CN103555751A - Method for enhancing stability of escherichia coli T7 expression system on basis of double-repression strategy - Google Patents

Abstract

The invention relates to a method for enhancing the stability of an escherichia coli T7 expression system on the basis of a double-repression strategy, and belongs to the field of regulation and control of escherichia coli expression. The method disclosed by the invention can be used for constructing the recombinant escherichia coli which expresses pullulanase by utilizing a gene engineering technology and taking the pullulanase of a bacteria source as an expression protein and constructing the recombinant bacteria E.coliBL21/pET-22b(+)-pul and E.coliBL21/pET-28a(+)-PelB-pul by using PET expression vectors pET-22b(+) and pET-28a(+) which contain lac operons and repressor genes lacI, basically eliminates the background expression of a toxic foreign protein by utilizing the precise double-repression regulation and control strategy of the lac operons, enhances the expression stability of a frozen glycerol strain and the expression quantity of target proteins with lactose self-induced and enhances the stability of the escherichia coli T7 expression system. According to the invention, the common problem of system stability of the escherichia coli T7 expression system is solved, and the method for enhancing the stability of the escherichia coli T7 expression system is developed.

Description

A kind of based on two methods that check strategy enhancing intestinal bacteria T7 expression system stability

Technical field

Based on two methods that check strategy enhancing 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.In the multiple system that can be used for heterologous protein expression, intestinal bacteria system is the most frequently used expressive host all the time.Compare with other expressive hosts, intestinal bacteria have numerous advantages, as clear in genetic background, growth rapidly, can in cheap substratum, reach high-density and have the ability of overexpression target protein.The target protein amount of the T7 system expression based on T7 RNA polymerase can, up to 50% of total protein of cell amount, therefore show the advantage with other intestinal bacteria systems.

The speed of the synthetic mRNA of T7 RNA polymerase is 5 times of intestinal bacteria self RNA polymerase, and its high vigor has been given the ability of the synthetic recombinant protein that T7 system is powerful.But the high vigor of T7 RNA polymerase has been proved to be expression system has been existed to some negative effects, and main manifestations is: although the encoding gene of T7 RNA polymerase is 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 because T7 RNA polymerase vigor is too high, even without inductor, exist, and transcribing also of target gene can be initiated, and produces background and expresses.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.

More existing researchs are devoted to reduce the background expression of intestinal bacteria T7 expression system.A kind of method that reduces background expression is to use to contain and the pLysS of pET carrier compatibility or the Host Strains of pLysE plasmid.The T7 N,O-Diacetylmuramidase of these two kinds of vector expressions, is the natural inhibitor of T7 RNA polymerase, can be combined and suppress the latter's activity with T7 RNA polymerase.Yet 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 and is existed, and still may cause the unsettled problem of system.And T7 N,O-Diacetylmuramidase is a kind of bifunctional albumen, it has destruction to Bacillus coli cells wall, and the strain growth that therefore contains pLysS or pLysE plasmid is slower.After induction, T7 N,O-Diacetylmuramidase will weaken expression, make target protein expression amount very low in addition.In substratum, add 0.5-1% glucose, the metabolism reptation behavior of generation also can reduce the background of T7 RNA polymerase expresses.

Above method is all directly for the background of T7 RNA polymerase, express and design, and target is all that the background that reduces T7 RNA polymerase is expressed.The present invention develops a kind of background that does not directly weaken T7 RNA polymerase and expresses, but can stop T7 RNA polymerase to cause the method for transcription of foreign genes on expression plasmid.In the T7 of pET carrier promotor downstream, insert lacoperon, forms T7- lacpromotor, can effectively reduce the background of target protein and express.T7- lacpromotor can provide one lacthe binding site of repressor, while there is not inductor in system, lacrepressor can be incorporated into T7-tightly lacpromotor lacin operon sequence, stop and by background, to express the T7 RNA polymerase producing and pass through, disturbed the extension of mRNA chain, thereby reduce the background of foreign gene under induction state not, transcribe.If there is q.s in system lacrepressor is all to occupy in cell lacoperon site, in inducing cell, the background of target protein is not expressed and will almost fully be eliminated, and expression system also will obtain stable.

