CN106480079B - It is a kind of based on λ cI857/pL to thermostabilization temperature control cracking system and its construction method and application - Google Patents
It is a kind of based on λ cI857/pL to thermostabilization temperature control cracking system and its construction method and application Download PDFInfo
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Abstract
The invention belongs to bioengineering fields, and in particular to it is a kind of based on λ cI857/pL to thermostabilization temperature control cracking system and its construction method and application.It is disclosed by the invention based on λ cI857/pL to thermostabilization temperature control cracking system, i.e. plasmid pKF396ML, sequence is as shown in SEQ ID NO.1.The plasmid pKF396ML is to be cloned into Lysis gene E between EcoRI the and KpnI restriction enzyme site in plasmid pKF396M-5 after promoter pL, then obtained -35 bit bases of promoter pL for C by T rite-directed mutagenesis by over-lap PCR.The system remains to the expression of stabilization checking gene E at a temperature of 37 DEG C, and when temperature is more than to remain to the cracking of normal Induction of bacterial when its highest checks temperature, be conducive to the holding of the important antigenic determinant of fast-growth and surface of bacterium, and heat shock response caused by traditional induction mode (28 DEG C → 42 DEG C) can be reduced.
Description
Technical field
The invention belongs to bioengineering fields, and in particular to a kind of to crack system to thermostabilization temperature control based on λ cI857/pL
System, also relates to its construction method and application.
Background technique
Ghost is that Gram-negative bacteria is formed after the inducing expression product cracking of Phage PhiX174 Lysis gene E
The intact bacterial ghost without Cytoplasmic inclusions.The Lysis gene E encodes a kind of memebrane protein containing 91 amino acid, can merge
The specific transmembrane channel of a diameter about 40~200nm is formed on the interior outer membrane of Gram-negative bacteria and on cell membrane,
The Cytoplasmic inclusions of gram-negative bacteria are discharged by duct, thus formed without nucleic acid, ribosomes and
Empty bacterial (Witte the et al., 1990a of other components；Witte et al.,1990b；Witte et al.,1992).
Due to remaining the original surface texture of bacterium and characteristic, ghost has the dual function of vaccine and adjuvant, can pierce
Swash body and generate stronger immune response, thus compared with conventional vaccine have better immune protective effect (Mayr et al.,
2012；Jawale and Lee,2016；Liu et al.,2015；Hajam et al., 2015), further advantages
Be can be used as antigen, nucleic acid and drug advanced delivery vector (Muhammad et al., 2012；Ganeshpurkar et
al.,2014；Langemann et al.,2010).Just because of having the advantages that these, so that ghost has become current vaccines
The hot spot of research.Currently, many other source of people and animal derived Gram-negative bacteria also can crack in addition to Escherichia coli
Ghost is generated under the action of gene E, the advantage compared with traditional vaccine is also confirmed in related experiment (Eko et again and again
al.,2003；Hu et al.,2013；Zhu et al.,2012；Kwon et al.,2007).
Currently, ghost production or preparation mainly by Phage PhiX174 Lysis gene E in Gram-negative bacteria place
Stringent expression regulation in main bacterium is realized.Lysis gene E can be controlled by multiple expression systems, such as pL/pR-cI857,
LacpO-LacIq or tol etc., wherein again to be originated from λ bacteriophage dextrad promoter pR or/and left-hand promoter pL and its temperature sensitive
Repressor cI857 built-up Human liver glutathione system be most widely used (Langemann et al., 2010；Kloos et
al.,1994；Szostak et al.,1996).Within the system, Lysis gene E is placed in the joint control of pR or pR or both
Under system, by the regulation of temperature sensitive aporepressor cI857, when temperature is lower than 30 DEG C (usually 28 DEG C), gene E is stablized suppression
System, and when temperature is higher than 30 DEG C (usually 42 DEG C), since aporepressor cI857 heat shock inactivated gene E starts to express.
