CN109370936A - Escherichia coli BL21 (the DE3)-PR of one plant of wide spectrum antiphagin and its application - Google Patents
Escherichia coli BL21 (the DE3)-PR of one plant of wide spectrum antiphagin and its application Download PDFInfo
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Abstract
The invention discloses E.coli BL21- (the DE3)-PR of one plant of wide spectrum antiphagin and its applications.The present invention is that parent plant successfully screens plant weight histone expression bacterial strain escherichia coli E.coli BL21 (DE3)-PR with engineered strain escherichia coli E.coli BL21 (DE3), which can resist infecting for the bacteriophage that can crack escherichia coli E.coli BL21 (DE3).The present invention carries out growth curve to recombinant protein expression bacterial strain escherichia coli E.coli BL21 (DE3)-PR and protein expression measures, the results showed that the growth ability and protein expression ability and original starting strain E.coli BL21 (DE3) of the bacterial strain are almost the same.
Description
Technical field
The present invention relates to E.coli BL21- (the DE3)-PR of one plant of wide spectrum antiphagin and its applications, and in particular to one plant
Bioengineering neck is not belonged to vulnerable to E.coli BL21- (DE3)-PR of phage-infect during growth and protein expression
Domain.
Background technique
Bacteriophage is the virus of the microorganisms such as a kind of bacterial infection, fungi, actinomyces or conveyor screw, is widely present in nature
In boundary.Due to bacteriophage have quantity it is more, it is small in size, breeding fastly and genome highly plastic the features such as so that industry send out
Ferment process is highly prone to the infection of bacteriophage.Phage-infect has generality and intractable, and fermentation enterprise is once by its sense
Dye, loss is immeasurable, and so far still without reply good plan.
PET system uses T7 promoter, is that the function of the clonal expression recombinant protein in Escherichia coli since the dawn of human civilization is most strong
Big system, E.coli BL21 (DE3) are the common expression host strains of pET system, and commercial prod has been used as to be answered extensively
With.E.coli BL21 (DE3) is often subject to phage-infect in factory and laboratory, therefore prevents and treats phage-infect problem urgently
It needs to solve.
The common measures taken of prevention and treatment phage-infect mainly includes the control source of infection, bacterial strain rotation, traditional gene work at present
Journey strategy and the bacteriophage control strategy based on CRISPR/Cas system etc..Phage-resistant bacteria is wherein screened by natural mutation
The method of strain since its is easy to operate, significant effect and be widely used.But what current each factory or laboratory used
Phage resistant strains can only resist the infection of a certain strain or certain a kind of bacteriophage mostly, can resist the work of more plants of phage-infects
Journey bacterial strain is still worth excavating.
Summary of the invention
It is an object of the present invention to provide one plant of escherichia coli Escherichia coli BL21 (DE3)-PR.
The deposit number of escherichia coli Escherichia coli BL21 (DE3)-PR provided by the invention is
CGMCC No.16406。
The classification naming of escherichia coli Escherichia coli BL21 (DE3)-PR provided by the invention is large intestine
Escherichia Escherichia coli, the bacterial strain were preserved in Chinese microorganism strain preservation management on August 30th, 2018
(abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences are micro- for committee's common micro-organisms center
Biological study institute, postcode 100101), deposit number is CGMCC No.16406.
It is a further object to provide above-mentioned escherichia coli Escherichia coli BL21 (DE3)-PR
Or the new application of its bacterium solution or its metabolism liquid or its culture solution.
The present invention provides above-mentioned escherichia coli Escherichia coli BL21 (DE3)-PR or its bacterium solution or its
It is metabolized liquid or its culture solution and infects and/or prevent and treat the application in phage-infect in phage resistance.
The present invention also provides above-mentioned escherichia coli Escherichia coli BL21 (DE3)-PR or its bacterium solution or
It is metabolized liquid or its culture solution prepare phage resistance infect and/or prevent and treat phage-infect product in application.
