CN103555751B - A kind of based on two method checking strategy enhancing intestinal bacteria T7 expression system stability - Google Patents

A kind of based on two method checking strategy enhancing intestinal bacteria T7 expression system stability Download PDF

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CN103555751B
CN103555751B CN201310505664.9A CN201310505664A CN103555751B CN 103555751 B CN103555751 B CN 103555751B CN 201310505664 A CN201310505664 A CN 201310505664A CN 103555751 B CN103555751 B CN 103555751B
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聂尧
徐岩
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Jiangnan University
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Abstract

Check based on two the method that strategy strengthens intestinal bacteria T7 expression system stability, belong to escherichia coli expression regulation and control field.The present invention utilizes genetic engineering technique, with the Pullulanase of bacterial origin for expressing protein, constructs the recombination bacillus coli of expressing Pullulanase.By using pET expression vector pET-22b (+) and pET-28a (+) that contain lac operon and repressor gene lacI, construct recombinant bacterium E.coliBL21/pET-22b (+)-pul and E.coliBL21/pET-28a (+)-PelB-pul, rigorous two of lac operon are utilized to check regulating strategy, the background essentially eliminating toxicity foreign protein is expressed, improve the target protein expression amount after the expression stability of freezing glycerine bacterial classification and lactose self-induction, enhance escherichia expression system stability.The invention solves system instability common in intestinal bacteria T7 expression system, develop the method strengthening intestinal bacteria T7 expression system stability.

Description

A kind of based on two method checking strategy enhancing intestinal bacteria T7 expression system stability
Technical field
Check based on two the method that strategy strengthens intestinal bacteria T7 expression system stability, belong to escherichia coli expression regulation and control field.
Background technology
Recombinant technology has been widely used in the high level expression of target protein.Can be used in the system of heterologous protein expression multiple, E. coli system is the most frequently used expressive host all the time.Compared with other expressive hosts, intestinal bacteria have numerous advantage, as clear in genetic background, to grow rapidly, can reach high-density in cheap substratum and have overexpression target protein ability.Therefore target protein amount based on the T7 system expression of T7 RNA polymerase up to 50% of total protein of cell amount, can show the advantage with other E. coli system.
The speed of T7 RNA polymerase synthesis mRNA is 5 times of intestinal bacteria self RNA polymerase, and its high vigor imparts the ability of the powerful synthesis recombinant protein of T7 system.But the high vigor of T7 RNA polymerase has been proved to be exists some negative effects to expression system, and main manifestations is: although the encoding gene of T7 RNA polymerase, be positioned at escherichia coli host BL21(DE3) T7 gene on karyomit(e) is subject to lacuV5 promotor and lacthe regulation and control of operon sequence, only have the T7 RNA polymerase " seepage " of minute quantity to express, but due to T7 RNA polymerase vigor too high, even without inductor exist, transcribing of target gene also can be initiated, produce background express.If target protein is toxic to Bacillus coli cells, this background is expressed will affect the stability of expression system and the normal expression of target protein, even expression strain potentially unstable or accumulation detrimental mutation.
The background that more existing researchs are devoted to reduce intestinal bacteria T7 expression system is expressed.A kind of method reducing background expression uses the Host Strains containing with pLysS or the pLysE plasmid of pET carrier compatibility.The T7 N,O-Diacetylmuramidase of these two kinds of vector expressions is natural inhibitors of T7 RNA polymerase, can be combined and suppress the activity of the latter with T7 RNA polymerase.But although the background expression level of BL21 (DE3) pLysS bacterial strain almost only has 1/10th of BL21 (DE3) bacterial strain, the former background is expressed still to be existed, and still may cause the problem of system instability.And T7 N,O-Diacetylmuramidase is a kind of bifunctional albumen, and it has destruction to Bacillus coli cells wall, the strain growth therefore containing pLysS or pLysE plasmid is slower.In addition after induction, T7 N,O-Diacetylmuramidase will weaken expression, make target protein expression amount very low.In substratum, add 0.5-1% glucose, the metabolism reptation behavior of generation also can reduce the background expression of T7 RNA polymerase.
Above method is all that the direct background for T7 RNA polymerase is expressed and designs, and target is all that the background reducing T7 RNA polymerase is expressed.The present invention develops a kind of background directly not weakening T7 RNA polymerase and expresses, but T7 RNA polymerase can be stoped to cause the method for transcription of foreign genes on expression plasmid.Insert in the T7 promotor downstream of pET carrier lacoperon, forms T7- lacpromotor, the background that effectively can reduce target protein is expressed.T7- lacpromotor can provide one lacthe binding site of repressor, when there is not inductor in system, lacrepressor can be incorporated into T7-tightly lacpromotor lacin operon sequence, stop the T7 RNA polymerase produced by background expression to be passed through, disturb the extension of mRNA chain, thus reduce the basal transcript of foreign gene under non-induction state.If to there is q.s in system lacrepressor is all to occupy in cell lacoperon site, in non-inducing cell, the background of target protein is expressed and will be almost completely eliminated, and acquisition is also stablized by expression system.
Summary of the invention
(1) technical problem that will solve
The object of this invention is to provide the method strengthening intestinal bacteria T7 expression system stability.The present invention for expressing protein with the Pullulanase of bacterial origin, employs and contains lacoperon and repressor gene lacIpET expression vector, utilize lacrigorous two of operon check regulating strategy, and the background essentially eliminating toxicity report albumen is expressed, and improves escherichia expression system stability, thus develops the method strengthening intestinal bacteria T7 expression system stability.
(2) technical scheme
Check based on two the method that strategy strengthens intestinal bacteria T7 expression system stability, first construct express Nagano bacillus ( bacillus naganoensis) recombination bacillus coli of CCTCC M 2012388 Pullulanase e.colibL21 (DE3)/pET-20b (+)- pul, the expression vector pET-20b (+) that this recombinant bacterium carries does not contain lacoperon and repressor gene lacI.In abduction delivering process, find that report albumen exists background and expresses, and freezing glycerine pipe expression level in frozen process of recombinant bacterium constantly declines, and shows that expression system exists unstable phenomenon.Therefore use and contain lacoperon and repressor gene lacIcarrier pET-22b (+) and pET-28a (+), utilize lacrigorous two of operon check regulating strategy, construct recombinant bacterium e.colibL21/ pET-22b (+)- pulwith e.colibL21/pET-28a (+)-PelB- pul.Compare the target protein expression amount after the background expression level of three strain recombination bacillus colis, freezing glycerine pipe expression amount stability and lactose self-induction.