This Human liver glutathione system may act on most bacteriums, and induction can be completed by simply heating, without making
With expensive chemical inducer such as IPTG etc., this nothing is suspected to have its convenient place, but the expression system also has its shortcoming.It is first
First, the optimum growth temperature of many animals and humans pathogens is not 28 DEG C, and the temperature is also unfavorable for a large amount of lifes of bacterium
Produce the holding with the important antigenic determinant of some bacterium surfaces.Secondly, the moment fast lifting of temperature can also Induction of bacterial generation
The splitting action of E protein mediation is answered and is inhibited in thermal excitation.Finally, when carrying out large scale fermentation production temperature significantly quickly
It converts also difficult far beyond laboratory test.Therefore just seeming very necessary to thermal stability for this temperature control system is improved.
It is repaired for this purpose, there is researcher once to carry out mutation to pR promoter by random mutation screening and rite-directed mutagenesis verifying
- 32 A by the way that λ pR promoter maneuvering area -41 T are sported C, or are sported G, the system can be made 37 by decorations
DEG C, the expression of stabilization checking gene E is remained at a temperature of 38 DEG C, the joint mutation of the two is even more can be under 39 DEG C of " high temperature " still
The expression of energy stabilization checking gene E, and when temperature is more than to remain to the cracking of normal Induction of bacterial when its highest checks temperature
(Jechlinger et al.,1999；Jechlinger et al., 2005), but the research is currently limited to pR promoter
Single expression vector, the related report that there is no the mutation modification about pL promoter and the temperature control cracking system based on this to construct at present
Road, and the temperature control system in practical applications based on λ cI857/pL is also very common.In addition to this, involved in aforementioned research
Plasmid volume is larger, is unfavorable for the efficient preparation of ghost；Also " mistake is arranged in temperature spacing when carrying out screen mutation to promoter pR
Greatly ", and there is overlapping in the screening section of setting, respectively 37 DEG C~42 DEG C and 38 DEG C~42 DEG C, theoretically will lead to screening
Task is heavy, is also further improved.
Summary of the invention
Human liver glutathione carrier based on λ bacteriophage promoter pL/pR has been widely used for the protokaryon table of all kinds of recombinant proteins
It reaches, novel bacteria can also be used in by the Lysis gene E or/and Staphylococcus nuclease A gene of clonal expression bacteriophage Phix174
Slough off the preparation of vaccine.But as previously mentioned, this system still has many technology barriers in practical applications, although having to λ
CI857/pR temperature control system is transformed to improve it to the research of thermal stability and report, but is there is no so far about pL promoter
The relevant report of mutation modification and the temperature control cracking system building based on this, and the temperature in practical applications based on λ cI857/pL
Control system is also very common, therefore it is steady to heat mainly for realizing the temperature control system transformation raising based on cI857/pL by the present invention
Preparation that is qualitative and then realizing efficient ghost vaccine, while the screening technique of random mutation is improved to reduce the labour of such work
Intensity.
The present invention relates to plasmid pKF396ML, and sequence is as shown in SEQ ID NO.1.Wherein:
The plasmid includes the duplication subsequence from plasmid pKF396M-5, and chloramphenicol acetyl transferasegene is temperature sensitive to check
Albumen cI857 sequence, lambda dextrad promoter pR, enhancer T7g10 and SD sequence, ribosomal RNA operon T1T2 are terminated
Son and the elements such as lambda left-hand promoter mutation body pLm and bacteriophage phiX174 Lysis gene E, physical map is as schemed
1。
In plasmid pKF396ML, common regulation of the promoter pLm and pR by temperature sensitive aporepressor cI857.Lysis gene E
Controlled by promoter pLm, the downstream promoter pR be multiple cloning sites, including XhoI, AfeI, NheI, ClaI, NaeI, SacII,
The restriction endonuclease sites such as BstZI, SphI and PstI, strong terminator --- ribosomal RNA operon T1T2 terminator then by
Be placed in after this multiple cloning sites, to prevent the appearance of promoter blocking effect, that is, avoid by promoter pR transcription pair
The transcription and translation for the Lysis gene E that downstream promoter pLm is instructed impacts.
