ︰ chloroform (1 ︰ 1) mixed liquor extracting is added once, room temperature
After lower mild mixing 10min, 12,000rpm, 4 DEG C of centrifugation 10min take supernatant to a new sterile EP tube, and RNase A is added extremely
Final concentration 100 μ L/ml, 37 DEG C of incubation 1h；It is primary to add isometric Fen ︰ chloroformic solution extracting, 12,000rpm, 4 DEG C of centrifugations
10min takes supernatant that isometric Fen ︰ Lv Fang ︰ isoamyl alcohol (25 ︰, 24 ︰ 1) is added to a new sterile EP tube, and after mixing 12,
000rpm, 4 DEG C of centrifugation 10min move supernatant to a new EP pipe, mix -20 DEG C of refrigerator precipitatings with the pre-cooling dehydrated alcohol of 2 times of volumes
Overnight.Take out mixture, 10,000rpm, 4 DEG C of centrifugation 10min；Supernatant is abandoned, 70% ethanol washing is added to precipitate, room temperature is mild up and down
Mix 10min；10,000rpm, 4 DEG C of centrifugation 10min；Supernatant is abandoned, it is molten that appropriate 1 × TE buffer is added after sufficiently drying in air
Liquid, 4 placement 3h take agarose gel electrophoresis identification and UV spectrophotometer measuring analysis DNA of a small amount of solution for 1% concentration
Concentration and purity, remaining be placed in -20 DEG C of refrigerators store it is spare.
As a result: the electrophoresis detection of tubercle bacillus H37Rv strain gene group DNA shows DNA without degradation, and no RNA and albumen are residual
It stays, sees Fig. 1.
1.2.3 PCR primer design and synthesis
According to mycobacterium tuberculosis international standard velogen strain H37Rv bacterial strain Pup gene upstream and downstream homology arm gene in GenBank
Sequence, using 5.0 software designed, designed amplimer (table 1) of primer premier, primer is public by the raw work bioengineering in Shanghai
Department's synthesis.
1 PCR primer of table
1.2.4 the PCR amplification and purifying of Pup gene upstream and downstream homology arm gene
Identify that successful H37Rv bacterial strain complete genome DNA as reaction template, uses primer Pup-N-F and Pup-N-R using extraction
The N-terminal homology arm for expanding Pup gene is the reduction base mispairing of maximum possible because this amplified fragments is used for the building of SOE-PCR
Rate need to use 2xPfu PCR MasterMix to be used for the PCR amplification of this segment, reaction system are as follows: H37Rv bacterial strain full-length genome
DNA 3 μ L, Primer1(Pup-N-F) 1 μ L, Primer2(Pup-N-R) 1 μ L, 2xPfu PCR MasterMix, 12.5 μ L,
ddH2o 7.5μL；Reaction condition are as follows: 94 DEG C of initial denaturations 3min, 94 DEG C of denaturation 30sec, anneal 30sec, 72 DEG C of extension 1min,
Totally 30 circulation, last 72 DEG C extend 8min eventually, and agarose gel electrophoresis of the 5 μ L pcr amplification products for 1.5% concentration is taken to reflect
It is fixed, it will identify that successfully this segment is named as Pup-N；C-terminal using primer Pup-C-F and Pup-C-R amplification Pup gene is homologous
Arm is the reduction base mispairing rate of maximum possible, need to use 2xPfu equally because this amplified fragments is used for the building of SOE-PCR
PCR MasterMix is used for the PCR amplification of this segment, reaction system are as follows: H37Rv bacterial strain complete genome DNA 3 μ L, Primer1
(Pup-C-F) 1 μ L, Primer2(Pup-C-R) 1 μ L, 2xPfu PCR MasterMix 12.5 μ L, ddH2o 7.5μL；Reaction
Condition are as follows: 94 DEG C of initial denaturations 3min, 94 DEG C of denaturation 30sec, anneal 30sec, 72 DEG C of extension 1min, totally 30 circulation, and last 72 DEG C
Extend 8min eventually, takes agarose gel electrophoresis of the 5 μ L pcr amplification products for 1.5% concentration to identify, will identify successful PCR
Amplified production is named as Pup-C.
As a result: using tubercle bacillus H37Rv pnca gene group DNA as template, amplifying size respectively is 508 bp and 498 bp
Pup-N, Pup-C, it is small with it is expected consistent, see Fig. 2.