(1) acquisition of Pullulanase gene
Nagano genus bacillus ( bacillus naganoensis) CCTCC M 2012388 substratum: CaCl 20.25 g/L, MgSO 47H 2o 0.5 g/L, (NH 4) 2sO 40.2 g/L, yeast extract 2 g/L, dextrose anhydrous 5 g/L, KH 2pO 43 g/L, inorganic salt solution 1 mL/L, pH 5.0, distilled water is prepared.
Inorganic salt solution: ZnSO 47H 2o 0.1 g/L, MnCl 2h 2o 0.03 g/L, H 3bO 30.3 g/L, CoCl 26H 2o 0.2 g/L, CuCl 22H 2o 0.01 g/L, NiCl 26H 2o 0.02 g/L, Na 2moO 42H 2o 0.03 g/L, distilled water is prepared.
By Nagano genus bacillus ( bacillus naganoensis) CCTCC M 2012388 strain inoculation in containing in 250 mL shaking flasks of 25 mL substratum, in 37 DEG C, 200 rpm shaking culture 72 hours.After cultivation terminates, thalline is centrifugal and with brine twice, collecting cell utilizes genome DNA extracting reagent kit Genomic DNA Extraction Miniprep System(VIOGENE company) extract genome.
Primer 1:5 '-gaaca gGATCCagatgggaacaccacaaaC-3 ',
Primer 2: 5 '-attcc ctcgagtttaccatcagatgggct-3 '.
Primer 1 contains bamHi restriction enzyme site, primer 2 contains xhoi restriction enzyme site.
PCR reaction system: ddH 2o 37 μ L, 10 × Reaction Buffer 5 μ L, dNTP(25 mmol/L) 0.5 μ L, primer 1(50 pmol/ μ L) 1 μ L, primer 2 (50 pmol/ μ L) 1 μ L, genomic dna 5 μ L, Taq DNA polymerase(5 U/ μ L) 0.5 μ L.
With Nagano genus bacillus ( bacillus naganoensis) CCTCC M 2012388 genome is template, adopts PCR method amplification Pullulanase gene.PCR reaction process: 95 DEG C of denaturation 5 min; 95 DEG C of 1 min, 60 DEG C of 0.5 min, 72 DEG C of 2 min, carry out 30 circulations; 72 DEG C extend 10 min.
Utilize 3S Spin Agarose Gel DNA Purification Kit(Shanghai Shenergy Biocolor BioScience & Technology Company) purify DNA segment.
Add sodium acetate solution (3 mol/L, pH 5.2) and 2 times of volume dehydrated alcohols of 1/10 volume in DNA solution ,-20 DEG C precipitate 1 hour.12000 rpm are in 4 DEG C of centrifugal 30 min.Add 75% ethanol 500 μ L to wash, 12000 rpm, in 4 DEG C of centrifugal 30 min, are dissolved in appropriate TE damping fluid after aseptic operating platform dries up, and use immediately or-20 DEG C of preservations.
(2) structure of the recombination bacillus coli containing Pullulanase gene
A, pET-20b (+)- pulstructure
Utilize the vast Tyke biological gene Technology Co., Ltd. in plasmid extraction kit Mini-Plasmid Rapid Isolation Kit(Beijing) extract plasmid pET20b (+).
Be added in Eppendorf pipe according to the order of water, damping fluid, PCR primer or plasmid DNA, enzyme, build pipe lid, vibration makes liquid fully mix, be placed in and make liquid concentrate at the bottom of pipe centrifugal 2 seconds in whizzer, 37 DEG C of water-baths 3 hours, pipe is maybe placed in 65 DEG C of insulations 10 minutes by the Loading Buffer adding 1/10 volume in pipe, stops endonuclease reaction.Digestion products carries out agarose gel electrophoresis analysis and cuts glue reclaiming object fragment, concentrated.
Reaction system forms: 10 × H Buffer 4 μ L, DNA 10 μ L, bamHi 2 μ L, xhoi 2 μ L, ddH 2system is supplied 40 μ L by O.
The connection of goal gene and plasmid pET20b (+)
Be connected with plasmid pET20b (+) by Pullulanase gene, reaction system is composed as follows: plasmid pET-20b (+) 0.8 μ L, Pullulanase gene 4.2 μ L, Ligation Solution 5 μ L.Hybrid connections liquid, to be placed in 16 DEG C of incubators ligation 12 hours.
Recombinant plasmid transformed intestinal bacteria
100 μ L intestinal bacteria e. coliadd 10 μ L in BL21 (DE3) competent cell suspension and connect product, mix gently, in ice bath, leave standstill 30 minutes.Proceed in 42 DEG C of water-baths, thermal shock 90 seconds.Fast transfer, in ice bath, cools 2 minutes.Add the LB liquid nutrient medium of 700 μ L, 37 DEG C, 100 rpm shaking table incubations cultivate 1 hour.After cultivating, centrifugal 2 minutes of bacterium liquid 3000 rpm, abandons supernatant 600 μ L, is applied to containing on the antibiotic LB flat board of 50 μ g/mL ammonia benzyl, is inverted for 37 DEG C and cultivates after the mixing of residue bacterium liquid.Obtain the recombination bacillus coli containing object Pullulanase gene e. colibL21 (DE3)/pET-20b (+)- pul.