Plasmid pKF396ML of the present invention can be constructed by following methods and be mentioned:
1) by Lysis gene E be cloned into EcoRI the and KpnI restriction enzyme site in plasmid pKF396M-5 after promoter pL it
Between, it is built into the recombinant plasmid pKF396ME containing Lysis gene E；2) by over-lap PCR by plasmid pKF396ME promoter pL-
35 (operation subregion OL2 the 15th) base by T rite-directed mutagenesis be C.
The invention also discloses the plasmid pKF396ML as based on λ cI857/pL to thermostabilization temperature control cracking system
Application.
It constructs the present invention is based on the screening to thermostabilization temperature control cracking system of λ cI857/pL with plasmid pKF396M-5 and is
Basis, it then follows following steps carry out:
1) by Lysis gene E be cloned into EcoRI the and KpnI restriction enzyme site in plasmid pKF396M-5 after promoter pL it
Between, it is built into the recombinant plasmid pKF396ME containing Lysis gene E.
2) plasmid pKF396ME is passed through into the heat-shock transformed E. coli mutant bacterial strain ES1578 competent cell to preparation.
3) screening conditions are set, the clone grown is screened by panel photocopy technology.
4) sequencing and sequence alignment are carried out to the clone of screening confirmation to determine that left-hand promoter pL manipulates subregion
Mutating alkali yl sequence.
5) according to comparison result design primer, sub-district is manipulated to plasmid pKF396ME left-hand promoter pL by over-lap PCR
The corresponding base position in domain carries out rite-directed mutagenesis and is verified.- 35 (operation subregion O of pKF396ME promoter pLL2 the tenth
Five) base by T sports C, it is named as pKF396ML.
6) plasmid pKF396ML successful to building carries out the test of the bacteriolyze under different inducing temperatures.
Finally constructing successful plasmid pKF396ML volume is 4135bp, all kinds of based on λ less than what is reported so far
CI857/pR and the temperature control cracking system modified by mutation.In addition, despite the presence of dextrad promoter pR element in carrier, but by
It is multiple cloning sites after pR, and has strong terminator to be placed in thereafter, therefore this system only cracks thermal stability with control
The pLm of gene E is related, and the multiple cloning sites after pR can be used for the orientation insertion of foreign gene, be conducive to expand the plasmid
Application range, such as the building of dual-expression vector.This completely new existing to thermostabilization temperature control cracking system based on λ cI857/pL
37 DEG C are able to maintain stabilization, and are remained at 42 DEG C by normal inducing lysis host strain, to 2h the and 4h bacteriolyze rate point of Escherichia coli
Not Wei 99.84% and 99.96%, higher than other similar systems Jing Guo temperature sensitive transformation, while the heat shock that can reduce host strain is anti-
It answers, therefore can be used for the preparation of efficient ghost vaccine, also may replace expression of traditional ultrasonic wave bacterial cell disruption for protein
Recycling.
Finally, when carrying out random mutation screening to promoter pL in the present invention temperature range that sets as 37 DEG C~39 DEG C,
37 DEG C~42 DEG C or 38 DEG C~42 DEG C of temperature range of setting when less than in aforementioned research to promoter pR progress screen mutation,
The application order of panel photocopy is also clearly defined simultaneously, these corrective measures are expected to be can effectively reduce similar to work
Proof strength.
Detailed description of the invention
The physical map of Fig. 1 plasmid pKF396ML
CI857: temperature sensitive aporepressor cI857；PR:lambda bacteriophage dextrad promoter；RrnbT1T2: rRNA
Operon T1T2 terminator；PLm:lambda bacteriophage left-hand promoter mutation body；E: bacteriophage phiX174 Lysis gene E；
Ori: copy starting area；Cat: chloramphenicol acetyl transferasegene.
The PCR amplification of Fig. 2 Lysis gene E
M:DNA marker DL-5000；1-3: the pcr amplification product of Lysis gene E；4: blank.
The PCR of Fig. 3 plasmid pKF396ML is identified
M:DNA marker DL-5000；1:pKF396ML；2:pKF396M-5；3: blank.
Operator/promoter sequence of Fig. 4 λ pL and its mutational site.
