CN110093365A - A kind of preparation and its application of tubercle bacillus PUP protein overexpression bacterial strain - Google Patents
A kind of preparation and its application of tubercle bacillus PUP protein overexpression bacterial strain Download PDFInfo
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- CN110093365A CN110093365A CN201910254167.3A CN201910254167A CN110093365A CN 110093365 A CN110093365 A CN 110093365A CN 201910254167 A CN201910254167 A CN 201910254167A CN 110093365 A CN110093365 A CN 110093365A
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Abstract
The invention discloses preparations and its application that a kind of building tubercle bacillus ubiquitin-like sample albumen PUP is overexpressed bacterial strain, cultivate BCG bacterial strain and extract its genomic DNA.Using BCG strain gene group DNA as template, PCR amplification tubercle bacillus Pup protein gene；Sequencing, identification.Using Escherichia coli-mycobacterium tuberculosis shuttle plasmid pMV361 as carrier, building and identification recombinant shuttle plasmid pMV361-Pup:BCG.Using electroporation technology by the recombination bacillus coli of building-mycobacterium tuberculosis shuttle plasmid pMV361 carrier (recombinant shuttle plasmid pMV361-Pup:BCG), it is transferred to BCG bacterial strain, building and identification tubercle bacillus Pup protein overexpression bacterial strain, are named as BCG:Pup bacterial strain.The bacterial strain can be used for studying tubercle bacillus ubiquitin-like sample proteasomal system GAP-associated protein GAP PUP adjust in growth of the mycobacterium tuberculosis in host, drug resistance, permeability, acid resistance in terms of effect, and R and D for intervening and treating new generation vaccine lungy.
Description
Technical field
The present invention relates to genetic engineerings, vaccine research field, are related to a kind of system of tubercle bacillus PUP protein overexpression bacterial strain
Standby and its application.
Background technique
Tuberculosis is a kind of world caused by being infected by knot branch core bacillus (Mycobacterium Tuberculosis)
Sexually transmitted disease and single pathogenic infection lead to the highest infectious diseases of the death rate.Tuberculosis is still global sense at present
The number one killer of infectious diseases.The new data that " the 2017 global tuberculosis report " of World Health Organization's publication is announced is shown:
10,400,000 new cases are estimated to be in world wides in 2017, wherein male 5,900,000 (56%), women 3,500,000 (34%), children
1000000 (34%).This six countries of India, Indonesia, China, Nigeria, Pakistan and South Africa account for new cases
The 60% of sum.Tuberculosis epidemic situation fails to obtain control, and reason of searching to the bottom is a lack of effective vaccine prevention hair lungy
It is raw.
Pup- proteasomal system (prokaryotic ubiquitin like protein-proteasome
System, PPS) it is mainly responsible for the choosing for mediating protein in tubercle bacillus (Mycobacterium Tuberculosis, Mtb)
The degradation of selecting property, and play an important role to the regulation of the processes such as the pathogenic and its caused host immune response of Mtb.Wherein ubiquitin
Sample albumen (prokaryotic ubiquitin-like protein, Pup) can be to protein to be degraded in tubercle bacillus
It is identified and is modified, be then fed into tubercle bacillus proteasome and degrade.PUP albumen in the system is to tubercle bacillus pair
The infection of host body is cured the disease, and is stayed living in host for a long time and generated the effect that drug resistance all plays key.
In recent years, since new and effective anti-tubercular drug and novel vaccine Recent Progresses In The Development are slow, so that tuberculosis
Preventing and controlling it is more acute, the novel strain that the present invention constructs be further investigation and tubercle bacillus Pup- proteasomal system phase
The pathogenic mechanism offer of pass is possible, can also further research and develop intervention and treatment new and improved bacterial strain vaccine lungy.
Summary of the invention
The preparation and its application for being designed to provide a kind of tubercle bacillus PUP protein overexpression bacterial strain of invention, for mesh
The sternness of preceding Tuberculosis and control situation and prevention tuberculosis develop the urgency of novel vaccine, consider tubercle bacillus
Pup- proteasomal system is pathogenic for tuberculosis and its regulation of the processes such as caused host immune response plays an important role,
The present invention, by PUP protein overexpression channel genes BCG vaccine (BCG), prepares a kind of tuberculosis bar with recombinant shuttle plasmid technology
Bacterium PUP protein overexpression bacterial strain, providing for further investigation pathogenic mechanism relevant to tubercle bacillus Pup- proteasomal system can
Can, it can also further research and develop intervention and treatment new and improved bacterial strain vaccine lungy.