B, pET-22b (+)- pulwith pET-28a (+)-PelB- pulstructure
By recombinant plasmid pET-20b (+)- pulrestriction enzyme is used respectively with expression vector pET-28a (+), pET-22b (+) xbai and xhoi substep single endonuclease digestion.Be added in Eppendorf pipe according to the order of water, damping fluid, plasmid, enzyme, build pipe lid, vibration makes liquid fully mix, be placed in and make liquid concentrate at the bottom of pipe in whizzer centrifugal 2 seconds, 37 DEG C of water-baths 3 hours, pipe is maybe placed in 65 DEG C of insulations 10 minutes by the Loading Buffer adding 1/10 volume in pipe, stops endonuclease reaction.Digestion products carries out agarose gel electrophoresis analysis and cuts glue reclaiming object fragment, concentrated.Reaction system forms: 10 × H Buffer 4 μ L, DNA 10 μ L, xbai 2 μ L, xhoi 2 μ L, ddH 2system is supplied 40 μ L by O.
Glue is reclaimed linearizing target fragment to be connected with pET-28a (+) and pET-22b (+) carrier respectively, reaction system is composed as follows: plasmid vector 1 μ L, target fragment 4 μ L, Ligation Solution 5 μ L.Hybrid connections liquid, to be placed in 16 DEG C of incubators ligation 12 hours.
Recombinant plasmid transformed intestinal bacteria
100 μ L intestinal bacteria e. coliadd 10 μ L in BL21 (DE3) competent cell suspension and connect product, mix gently, in ice bath, leave standstill 30 minutes.Proceed in 42 DEG C of water-baths, thermal shock 90 seconds.Fast transfer, in ice bath, cools 2 minutes.Add the LB liquid nutrient medium of 700 μ L, 37 DEG C, 100 rpm shaking table incubations cultivate 1 hour.After cultivating, centrifugal 2 minutes of bacterium liquid 3000 rpm, abandons supernatant 600 μ L, is applied on the LB flat board containing 50 μ g/mL kantlex, is inverted for 37 DEG C and cultivates after the mixing of residue bacterium liquid.Obtain the recombination bacillus coli containing object Pullulanase gene e. colibL21 (DE3)/pET-28a (+)-PelB- puland e. colibL21 (DE3)/pET-22b (+)- pul.
The background that two reptation behavior substantially eliminates recombination bacillus coli expresses phenomenon, and the two reptation behavior of expression display of the freezing glycerine pipe of recombination bacillus coli can maintain the stability of expression system, and the stability that recombination bacillus coli self-induction is expressed is improved.
(3), the background expression of recombination bacillus coli
LB substratum: Tryptones 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH7.0, distilled water is prepared.Add penbritin (100 μ g/mL) or kantlex (50 μ g/mL) before using when needing, solid medium adds 15 g/L agar powders.
The recombination bacillus coli list colony inoculation that picking newly transforms in 3 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 DEG C, 200 rpm shaking culture spend the night.Getting 0.5 mL nutrient solution transfers in 50 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 DEG C, 200 rpm shaking culture are to OD 600be about 1.2.Nutrient solution proceeds in 20 DEG C and continues cultivation 12 hours.After cultivation terminates, by fermented liquid in 10000 rpm centrifugal 10 minutes, bacterial sediment ultrasonication, broken liquid centrifugal 20 minutes in 12000 rpm, got supernatant liquor for Pullulanase enzyme activity determination in born of the same parents.
Through Enzyme activity assay, e. colibL21 (DE3)/pET-20b (+)- pulborn of the same parents in component enzymes live be about 1.9 U/mL, and e. colibL21 (DE3)/pET28a (+)-PelB- puland e. colibL21 (DE3)/pET-22b (+)- pulborn of the same parents in component then do not measure enzyme and live, show due in expression vector pET-22b (+) and pET-28a (+) lacoperon and repressor gene lacIexistence, repressor can be incorporated into T7 upstream region of gene on escherichia coli chromosome simultaneously lacoperon and expression vector lacon operon, this pair of reptation behavior substantially eliminates background and expresses phenomenon.
Pullulanase measuring method
Get enzyme liquid that the 100 μ L acetate buffer solution of 100 mM, pH 5.0 suitably dilutes to be mixed in mutually in 50 DEG C of water-baths to be incubated 30 minutes with the isopyknic 10 g/L Propirams of 100 mM, pH 5.0 in acetate buffer solution that are dissolved in.Add DNS reagent 300 μ L to shake up, be placed in boiling water and boil 15 minutes, take out after cooling rapidly with flowing water, in 540 nm wavelength places, with 0.5 cm cuvette, the absorbance of assaying reaction liquid.
Enzyme unit definition alive: under above-mentioned condition of specifying, it is 1 Ge Meihuo unit (U) that per minute catalytic decomposition pulullan polysaccharide generates the enzyme needed for reducing sugar being equivalent to 1 μm of ol glucose.
(4), the expression stability of the freezing glycerine pipe of recombination bacillus coli compares
Three kinds of recombinant expression plasmids transform e.colibL21 (DE3) competent cell, is inoculated in 3 mL at once containing in corresponding antibiotic LB liquid nutrient medium after transformant grows, in 37 DEG C, 200 rpm shaking culture spend the night.Getting 0.5 mL nutrient solution transfers in 50 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 DEG C, 200 rpm shaking culture are to OD 600be about 1.2.Nutrient solution proceeds in 20 DEG C and continues cultivation 12 hours.After cultivation terminates, by fermented liquid in 10000 rpm centrifugal 10 minutes, supernatant kept sample for extracellular fraction.Bacterial sediment ultrasonication, broken liquid centrifugal 20 minutes in 12000 rpm, gets supernatant liquor for component in born of the same parents.Measure the IPTG abduction delivering result that exoenzyme slip-knot fruit in born of the same parents is first.Residue test tube seed liquor is for preserving freezing glycerine pipe, freezen protective in-70 DEG C.Take out afterwards frozen 2 days and 4 days respectively and be used for second, third batch of abduction delivering.The inside and outside total enzyme of born of the same parents measuring more each engineering bacteria is lived.