Fig. 5 PCR amplification Δ rrnbT1T2-pLm- Δ E
M:DNA marker DL-5000；1: the PCR product of Δ rrnbT1T2-pLm；The PCR product of 3:pLm- Δ E；5:
The PCR product of Δ rrnbT1T2-pLm- Δ E；2,4,6: blank.
Fig. 6 pKF396ML containing plasmid (A) and pKF396ME (B) bacillus coli DH 5 alpha bacteriolyze curve.
Specific embodiment
Below in an example, the various processes and method being not described in detail are routine side as known in the art
Method.Meanwhile term used in the present invention is generally generally understood with those of ordinary skill in the art unless otherwise indicated
Meaning.
(1) experimental material
1. bacterial strain and plasmid
Bacillus coli DH 5 alpha Competent cell purchased from Nanjing Vazyme Biotechnology Co., Ltd. (product number:
C502-02), E. coli mutant bacterial strain ES1578 (CGSC#:6485) saves center by Yale University's Escherichia coli genetic resources
(The E.coli Genetic Stock Center) Narinder doctor Whitehead give.Plasmid pBV220-E,
PKF396M-5 saves (Fu LX, Lu.CP.A Novel Dual Vector Coexpressing by this experimental construction
PhiX174Lysis E Gene and Staphylococcal Nuclease A Gene on the Basis of Lambda
Promoter pR and pL,Respectively.Mol Biotechnol,2013,54(2):436-444)。
2. main agents
Restriction enzyme EcoRI (Code No.1611), KpnI (Code No.1618), XbaI (Code
No.1634), T4DNA ligase (Code No.2011A), PrimeSTAR HS (Premix) (Code No.R040A),
Premix Taq (Code No.RR901A), DL5000DNA Marker (Code No.3428A) etc. are purchased from Takara treasured
Bioengineering (Dalian) Co., Ltd.DNA Mini Kit (Code No.RR901A), Presto Mini Plasmid Kit
(Cat No.PDH100) and Gel/PCR DNA Fragments Extraction Kit (Cat No.DF100) etc. are
Geneaid product.All primers are by the limited public affairs of Invitrogen (Shanghai) trade under the silent winged generation your (Thermo Fisher) of match
Department's synthesis.
(2) building to thermostabilization temperature control cracking system pKF396ML based on λ cI857/pL
1. the building of plasmid pKF396ME
According to the primer P1 and P2 (table of a pair of restriction enzyme site containing EcoRI and KpnI respectively of Lysis gene E sequence design synthesis
1), using plasmid pBV220-E as template, amplification gene E.Reaction system is 50 μ l, wherein forward and reverse primer and template are respectively 1 μ l,
25 μ l of PrimerStar HS Premix, sterilize 22 μ l of distilled water.PCR amplification condition are as follows: 98 DEG C of 10s, 60 DEG C of 15s, 72 DEG C
30s, 30 circulations.After reaction, PCR reaction product is enterprising in 0.8% low melting-point agarose gel after bromophenol blue dyes
Row electrophoresis, cuts target fragment in uv analyzer, with plastic recovery kit Gel/PCR DNA Fragments
Extraction Kit recycles target fragment.
Double digestion is carried out to recycling target fragment with EcoRI and KpnI.Digestion system is 50 μ l: public 5 μ of enzyme cutting buffering liquid
L, 20 each 1.5 μ l of μ l, EcoRI and KpnI of target fragment, complements to 50 μ l with sterilizing distilled water.By each component be packed into 1.5ml from
After heart pipe mixes, in 37 DEG C of water-bath 2h, then electrophoresis and gel extraction gene E segment, are connected into same warp by T4DNA ligase
In the pKF396M-5 carrier of EcoRI and KpnI double digestion.Linked system is 25 μ l, keeps gene E segment to rub in linked system
Your the ratio between number and pBV220 molal quantity are about 3-10:1, after each component is mixed gently overnight in 16 DEG C of connections.