In above-mentioned application, the bacteriophage is Ji Wei section bacteriophage and/long-tail section bacteriophage.
Further, Ji Wei section bacteriophage can be T4, vB_EcoM_IME281, vB_EcoM_IME338, vB_
EcoM_IME339, vB_EcoM_IME340, vB_EcoM_IME341 Deng Ji tail section bacteriophage, long-tail section bacteriophage can be
The long-tails such as T1, vB_EcoS_IME18, vB_EcoS_IME253, vB_EcoS_IME347, JMPW1, EEP section bacteriophage.
The present invention also provides above-mentioned escherichia coli Escherichia coli BL21 (DE3)-PR to be used as host strain
Application in expression recombinant protein.
The present invention also provides above-mentioned escherichia coli Escherichia coli BL21 (DE3)-PR to be used as host strain
Application in the product of preparation expression recombinant protein.
The present invention also provides above-mentioned escherichia coli Escherichia coli BL21 (DE3)-PR to be used as host strain
Expression recombinant protein and phage resistance infect in application.
The present invention also provides above-mentioned escherichia coli Escherichia coli BL21 (DE3)-PR to be used as host strain
Application in the product that preparation expression recombinant protein and phage resistance infect.
The present invention also provides above-mentioned escherichia coli Escherichia coli BL21 (DE3)-PR or its bacterium solution or
It is metabolized the application of liquid or its culture solution in expression recombinant protein.
The present invention also provides escherichia coli Escherichia coli BL21 (DE3)-PR or its bacterium solution or its generations
Thank to the application of liquid or its culture solution in the product of preparation expression recombinant protein.
It is a still further object of the present invention to provide a kind of expressions of recombinant protein.
The expression of recombinant protein provided by the invention includes the following steps: to import the encoding gene of recombinant protein
It states in escherichia coli Escherichia coli BL21 (DE3)-PR, obtains recombinant bacterium；The recombinant bacterium is induced
Expression, obtains the recombinant protein.
In the above method, the encoding gene of the recombinant protein imports above-mentioned escherichia coli by recombinant plasmid
In Escherichia coli BL21 (DE3)-PR.The recombinant plasmid is that the encoding gene of recombinant protein is cloned into expression
It is obtained on plasmid.
The present invention successfully screens plant weight histone expression bacterial strain using escherichia coli E.coli BL21 (DE3)
Escherichia coli E.coli BL21 (DE3)-PR, which, which can resist, can crack escherichia coli E.coli BL21
(DE3) bacteriophage is infected.The present invention to recombinant protein express bacterial strain escherichia coli E.coli BL21 (DE3)-PR into
Row growth curve and protein expression measurement, the results showed that the growth ability and protein expression ability of the bacterial strain and former starting strain
E.coli BL21 (DE3) is almost the same.
Detailed description of the invention
Fig. 1 is bacteriophage plaques figure.The bacteriophage plaques figure of A:E.coli BL21 (DE3)；B:E.coli BL21
(DE3) the bacteriophage plaques figure of-PR.First row is myovirus, and secondary series is siphovirus.
Fig. 2 is the growth curve chart of E.coli BL21 (DE3) and E.coli BL21 (DE3)-PR.
The protein expression SDS-PAGE that Fig. 3 is E.coli BL21 (DE3) and E.coli BL21 (DE3)-PR schemes.M:
Maker；The pET-28a empty plasmid of 1:E.coli BL21 (DE3)；The pET-28a-EGFP matter of 2:E.coli BL21 (DE3)
Grain, IPTG induce 8h；The pET-28a-EGFP plasmid of 3:E.coli BL21 (DE3), IPTG induce 10h；4:E.coli BL21
(DE3) pET-28a-EGFP plasmid, IPTG induce 12h；The pET-28a-EGFP plasmid of 5:E.coli BL21 (DE3)-PR,
IPTG induces 8h；The pET-28a-EGFP plasmid of 6:E.coli BL21 (DE3)-PR, IPTG induce 10h；7:E.coli BL21
(DE3) the pET-28a-EGFP plasmid of-PR, IPTG induce 12h.Wherein, arrow meaning is purpose band.