Relatively the enzyme of different fermentations batch is lived and be found that, along with the increase of number of days frozen in-70 DEG C, e. colibL21 (DE3)/pET-20b (+)- pulenzymatic productivity constantly decline.Bacterial classification in-70 DEG C frozen 4 days, in LB substratum after fermentation intracellular enzyme live drop to new transform that bacterial classification fermenting enzyme lives 38%, this project bacterium unstable properties under frozen state is described, and the ability to express of target protein can decline rapidly.And e. colibL21 (DE3)/pET-22b (+)- pulwith e. colibL21 (DE3)/pET-28a (+)-PelB- pulenzyme running water flat be then about 15 U/mL always, namely remain initial enzymatic productivity always, illustrate in carrier lactwo reptation behaviors that operon produces can maintain the stability of expression system, make this two strains engineering bacteria can keep producing enzyme performance normally under frozen state.
(5), the comparison of recombination bacillus coli self-induction expression
Self-induction substratum (g/L): beta lactose 1 ~ 50, dextrose anhydrous 0.5, glycerine 5, KH 2pO 46.8, MgSO 40.24, Tryptones 10, yeast extract 5, Na 2hPO 47.1, Na 2sO 40.71, NH 4cl 2.67, trace element solution 400 μ L/L, pH 7.5 ~ 8.0, distilled water is prepared.
Trace element solution (g/L): FeCl 38.125, CaCl 22.22, MnCl 22.52, ZnSO 41.61, CoCl 20.26, CuCl 20.27, NiCl 20.26, Na 2moO 40.41, Na 2seO 30.346, H 3bO 30.124, HCl 2.19, distilled water is prepared.
Three kinds of recombinant expression plasmids transform e.colibL21 competent cell, is inoculated in 3 mL at once containing in corresponding antibiotic LB liquid nutrient medium after transformant grows, in 37 DEG C, 200 rpm shaking culture spend the night.Get 2mL nutrient solution and be inoculated in 50mL containing in corresponding antibiotic self-induction substratum, 37 DEG C, cultivate under 200rpm to proceed in 20 DEG C after 2 hours and continue cultivation 70 hours.Measure exoenzyme running water in born of the same parents respectively to put down.
After fermentation ends, e. colibL21 (DE3)/pET-20b (+)- pulthe inside and outside total enzyme work of born of the same parents be 23 U/mL, and e. colibL21 (DE3)/pET-22b (+)- puland e. colibL21 (DE3)/pET-28a (+)-PelB- pultotal enzyme live and be 550 U/mL, show due in carrier lactwo reptation behaviors of operon eliminate the background expression of target protein, and the stability of expression system is improved, and makes bacterial classification also can keep the ability to express of target protein during the fermentation, can not degenerate.
(3) beneficial effect
Successful clone Nagano genus bacillus ( bacillus naganoensis) CCTCC NO:M 2012388 Pullulanase encoding gene, this full length gene 2781 bp, 926 amino-acid residues of encoding, the nucleotides sequence of its gene is classified as: SEQ ID NO:1, and its amino acid consists of SEQ ID NO:2.Utilize and do not contain lacoperon and repressor gene lacIcarrier pET-20b (+) construct recombination bacillus coli containing Pullulanase gene e. colibL21 (DE3)/pET-20b (+)- pul, utilization contains lacoperon and repressor gene lacIcarrier pET-28a (+) and pET-22b (+) construct recombination bacillus coli containing Pullulanase gene respectively e. colibL21 (DE3)/pET-28a (+)-PelB- pulwith e. colibL21 (DE3)/pET-22b (+)- pul.
Use and do not contain lacthe pET carrier system of operon and repressor gene e. colibL21 (DE3)/pET-20b (+)- pulwhen expressing toxicity report albumen, find that there is obvious background expresses the phenomenon with the expression system instability caused thereupon.The instability of expression system causes the problem that expression level declines and expression amount is on the low side in self-induction substratum of recombinant bacterium freezing glycerine pipe.Contain by using lacthe pET carrier system of operon and repressor gene e. colibL21 (DE3)/pET-22b (+)- pulwith e. colibL21 (DE3)/pET-28a (+)-PelB- pul, the background of toxicity report albumen is expressed and is substantially eliminated, and the protein expression ability of the freezing glycerine pipe of recombinant bacterium is stablized, and the expressing quantity in self-induction substratum significantly improves.Show lacrigorous two of operon check regulating strategy, enhance the stability of escherichia expression system.These work are that the system stability strengthening recombination bacillus coli provides available strategy, significant to high expression target protein.
This Nagano genus bacillus ( bacillus naganoensis) CCTCC M 2012388; Depositary institution: China typical culture collection center, writes a Chinese character in simplified form CCTCC, address: Wuhan, China Wuhan University, deposit number CCTCC NO:M 2012388, and preservation date is on September 28th, 2012.Used in former patent application, " a kind of method of gene recombined escherichia coli and High-efficient Production Pullulanase thereof ", application number 201210481749.3, November 24 2012 applying date.
Embodiment
Embodiment 1
LB substratum: Tryptones 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH7.0, distilled water is prepared.Add penbritin (100 μ g/mL) or kantlex (50 μ g/mL) before using when needing, solid medium adds 15 g/L agar powders.
Picking newly transforms e. colibL21 (DE3)/pET-20b (+)- pulsingle colony inoculation in 3 mL containing in the LB liquid nutrient medium of kantlex (50 μ g/mL), in 37 DEG C, 200 rpm shaking culture spend the night.Getting 0.5 mL nutrient solution transfers in 50 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 DEG C, 200 rpm shaking culture are to OD 600be about 1.2.Nutrient solution proceeds in 20 DEG C and continues cultivation 12 hours.After cultivation terminates, by fermented liquid in 10000 rpm centrifugal 10 minutes, bacterial sediment ultrasonication, broken liquid centrifugal 20 minutes in 12000 rpm, get supernatant liquor for component in born of the same parents, the work of Pullulanase enzyme was 1.9 U/mL.And e. colibL21 (DE3)/pET28a (+)-PelB- puland e. colibL21 (DE3)/pET-22b (+)- pulborn of the same parents in component then do not measure enzyme live.
Embodiment 2
LB substratum: Tryptones 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH7.0, distilled water is prepared.Add penbritin (100 μ g/mL) or kantlex (50 μ g/mL) before using when needing, solid medium adds 15 g/L agar powders.