Summary of the invention

(1) technical problem that will solve

The object of this invention is to provide the method that strengthens intestinal bacteria T7 expression system stability.It is expressing protein that the Pullulanase of bacterial origin is take in the present invention, has used and has contained lacoperon and repressor gene lacIpET expression vector, utilize lactwo regulating strategies that check that operon is rigorous, basically eliminate the background of toxicity report albumen express, improved escherichia expression system stability, thereby developed the method that strengthens intestinal bacteria T7 expression system stability.

(2) technical scheme

Based on two, check the methods that strategy strengthens intestinal bacteria T7 expression system stability, first built expression Nagano bacillus ( bacillus naganoensis) recombination bacillus coli of CCTCC M 2012388 Pullulanases 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 to express, and freezing glycerine pipe expression level in frozen process of recombinant bacterium constantly declines, show that expression system exists unsettled phenomenon.Therefore use and contain lacoperon and repressor gene lacIcarrier pET-22b (+) and pET-28a (+), utilize lacrigorous two of operon check regulating strategy, have built recombinant bacterium e.colibL21/ pET-22b (+)- pulwith e.colibL21/pET-28a (+)-PelB- pul.Compared 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 preparation.

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 preparation.

By Nagano genus bacillus ( bacillus naganoensis) CCTCC M 2012388 bacterial classifications are inoculated in the 250 mL shaking flasks that contain 25 mL substratum, in 37 ℃, 200 rpm shaking culture 72 hours.After cultivation finishes, thalline is centrifugal and use physiological saline washed twice, collecting cell to utilize 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 genomes are template, adopt PCR method amplification Pullulanase gene.PCR reaction process: 95 ℃ of denaturation 5 min; 95 ℃ of 1 min, 60 ℃ of 0.5 min, 72 ℃ of 2 min, carry out 30 circulations; 72 ℃ are extended 10 min.

Utilize 3S Spin Agarose Gel DNA Purification Kit(Shanghai Shenergy Biocolor BioScience & Technology Company) purify DNA segment.

The sodium acetate solution (3 mol/L, pH 5.2) and the 2 times of volume dehydrated alcohols that in DNA solution, add 1/10 volume ,-20 ℃ precipitate 1 hour.12000 rpm are in 4 ℃ of centrifugal 30 min.Add 75% ethanol 500 μ L washings, 12000 rpm, in 4 ℃ of centrifugal 30 min, are dissolved in after aseptic operating platform dries up in appropriate TE damping fluid, use immediately or-20 ℃ of preservations.

(2) structure of the recombination bacillus coli that contains Pullulanase gene

A, pET-20b (+)- pulstructure

Utilize vast Tyke, plasmid extraction kit Mini-Plasmid Rapid Isolation Kit(Beijing biological gene technology company limited) extraction plasmid pET20b (+).

According to the order of water, damping fluid, PCR product or plasmid DNA, enzyme, be added in Eppendorf pipe, build pipe lid, vibration fully mixes liquid, be placed in whizzer and centrifugal 2 seconds liquid concentrated on to manage at the end, 37 ℃ of water-baths 3 hours, in pipe, add the Loading Buffer of 1/10 volume maybe pipe to be placed in to 65 ℃ of insulations 10 minutes, stop endonuclease reaction.Enzyme is cut product and is carried out agarose gel electrophoresis analysis and cut glue and reclaim object fragment, concentrated.

Reaction composition: 10 * H Buffer, 4 μ L, DNA 10 μ L, bamHi 2 μ L, xhoi 2 μ L, ddH ₂o supplies 40 μ L by system.

Goal gene is connected with plasmid pET20b's (+)

Pullulanase gene is connected with plasmid pET20b (+), and reaction composition is as follows: plasmid pET-20b (+) 0.8 μ L, Pullulanase gene 4.2 μ L, Ligation Solution 5 μ L.Mix connecting fluid, be placed in 16 ℃ of incubators ligation 12 hours.