The object of the invention is overexpressed bacterial strain first is that preparing a kind of tubercle bacillus ubiquitin-like sample albumen PUP, dry for tuberculosis
Pre- and treatment new generation vaccine R and D.
The object of the invention is second is that be overexpressed Strain Designation for the tubercle bacillus ubiquitin-like sample albumen PUP of building are as follows: " BCG:
Pup bacterial strain "
The object of the invention three is to provide the method that preparation tubercle bacillus ubiquitin-like sample albumen PUP is overexpressed bacterial strain.
The object of the invention is fourth is that be overexpressed the expression of strain protein PUP by detection tubercle bacillus ubiquitin-like sample albumen PUP
Amount analyzes difference and its corresponding function feature of this albumen to bacterial strain in the variation of drug resistance, acid resistance and permeability.
Its technical solution is as follows:
A kind of tubercle bacillus ubiquitin-like sample albumen PUP is overexpressed the preparation of bacterial strain, comprising the following steps:
1) according to BCG bacterial strain design primer.
Pup gene primer is
Sequence(5′-3′) F:CATAAGCTTATGGCGCAAGAGCAGACCAAGCG
R:TGAGAATTCTCACTGTCCGCCCTTTTGGACGT
2) PCR amplification and identification of target gene.
3) connection of target gene and carrier T and it is transformed into E.coli DH5 α competent cell.
4) extraction and sequencing identification of purpose plasmid, by the raw work biology Co., Ltd sequencing in Shanghai.
5) construct and identify recombinant shuttle plasmid pMV361- Pup:BCG.
6) utilize electroporation technology by recombinant shuttle plasmid pMV361- Pup:BCG, electrotransformation to BCG.
The present invention, by PUP protein overexpression channel genes BCG vaccine (BCG), is prepared with recombinant shuttle plasmid technology
Tubercle bacillus PUP protein overexpression bacterial strain, to further investigate pathogenic mechanism relevant to tubercle bacillus Pup- proteasomal system
Offer is possible, can also further research and develop intervention and treatment new and improved bacterial strain vaccine lungy.
Detailed description of the invention
Fig. 1 tubercle bacillus BCG(1) strain gene group DNA electrophoretic analysis
The PCR amplification of Fig. 2 Pup gene:
The PCR product of 2000 1:Pup gene of M:DNA Marker
Fig. 3 Escherichia coli-tubercle bacillus shuttle expression carrier pMV361 physical map
The sequencing of Fig. 4 recombinant shuttle plasmid pMV361/Pup is identified
Specific embodiment
Technical solution of the present invention is described in more detail with reference to the accompanying drawings and detailed description.
The preparation of 1 tubercle bacillus ubiquitin-like sample albumen PUP of embodiment overexpression bacterial strain
1. material:
1.1 main materials:
Main material: strain of BCG vaccine (abbreviation BCG bacterial strain) is provided by Chinese drug biological products assay institute, this laboratory is protected
It deposits；Escherichia coliE.coliDH5 α is provided by this laboratory.Escherichia coli-tubercle bacillus shuttle expression carrier pMV361 is by this
Laboratory saves.
1.2 main agents
1) NEB biotech firm, the U.S. restriction enzyme EcoRI
2) NEB biotech firm, the U.S. restriction enzyme Hind III
3) NEB biotech firm, the T4 DNA ligase U.S.
4) 2 × pfu Master Mix Beijing health is ShiJi Co., Ltd
5) DNA Marker Beijing Tiangeng biochemical technology Co., Ltd
6) Ago-Gel QIAquick Gel Extraction Kit (centrifugal column type) Beijing Tiangeng biochemical technology Co., Ltd
7) the small extraction reagent kit of plasmid (centrifugal column type) Beijing Tiangeng biochemical technology Co., Ltd
8) hundred Tyke Bioisystech Co., Ltd of the Beijing nucleic acid dye Gelview
9) Takara company, Loading buffer Japan
10) original-pack agarose Shanghai Ai Yan Biotechnology Co., Ltd, Spain
11) the raw work biology Co., Ltd in the Shanghai 50 × TAE
12) the extensive and profound in meaning star Bioisystech Co., Ltd in agar powder Beijing
13) Tryptone company, OXOID tryptone Britain
14) Tryptone company, OXOID yeast extract Britain
15) deionized water Beijing health is ShiJi Co., Ltd
16) Shanghai NaCL bioengineering Co., Ltd
17) Proteinase K Shanghai bioengineering Co., Ltd
18) lysozyme Shanghai bioengineering Co., Ltd
19) the triumphant rich biological Co., Ltd in the Shanghai Rnase
20) SDS Sigma Co., USA
21) phenol, chloroform, isoamyl alcohol (25:24:1) Suo Laibao Science and Technology Ltd.