Preservation explanation
Strain name: escherichia coli
Latin name: Escherichia coli
Strain number: BL21 (DE3)-PR
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on August 30th, 2018
Collection is registered on the books number: CGMCC No.16406
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
E.coli BL21 (DE3) in following embodiments is Beijing Quan Shijin biotech firm product, BL21 (DE3)
Chemically Competent Cell, article No. CD601-01.
The solvent of LB liquid medium in following embodiments is water, and solute and its concentration difference are as follows: tryptone
10g/L, yeast extract 5g/L, sodium chloride 10g/L.LB semisolid culturemedium is obtained after agar is added in LB liquid medium
It arrives, mass fraction of the agar in LB semisolid culturemedium is 0.7%.LB solid medium is added in LB liquid medium
Enter and obtain after agar, mass fraction of the agar in LB solid medium be 1.5%.
Bacteriophage vB_EcoM_IME281, vB_EcoM_IME338, vB_EcoM_IME339 in following embodiments are
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.In the preservation of bacteriophage vB_EcoM_IME281
The heart is registered on the books number: CGMCC No.16291；The collection of bacteriophage vB_EcoM_IME338 is registered on the books number:
CGMCC No.16292；The collection of bacteriophage vB_EcoM_IME339 is registered on the books number: CGMCC No.16293.
Bacteriophage T4 in following embodiments is recorded in document " Chen Zhaobin, Xu Xin, Dai little Ying, Zeng Zhongming, Zhang Chaowu .MS2
With T4 bacteriophage to comparative studies [J] modern preventive medicine of short wave ultraviolet resistance, 2007 (03): in 469-471. ",
The public can obtain from applicant, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes
It uses.
Bacteriophage T1 in following embodiments be recorded in document " Wietzorrek A, Schwarz H, Herrmann C,
Braun V.The genome of the novel phage Rtp,with a rosette-like tail tip,is
homologous to the genome of phage T1.J Bacteriol.2006Feb；188 (4): in 1419-36. ",
The public can obtain from applicant, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes
It uses.
Bacteriophage vB_EcoS_IME253 in following embodiments is recorded in document " Xing Shaozhen, Zhao Feiyang, Sun Qiang, meter Zhi
By force, An little Ping, Tong Yigang, Zhao treasured China utilize comparative genomics Rapid identification Escherichia coli bacteriophages tolerance gene [J]
Chinese antibiotic magazine, 2017,42 (10): in 871-879. ", the public can obtain from applicant, which only attaches most importance to
Used in the related experiment of duplicate invention, it not can be used as other purposes and use.
Bacteriophage vB_EcoS_IME18 in following embodiments is recorded in document " Li Ping, Lin Hong, Tong Yigang, Wang Jingxue.
The identification and its receptor assay [J/OL] Food Science of one plant of T1 sample bacteriophage vB_EcoS_IME18: 1-11 [2018-10-
22] ", the public can obtain from applicant, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as it
Its purposes uses.
Bacteriophage vB_EcoS_IME347 in following embodiments is recorded in document: " Ping Li, Hong Lin,
Zhiqiang Mi,Yigang Tong,Jingxue Wang.vB_EcoS_IME347a novel T1-like
Escherichia coli bacteriophage.Journal of Basic Microbiology.doi:10.1002/
In jobm.201800271 ", the public can obtain from applicant, the biomaterial only attach most importance to duplicate invention related experiment institute
With not can be used as other purposes and use.
Bacteriophage JMPW1 in following embodiments is recorded in document " Shen M, Zhu H, Lu S, Le S, Li G, Tan
Y,Zhao X,Shen W,Hu F,Wang J.Complete Genome Sequences of T1-Like Phages
JMPW1and JMPW2.Genome Announc.2016Jun 23；4(3).pii:e00601-16.doi:10.1128/
GenomeA.00601-16. in ", the public can obtain from applicant, the biomaterial only attach most importance to duplicate invention related experiment
It is used, it not can be used as other purposes and use.