Recombinant plasmid transformed intestinal bacteria

100 μ L intestinal bacteria e. coliin BL21 (DE3) competent cell suspension, add 10 μ L to connect products, mix gently, in ice bath standing 30 minutes.Proceed in 42 ℃ of water-baths thermal shock 90 seconds.Fast transfer to ice bath, cooling 2 minutes.The LB liquid nutrient medium that adds 700 μ L, 37 ℃, 100 rpm shaking table incubations are cultivated 1 hour.After cultivating, bacterium liquid 3000 rpm centrifugal 2 minutes, abandon supernatant 600 μ L, after residue bacterium liquid mixes, are applied to and contain on the antibiotic LB flat board of 50 μ g/mL ammonia benzyl, are inverted for 37 ℃ and cultivate.The recombination bacillus coli that acquisition contains object Pullulanase gene e. colibL21 (DE3)/pET-20b (+)- pul.

B, pET-22b (+)- puland pET-28a (+)-PelB- pulstructure

By recombinant plasmid pET-20b (+)- puluse respectively restriction enzyme with expression vector pET-28a (+), pET-22b (+) xbai and xhoi substep single endonuclease digestion.According to the order of water, damping fluid, plasmid, enzyme, be added in Eppendorf pipe, build pipe lid, vibration fully mixes liquid, be placed in and liquid concentrated in centrifugal 2 seconds in whizzer to manage at the end, 37 ℃ of water-baths 3 hours, in pipe, add the Loading Buffer of 1/10 volume maybe pipe to be placed in to 65 ℃ of insulations 10 minutes, stop endonuclease reaction.Enzyme is cut product and is carried out agarose gel electrophoresis analysis and cut glue and reclaim object fragment, concentrated.Reaction composition: 10 * H Buffer, 4 μ L, DNA 10 μ L, xbai 2 μ L, xhoi 2 μ L, ddH ₂o supplies 40 μ L by system.

Glue is reclaimed to linearizing target fragment and be connected with pET-28a (+) and pET-22b (+) carrier respectively, reaction composition is as follows: plasmid vector 1 μ L, target fragment 4 μ L, Ligation Solution 5 μ L.Mix connecting fluid, be placed in 16 ℃ of incubators ligation 12 hours.

Recombinant plasmid transformed intestinal bacteria

100 μ L intestinal bacteria e. coliin BL21 (DE3) competent cell suspension, add 10 μ L to connect products, mix gently, in ice bath standing 30 minutes.Proceed in 42 ℃ of water-baths thermal shock 90 seconds.Fast transfer to ice bath, cooling 2 minutes.The LB liquid nutrient medium that adds 700 μ L, 37 ℃, 100 rpm shaking table incubations are cultivated 1 hour.After cultivating, bacterium liquid 3000 rpm centrifugal 2 minutes, abandon supernatant 600 μ L, after residue bacterium liquid mixes, are applied on the LB flat board that contains 50 μ g/mL kantlex, are inverted for 37 ℃ and cultivate.The recombination bacillus coli that acquisition contains object Pullulanase gene e. colibL21 (DE3)/pET-28a (+)-PelB- puland e. colibL21 (DE3)/pET-22b (+)- pul.

Two reptation behaviors have substantially been eliminated the background of recombination bacillus coli and have been expressed phenomenon, and the expression of the freezing glycerine pipe of recombination bacillus coli shows that two reptation behaviors 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 preparation.Before using while needing, add penbritin (100 μ g/mL) or kantlex (50 μ g/mL), solid medium adds 15 g/L agar powders.

The new recombination bacillus coli list colony inoculation transforming of picking, spends the night in 37 ℃, 200 rpm shaking culture containing in corresponding antibiotic LB liquid nutrient medium in 3 mL.Get 0.5 mL nutrient solution and transfer in 50 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 ℃, 200 rpm shaking culture to OD ₆₀₀be about 1.2.Nutrient solution proceeds in 20 ℃ and continues to cultivate 12 hours.After cultivation finishes, by fermented liquid in 10000 rpm centrifugal 10 minutes, bacterial sediment ultrasonication, broken liquid centrifugal 20 minutes in 12000 rpm, gets supernatant liquor for Pullulanase enzyme activity determination in born of the same parents.