It takes 10 μ l connection products to be added in the DH5 α competent cell melted in advance, gently rotates to mix content, set
Then 1.5ml EP pipe is put into heat shock 60s in the circulator bath of pre-heating to 42 DEG C, quickly by EP by ice bath 30min on trash ice
Pipe, which is transferred in ice bath, keeps cell 2-3 minutes cooling, 890 μ l SOC fluid nutrient mediums is added, in 28 DEG C of constant-temperature shaking cultures
60min, 12000rpm/min are centrifuged 15s, remove supernatant, retain 100 μ l liquid and precipitating thallus is resuspended, be spread evenly across chloramphenicol
(35 μ g/ml) resistant panel, is put into incubator and is incubated overnight in 28 DEG C.After growing bacterium colony on plate, with sterilizing toothpick picking
Single bacterium colony is inoculated in the LB liquid medium containing chloramphenicol, and 28 DEG C of shake cultures are stayed overnight, and carries out bacterium using primer P1 and P7
Liquid PCR identification, PCR are accredited as positive clone and raw work bioengineering Shanghai (share) Co., Ltd are sent to carry out sequence verification, will
Correct carrier is sequenced and is named as pKF396ME (Fig. 1).
Experimental result: using plasmid pBV220-E as template, P1/P2 primer pair Successful amplification goes out 292bp (containing restriction enzyme
Enzyme and protection base sequence) Lysis gene E expected purpose segment (Fig. 2).To construct successful plasmid with P1 and P7 primer into
Row PCR identifies the target fragment that amplifiable size expected out is 351bp, and the pKF396M-5 and blank as reference then fail
Amplify target fragment (Fig. 3).Final sequencing result also indicates that, successfully constructs plasmid pKF396ME.
The primer in 1 present invention of table
2. the random mutation of plasmid pKF396ME
Escherichia coli ES1578 Competent cell, specific steps are as follows: be inoculated with Escherichia coli ES1578 are prepared first
In 5ml LB liquid medium, in 37 DEG C of constant temperature oscillator overnight incubations.Next day takes 100 μ l inoculums in the LB of 10ml
Shake culture 3-4h in fluid nutrient medium takes out culture bottle when OD value is to 0.3~0.4, is sub-packed in 1.5ml centrifuge tube,
8000rpm is centrifuged 30s, discards supernatant liquid, and the 0.1mol/LCaCl of 500 μ l pre-cooling is added into precipitating2–MgCl2Solution
(80mmol/L CaCl2,20mmol/L MgCl2) every part of cell precipitation is resuspended, set ice bath 30min on trash ice, 10000rpm centrifugation
The 0.1mol/L CaCl containing 15% glycerol of 100 μ l pre-cooling is added into precipitating by 15s2Thallus is resuspended in solution, sets -40 DEG C of refrigerators
It saves backup.
It is thin that plasmid pKF396ME by aforementioned heat-shock transformed method is transferred to E. coli mutant bacterial strain ES1578 competence
Then conversion fluid is spread evenly across the LB resistant panel containing chloramphenicol (34 μ g/ml) by born of the same parents, after bacterium colony is grown, carry out plate
The plate of first time photocopy is placed in 39 DEG C of cultures by photocopy, and the plate of second of photocopy is placed in 37 DEG C of cultures, and filtering out can be 37
DEG C growth and the clone that cannot be grown at 39 DEG C, sample presentation carry out sequencing, region overlay cI857 is to left-hand promoter for sequencing
pL。
The result shows that: the bacillus coli DH 5 alpha containing mutant plasmid can be grown at 37 DEG C, and cannot be grown at 42 DEG C.Sequencing knot
Fruit shows -35 (operation subregion O of promoter pLL2 the 15th) base sports C (Fig. 4) by T, cI857, pR and
The regional sequences such as rrnbT1T2 are without mutation.
3. the building of plasmid pKF396ML
According to random mutation as a result, synthetic primer P3, P4, P5, P6 (table 1) is designed, by over-lap PCR to promoter pL's
Operate subregion OL2 the 15th bit bases carry out rite-directed mutagenesis.To avoid pcr amplified fragment is too small from being unfavorable for recycling, primer P3,
The targeting sequence of P6 is located in the region rrnbT1T2 of left-hand promoter upstream and the Lysis gene E in downstream, only primer
P4, P5 targeting sequence are located in promoter pL, site containing targeted mutagenesis and complementation, contain XbaI in the segment finally amplified
With EcoRI restriction enzyme site.