Phage E EP in following embodiments be recorded in document " Li S, Liu L, Zhu J, Zou L, Li M, Cong Y,
Rao X,Hu X,Zhou Y,Chen Z,Hu F.Characterization and genome sequencing of a
novel coliphage isolated from engineered Escherichia coli.Intervirology.2010；
53 (4): in 211-20.doi:10.1159/000299063.Epub 2010Mar 23. ", the public can obtain from applicant,
The biomaterial is only attached most importance to used in the related experiment of duplicate invention, not can be used as other purposes and is used.
The screening and its preservation of embodiment 1, phage resistant strains E.coli BL21 (DE3)-PR
One, the screening of phage resistant strains E.coli BL21 (DE3)-PR
1, the preparation of BL21 (DE3) bacterium solution
E.coli BL21 (DE3) is inoculated in LB liquid medium, at 37 DEG C, is incubated overnight, obtains under the conditions of 220rpm
To BL21 (DE3) bacterium solution.
2, the separation of bacteriophage
It taking sewage (sewage source is in the waste water of 307 hospital of the Chinese People's Liberation Army), 10 000r/min are centrifuged 10min,
Collect supernatant.0.22 μm of filter of supernatant is filtered, sewage filtrate is obtained.Then 200 μ L sewage filtrates and 4mL are walked
BL21 (DE3) bacterium solution of rapid 1 preparation is added in 3 × LB liquid medium of 2mL, and 37 DEG C, 220rpm cultivates 6h, obtains bacteriophage
Stoste.It again by 0.22 μm of filtering with microporous membrane of bacteriophage stoste, obtains bacteriophage and saves liquid, and saved in 4 DEG C.
3, the purifying of bacteriophage
With BL21 (DE3) for indicator bacteria, the bacteriophage in the above-mentioned bacteriophage stoste of double-layer agar technique culture is used.Specific step
It is rapid as follows:
1) bacteriophage saves the dilution of liquid
Step 2 is made with Gibco phosphate buffer PBS (Thermo Fisher Scientific, C10010500BT)
Standby bacteriophage saves liquid and carries out gradient dilution, obtains different dilutions (101-108Pfu/mL bacteriophage) saves liquid.
2) double-layer agar technique culture bacteriophage
The different dilutions (10 that BL21 (DE3) bacterium solution prepared by 300 μ L steps 1 is prepared from 100 μ L steps 1) respectively1-
108Pfu/mL bacteriophage) saves liquid mixing, and room temperature adsorbs 5min.Then the training of 5mL LB semisolid is added in mixed liquor respectively
It supports in base, is uniformly mixed, is laid in the solid medium tablets of LB containing lower layer, room temperature dries 5min.Plate is trained in 37 DEG C again
8h is supported, plaque is observed.
3) purifying of bacteriophage
5mL LB is added in single plaque and BL21 (DE3) bacterium solution of 300 μ L steps 1 preparation on the above-mentioned double-layer plate of picking
In fluid nutrient medium, 37 DEG C, 220rpm cultivates 6h, obtains culture solution.Then by culture solution with 0.22 μm of filtering with microporous membrane simultaneously
It is saved in 4 DEG C.
4) according to above step 2) -3) in triplicate, the bacteriophage purified saves liquid.
4, the screening of phage resistant strains
The different dilutions for the purifying that BL21 (DE3) bacterium solution prepared by 300 μ L steps 1 is prepared from 100 μ L steps 3 respectively
(101-108Pfu/mL bacteriophage) saves liquid mixing, and room temperature adsorbs 5min.Then the training of 5mL LB semisolid is added in mixed liquor
It supports in base, is uniformly mixed, is laid in the solid medium tablets of LB containing lower layer, room temperature dries 5min.Plate is trained in 37 DEG C again
24-48h is supported, until being that the bright plate of plaque grows the smooth colony that diameter is 2~3mm entirely.Finally by bacterium colony into
Row plate streaking culture, obtains phage resistant strains monoclonal.