Through enzyme biopsy, survey, e. colibL21 (DE3)/pET-20b (+)- pulborn of the same parents in component enzymes work 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 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 eliminated background and expressed phenomenon.

Pullulanase measuring method

The enzyme liquid of getting the suitable dilution of acetate buffer solution of 100 100 mM for μ L, pH 5.0 is mixed in mutually in 50 ℃ of water-baths and is incubated 30 minutes with isopyknic 10 g/L Propiram that are dissolved in 100 mM, pH 5.0 acetate buffer solutions.Add DNS reagent 300 μ L to shake up, be placed in boiling water and boil 15 minutes, take out cooling rapidly with flowing water after, in 540 nm wavelength places, with 0.5 cm cuvette, the absorbance of assaying reaction liquid.

Enzyme unit definition alive: under the condition of above-mentioned appointment, the required enzyme of reducing sugar that the generation of per minute catalytic decomposition pulullan polysaccharide is equivalent to 1 μ mol glucose is 1 Ge Meihuo unit (U).

(4), the expression stability comparison of the freezing glycerine pipe of recombination bacillus coli

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 ℃, 200 rpm shaking culture, spend the night.Get 0.5 mL nutrient solution and transfer in 50 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 ℃, 200 rpm shaking culture to OD ₆₀₀be about 1.2.Nutrient solution proceeds in 20 ℃ and continues to cultivate 12 hours.After cultivation finishes, 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.Measuring exoenzyme slip-knot in born of the same parents is really first IPTG abduction delivering result.Residue test tube seed liquor is used for preserving freezing glycerine pipe, freezing preservation in-70 ℃.At frozen 2 days and 4 days, take out for second, third batch of abduction delivering afterwards respectively.Measuring the relatively inside and outside total enzyme of born of the same parents of each engineering bacteria lives.

Relatively the enzyme work of different fermentations batch found that, along with the increase of frozen number of days in-70 ℃, e. colibL21 (DE3)/pET-20b (+)- pulenzymatic productivity constantly decline.Bacterial classification in-70 ℃ frozen 4 days, in LB substratum, after fermentation, intracellular enzyme work drops to that new conversion bacterial classification fermenting enzyme lives 38%, and this project bacterium unstable properties under frozen state be described, and the ability to express of target protein is decline rapidly.And e. colibL21 (DE3)/pET-22b (+)- pulwith e. colibL21 (DE3)/pET-28a (+)-PelB- pulenzyme running water flat be about 15 U/mL always, keeping 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 under frozen state, can keep producing normally enzyme performance.

(5) comparison that, recombination bacillus coli self-induction is expressed

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, and pH 7.5 ~ 8.0, distilled water preparation.

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 preparation.

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 ℃, 200 rpm shaking culture, spend the night.Get 2mL nutrient solution and be inoculated in 50mL containing in corresponding antibiotic self-induction substratum, under 37 ℃, 200rpm, cultivate after 2 hours and proceed in 20 ℃ and continue to cultivate 70 hours.Measuring respectively exoenzyme running water in born of the same parents puts 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 work be 550 U/mL, show due in carrier lactwo reptation behaviors of operon have been eliminated the background of target protein and have been expressed, and the stability of expression system is improved, and makes bacterial classification also can keep during the fermentation the ability to express of target protein, can not degenerate.