Recombinant plasmid pET-20b (+)- pultransform e.colibL21 competent cell, is inoculated in 3 mL at once containing in corresponding antibiotic LB liquid nutrient medium after transformant grows, in 37 DEG C, 200 rpm shaking culture spend the night.Getting 0.5 mL nutrient solution transfers in 50 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 DEG C, 200 rpm shaking culture are to OD 600be about 1.2.Nutrient solution proceeds in 20 DEG C and continues cultivation 12 hours.After cultivation terminates, by fermented liquid in 10000 rpm centrifugal 10 minutes, supernatant kept sample for extracellular fraction.Bacterial sediment ultrasonication, broken liquid centrifugal 20 minutes in 12000 rpm, gets supernatant liquor for component in born of the same parents.The inside and outside total enzyme work of born of the same parents is 15 U/mL, as first IPTG abduction delivering result.Residue test tube seed liquor is for preserving freezing glycerine pipe, freezen protective in-70 DEG C.Take out afterwards frozen 2 days and 4 days respectively and be used for second, third batch of abduction delivering, enzyme slip-knot fruit is respectively 8.6 U/mL and 5.1 U/mL.And e. colibL21 (DE3)/pET-22b (+)- pulwith e. colibL21 (DE3)/pET-28a (+)-PelB- pulenzyme running water flat be then about 15 U/mL always.
Embodiment 3
Self-induction substratum (g/L): beta lactose 1 ~ 50, dextrose anhydrous 0.5, glycerine 5, KH 2pO 46.8, MgSO 40.24, Tryptones 10, yeast extract 5, Na 2hPO 47.1, Na 2sO 40.71, NH 4cl 2.67, trace element solution 400 μ L/L, pH 7.5 ~ 8.0, distilled water is prepared.
Trace element solution (g/L): FeCl 38.125, CaCl 22.22, MnCl 22.52, ZnSO 41.61, CoCl 20.26, CuCl 20.27, NiCl 20.26, Na 2moO 40.41, Na 2seO 30.346, H 3bO 30.124, HCl 2.19, distilled water is prepared.
Recombinant plasmid pET-22b (+)- pultransform e.colibL21 competent cell, is inoculated in 3 mL at once containing in corresponding antibiotic LB liquid nutrient medium after transformant grows, in 37 DEG C, 200 rpm shaking culture spend the night.Get 2mL nutrient solution and be inoculated in 50mL containing in corresponding antibiotic self-induction substratum, 37 DEG C, cultivate under 200rpm to proceed in 20 DEG C after 2 hours and continue cultivation 70 hours.Recording the inside and outside total enzyme work of born of the same parents is 23 U/mL.And e. colibL21 (DE3)/pET-22b (+)- puland e. colibL21 (DE3)/pET-28a (+)-PelB- pultotal enzyme live and be 550 U/mL.
<210> SEQ ID NO: 1
<211> 2781
<212> DNA
<213> Nagano genus bacillus ( bacillus naganoensis) CCTCC NO:M 2012388
 
<214>
gatgggaaca ccacaaacat cgtagtccat tattttcgtc ctagtgggga ttatacggat 60
tggaatcttt ggatgtggcc ggagaacggt gatggggctg agtatgattt taatcaaccg 120
actgattctt atggggaggt tgcaagtgtg gacattcctg gaaacccaag tcaagtaggg 180
attattgtcc gtaaaggaaa ttgggatgcg aaagacattg atagtgaccg ctacatcgat 240
ttaagcaaag ggcatgagat ttggctcgtc caaggaaaca gccagatttt ctatagtgaa 300
aaggatgctg aggcagccgc acaacctgct gtaagtaacg cttatttaga tgcttccaac 360
caagtgttgg tcaagcttag ccagccgttt actcttggtg aaggttcaag cggttttacg 420
gttcatgatg acacagcaaa taaggatatt ccagttacat ctgttagtga tgccaatcag 480
gtaacggctg ttttagcagg tactttccag catatttttg gggggagtga ttgggcaccg 540
gataatcaca atactttact aaaaaaggtg aatagcaatc tctatcaatt ttcaggaaat 600
cttcctgaag gaaactacca atataaagtg gctttaaatg atagctggaa taatccgagc 660
tacccatctg ataacattaa tttgacagtg ccagctggtg gtgcccatgt tacattttct 720
tatatcccat ccacccatgc tgtttatgac acgattaaca atcctaatgc ggatttacaa 780
gtagatagca gcggtgttaa gacggatctc gtggcggtta ctcttggaga aaatcctgat 840
gtaagccata ccctgtccat tcaaacagag gactatcagg caggacaggt catacctcgt 900
aaggtgcttg attcatccca gtactactat tccggagatg atctcgggaa tacctataca 960
aagaatgcaa ctacctttaa ggtctgggcg cctacatcca ctcaagtaaa tgtccttctt 1020
tataatagtg caaccggcgc ggtaactaaa acggttccaa tgaccgcatc aggccatggt 1080
gtatgggaag caacagtcaa ccaagacctt gaaaattggt attacatgta tgaggtaaca 1140
ggacaaggct caacccgaac ggctgttgat ccgtatgcaa cagctattgc accaaacgga 1200
acgagaggca tgattgtgga cctagccaaa acagacccgg ccggatggga gagtgacaaa 1260
catattacgc caaagaatat agaagatgaa gtcatctatg aaatggatgt tcgtgacttt 1320
tccatcgact ctaattcggg tatgaaaaat aaaggaaagt atttggcact tacagaaaaa 1380
ggaactaaag gccctgacaa tgtaaagaca ggggtagatt ccttaaaaca acttgggatt 1440
actcatgttc agcttcagcc tgttttcgca tttaatagtg tcaatgaaaa cgatccaact 1500