Using plasmid pKF396ME as template, is matched respectively with P3 and P4, P5 and P6, utilize high-fidelity DNA polymerase
PrimerStar HS Premix carries out PCR amplification.After reaction, agarose gel electrophoresis is carried out, Gel/PCR DNA is utilized
Target fragment Δ rrnbT1T2-pLm and pLm- Δ E is separately recovered in Fragments Extraction Kit.Then by recycling
Target fragment, which carries out mixing, makes its template and primer each other, and primer P3 and P6 is added, and utilizes high-fidelity DNA polymerase again
PrimerStar HS Premix carries out the complete sequence Δ rrnbT1T2-pLm- Δ E that PCR amplification provides mutational site.With
XbaI/KpnI carries out double digestion to recycling PCR product, is connected into the equally pKF396ME plasmid through XbaI/EcoRI double digestion,
Then bacillus coli DH 5 alpha is converted, is screened with the LB solid medium containing 35 μ g/mL chloramphenicol.PCR is accredited as the positive
Clone's sample presentation sequencing, correct plasmid will be sequenced and be named as pKF396ML.
Experimental result: using plasmid pKF396ME as template, P3/P4 and P5/P6 primer pair Successful amplification goes out size and is respectively
The expected purpose segment of 145bp and 221bp.It is to draw with P3/P6 with overlapping pcr to recycle segment as hybrid template
Object, then Successful amplification goes out expected fusion segment (Fig. 5) that size is about 346bp.Sequencing result shows the operation in pL promoter
Sub- OL2 the 15th bit bases are successfully C (T → C) by T mutagenesis.
(3) the application test of pKF396ML
The application test of plasmid pKF396ML is carried out by bacteriolytic test, and plasmid pKF396ME is as control, concrete operations
For 100ml (35 μ g/ml) containing chloramphenicol will be seeded to respectively containing the bacillus coli DH 5 alpha of plasmid pKF396ME and pKF396ML
LB liquid medium in, in 28 DEG C of shake cultures.When culture solution OD600 reaches 0.3~0.4, temperature is risen to 37 respectively
DEG C, 38 DEG C, 39 DEG C, 40 DEG C, the OD600 of culture is measured by sampling in the expression of 42 DEG C of induced genes at regular intervals, and monitoring is thin
The growing state of bacterium.To 0h after induction, after the bacterium solution of 2h and 4h do appropriate dilution, viable plate count is carried out, calculates cracking base
The bacteriolyze efficiency mediated by E.Calculation formula are as follows:
Bacteriolyze rate=(CFU before CFU/ is induced after 1- induction) × 100%.
Test result: conversion has the bacillus coli DH 5 alpha of plasmid pKF396ML under 37 DEG C of inducing temperatures, and OD600 is being passed through
It tends towards stability after going through about 2h rapid growth, under the inducing temperature higher than 37 DEG C, OD600 after experience increases then in the case where tending to
Drop, and as the increase of inducing temperature, fall increase, the time also shifts to an earlier date, wherein the OD600 range of decrease under 42 DEG C of inducing temperatures
Maximum is finally stable at about 0.15 or so (Fig. 6 A).Bacillus coli DH 5 alpha (pKF396ME) as blank control is then all
Test inducing temperature under OD600 value show as first rising declining afterwards, finally tend to be steady, wherein again with 39 DEG C of inducing temperatures
Lower OD600 be it is minimum, be finally stable at about 0.12 or so, and under 42 DEG C of inducing temperatures OD600 be finally stable at about 0.28 a left side
Right (Fig. 6 B).
Viable plate count reacted from another point of view Lysis gene E function and effect and system to thermal stability.From
After 28 DEG C are warming up to 40 DEG C and 42 DEG C, bacillus coli DH 5 alpha (pKF396ML) viable count is on a declining curve always, relatively induces front lower
The 2-3 order of magnitude has dropped, wherein the bacteriolyze rate of 2h and 4h are respectively 99.84% and 99.96% under 42 DEG C of inducing temperatures；And it rises
Temperature is to after 37 DEG C, 38 DEG C and 39 DEG C, and viable count increases 3.65,1.82 and 1.77 times before relatively inducing respectively after 2h, but 38 after 4h
DEG C and 39 DEG C of induction group viable counts then have dropped the 1-2 order of magnitude respectively, and 37 DEG C of induction groups then only have dropped 3.07 times, substantially
Maintain stabilization.Viable count one of the bacillus coli DH 5 alpha (pKF396ME) under all test inducing temperatures as blank control
Straight on a declining curve, viable count has dropped the 3-5 order of magnitude (table 2) before relatively inducing respectively after 4h.