(3) beneficial effect

Successfully cloned Nagano genus bacillus ( bacillus naganoensis) CCTCC NO:M 2012388 Pullulanase encoding genes, 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, its amino acid consists of SEQ ID NO:2.Utilize and do not contain lacoperon and repressor gene lacIcarrier pET-20b (+) built the recombination bacillus coli that contains Pullulanase gene e. colibL21 (DE3)/pET-20b (+)- pul, utilization contains lacoperon and repressor gene lacIcarrier pET-28a (+) and pET-22b (+) built respectively the recombination bacillus coli that contains Pullulanase gene 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 (+)- pulwhile expressing toxicity report albumen, find the unsettled phenomenon of expression system that exists obvious background to express and cause thereupon.The unstable of expression system caused that the expression level of the freezing glycerine pipe of recombinant bacterium declines and expression amount problem on the low side in self-induction substratum.By use, contain 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, have strengthened the stability of escherichia expression system.These work provide available strategy for strengthening the system stability of recombination bacillus coli, significant to high efficient expression target protein.

This Nagano genus bacillus ( bacillus naganoensis) CCTCC M 2012388; Depositary institution: Chinese Typical Representative culture collection center, write a Chinese character in simplified form CCTCC, address: Wuhan, China Wuhan University, deposit number CCTCC NO:M 2012388, preservation date is on September 28th, 2012.In former patent application, used " 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 preparation.Before using while needing, add penbritin (100 μ g/mL) or kantlex (50 μ g/mL), solid medium adds 15 g/L agar powders.

The new conversion of picking e. colibL21 (DE3)/pET-20b (+)- pulsingle colony inoculation, spends the night in 37 ℃, 200 rpm shaking culture containing in the LB liquid nutrient medium of kantlex (50 μ g/mL) in 3 mL.Get 0.5 mL nutrient solution and transfer in 50 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 ℃, 200 rpm shaking culture to OD ₆₀₀be about 1.2.Nutrient solution proceeds in 20 ℃ and continues to cultivate 12 hours.After cultivation finishes, by fermented liquid in 10000 rpm centrifugal 10 minutes, bacterial sediment ultrasonication, broken liquid centrifugal 20 minutes in 12000 rpm, gets supernatant liquor for components in born of the same parents, and the work of Pullulanase enzyme is 1.9 U/mL.And e. colibL21 (DE3)/pET28a (+)-PelB- puland e. colibL21 (DE3)/pET-22b (+)- pulborn of the same parents in component do not measure enzyme and live.

Embodiment 2

LB substratum: Tryptones 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH7.0, distilled water preparation.Before using while needing, add penbritin (100 μ g/mL) or kantlex (50 μ g/mL), 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 ℃, 200 rpm shaking culture, spend the night.Get 0.5 mL nutrient solution and transfer in 50 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 ℃, 200 rpm shaking culture to OD ₆₀₀be about 1.2.Nutrient solution proceeds in 20 ℃ and continues to cultivate 12 hours.After cultivation finishes, 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 used for preserving freezing glycerine pipe, freezing preservation in-70 ℃.At frozen 2 days and 4 days, take out for second, third batch of abduction delivering afterwards respectively, enzyme slip-knot is really respectively 8.6 U/mL and 5.1 U/mL.And e. colibL21 (DE3)/pET-22b (+)- pulwith e. colibL21 (DE3)/pET-28a (+)-PelB- pulflat 15 U/mL that are always about of enzyme running water.

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, and pH 7.5 ~ 8.0, distilled water preparation.

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 preparation.

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 ℃, 200 rpm shaking culture, spend the night.Get 2mL nutrient solution and be inoculated in 50mL containing in corresponding antibiotic self-induction substratum, under 37 ℃, 200rpm, cultivate after 2 hours and proceed in 20 ℃ and continue to cultivate 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 work 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 (4)

1. based on two, check the methods 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, have built recombinant bacterium e.colibL21/pET-22b (+)- puland e.colibL21/pET-28a (+)-PelB- pul, in contrast, do not built and contained 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 preparation;

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 preparation;

By Nagano genus bacillus ( bacillus naganoensis) CCTCC M 2012388 bacterial classifications are inoculated in the 250 mL shaking flasks that contain 25 mL substratum, in 37 ℃, 200 rpm shaking culture 72 hours, after cultivation finishes, thalline is centrifugal and use physiological saline washed twice, collecting cell to utilize 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 ₂o 37 μ L, 10 * Reaction Buffer, 5 μ L, the dNTP 0.5 μ L of 25 mmol/L, the primer 11 μ L of 50 pmol/ μ L, the primer 21 μ L of 50 pmol/ μ L, genomic dna 5 μ L, the Taq DNA polymerase 0.5 μ L of 5 U/ μ L;