caatataatt ggggttatga ccctcgcaac tacaatgttc ctgagggaca atatgctact 1560
aatgcaaacg gaacaactcg gattaaagag tttaaggaaa tggttctttc actccatcag 1620
gaccacattg gggttaatat ggatgttgtt tataatcata cctttgccac gcaaatctct 1680
gacttcgata agattgtacc agaatattac taccgcacgg atgatgctgg taactacact 1740
aacggctcag gtactggaaa cgaaatcgca gccgaaagac caatggttca aaaatttatt 1800
atcgattcac ttaagttttg ggtcaatgag taccacgttg acggtttccg ttttgactta 1860
atggcgttgc ttggaaaaga tacaatgtct aaagctgcca cgcagcttca tgccattgat 1920
ccaggaattg ctctctacgg tgagccatgg acaggaggaa catccgcgct gccagccgat 1980
cagcttttaa caaaaggagc tcaaaaaggc atgggagtgg ctgtatttaa tgacaatctg 2040
cgaaacggtt tggacggcag tgtctttgat tcatctgctc aaggttttgc gacaggtgct 2100
actggtttaa cggatgctat taaaaatgga gttgaaggaa gtattaatga cttcaccgct 2160
tcaccaggcg agacgatcaa ctatgtcaca agtcatgata actataccct ttgggacaag 2220
attgcccaaa gcaatccaaa cgattctgaa gcggatcgaa ttaaaatgga tgagctcgct 2280
caagcgatcg tcatgacctc acaaggcatt cctttcatgc agggcgggga agaaatgctt 2340
cgtacgaaag gcggcaacga caatagctat aatgctggtg atgtagtgaa cgagtttgat 2400
tggagcagaa aagctcaata tccagatgtt ttcaattatt atagcgggct gattcatctt 2460
cgtcttgatc acccagcctt ccgcatgacg acagctaatg aaatcaatag ccacctccaa 2520
ttcctaaata gcccagagaa cacagtggcc tatgaattat ctgatcatgc aaataaagat 2580
acatggggta atattgtggt tatttataat ccaaataaaa cggcagaaac cattaatttg 2640
ccaagcggga aatgggaaat caatgcgacg agcggtaagg tgggagaatc cacacttggt 2700
caagcagagg gcagtgttca agttccaggc atatctatga tgattcttca tcaagaagta 2760
agcccatctg atggtaaata g 2781
 
<210> SEQ ID NO: 2
<211> 926
<212> PRT
<213> Nagano genus bacillus ( bacillus naganoensis) CCTCC NO:M 2012388
 
<400>1
Asp Gly Asn Thr Thr Asn Ile Val Val His Tyr Phe Arg Pro Ser
1 5 10 15
Gly Asp Tyr Thr Asp Trp Asn Leu Trp Met Trp Pro Glu Asn Gly
20 25 30
Asp Gly Ala Glu Tyr Asp Phe Asn Gln Pro Thr Asp Ser Tyr Gly
35 40 45
Glu Val Ala Ser Val Asp Ile Pro Gly Asn Pro Ser Gln Val Gly
50 55 60
Ile Ile Val Arg Lys Gly Asn Trp Asp Ala Lys Asp Ile Asp Ser
65 70 75
Asp Arg Tyr Ile Asp Leu Ser Lys Gly His Glu Ile Trp Leu Val
80 85 90
Gln Gly Asn Ser Gln Ile Phe Tyr Ser Glu Lys Asp Ala Glu Ala
95 100 105
Ala Ala Gln Pro Ala Val Ser Asn Ala Tyr Leu Asp Ala Ser Asn
110 115 120
Gln Val Leu Val Lys Leu Ser Gln Pro Phe Thr Leu Gly Glu Gly
125 130 135
Ser Ser Gly Phe Thr Val His Asp Asp Thr Ala Asn Lys Asp Ile
140 145 150
Pro Val Thr Ser Val Ser Asp Ala Asn Gln Val Thr Ala Val Leu
155 160 165
Ala Gly Thr Phe Gln His Ile Phe Gly Gly Ser Asp Trp Ala Pro
170 175 180
Asp Asn His Asn Thr Leu Leu Lys Lys Val Asn Ser Asn Leu Tyr
185 190 195
Gln Phe Ser Gly Asn Leu Pro Glu Gly Asn Tyr Gln Tyr Lys Val
200 205 210
Ala Leu Asn Asp Ser Trp Asn Asn Pro Ser Tyr Pro Ser Asp Asn
215 220 225
Ile Asn Leu Thr Val Pro Ala Gly Gly Ala His Val Thr Phe Ser
230 235 240
Tyr Ile Pro Ser Thr His Ala Val Tyr Asp Thr Ile Asn Asn Pro
245 250 255
Asn Ala Asp Leu Gln Val Asp Ser Ser Gly Val Lys Thr Asp Leu
260 265 270
Val Ala Val Thr Leu Gly Glu Asn Pro Asp Val Ser His Thr Leu
275 280 285
Ser Ile Gln Thr Glu Asp Tyr Gln Ala Gly Gln Val Ile Pro Arg
290 295 300
Lys Val Leu Asp Ser Ser Gln Tyr Tyr Tyr Ser Gly Asp Asp Leu
305 310 315
Gly Asn Thr Tyr Thr Lys Asn Ala Thr Thr Phe Lys Val Trp Ala
320 325 330
Pro Thr Ser Thr Gln Val Asn Val Leu Leu Tyr Asn Ser Ala Thr
335 340 345
Gly Ala Val Thr Lys Thr Val Pro Met Thr Ala Ser Gly His Gly
350 355 360
Val Trp Glu Ala Thr Val Asn Gln Asp Leu Glu Asn Trp Tyr Tyr
365 370 375
Met Tyr Glu Val Thr Gly Gln Gly Ser Thr Arg Thr Ala Val Asp
380 385 390
Pro Tyr Ala Thr Ala Ile Ala Pro Asn Gly Thr Arg Gly Met Ile
395 400 405
Val Asp Leu Ala Lys Thr Asp Pro Ala Gly Trp Glu Ser Asp Lys
410 415 420
His Ile Thr Pro Lys Asn Ile Glu Asp Glu Val Ile Tyr Glu Met
425 430 435
Asp Val Arg Asp Phe Ser Ile Asp Ser Asn Ser Gly Met Lys Asn
440 445 450
Lys Gly Lys Tyr Leu Ala Leu Thr Glu Lys Gly Thr Lys Gly Pro
455 460 465
Asp Asn Val Lys Thr Gly Val Asp Ser Leu Lys Gln Leu Gly Ile
470 475 480
Thr His Val Gln Leu Gln Pro Val Phe Ala Phe Asn Ser Val Asn
485 490 495
Glu Asn Asp Pro Thr Gln Tyr Asn Trp Gly Tyr Asp Pro Arg Asn
500 505 510
Tyr Asn Val Pro Glu Gly Gln Tyr Ala Thr Asn Ala Asn Gly Thr
515 520 525
Thr Arg Ile Lys Glu Phe Lys Glu Met Val Leu Ser Leu His Gln
530 535 540
Asp His Ile Gly Val Asn Met Asp Val Val Tyr Asn His Thr Phe
545 550 555
Ala Thr Gln Ile Ser Asp Phe Asp Lys Ile Val Pro Glu Tyr Tyr
560 565 570
Tyr Arg Thr Asp Asp Ala Gly Asn Tyr Thr Asn Gly Ser Gly Thr