22) Shanghai 1 × TE bioengineering Co., Ltd
23) QIAGEN company, RNeasy Plus Universal Mini Kit Germany
24) QIAGEN company, QuantiTect Reverse Transcription Germany
25) Thermo company, the U.S. RevertAid First Strand cDNA Synthesis Ki
26) the raw work biology Co., Ltd in the Shanghai 5 × TBE
1.3 major experimental instrument and equipments
1) TECHNE company, TC3000 PCR instrument Britain
2) Bio-RAD grads PCR instrument Bio Rad Laboratories
3) DYY-6B type voltage stabilization and current stabilization electrophoresis apparatus Beijing Liuyi Instrument Factory
4) large capacity total temperature constant-temperature table Shanghai Zhi Cheng Analytical Instrument Co., Ltd
5) digital display electric heating incubator Shanghai Boxun Industrial Co., Ltd.
6) electric-heated thermostatic water bath Shanghai Boxun Industrial Co., Ltd.
7) safe and sound air technique Co., Ltd, superclean bench Su Jing group
8) DEVEN instrument company, the electronic balance U.S.
9) Eppendorf company, micro sample-adding rifle Germany
10) Eppendorf company, electroporation Germany
11) magnetic stirring apparatus Jiangsu medical apparatus and instruments factory
12) Biofage stratos company, D-37520 high speed room temperature centrifuge Germany
13) -80 degree Thermo company, the ultra low temperature freezer U.S.
14) micro-wave oven Matsushita Corporation of Japan
15) HERMILE company, Z323K refrigerated centrifuge Germany
16) millipore company, the MilliQ ultrapure water machine U.S.
17) Techne company, ultraviolet specrophotometer Nanodrop2000 Britain
18) Bio-RAD company, the Gel Doc2000 gel imaging system U.S.
19) I real-time fluorescence quantitative PCR instrument Roche Holding Ag of SYBR Green
20) HIRAYAMA company, HVE-50 autoclave sterilizer Japan
2 experimental methods
2.1 BCG inoculation and culture
With improvement Roche solid medium difference bcg vaccination bacterial strain (hereinafter referred to as BCG), it is placed in 37 DEG C of incubators and trains
It supports 3-4 weeks.
2.2 building tubercle bacillus ubiquitin-like sample proteasomal system PUP protein overexpression bacterial strains
The recombinant shuttle plasmid of 2.3 buildings and identification tubercle bacillus Pup- proteasomal system
2.3.1 the preparation of BCG strain gene group DNA
Appropriate BCG bacterial strain is picked them separately into sterile EP tube, 400 μ L 1 × TE buffer are added, after mixing fullys shake, put
In inactivating 30min in 80 DEG C of water baths；Be added 25% sucrose solution, 50 500 μ L, 125g/L lysozyme of μ L, 10%SDS, 80 μ L and
20 μ L of 20g/L Proteinase K, 37 DEG C of water-baths are stayed overnight after mixing well；Second day takes out EP pipe, and 12000rpm, is centrifuged by 4 DEG C
10min is moved in supernatant to a new sterile EP tube；Fen ︰ chloroform is added: about 700 μ L of isoamyl alcohol (25:24:1), room temperature are mild up and down
After mixing 10min, 12000rpm, 4 DEG C of centrifugation 10min；Supernatant is moved to a new EP pipe, is added 50 μ L of 10g/L Rnase, 37 DEG C
Incubate 1h；Fen ︰ chloroform: isoamyl alcohol (25:24:1) is added, after room temperature mildly mixes 10min up and down, 12000rpm, 4 DEG C of centrifugations
10min；It moves supernatant to manage to a new EP, isometric Fen ︰ Lv Fang ︰ isoamyl alcohol (25 ︰, 24 ︰ 1) is added, after mixing well 10min,
12000rpm, 4 DEG C of centrifugation 10min；Supernatant is moved to a new EP pipe, isometric pre-cooling dehydrated alcohol (about 700 μ L) is added and mixes
After 10min, it is placed in -20 DEG C of refrigerator precipitates overnights；EP pipe, 12000rpm, 4 DEG C of centrifugation 10min are taken out from refrigerator；Supernatant is abandoned,
Add 70% ethanol washing to precipitate, mixes 10min up and down；12000rpm, 4 DEG C of centrifugation 10min；Supernatant is abandoned, drying at room temperature 30min makes
Ethyl alcohol volatilization is complete；1 × TE buffer 50-100 μ L is added, piping and druming mixes, and it is spare that 4 DEG C of placement 3h are placed on -20 DEG C of storages.