Nagano genus bacillus CCTCC M 2012388 genomes of take are template, adopt PCR method amplification Pullulanase gene, PCR reaction process: 95 ℃ of denaturation 5 min; 95 ℃ of 1 min, 60 ℃ of 0.5 min, 72 ℃ of 2 min, carry out 30 circulations; 72 ℃ are extended 10 min;

Utilize the 3S Spin Agarose Gel DNA Purification Kit purify DNA segment of Shanghai Shenergy Biocolor BioScience & Technology Company;

The sodium acetate solution and the 2 times of volume dehydrated alcohols that in DNA solution, add pH 5.2,3 mol/L of 1/10 volume,-20 ℃ precipitate 1 hour, 12000 rpm are in 4 ℃ of centrifugal 30 min, add 75% ethanol 500 μ L washings, 12000 rpm are in 4 ℃ of centrifugal 30 min, after aseptic operating platform dries up, be dissolved in appropriate TE damping fluid, use immediately or-20 ℃ of preservations;

(2) structure of the recombination bacillus coli that contains Pullulanase gene

A, pET-20b (+)- pulstructure

Utilize the plasmid extraction kit Mini-Plasmid Rapid Isolation Kit of vast Tyke, Beijing biological gene technology company limited to extract plasmid pET-20b (+);

According to the order of water, damping fluid, PCR product or plasmid DNA, enzyme, be added in Eppendorf pipe, build pipe lid, vibration fully mixes liquid, be placed in whizzer and centrifugal 2 seconds liquid concentrated on to manage at the end, 37 ℃ of water-baths 3 hours add the Loading Buffer of 1/10 volume maybe pipe to be placed in to 65 ℃ of insulations 10 minutes in pipe, stop endonuclease reaction, enzyme is cut product and is carried out agarose gel electrophoresis analysis and cut glue and reclaim object fragment, concentrated;

Reaction composition: 10 * H Buffer, 4 μ L, DNA 10 μ L, bamHi 2 μ L, xhoi 2 μ L, ddH ₂o supplies 40 μ L by system;

Goal gene is connected with plasmid pET-20b's (+)

Pullulanase gene is connected with plasmid pET-20b (+), and reaction composition is as follows: plasmid pET-20b (+) 0.8 μ L, Pullulanase gene 4.2 μ L, Ligation Solution 5 μ L; Mix connecting fluid, be placed in 16 ℃ of incubators ligation 12 hours;

Recombinant plasmid transformed intestinal bacteria

100 μ L intestinal bacteria e. coliin BL21 (DE3) competent cell suspension, add 10 μ L to connect product, mix gently, in ice bath standing 30 minutes, proceed in 42 ℃ of water-baths, thermal shock 90 seconds, fast transfer is to ice bath, cooling 2 minutes, the LB liquid nutrient medium that adds 700 μ L, 37 ℃, 100 rpm shaking table incubations are cultivated 1 hour, and after cultivating, bacterium liquid 3000 rpm centrifugal 2 minutes, abandon supernatant 600 μ L, after residue bacterium liquid mixes, be applied to and contain on the antibiotic LB flat board of 50 μ g/mL ammonia benzyl, be inverted for 37 ℃ and cultivate, obtain the recombination bacillus coli that contains object Pullulanase gene e. colibL21 (DE3)/pET-20b (+)- pul;