5, plaque ethods identify phage resistant strains
The above-mentioned steps 4 that parent plant E.coli BL21 (DE3) bacterium solution and 300 μ L for taking 300 μ L to be incubated overnight are incubated overnight
The phage resistant strains bacterium solution of preparation is separately added into 5mL LB semisolid culturemedium, is uniformly mixed, and it is solid to be laid in LB containing lower layer
On body culture medium flat plate, room temperature dries 5min；The bacteriophage by the purifying of 1 drop (2 μ L) step 3 preparation saves liquid respectively
(108Pfu/mL) drop is on bacterial layer culture medium, 37 DEG C of culture 8h, observes plaque, and selection can not form plaque and life
Long bacterial strain in order is phage resistant strains.
6, the screening of phage resistant strains E.coli BL21 (DE3)-PR
Successively according to method described in above-mentioned steps 1-5, bacteriophage IME18 (step 1-3) and tolerance bacteriophage are obtained
The bacterial strain R1 (step 4 and 5) of IME18.Then it using bacterial strain R1 as parent plant, successively according to method described in above-mentioned steps 1-5, obtains
To the bacterial strain R2 of bacteriophage IME253 and tolerance bacteriophage IME253.And so on, it is screened by 7 wheels, successively obtains bacteriophage
IME18, IME253, IME281, IME338, IME339, IME340, IME347 finally obtain the bacterium of one plant of wide spectrum antiphagin
Strain, and it is denoted by BL21 (DE3)-PR.
Two, the Molecular Identification of phage resistant strains BL21 (DE3)-PR
It is expanded, is gone forward side by side using 16S rDNA gene order of the universal primer to phage resistant strains BL21 (DE3)-PR
Row sequencing.The result shows that: the 16S rDNA gene order of resistant strain BL21 (DE3)-PR is as shown in sequence 1.
Three, the preservation of phage resistant strains BL21 (DE3)-PR
The classification naming of phage resistant strains BL21 (DE3)-PR is escherichia coli Escherichia coli, the bacterium
Strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as on August 30th, 2018
CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), it protects
Hiding number is CGMCC No.16406.
The performance detection of embodiment 2, phage resistant strains BL21 (DE3)-PR
One, the bacteriophage plaques test of phage resistant strains BL21 (DE3)-PR
It is (hereafter simple in order to detect phage resistant strains escherichia coli Escherichia coli BL21 (DE3)-PR
It is denoted as carrying out bacteriophage plaques test for the antiphagin performance of E.coli BL21 (DE3)-PR).Specific step is as follows: taking 300
The phage resistant strains of 6 screenings of 1 step 1 of BL21 bacterium solution and 300 μ L embodiment prepared in the 1 of 1 step 1 of μ L embodiment
BL21 (DE3)-PR bacterium solution is separately added into 5mL LB semisolid culturemedium, is uniformly mixed, is laid in the solid culture of LB containing lower layer
On base plate, room temperature dries 5min；It is respectively 10 by 1 drop (2 μ L) concentration8The bacteriophage of pfu/mL saves liquid (T4, vB_EcoM_
IME281、vB_EcoM_IME338、vB_EcoM_IME339、vB_EcoM_IME340、vB_EcoM_IME341、T1、vB_
EcoS_IME18, vB_EcoS_IME253, vB_EcoS_IME347, JMPW1, EEP) drop is on bacterial layer culture medium, 37 DEG C of trainings
8h is supported, whether there is or not plaque appearance for observation.