And 2ul is taken from each sample DNA, it is analyzed using the agarose gel electrophoresis identification of 1% concentration and UV spectrophotometer measuring
DNA concentration and purity.As shown in Figure 1.
2.3.2 the design of primer
By finding BCGPup gene order in GenBank database, suitable enzyme is selected according to plasmid pMV361 map (see figure 2)
Enzyme site, and design primer.Pup gene primer is
Sequence(5′-3′) F:CATAAGCTTATGGCGCAAGAGCAGACCAAGCG
R:TGAGAATTCTCACTGTCCGCCCTTTTGGACGT
2.3.3 the PCR amplification of target gene
The PCR amplification of Pup gene.As shown in Figure 2
2.3.4 the recycling of gene PCR amplified production
2.3.5 the extraction of plasmid pMV361
The Escherichia coli bacteria liquid of pMV361 containing plasmid for drawing 5mL overnight incubation is added in 2mL EP pipe by several times, 12000rpm centrifugation
1min, as far as possible absorption supernatant；For equilibrium adsorption column, 500 μ L, 12000rpm centrifugation of equilibrium liquid BL is added into adsorption column CP3
1min；It is added in the 2mL EP pipe that 250 μ L of P1 solution is precipitated to mycetome, is acutely shaken using turbula shaker, keep thallus heavy
It forms sediment and sufficiently suspends；250 μ L of P2 solution is added thereto again, mildly spins upside down at once for several times sufficiently to crack thallus；Thereto
350 μ L of P3 solution is added, leniently spins upside down at once for several times, EP bottom of the tube will appear white flock precipitate, 12000rpm from
Heart 10min；The supernatant of collection is transferred in adsorption column, 12000rpm is centrifuged 45s, abandons waste liquid；600 μ of rinsing liquid PW is added
For L into adsorption column, 12000rpm is centrifuged 1min；It repeats to rinse primary；Use up rinsing liquid from 2min adsorption column 12000rpm sky
Amount eliminates；Subsequent experimental may be influenced to prevent the rinsing liquid containing ethyl alcohol from remaining, adsorption column CA2 is uncapped and is put to a sterilizing EP
In, super-clean bench places 15min；50 μ L of elution buffer EB is vacantly added dropwise to the adsorbed film central location of adsorption column CA2, stands
After 2min, 12000rpm is centrifuged 2min；Solution in EP pipe is rejoined in adsorption column, stand 2min after, 12000rpm from
It is plasmid pMV361 in heart 2min, EP pipe, is placed in -20 DEG C of refrigerators and saves.
Escherichia coli-tubercle bacillus shuttle expression carrier pMV361 physical map.As shown in Figure 3.
2.3.6 the extraction of plasmid pMV361
The Escherichia coli bacteria liquid of pMV361 containing plasmid for drawing 5mL overnight incubation is added in 2mL EP pipe by several times, 12000rpm centrifugation
1min, as far as possible absorption supernatant；For equilibrium adsorption column, 500 μ L, 12000rpm centrifugation of equilibrium liquid BL is added into adsorption column CP3
1min；It is added in the 2mL EP pipe that 250 μ L of P1 solution is precipitated to mycetome, is acutely shaken using turbula shaker, keep thallus heavy
It forms sediment and sufficiently suspends；250 μ L of P2 solution is added thereto again, mildly spins upside down at once for several times sufficiently to crack thallus；Thereto
350 μ L of P3 solution is added, leniently spins upside down at once for several times, EP bottom of the tube will appear white flock precipitate, 12000rpm from
Heart 10min；The supernatant of collection is transferred in adsorption column, 12000rpm is centrifuged 45s, abandons waste liquid；600 μ of rinsing liquid PW is added
For L into adsorption column, 12000rpm is centrifuged 1min；It repeats to rinse primary；Use up rinsing liquid from 2min adsorption column 12000rpm sky
Amount eliminates；Subsequent experimental may be influenced to prevent the rinsing liquid containing ethyl alcohol from remaining, adsorption column CA2 is uncapped and is put to a sterilizing EP
In, super-clean bench places 15min；50 μ L of elution buffer EB is vacantly added dropwise to the adsorbed film central location of adsorption column CA2, stands
After 2min, 12000rpm is centrifuged 2min；Solution in EP pipe is rejoined in adsorption column, stand 2min after, 12000rpm from
It is plasmid pMV361 in heart 2min, EP pipe, is placed in -20 DEG C of refrigerators and saves.