Conclusion: this completely new thermostabilization temperature control cracking system can be stablized in 37 DEG C of holdings based on λ cI857/pL, and
It is remained at 42 DEG C by normal inducing lysis host strain, while volume is smaller, the bacteriolyze rate of Escherichia coli is passed through higher than other
The similar system of temperature sensitive transformation can also reduce the heat shock response of host strain.
The induction of 2 pKF396ME containing plasmid of table and pKF396ML bacillus coli DH 5 alpha inactivates
Claims (3)
1. a kind of plasmid pKF396ML, sequence is as shown in SEQ ID NO.1.
2. the construction method of plasmid pKF396ML described in claim 1, it is characterised in that the following steps are included: 1) by lysis genes
E is cloned between EcoRI the and KpnI restriction enzyme site in plasmid pKF396M-5 after promoter pL, is built into containing Lysis gene E
Recombinant plasmid pKF396ME；2) pass through over-lap PCR for the operation subregion O of plasmid pKF396ME promoter pLL2 the 15th alkali
Base is C by T rite-directed mutagenesis；Specifically: using plasmid pKF396ME as template, being matched with P3 and P4, P5 and P6, protected using height respectively
True archaeal dna polymerase PrimerStar HS Premix carries out PCR amplification；After reaction, agarose gel electrophoresis is carried out, is utilized
Target fragment Δ rrnbT1T2-pLm and pLm- Δ E is separately recovered in Gel/PCR DNA Fragments Extraction Kit；
Then the target fragment of recycling is carried out mixing makes its template and primer each other, and primer P3 and P6 is added, and is protected again using high
True archaeal dna polymerase PrimerStar HS Premix carries out the complete sequence Δ rrnbT1T2- that PCR amplification provides mutational site
pLm-ΔE；Double digestion is carried out to recycling PCR product with XbaI/KpnI, is connected into equally through XbaI/EcoRI double digestion
In pKF396ME plasmid, bacillus coli DH 5 alpha is then converted, is screened with the LB solid medium containing 35 μ g/mL chloramphenicol；
PCR is accredited as to positive clone's sample presentation sequencing, correct plasmid will be sequenced and be named as pKF396ML；Described P3, P4, P5 and P6
Sequence respectively as shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7.
3. application of the plasmid pKF396ML described in claim 1 as the thermostabilization temperature control cracking system based on λ cI857/pL.
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| CN103497965A (en) * | 2013-10-17 | 2014-01-08 | 扬州大学 | Temperature-controlled zero background T-vector precursor and construction method and application thereof |
| WO2014035457A1 (en) * | 2012-08-29 | 2014-03-06 | Nature Technology Corporation | Dna plasmids with improved expression |
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| WO2014035457A1 (en) * | 2012-08-29 | 2014-03-06 | Nature Technology Corporation | Dna plasmids with improved expression |
| CN103497965A (en) * | 2013-10-17 | 2014-01-08 | 扬州大学 | Temperature-controlled zero background T-vector precursor and construction method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Modulation of gene expression by promoter mutants of the λ cI857 pRM pR system;Wolfgang Jechlinger等;《Journal of Biotechnology》;20050302;第11卷(第1期);11-20 * |
| λpL/pR-cI857温控系统的改造及其对大肠杆菌菌蜕制备的影响;董洪亮等;《生物工程学报》;20121225;第28卷(第12期);1423-1430 * |
| 嗜水气单胞菌非质粒依赖性菌蜕疫苗的研制及评价;付立霞;《中国博士学位论文全文数据库（农业科技辑）》;20131215(第12期);D052-4 * |
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