575 580 585
Gly Asn Glu Ile Ala Ala Glu Arg Pro Met Val Gln Lys Phe Ile
590 595 600
Ile Asp Ser Leu Lys Phe Trp Val Asn Glu Tyr His Val Asp Gly
605 610 615
Phe Arg Phe Asp Leu Met Ala Leu Leu Gly Lys Asp Thr Met Ser
620 625 630
Lys Ala Ala Thr Gln Leu His Ala Ile Asp Pro Gly Ile Ala Leu
635 640 645
Tyr Gly Glu Pro Trp Thr Gly Gly Thr Ser Ala Leu Pro Ala Asp
650 655 660
Gln Leu Leu Thr Lys Gly Ala Gln Lys Gly Met Gly Val Ala Val
665 670 675
Phe Asn Asp Asn Leu Arg Asn Gly Leu Asp Gly Ser Val Phe Asp
680 685 690
Ser Ser Ala Gln Gly Phe Ala Thr Gly Ala Thr Gly Leu Thr Asp
695 700 705
Ala Ile Lys Asn Gly Val Glu Gly Ser Ile Asn Asp Phe Thr Ala
710 715 720
Ser Pro Gly Glu Thr Ile Asn Tyr Val Thr Ser His Asp Asn Tyr
725 730 735
Thr Leu Trp Asp Lys Ile Ala Gln Ser Asn Pro Asn Asp Ser Glu
740 745 750
Ala Asp Arg Ile Lys Met Asp Glu Leu Ala Gln Ala Ile Val Met
755 760 765
Thr Ser Gln Gly Ile Pro Phe Met Gln Gly Gly Glu Glu Met Leu
770 775 780
Arg Thr Lys Gly Gly Asn Asp Asn Ser Tyr Asn Ala Gly Asp Val
785 790 795
Val Asn Glu Phe Asp Trp Ser Arg Lys Ala Gln Tyr Pro Asp Val
800 805 810
Phe Asn Tyr Tyr Ser Gly Leu Ile His Leu Arg Leu Asp His Pro
815 820 825
Ala Phe Arg Met Thr Thr Ala Asn Glu Ile Asn Ser His Leu Gln
830 835 840
Phe Leu Asn Ser Pro Glu Asn Thr Val Ala Tyr Glu Leu Ser Asp
845 850 855
His Ala Asn Lys Asp Thr Trp Gly Asn Ile Val Val Ile Tyr Asn
860 865 870
Pro Asn Lys Thr Ala Glu Thr Ile Asn Leu Pro Ser Gly Lys Trp
875 880 885
Glu Ile Asn Ala Thr Ser Gly Lys Val Gly Glu Ser Thr Leu Gly
890 895 900
Gln Ala Glu Gly Ser Val Gln Val Pro Gly Ile Ser Met Met Ile
905 910 915
Leu His Gln Glu Val Ser Pro Ser Asp Gly Lys ***
920 925 926

Claims (1)

1. check based on two the method that strategy strengthens intestinal bacteria T7 expression system stability, it is characterized in that: with derive from Nagano genus bacillus ( bacillus naganoensis) Pullulanase of CCTCC M 2012388 is expressing protein, use contains lacoperon and repressor gene lacIpET expression vector pET-22b (+) and pET-28a (+), utilize lacrigorous two of operon check regulating strategy, construct recombinant bacterium e.colibL21/pET-22b (+)- puland e.colibL21/pET-28a (+)-PelB- pul, in contrast, construct and do not contain lacoperon and repressor gene lacIexpression system e.colibL21 (DE3)/pET-20b (+)- pul; Step is:
(1) acquisition of Pullulanase gene
Nagano genus bacillus ( bacillus naganoensis) CCTCC M 2012388 substratum: CaCl 20.25 g/L, MgSO 47H 2o 0.5 g/L, (NH 4) 2sO 40.2 g/L, yeast extract 2 g/L, dextrose anhydrous 5 g/L, KH 2pO 43 g/L, inorganic salt solution 1 mL/L, pH 5.0, distilled water is prepared;
Inorganic salt solution: ZnSO 47H 2o 0.1 g/L, MnCl 2h 2o 0.03 g/L, H 3bO 30.3 g/L, CoCl 26H 2o 0.2 g/L, CuCl 22H 2o 0.01 g/L, NiCl 26H 2o 0.02 g/L, Na 2moO 42H 2o 0.03 g/L, distilled water is prepared;
By Nagano genus bacillus ( bacillus naganoensis) CCTCC M 2012388 strain inoculation in containing 25 mL substratum 250 mL shaking flasks in, in 37 DEG C, 200 rpm shaking culture 72 hours, after cultivation terminates, thalline is centrifugal and with brine twice, collecting cell utilizes the genome DNA extracting reagent kit Genomic DNA Extraction Miniprep System of VIOGENE company to extract genome;
Primer 1:5 '-gaa ca g GAT CCa gat ggg aac acc aca aaC-3 ',
Primer 2: 5 '-att cc c tcg agt tta cca tca gat ggg ct-3 ';
Primer 1 contains bamHi restriction enzyme site, primer 2 contains xhoi restriction enzyme site;
PCR reaction system: ddH 2the dNTP 0.5 μ L of O 37 μ L, 10 × Reaction Buffer 5 μ L, 25 mmol/L, the primer 11 μ L of 50 pmol/ μ L, the Taq DNA polymerase 0.5 μ L of the primer 21 μ L of 50 pmol/ μ L, genomic dna 5 μ L, 5 U/ μ L;
With Nagano genus bacillus CCTCC M 2012388 genome for template, adopt PCR method amplification Pullulanase gene, PCR reaction process: 95 DEG C of denaturation 5 min; 95 DEG C of 1 min, 60 DEG C of 0.5 min, 72 DEG C of 2 min, carry out 30 circulations; 72 DEG C extend 10 min;
Utilize the 3S Spin Agarose Gel DNA Purification Kit purify DNA segment of Shanghai Shenergy Biocolor BioScience & Technology Company;
Sodium acetate solution and 2 times of volume dehydrated alcohols of pH 5.2,3 mol/L of 1/10 volume are added in DNA solution,-20 DEG C precipitate 1 hour, 12000 rpm are in 4 DEG C of centrifugal 30 min, add 75% ethanol 500 μ L to wash, 12000 rpm are in 4 DEG C of centrifugal 30 min, be dissolved in appropriate TE damping fluid after aseptic operating platform dries up, use immediately or-20 DEG C of preservations;