2.3.7 the digestion, purifying of plasmid pMV361
It is separately added into restriction enzyme HindIII and EcoRI digested plasmid pMV361,3h under 37 DEG C of water bath conditions utilizes 2%
After concentration agarose gel electroresis appraisal, gel extraction DNA fragmentation.
2.3.8 the digestion, purifying of target gene amplified production
Using restriction enzyme HindIII and EcoRI double digestion target gene (Pup, Mpa, Dop and PafA) amplified production,
3h under 37 DEG C of water bath conditions, after 2% concentration agarose gel electroresis appraisal, gel extraction DNA fragmentation.
2.3.9 the connection of target gene and plasmid pMV361
2.3.10 the preparation of E. coli DH5 α competence
1. taking out E. coli DH5 α clone's strain that laboratory has been stored in -80 DEG C of refrigerators, (be free of in LB plate
Antibiotic) on streak inoculation, 37 DEG C of inversion overnight incubations；2. picking single colonie is inoculated into 5mL LB Liquid Culture in super-clean bench
In base (being free of antibiotic), 37 DEG C of shake cultures of 180rpm/min shaking table are stayed overnight；3. the bacterium solution for drawing 1000 μ L overnight incubations adds
Enter in 100mL LB liquid medium (without antibiotic), 37 DEG C of shake cultures of 180rpm/min shaking table to OD600 value are
0.4-0.6(about 3h)；4. drawing 1.5mL bacterium solution to the 2mL centrifuge tube being pre-chilled in advance, 10min is placed on ice, is placed in 4 DEG C of centrifugations
In machine, 3000rpm is centrifuged 10min；5. discarding supernatant and exhausting wherein raffinate, the 0.1mol/ of 750 μ L pre-cooling is added into EP pipe
L CaCl2 solution, places 15min on ice, is placed in 4 DEG C of centrifuges, and 3000rpm is centrifuged 10min；
2.3.11 the conversion of plasmid pMV361/PCR recovery product connection liquid
1. 10 μ L connection liquid are added in 100 μ L E.coli DH5 α competent cells, mix gently, ice bath 30min.②42
DEG C water-bath heat shock 90sec(avoids shaking) after, ice bath 10min at once.3. the not antibiotic LB Liquid Culture of 800 μ L is added
Base.4. 37 DEG C of shake culture 45min of 150rpm/min shaking table.5. 3000rpm is centrifuged 20sec, part supernatant is abandoned, 150 μ L are only stayed
Thallus is resuspended in liquid, is respectively coated on the LB plate containing 0.05g/L Kana with sterile spreading rod, and first just setting 30min makes bacterium
Liquid absorbs completely, then is placed in 37 DEG C of culture carton upside down overnight incubations.
2.3.12 the identification of recombinant shuttle plasmid
Picking single colonie carries out bacterium solution PCR preliminary screening, and positive clone molecule is inoculated in the LB liquid of the Kana containing 0.05g/L
In culture medium, 37 DEG C of shakings are incubated overnight, and row double digestion is identified after next day extracts plasmid, and recombinant shuttle plasmid is named as
PMV361/Pup send Huada gene company to be sequenced.
The sequencing of recombinant shuttle plasmid pMV361/Pup is identified.As shown in Figure 4.
The building and identification of the BCG bacterial strain of 2.4 Pup gene overexpressions
2.4.1 the preparation of tubercle bacillus competence
With the logarithmic growth phase BCG bacterial strain of the appropriate 37 DEG C of cultures of oese picking, it is placed in sterile glass beads, uses vortex oscillator
It shakes 30min and smashes mycoderm, draw thallus respectively with liquid-transfering gun in 2mL centrifuge tube, subglacial stands 30min, 8000r/ at 4 DEG C
Min centrifugation 5min collects thallus respectively, abandons supernatant, is resuspended with 500uL ice-cold sterile deionized water, stands 5min on ice, repeats
Above-mentioned washing is stood process 4 times, abandons supernatant, is resuspended with 10% pre- cold glycerol.With standard Maxwell opacity tube than turbid, add 10% it is pre-
Cold glycerol is configured to the outstanding bacterium solution of about 15.0 bacterial populations/ml (× 108).- 80 DEG C save backup.
2.4.2 the extraction of recombinant shuttle plasmid
Plasmid pMV361/Pup is extracted according to the small extraction reagent kit step of plasmid and utilizes 1% concentration agarose gel electrophoresis and ultraviolet
Line spectrophotometer detects the concentration and purity of plasmid, -20 DEG C of preservation plasmids.