1.2.5 the PCR amplification of kalamycin resistance gene
With the PUC19K plasmid gene group DNA after dilution for PCR reaction template, upstream primer Kana-F and downstream primer are used
Kana-R expands kalamycin resistance gene, is the reduction base of maximum possible because this amplified fragments is used for the building of SOE-PCR
Mismatch rate need to use 2xPfu PCR MasterMix to be used for the PCR amplification of this segment, reaction system are as follows: PUC19K Plasmid DNA
3 μ L, Primer1(Kana-F) 0.5 μ L, Primer2(Kana-R) 0.5 μ L, 2xPfu PCR MasterMix, 12.5 μ L,
ddH2o 8.5μL；Reaction condition are as follows: 94 DEG C of initial denaturations 3min, 94 DEG C of denaturation 30sec, anneal 30sec, 72 DEG C of extensions
2.min30sec, totally 30 circulation, last 72 DEG C whole extension 8min take agarose of the 5 μ L pcr amplification products for 1.5% concentration
Gel electrophoresis identification, will identify that successfully the genetic fragment is named as K.
As a result: using pUC19K Plasmid DNA as template, amplifying the kalamycin resistance gene of 1 093 bp, size and pre-
Phase is consistent, sees Fig. 3.
1.2.6 the purifying and recycling of Pup gene upstream and downstream homology arm gene and kalamycin resistance gene
Use DP209-- centrifugal column type -- common DNA gel QIAquick Gel Extraction Kit, by Pup gene upstream and downstream homology arm genetic fragment
Purification and recovery is carried out with kalamycin resistance gene segment.Purpose is cut from Ago-Gel rapidly under ultraviolet light irradiation
Band Pup-N, Pup-C, time too long cannot cause DNA double chain to be broken in order to avoid ultraviolet light irradiates for a long time, the gel that will be cut
Segment is respectively put into 1.5mL sterile EP tube and weighs weight respectively；Isometric PN sol solution is added into EP pipe, is placed in
10-20 min in 50 DEG C of thermostat water baths mildly should spin upside down EP during water-bath during which to dissolve gel piece sufficiently
Pipe；Equilibrium liquid BL 500 μ L, 12,000rpm centrifugation 1min, with equilibrium adsorption column is added into adsorption column CA2 at the same time；To
A batch or the gel piece lysate being cooled to room temperature is added portionwise in adsorption column CA2, after standing 2min, 12,000rpm centrifugations
45sec abandons waste liquid；Rinsing liquid PW 600 μ L, 12,000rpm centrifugation 45sec containing dehydrated alcohol are added into adsorption column CA2,
Abandon waste liquid；Repeat previous action；12,000rpm centrifugation 2min, eliminate rinsing liquid as far as possible, to prevent the rinsing liquid containing ethyl alcohol
Adsorption column CA2 is uncapped and is put into a sterilizing EP to influence subsequent experimental by residual, and super-clean bench places 15min；To adsorption column
40 μ L of elution buffer EB is vacantly added dropwise in the adsorbed film central location of CA2, after standing 2min, 12,000rpm centrifugation 2min, EP
Purified DNA solution needed for being in pipe, is placed in -20 DEG C of refrigerators for the DNA solution of purifying and saves backup.
1.2.7 the synthesis and identification of deletion mutation box Pup-N-K-Pup-C
Through repetition test, using three segment fusion method success rate highests, specific steps are as follows: respectively by Pup-N, K, Pup- of purifying
C, the reaction template after 1: 2: 1 ratio mixing as SOE-PCR, is not added any primer and carries out the first run one circulation in molar ratio
SOE-PCR amplification, because fusion segment to be used for TA clone, so should select 2xTaq PCR MasterMix for this wheel
PCR amplification, reaction system are as follows: 10 20 10 μ L, 2xPfu PCR MasterMix of μ L, Pup-C of μ L, K of Pup-N, 50 μ L,
ddH2O 40μL；Reaction condition are as follows: 95 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30sec, 45 DEG C of annealing 5min, 72 DEG C extend
2.min30sec, totally 1 recycles, and last 72 DEG C extend 10min eventually；When first round PCR after reaction, in the reaction product plus
Enter Primer1(Pup-N-F) 4 μ L, Primer2(Pup-C-R) 4 μ L carry out the pcr amplification reaction of the second wheel, and reaction condition is
94 DEG C of initial denaturations 4min, 94 DEG C of denaturation 30sec, 59 DEG C of annealing 30sec, 72 DEG C of extension 3min, totally 30 recycle, and last 72 DEG C
Extend 5min eventually；Agarose gel electrophoresis of the 5 μ L PCR reaction products for 1.5% concentration is taken to identify after reaction to PCR,
It will identify that successfully this segment is named as Pup-N-K-Pup-C.