As a result as shown in Figure 1.Figure 1A is the bacteriophage plaques figure of E.coli BL21 (DE3)；Figure 1B is E.coli BL21
(DE3) the bacteriophage plaques figure of-PR.Wherein, first row is myovirus, is from top to bottom followed successively by T4 bacteriophage, vB_
EcoM_IME281 bacteriophage, vB_EcoM_IME338 bacteriophage, vB_EcoM_IME339 bacteriophage, vB_EcoM_IME340 bite
Thallus, vB_EcoM_IME341 bacteriophage；Secondary series is siphovirus, is from top to bottom followed successively by T1 bacteriophage, vB_
EcoS_IME18 bacteriophage, vB_EcoS_IME253 bacteriophage, vB_EcoS_IME347 bacteriophage, JMPW1 bacteriophage, EEP bite
Thallus.As can be seen from the figure: all equal cleavable E.coli BL21 (DE3) bacterial strains of bacteriophage form plaque, and
E.coli BL21 (DE3)-PR bacterial strain occurs without plaque, and illustrating that E.coli BL21 (DE3)-PR bacterial strain can be resisted can be with
Infecting for the bacteriophage of E.coli BL21 (DE3) is cracked, including resists Duo Zhuji tail section bacteriophage (T4, vB_EcoM_
IME281, vB_EcoM_IME338, vB_EcoM_IME339, vB_EcoM_IME340, vB_EcoM_IME341) infection and support
Kang Duozhu long-tail section bacteriophage (T1, vB_EcoS_IME18, vB_EcoS_IME253, vB_EcoS_IME347, JMPW1, EEP)
Infection.
Two, the growth curve measurement of phage resistant strains E.coli BL21 (DE3)-PR
Measure the growth curve of E.coli BL21 (DE3) and E.coli BL21 (DE3)-PR.Specific step is as follows: will
E.coli BL21 (DE3) and E.coli BL21 (DE3)-PR are inoculated in respectively in 5mL LB culture medium, and 37 DEG C, 220rpm oscillation
Cultivate 12h.Next day takes 500 μ L in 50mL LB culture medium respectively, and 37 DEG C, 220rpm shaken cultivation 12h.Culture 0h,
1mL is taken out when 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h respectively, is measured using ultraviolet specrophotometer
OD600。
As a result as shown in Figure 2.As can be seen from the figure: E.coli BL21's (DE3) and E.coli BL21 (DE3)-PR
Upgrowth situation is almost the same.
Three, the protein expression level measurement of phage resistant strains E.coli BL21 (DE3)-PR
Measure the protein expression level of E.coli BL21 (DE3) and E.coli BL21 (DE3)-PR.Specific step is as follows:
1, the preparation of recombinant plasmid
DNA molecular shown in sequence 2 is inserted into EcoRI the and XhoI digestion of pET-28a carrier (excellent precious biology, VT1207)
Between site, pET-28a-EGFP recombinant plasmid is obtained.PET-28a-EGFP expression of recombinant plasmid EGFP albumen.
2, the preparation of recombinant bacterium
PET-28a-EGFP recombinant plasmid prepared by step 1 is directed respectively into E.coli BL21 (DE3) and E.coli
In BL21 (DE3)-PR, the E.coli BL21 (DE3) containing pET-28a-EGFP is respectively obtained and containing pET-28a-EGFP's
E.coli BL21(DE3)-PR。
3, the inducing expression of recombinant bacterium
E.coli BL21 (DE3) containing pET-28a-EGFP prepared by step 2 and containing pET-28a-EGFP's
E.coli BL21 (DE3)-PR is inoculated in 5mL respectively and contains in the LB culture medium of 50 μ g/mL kanamycins, and 37 DEG C, 220rpm vibration
Swing culture 12h.Next day respectively transfers the above-mentioned bacterium solution of 500 μ L in the LB culture medium that 50mL contains 50 μ g/mL kanamycins, and 37
DEG C, 220rpm shaken cultivation.OD is measured using ultraviolet specrophotometer600, work as OD600It is (dense eventually using IPTG induction when=0.6
Degree is 0.8mM), the separately sampled 1mL in Fiber differentiation 8h, 10h, 12h.1mL bacterium solution 12000rpm is centrifuged 1min, is used
1mL PBS is resuspended, and 50 μ L bacterium solutions is taken to add 10 μ 6 × sample-loading buffers of L, boils 7min, carries out SDS-PAGE electrophoresis detection.