2.4.3 recombinant shuttle plasmid electrotransformation is to BCG bacterial strain
Construct the tubercle bacillus of Pup protein overexpression.10uL recombinant shuttle plasmid is felt with the freshly prepared BCG of 200uL respectively
It is mixed, is added in the 1mm electric shock cup of pre-cooling, under the conditions of 2100-2500kv/cm after pulse, rapidly by electrotransformation by state cell
Product ice bath 10min is gone in test tube of the 5mL without kanamycins 7H9 culture solution, 37 DEG C of culture 48h, after thalline were collected by centrifugation,
30ul is taken to be coated in the modified Russell medium of the 0.03mg/L containing kanamycins, 37 DEG C are cultivated 3-4 weeks, and resistance screening is carried out.
Passage no less than 3 times, stablize to each overexpression bacterial strain and express in modified Russell medium containing kanamycins, carry out next step inspection
It surveys.
2.4.4 the BCG bacterial strain for constructing successful Pup gene overexpression is detected and identified using Realtime-PCR technology.
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. prepared by the recombinant shuttle plasmid of tubercle bacillus Pup- proteasomal system, which comprises the following steps:
1.1 BCG inoculation and culture
With improvement Roche solid medium difference bcg vaccination bacterial strain (hereinafter referred to as BCG), it is placed in 37 DEG C of incubators and trains
It supports 3-4 weeks；
The recombinant shuttle plasmid of 1.2 buildings and identification tubercle bacillus Pup- proteasomal system
1.2.1 the preparation of BCG strain gene group DNA
Appropriate BCG bacterial strain is picked them separately into sterile EP tube, 400 μ L 1 × TE buffer are added, after mixing fullys shake, put
In inactivating 30min in 80 DEG C of water baths；Be added 25% sucrose solution, 50 500 μ L, 125g/L lysozyme of μ L, 10%SDS, 80 μ L and
20 μ L of 20g/L Proteinase K, 37 DEG C of water-baths are stayed overnight after mixing well；Second day takes out EP pipe, and 12000rpm, is centrifuged by 4 DEG C
10min is moved in supernatant to a new sterile EP tube；Fen ︰ chloroform is added: about 700 μ L of isoamyl alcohol (25:24:1), room temperature are mild up and down
After mixing 10min, 12000rpm, 4 DEG C of centrifugation 10min；Supernatant is moved to a new EP pipe, is added 50 μ L of 10g/L Rnase, 37 DEG C
Incubate 1h；Fen ︰ chloroform: isoamyl alcohol (25:24:1) is added, after room temperature mildly mixes 10min up and down, 12000rpm, 4 DEG C of centrifugations
10min；It moves supernatant to manage to a new EP, isometric Fen ︰ Lv Fang ︰ isoamyl alcohol (25 ︰, 24 ︰ 1) is added, after mixing well 10min,
12000rpm, 4 DEG C of centrifugation 10min；Supernatant is moved to a new EP pipe, isometric pre-cooling dehydrated alcohol (about 700 μ L) is added and mixes
After 10min, it is placed in -20 DEG C of refrigerator precipitates overnights；EP pipe, 12000rpm, 4 DEG C of centrifugation 10min are taken out from refrigerator；Supernatant is abandoned,
Add 70% ethanol washing to precipitate, mixes 10min up and down；12000rpm, 4 DEG C of centrifugation 10min；Supernatant is abandoned, drying at room temperature 30min makes
Ethyl alcohol volatilization is complete；1 × TE buffer 50-100 μ L is added, piping and druming mixes, and it is spare that 4 DEG C of placement 3h are placed on -20 DEG C of storages,
And 2ul is taken from each sample DNA, it is analyzed using the agarose gel electrophoresis identification of 1% concentration and UV spectrophotometer measuring
DNA concentration and purity；
1.2.2 the design of primer
By finding BCGPup gene order in GenBank database, suitable restriction enzyme site is selected according to plasmid pMV361 map,
And design primer, Pup gene primer are
Sequence(5′-3′) F:CATAAGCTTATGGCGCAAGAGCAGACCAAGCG
R:TGAGAATTCTCACTGTCCGCCCTTTTGGACGT
1.2.3 the PCR amplification of target gene
1.2.4 the recycling of gene PCR amplified production
1.2.5 the extraction of plasmid pMV361
The Escherichia coli bacteria liquid of pMV361 containing plasmid for drawing 5mL overnight incubation is added in 2mL EP pipe by several times, 12000rpm centrifugation
1min, as far as possible absorption supernatant；For equilibrium adsorption column, 500 μ L, 12000rpm centrifugation of equilibrium liquid BL is added into adsorption column CP3