As a result: after using three segment fusion methods to mix 3 segments of N, K, C of each gene with the ratio of molar ratio 1: 2: 1
A circulation SOE-PCR amplification is carried out as reaction template, using amplified production as the template of the second wheel PCR reaction, uses primer
Pup-N-F and Pup-C-R amplify 2 060 bp deletion mutation box Pup-N-K-Pup-C and expection it is in the same size, see Fig. 4.
1.2.8 the building and identification of Pup deletion mutant carrier T-Pup-N-K-Pup-C
By after purification deletion mutation box Pup-N-K-Pup-C, T-Vector pMD 19 (Simple), efficiently connect liquid
Solution I mixing group is experimental group, reaction system are as follows: 2 μ L, T-Vector pMD 19 of Pup-N-K-Pup-C
(Simple) 1 μ L, dH22 μ L of O, efficiently connects 5 μ L of liquid Solution I, and reaction condition is 16 DEG C of 30min.With T-
1 μ L of Vector pMD 19 (Simple), efficiently connection 5 μ L mixing group of liquid Solution I is 1 group of negative control for examining
It surveys carrier T and occurs whether there is or not phenomenon is connected certainly, reaction condition is 16 DEG C of 30min；With 1 μ L of deionized water, efficiently connection liquid Solution
5 μ L mixing group of I is active when being 2 groups of the negative control antibiotic activities to detect kanamycins and ammonia benzyl mycin, instead
Answering condition is 16 DEG C of 30min.After each group carries out TA connection respectively, by each group connection product mixture withE. coliDH5 α sense
By state, it is coated in the LB solid medium tablets of the ammonia benzyl mycin containing kanamycins 100 mg/L and 50 mg/L, 37 DEG C of perseverances
Temperature is incubated overnight.Single positive colony bacterium colony on collarium picking each group plate is connect with disposable sterilized, is inoculated in containing kanamycins
In the LB liquid medium of the ammonia benzyl mycin of 100 mg/L and 50 mg/L, with 5 μ L bacterium solutions after 37 DEG C of constant-temperature table shaking overnights
For 1 μ L, Primer2(Pup-C-R of PCR reaction template, Primer1(Pup-N-F)), 1 μ L, 2xTaq PCR MasterMix
12.5 μ L, ddH25.5 μ L row bacterium solution PCR of o detection；Reaction condition are as follows: 94 DEG C of initial denaturations 3min, 94 DEG C of denaturation 30sec, annealing
30sec, 72 DEG C of extension 2.5min, totally 30 circulation, last 72 DEG C whole extension 5min take 5 μ L pcr amplification products dense for 1.5%
The agarose gel electrophoresis of degree is identified, and send Hua Da gene sequencing, will identify that successfully this carrier is named as T-Pup-N-K-
Pup-C.It will finally identify that successfully the bacterium solution of the T-Pup-N-K-Pup-C of carrier containing deletion mutation and glycerol are with certain 8:2 ratio
After mixing, it is spare to be put into storage in -80 DEG C of ultra low temperature freezers.
As a result: experimental group carries out TA connection, connection product transformed competence colibacillus DH5 α, and coating ammonia benzyl/block that resistant panel obtains
To several positive colony bacterium colonies, Fig. 5 is seen；1 group of carry out TA connection of negative control, connection product transformed competence colibacillus DH5 α are coated with ammonia
Benzyl/block that resistant panel is grown after culture without escherichia coli specificity bacterium colony, to confirm T-Vector pMD 19
(Simple) there is not carrier and connect phenomenon certainly；2 groups of carry out TA connections of negative control, connection product transformed competence colibacillus DH5 α are applied
Cloth ammonia benzyl/block that resistant panel is grown after culture without escherichia coli specificity bacterium colony, to confirm that antibiotic activity is good.
The single positive colony bacterium colony of picking experimental group is inoculated in the LB liquid of the ammonia benzyl mycin containing kanamycins 100 mg/L and 50 mg/L
It is upstream and downstream primer row bacterium colony PCR with Pup-N-F and Pup-C-R after 37 DEG C of shaking tables shake overnight incubation in culture medium, expands
The Pup-N-K-Pup-C segment for increasing 2 060 bp out, is shown in Fig. 6, send deletion mutation carrier T-Pup-N-K-Pup-C to genome company
Sequencing, sequencing result are consistent with implementation sequence, it was demonstrated that deletion mutation carrier T-Pup-N-K-Pup-C is constructed successfully.