As a result as shown in Figure 3.As can be seen from the figure: the E.coli BL21 (DE3) containing pET-28a-EGFP and containing
E.coli BL21 (the DE3)-PR bacterial strain expression EGFP protein band of pET-28a-EGFP is essentially identical, illustrates containing pET-
The E.coli BL21 (DE3) of 28a-EGFP and E.coli BL21 (DE3)-PR containing pET-28a-EGFP expresses EGFP albumen
Ability it is almost the same.
Sequence table
<110>Beijing University of Chemical Technology of PLA Academy of Military Sciences's military medical research institute
Escherichia coli BL21- (the DE3)-PR of<120>one plants of wide spectrum antiphagins and its application
<160>2
<170>PatentIn version 3.5
<210>1
<211>1355
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>1
gctacctact tcttttgcaa cccactccca tggtgtgacg ggcggtgtgt acaaggcccg 60
ggaacgtatt caccgtggca ttctgatcca cgattactag cgattccgac ttcatggagt 120
cgagttgcag actccaatcc ggactacgac gcactttatg aggtccgctt gctctcgcga 180
ggtcgcttct ctttgtatgc gccattgtag cacgtgtgta gccctggtcg taagggccat 240
gatgacttga cgtcatcccc accttcctcc agtttatcac tggcagtctc ctttgagttc 300
ccggccggac cgctggcaac aaaggataag ggttgcgctc gttgcgggac ttaacccaac 360
atttcacaac acgagctgac gacagccatg cagcacctgt ctcacggttc ccgaaggcac 420
cttcccatct ctgaaaactt ctgtggatgt caagaccagg taaggttctt cgcgttgcat 480
cgaattaaac cacatgctcc accgcttgtg cgggcccccg tcaattcatt tgagttttaa 540
ccttgcggcc gtactcccca ggcggtcgac ttaacgcgtt agctccggaa gccacgcctc 600
aagggcacaa cctccaagtc gacatcgttt acggcgtgga ctaccagggt atctaatcct 660
gtttgctccc cacgctttcg cacctgagcg tcagtcttcg tccagggggc cgccttcgcc 720
accggtattc ctccagatct ctacgcattt caccgctaca cctggaattc tacccccctc 780
tacgagactc aagcttgcca gtatcagatg cagttcccag gttgagcccg gggatttcac 840
atctgactta acaaaccgcc tgcgtgcgct ttacgcccag taattccgat taacgcttgc 900
accctccgta ttaccgcggc tgctggcacg gagttagccg gtgcttcttc tgcgggtaac 960
gtcaatgagc aaaggtatta actttactcc cttcctcccc gctgaaagta ctttacaacc 1020
cgaaggcctt cttcatacac gcggcatggc tgcatcaggc ttgcgcccat tgtgcaatat 1080
tccccactgc tgcctcccgt aggagtctgg accgtgtctc agttccagtg tggctggtca 1140
tcctctcaga ccagctaggg atcgtcgcct aggtgagccg ttaccccacc tactagctaa 1200
tcccatctgg gcacatccga tggcaagagg cccgaaggtc cccctctttg gtcttgcgac 1260
gttatgcggt attagctacc gtttccagta gttatccccc tccatcaggc agtttcccag 1320
acattactca cccgtccgcc actcgtcagc gaagc 1355
<210>2
<211>732
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>2
gaattcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 60
ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 120
acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 180
cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac 240
atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 300
atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 360
accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 420
gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 480
aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 540
ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 600
aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 660
atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 720
aagtaactcg ag 732
Claims (9)
1. one plant of escherichia coli Escherichia coli BL21 (DE3)-PR, deposit number CGMCC
No.16406。
2. escherichia coli Escherichia coli BL21 (DE3)-PR described in claim 1 or its bacterium solution or its generation
It thanks liquid or its culture solution and infects and/or prevent and treat the application in phage-infect in phage resistance.