1min；It is added in the 2mL EP pipe that 250 μ L of P1 solution is precipitated to mycetome, is acutely shaken using turbula shaker, keep thallus heavy
It forms sediment and sufficiently suspends；250 μ L of P2 solution is added thereto again, mildly spins upside down at once for several times sufficiently to crack thallus；Thereto
350 μ L of P3 solution is added, leniently spins upside down at once for several times, EP bottom of the tube will appear white flock precipitate, 12000rpm from
Heart 10min；The supernatant of collection is transferred in adsorption column, 12000rpm is centrifuged 45s, abandons waste liquid；600 μ of rinsing liquid PW is added
For L into adsorption column, 12000rpm is centrifuged 1min；It repeats to rinse primary；Use up rinsing liquid from 2min adsorption column 12000rpm sky
Amount eliminates；Subsequent experimental may be influenced to prevent the rinsing liquid containing ethyl alcohol from remaining, adsorption column CA2 is uncapped and is put to a sterilizing EP
In, super-clean bench places 15min；50 μ L of elution buffer EB is vacantly added dropwise to the adsorbed film central location of adsorption column CA2, stands
After 2min, 12000rpm is centrifuged 2min；Solution in EP pipe is rejoined in adsorption column, stand 2min after, 12000rpm from
It is plasmid pMV361 in heart 2min, EP pipe, is placed in -20 DEG C of refrigerators and saves；
1.2.6 the extraction of plasmid pMV361
The Escherichia coli bacteria liquid of pMV361 containing plasmid for drawing 5mL overnight incubation is added in 2mL EP pipe by several times, 12000rpm centrifugation
1min, as far as possible absorption supernatant；For equilibrium adsorption column, 500 μ L, 12000rpm centrifugation of equilibrium liquid BL is added into adsorption column CP3
1min；It is added in the 2mL EP pipe that 250 μ L of P1 solution is precipitated to mycetome, is acutely shaken using turbula shaker, keep thallus heavy
It forms sediment and sufficiently suspends；250 μ L of P2 solution is added thereto again, mildly spins upside down at once for several times sufficiently to crack thallus；Thereto
350 μ L of P3 solution is added, leniently spins upside down at once for several times, EP bottom of the tube will appear white flock precipitate, 12000rpm from
Heart 10min；The supernatant of collection is transferred in adsorption column, 12000rpm is centrifuged 45s, abandons waste liquid；600 μ of rinsing liquid PW is added
For L into adsorption column, 12000rpm is centrifuged 1min；It repeats to rinse primary；Use up rinsing liquid from 2min adsorption column 12000rpm sky
Amount eliminates；Subsequent experimental may be influenced to prevent the rinsing liquid containing ethyl alcohol from remaining, adsorption column CA2 is uncapped and is put to a sterilizing EP
In, super-clean bench places 15min；50 μ L of elution buffer EB is vacantly added dropwise to the adsorbed film central location of adsorption column CA2, stands
After 2min, 12000rpm is centrifuged 2min；Solution in EP pipe is rejoined in adsorption column, stand 2min after, 12000rpm from
It is plasmid pMV361 in heart 2min, EP pipe, is placed in -20 DEG C of refrigerators and saves；
1.2.7 the digestion, purifying of plasmid pMV361
It is separately added into restriction enzyme HindIII and EcoRI digested plasmid pMV361,3h under 37 DEG C of water bath conditions utilizes 2%
After concentration agarose gel electroresis appraisal, gel extraction DNA fragmentation；
1.2.8 the digestion, purifying of target gene amplified production
Using restriction enzyme HindIII and EcoRI double digestion target gene (Pup, Mpa, Dop and PafA) amplified production,
3h under 37 DEG C of water bath conditions, after 2% concentration agarose gel electroresis appraisal, gel extraction DNA fragmentation；
1.2.9 the connection of target gene and plasmid pMV361
1.2.10 the preparation of E. coli DH5 α competence
1. taking out E. coli DH5 α clone's strain that laboratory has been stored in -80 DEG C of refrigerators, (be free of in LB plate
Antibiotic) on streak inoculation, 37 DEG C of inversion overnight incubations；2. picking single colonie is inoculated into 5mL LB Liquid Culture in super-clean bench
In base (being free of antibiotic), 37 DEG C of shake cultures of 180rpm/min shaking table are stayed overnight；3. the bacterium solution for drawing 1000 μ L overnight incubations adds
Enter in 100mL LB liquid medium (without antibiotic), 37 DEG C of shake cultures of 180rpm/min shaking table to OD600 value are