The building of 2 tubercle bacillus Pup deletion mutant bacterial strain of embodiment and screening technique
1. experimental method
The extraction and purification of 1.1 deletion mutation carrier T-Pup-N-K-Pup-C
The bacterium solution containing deletion mutation carrier T-Pup-N-K-Pup-C for being stored in -80 DEG C of refrigerators is taken into 10 μ l, be coated on card that
The dual anti-solid medium of LB of mycin (100 μ g/ml) and ammonia benzyl mycin (50 μ g/ml), (at least 12h) training overnight of 37 DEG C of constant temperature
It supports.The single positive colony bacterium colony of picking is inoculated in the dual anti-liquid of LB of kanamycins (100 μ g/ml) and ammonia benzyl mycin (50 μ g/ml)
Body culture medium, 37 DEG C of shaking table concussions are incubated overnight, and simultaneously plasmid purification are extracted with miniplasmids extracts kit, with DNA gel electrophoresis
Purification Identification, at the same with ultraviolet specrophotometer measurement plasmid concentration and purity.The plasmid of purifying sets -20 DEG C of low temperature refrigerators
Interior storage is spare.
The preparation of 1.2 M. tuberculosis strains competent cells
Each drug resistance tubercle bacillus bacterial strain of appropriate logarithmic growth phase is picked them separately with 22SWG standard inoculation ring, is placed in disposable sterilized
In bead, 6 times of revolving speeds of eddy mixer shake 20~30 min and sufficiently smash mycoderm, and bacterium solution is moved in 2 ml centrifuge tubes,
30 min are stood on ice, are centrifuged radius 10cm in 4 DEG C with 8 000 r/min() centrifugation 8 min collection thallus, abandon supernatant；500 μ
L is pre-chilled aseptic deionized water and 1 min is resuspended, and stands 5 min on ice, and in 4 DEG C, ibid 8 min of centrifugation, collection thallus abandon supernatant；
Thallus is resuspended with the pre- cold glycerol of 500 μ l 10%, stands 5 min on ice.It repeats previous step process 3 times, abandons supernatant, with 10% pre-cooling
Thallus is resuspended in glycerol, and with standard Maxwell opacity tube than turbid rear plus 10% pre- cold glycerol, making into bacterial population is about 1.0 × 108A/ml
Bacteria suspension, it is spare to set storage in -20 DEG C of low temperature refrigerators.
1.3 deletion mutation carrier electrotransformations are to tubercle bacillus bacterial strain
The freshly prepared mycobacterium tuberculosis competent cell of 10 μ l deletion mutation carrier T-PhoPR and 100 μ l is added sterile
2mlEP pipe mixes, while the mycobacterium tuberculosis competent cell for taking 110 μ l that any plasmid is not added sets ice as blank control
30 min are bathed, are transferred in the 1 mm specification electric shock cup slot electrode of pre-cooling, are 1 700 V, 1 800 V, 1 in electric converter voltage
Electrotransformation (its is carried out immediately under the conditions of 900 V, 2 000 V, 2 100 V, 2 200 V, 2 300 V, 2 400 V, 2 500 V
In 1 700~1 900 V electric shock twice, 2 000~2 500 V shock by electricity 1 time).Deletion mutation carrier will be contained after electrotransformation
With 10 min of electrotransformation product ice bath of blank control, it is all coated on the improvement Roche culture containing 100 μ g/ml kanamycins
In base, 37 DEG C are observed result after stationary culture 4 weeks.
We successfully construct clinical each drug resistance tubercle bacillus Pup deletion mutant bacterial strain with this method respectively
The resistance to sharp good fortune Pup deletion mutant bacterial strain RFP- of the MTB Pup of resistance to isoniazid deletion mutant bacterial strain INH-MTB △ Pup, MTB
MTB △ Pup, MTB resistance to streptomycin Pup deletion mutant bacterial strain SM-MTB △ Pup, the MTB Pup of resistance to ethambutol gene delection are prominent
Become bacterial strain EB-MTB △ Pup, multi-drug resistant Pup deletion mutant bacterial strain MDR-MTB △ Pup and MTB the medicaments insensitive bacterial strain of MTB
Pup deletion mutant bacterial strain Drug-sensitive-MTB △ Pup.