(2) structure of the recombination bacillus coli containing Pullulanase gene
A, pET-20b (+)- pulstructure
The plasmid extraction kit Mini-Plasmid Rapid Isolation Kit of the vast Tyke biological gene Technology Co., Ltd. in Beijing is utilized to extract plasmid pET-20b (+);
Be added in Eppendorf pipe according to the order of water, damping fluid, PCR primer or plasmid DNA, enzyme, build pipe lid, vibration makes liquid fully mix, be placed in and make liquid concentrate at the bottom of pipe centrifugal 2 seconds in whizzer, 37 DEG C of water-baths 3 hours, pipe is maybe placed in 65 DEG C of insulations 10 minutes by the Loading Buffer adding 1/10 volume in pipe, stops endonuclease reaction, digestion products carries out agarose gel electrophoresis analysis and cuts glue reclaiming object fragment, concentrated;
Reaction system forms: 10 × H Buffer 4 μ L, DNA 10 μ L, bamHi 2 μ L, xhoi 2 μ L, ddH 2system is supplied 40 μ L by O;
The connection of goal gene and plasmid pET-20b (+)
Be connected with plasmid pET-20b (+) by Pullulanase gene, reaction system is composed as follows: plasmid pET-20b (+) 0.8 μ L, Pullulanase gene 4.2 μ L, Ligation Solution 5 μ L; Hybrid connections liquid, to be placed in 16 DEG C of incubators ligation 12 hours;
Recombinant plasmid transformed intestinal bacteria
100 μ L intestinal bacteria e. coliadd 10 μ L in BL21 (DE3) competent cell suspension and connect product, mix gently, 30 minutes are left standstill in ice bath, proceed in 42 DEG C of water-baths, thermal shock 90 seconds, fast transfer is in ice bath, cool 2 minutes, add the LB liquid nutrient medium of 700 μ L, 37 DEG C, 100 rpm shaking table incubations cultivate 1 hour, centrifugal 2 minutes of bacterium liquid 3000 rpm after cultivating, abandon supernatant 600 μ L, be applied to containing on the antibiotic LB flat board of 50 μ g/mL ammonia benzyl after the mixing of residue bacterium liquid, be inverted for 37 DEG C and cultivate, obtain the recombination bacillus coli containing object Pullulanase gene e. colibL21 (DE3)/pET-20b (+)- pul;
B, pET-22b (+)- pulwith pET-28a (+)-PelB- pulstructure
By recombinant plasmid pET-20b (+)- pulrestriction enzyme is used respectively with expression vector pET-28a (+), pET-22b (+) xbai and xhoi substep single endonuclease digestion, be added in Eppendorf pipe according to the order of water, damping fluid, plasmid, enzyme, build pipe lid, vibration makes liquid fully mix, and is placed in and makes liquid concentrate at the bottom of pipe in whizzer centrifugal 2 seconds, 37 DEG C of water-baths 3 hours, pipe is maybe placed in 65 DEG C of insulations 10 minutes by the Loading Buffer adding 1/10 volume in pipe, stop endonuclease reaction, digestion products carries out agarose gel electrophoresis analysis and cuts glue reclaiming object fragment, concentrated; Reaction system forms: 10 × H Buffer 4 μ L, DNA 10 μ L, xbai 2 μ L, xhoi 2 μ L, ddH 2system is supplied 40 μ L by O;
Glue is reclaimed linearizing target fragment to be connected with pET-28a (+) and pET-22b (+) carrier respectively, reaction system is composed as follows: plasmid vector 1 μ L, target fragment 4 μ L, Ligation Solution 5 μ L, Hybrid connections liquid, to be placed in 16 DEG C of incubators ligation 12 hours;
Recombinant plasmid transformed intestinal bacteria
100 μ L intestinal bacteria e. coliadd 10 μ L in BL21 (DE3) competent cell suspension and connect product, mix gently, leave standstill 30 minutes in ice bath, proceed in 42 DEG C of water-baths, thermal shock 90 seconds: fast transfer, in ice bath, cools 2 minutes; Add the LB liquid nutrient medium of 700 μ L, 37 DEG C, 100 rpm shaking table incubations cultivate 1 hour, centrifugal 2 minutes of bacterium liquid 3000 rpm after cultivating, abandon supernatant 600 μ L, be applied on the LB flat board containing 50 μ g/mL kantlex after the mixing of residue bacterium liquid, be inverted for 37 DEG C and cultivate, obtain the recombination bacillus coli containing object Pullulanase gene e. colibL21 (DE3)/pET-28a (+)-PelB- puland e. colibL21 (DE3)/pET-22b (+)- pul;
The stability that two reptation behavior makes recombination bacillus coli self-induction express is improved.
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