0.4-0.6(about 3h)；4. drawing 1.5mL bacterium solution to the 2mL centrifuge tube being pre-chilled in advance, 10min is placed on ice, is placed in 4 DEG C of centrifugations
In machine, 3000rpm is centrifuged 10min；5. discarding supernatant and exhausting wherein raffinate, the 0.1mol/ of 750 μ L pre-cooling is added into EP pipe
L CaCl2 solution, places 15min on ice, is placed in 4 DEG C of centrifuges, and 3000rpm is centrifuged 10min；
1.2.11 the conversion of plasmid pMV361/PCR recovery product connection liquid
1. 10 μ L connection liquid are added in 100 μ L E.coli DH5 α competent cells, mix gently, ice bath 30min；②42
DEG C water-bath heat shock 90sec(avoids shaking) after, ice bath 10min at once；3. the not antibiotic LB Liquid Culture of 800 μ L is added
Base；4. 37 DEG C of shake culture 45min of 150rpm/min shaking table；5. 3000rpm is centrifuged 20sec, part supernatant is abandoned, 150 μ L are only stayed
Thallus is resuspended in liquid, is respectively coated on the LB plate containing 0.05g/L Kana with sterile spreading rod, and first just setting 30min makes bacterium
Liquid absorbs completely, then is placed in 37 DEG C of culture carton upside down overnight incubations；
1.2.12 the identification of recombinant shuttle plasmid
Picking single colonie carries out bacterium solution PCR preliminary screening, and positive clone molecule is inoculated in the LB liquid of the Kana containing 0.05g/L
In culture medium, 37 DEG C of shakings are incubated overnight, and row double digestion is identified after next day extracts plasmid, and recombinant shuttle plasmid is named as
PMV361/Pup send Huada gene company to be sequenced；
The sequencing of recombinant shuttle plasmid pMV361/Pup is identified.
The building and identification of the BCG bacterial strain of 2.Pup gene overexpression
The preparation of 2.1 tubercle bacillus competence
With the logarithmic growth phase BCG bacterial strain of the appropriate 37 DEG C of cultures of oese picking, it is placed in sterile glass beads, uses vortex oscillator
It shakes 30min and smashes mycoderm, draw thallus respectively with liquid-transfering gun in 2mL centrifuge tube, subglacial stands 30min, 8000r/ at 4 DEG C
Min centrifugation 5min collects thallus respectively, abandons supernatant, is resuspended with 500uL ice-cold sterile deionized water, stands 5min on ice, repeats
Above-mentioned washing is stood process 4 times, abandons supernatant, is resuspended with 10% pre- cold glycerol, with standard Maxwell opacity tube than turbid, add 10% it is pre-
Cold glycerol is configured to the outstanding bacterium solution of about 15.0 bacterial populations/ml (× 108), and -80 DEG C save backup；
The extraction of 2.2 recombinant shuttle plasmids
Plasmid pMV361/Pup is extracted according to the small extraction reagent kit step of plasmid and utilizes 1% concentration agarose gel electrophoresis and ultraviolet
Line spectrophotometer detects the concentration and purity of plasmid, -20 DEG C of preservation plasmids；
2.3 recombinant shuttle plasmid electrotransformations are to BCG bacterial strain
It constructs the tubercle bacillus of Pup protein overexpression: 10uL recombinant shuttle plasmid is felt with the freshly prepared BCG of 200uL respectively
It is mixed, is added in the 1mm electric shock cup of pre-cooling, under the conditions of 2100-2500kv/cm after pulse, rapidly by electrotransformation by state cell
Product ice bath 10min is gone in test tube of the 5mL without kanamycins 7H9 culture solution, 37 DEG C of culture 48h, after thalline were collected by centrifugation,
30ul is taken to be coated in the modified Russell medium of the 0.03mg/L containing kanamycins, 37 DEG C are cultivated 3-4 weeks, resistance screening is carried out,
Passage no less than 3 times, stablize to each overexpression bacterial strain and express in modified Russell medium containing kanamycins, carry out next step inspection
It surveys；
2.4 detect and identify the BCG bacterial strain for constructing successful Pup gene overexpression using Realtime-PCR technology.
3. PUP gene order in claim 1 are as follows:
Sequence(5′-3′) F:CATAAGCTTATGGCGCAAGAGCAGACCAAGCG
R:TGAGAATTCTCACTGTCCGCCCTTTTGGACGT。
4. tubercle bacillus PUP protein overexpression Strain Designation is BCG:Pup bacterial strain in claim 2.
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