3. escherichia coli Escherichia coli BL21 (DE3)-PR described in claim 1 or its bacterium solution or its generation
Thank liquid or its culture solution prepare phage resistance infect and/or prevent and treat phage-infect product in application.
4. application according to claim 2 or 3, it is characterised in that: the bacteriophage is Ji Wei section bacteriophage and/or long-tail
Section bacteriophage.
5. escherichia coli Escherichia coli BL21 (DE3)-PR described in claim 1 is as host strain in table
Up to the application in recombinant protein；
Or, escherichia coli Escherichia coli BL21 (DE3)-PR described in claim 1 is making as host strain
Application in the product of standby expression recombinant protein.
6. escherichia coli Escherichia coli BL21 (DE3)-PR described in claim 1 is as host strain in table
Up to recombinant protein and phage resistance infect in application；
Or, escherichia coli Escherichia coli BL21 (DE3)-PR described in claim 1 is making as host strain
The application in product that standby expression recombinant protein and phage resistance infect.
7. escherichia coli Escherichia coli BL21 (DE3)-PR described in claim 1 or its bacterium solution or its generation
Thank to the application of liquid or its culture solution in expression recombinant protein.
8. escherichia coli Escherichia coli BL21 (DE3)-PR described in claim 1 or its bacterium solution or its generation
Thank to the application of liquid or its culture solution in the product of preparation expression recombinant protein.
9. a kind of expression of recombinant protein includes the following steps: to import the encoding gene of recombinant protein in claim 1
In described escherichia coli Escherichia coli BL21 (the DE3)-PR, recombinant bacterium is obtained；The recombinant bacterium is carried out
Inducing expression obtains the recombinant protein.
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| CN201811249585.5A CN109370936A (en) | 2018-10-25 | 2018-10-25 | Escherichia coli BL21 (the DE3)-PR of one plant of wide spectrum antiphagin and its application |
| CN201910140575.6A CN109762770B (en) | 2018-10-25 | 2019-02-26 | Escherichia coli BL21(DE3) -PR with broad-spectrum phage resistance and application thereof |
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| CN201910140575.6A Expired - Fee Related CN109762770B (en) | 2018-10-25 | 2019-02-26 | Escherichia coli BL21(DE3) -PR with broad-spectrum phage resistance and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114075521A (en) * | 2020-08-21 | 2022-02-22 | 安徽华恒生物科技股份有限公司 | Method for screening valine production strain with phage resistance and application thereof |
| CN117586920A (en) * | 2023-11-30 | 2024-02-23 | 南京诺唯赞生物科技股份有限公司 | Bacterial strain resistant to phage population infection, phage resistant element and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110396533B (en) * | 2019-07-04 | 2023-05-26 | 成都英普博集生物科技有限公司 | Escherichia coli strain with phage resistance, screening method and preparation method of recombinant engineering bacterium expressed recombinant protein |
| JP2023509565A (en) * | 2019-11-27 | 2023-03-09 | イノバシオン・イ・デサロージョ・デ・エネルヒーア・アルファ・スステンタブレ・ソシエダ・アノニマ・デ・カピタル・バリアブレ | phage-resistant microorganisms |
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| EP0497757B1 (en) * | 1989-10-31 | 1994-06-22 | Schering Corporation | E. coli secretory strains |
| CN102010848B (en) * | 2010-03-30 | 2012-02-08 | 江南大学 | L-phenylalanine producing strain having double phage resistance and breeding method thereof |
-
2018
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114075521A (en) * | 2020-08-21 | 2022-02-22 | 安徽华恒生物科技股份有限公司 | Method for screening valine production strain with phage resistance and application thereof |
| CN117586920A (en) * | 2023-11-30 | 2024-02-23 | 南京诺唯赞生物科技股份有限公司 | Bacterial strain resistant to phage population infection, phage resistant element and application thereof |
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| Publication number | Publication date |
|---|---|
| CN109762770A (en) | 2019-05-17 |
| CN109762770B (en) | 2021-05-11 |
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