Screening, the identification of 1.4 M. tuberculosis strains deletion mutation strains
The characteristics of being primarily based on T-Vector pMD 19 (Simple) carrier itself (cannot except escherichia coli Pseudomonas with
Replicated in outer bacterium, theoretically can be used as suicide vector) it is used for the building of tubercle bacillus gene-deleted strain；Secondly will contain scarce
Each plasmid vector electrotransformation of mutation box is lost into each drug resistance tubercle bacillus bacterial strain, using kanamycins as antibiotic-screening
Preliminary screening positive colony is marked, by the positive colony extraction genomic DNA of screening and as reaction template, respectively with Pup-
N-F is upstream primer, and Kan-R is downstream primer and using Kan-F as upstream primer, and Pup-C-R is that downstream primer carries out bacterium colony PCR
Identification detects specific splice segment Pup-N-K and K-Pup-C, to identify whether deletion mutant strain constructs success.
As a result: the equal nontuberculous mycobacteria of blank control group is grown, and screens number on recombinant tubercle bacillus group culture medium
The tubercle bacillus specific positive colony bacterium colony that amount does not wait.With the gene of the positive colony bacterium colony (INH-MTB △ Pup) screened
Group DNA is template, and using Pup-N-F as upstream primer, Kan-R is downstream primer and using Kan-F as upstream primer, and Pup-C-R is
Downstream primer carries out bacterium colony PCR respectively, amplifies the special of the specific splice segment Pup-N-K and 1591 bp of 1 601 bp
Property splice segment K-Pup-C；Using the original Clinical isolation of resistance to isoniazid DNA as template, using Pup-F, Pup-R as upstream and downstream
Primer row bacterium colony PCR obtains the Pup genetic fragment of 213 bp；It is with the Pup of resistance to isoniazid gene deletion mutants genomic DNA
Template carries out bacterium colony PCR by upstream and downstream primer of Pup-F, Pup-R, does not amplify target gene fragment, see Fig. 7.RFP-MTB
△ Pup, SM-MTB △ Pup, EB-MTB △ Pup, MDR-MTB △ Pup and Drug-sensitive-MTB △ Pup group experiment knot
Fruit is identical as INH-MTB △ Pup group (figure omits).
The foregoing is only a preferred embodiment of the present invention, the scope of protection of the present invention is not limited to this, it is any ripe
Know those skilled in the art within the technical scope of the present disclosure, the letter for the technical solution that can be become apparent to
Altered or equivalence replacement are fallen within the protection scope of the present invention.
Claims (4)
1. a kind of construction method of tubercle bacillus Pup deletion mutant bacterial strain, which comprises the following steps:
Step 1, the building of mycobacterium tuberculosis Pup gene substitution type deletion mutation carrier and identification method
By the purifying of SOE-PCR technology, Ago-Gel QIAquick Gel Extraction Kit and agarose gel electrophoresis method, constructs and identify tuberculosis
Mycobacteria deletion mutation segment Pup-N-K-Pup-C, by carrier T clone technology and gene sequencing technology will building identification at
The Mycobacterium tuberculosis Pup deletion mutant carrier of function is named as T-Pup-N-K-Pup-C, and is mentioned using plasmid extraction kit
It takes and plasmid purification, with DNA gel electroresis appraisal, while with the concentration and purity of ultraviolet specrophotometer measurement plasmid；
Step 2, the building of tubercle bacillus Pup deletion mutant bacterial strain and screening technique
10 μ l T-Pup-N-K-Pup-C are made to enter the freshly prepared MTB competent cell of 100 μ l by electrotransformation technology, together
When take 110 μ l that any plasmid is not added mycobacterium tuberculosis competent cell as blank control, recombinated using homology of chromosome
And multiplex screening, identification obtain the tubercle bacillus Pup deletion mutant bacterial strain of resistant gene containing kanamycin.
2. the construction method of tubercle bacillus Pup deletion mutant bacterial strain according to claim 1, which is characterized in that step
In rapid 1, mycobacterium tuberculosis deletion mutation segment Pup-N-K-Pup-C, Mycobacterium tuberculosis Pup gene substitution type deletion mutation are carried
The building and identification of body T-Pup-N-K-Pup-C.
3. the construction method of tubercle bacillus Pup deletion mutant bacterial strain according to claim 1, which is characterized in that step
In rapid 2, the tubercle bacillus Pup deletion mutant bacterial strain of building has knocked out the Pup gene in Pup- proteasomal system.
4. tubercle bacillus Pup deletion mutant bacterial strain described in claim 1, which is characterized in that in research tubercle bacillus Pup
The application of deletion mutant drug tolerance of strain variation.
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