B, pET-22b (+)- puland pET-28a (+)-PelB- pulstructure

By recombinant plasmid pET-20b (+)- puluse respectively restriction enzyme with expression vector pET-28a (+), pET-22b (+) xbai and xhoi substep single endonuclease digestion, according to the order of water, damping fluid, plasmid, enzyme, be added in Eppendorf pipe, build pipe lid, vibration fully mixes liquid, is placed in liquid to be concentrated in centrifugal 2 seconds in whizzer to manage at the end 37 ℃ of water-baths 3 hours, in pipe, add the Loading Buffer of 1/10 volume maybe pipe to be placed in to 65 ℃ of insulations 10 minutes, stop endonuclease reaction, enzyme is cut product and is carried out agarose gel electrophoresis analysis and cut glue and reclaim object fragment, concentrated; Reaction composition: 10 * H Buffer, 4 μ L, DNA 10 μ L, xbai 2 μ L, xhoi 2 μ L, ddH ₂o supplies 40 μ L by system;

Glue is reclaimed to linearizing target fragment to be connected with pET-28a (+) and pET-22b (+) carrier respectively, reaction composition is as follows: plasmid vector 1 μ L, target fragment 4 μ L, Ligation Solution 5 μ L, mix connecting fluid, be placed in 16 ℃ of incubators ligation 12 hours;

Recombinant plasmid transformed intestinal bacteria

100 μ L intestinal bacteria e. coliin BL21 (DE3) competent cell suspension, add 10 μ L to connect products, mix gently, in ice bath standing 30 minutes, proceed in 42 ℃ of water-baths thermal shock 90 seconds: fast transfer to ice bath, cooling 2 minutes; The LB liquid nutrient medium that adds 700 μ L, 37 ℃, 100 rpm shaking table incubations are cultivated 1 hour, after cultivating, bacterium liquid 3000 rpm centrifugal 2 minutes, abandon supernatant 600 μ L, after mixing, residue bacterium liquid is applied on the LB flat board that contains 50 μ g/mL kantlex, be inverted for 37 ℃ and cultivate, obtain the recombination bacillus coli that contains object Pullulanase gene e. colibL21 (DE3)/pET-28a (+)-PelB- puland e. colibL21 (DE3)/pET-22b (+)- pul;

Two reptation behaviors have substantially been eliminated the background of recombination bacillus coli and have been expressed phenomenon, and the expression of the freezing glycerine pipe of recombination bacillus coli shows that two reptation behaviors can maintain the stability of expression system, and the stability that recombination bacillus coli self-induction is expressed is improved.

2. according to claim 1 based on two methods that check strategy enhancing intestinal bacteria T7 expression system stability, it is characterized in that: the background expression of recombination bacillus coli;

The new recombination bacillus coli list colony inoculation transforming of picking, spends the night in 37 ℃, 200 rpm shaking culture containing in corresponding antibiotic LB liquid nutrient medium in 3 mL; Get 0.5 mL nutrient solution and transfer in 50 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 ℃, 200 rpm shaking culture to OD ₆₀₀be 1.2; Nutrient solution proceeds in 20 ℃ and to continue to cultivate 12 hours, and after cultivating and finishing, by fermented liquid in 10000 rpm centrifugal 10 minutes, bacterial sediment ultrasonication, broken liquid centrifugal 20 minutes in 12000 rpm, gets supernatant liquor for Pullulanase enzyme activity determination in born of the same parents;

Through enzyme biopsy, survey, e. colibL21 (DE3)/pET-20b (+)- pulborn of the same parents in component enzymes work be 1.9 U/mL, and e. colibL21 (DE3)/pET-28a (+)-PelB- puland e. colibL21 (DE3)/pET-22b (+)- pulborn of the same parents in component 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 eliminated background and expressed phenomenon.

3. according to claim 1 based on two methods that check strategy enhancing intestinal bacteria T7 expression system stability, it is characterized in that: the expression stability comparison of the freezing glycerine pipe of recombination bacillus coli;

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 ℃, 200 rpm shaking culture, spend the night; Get 0.5 mL nutrient solution and transfer in 50 mL containing in corresponding antibiotic LB liquid nutrient medium, in 37 ℃, 200 rpm shaking culture to OD ₆₀₀be about 1.2; Nutrient solution proceeds in 20 ℃ and continues to cultivate 12 hours; After cultivation finishes, 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; Measuring exoenzyme slip-knot in born of the same parents is really first IPTG abduction delivering result, residue test tube seed liquor is used for preserving freezing glycerine pipe, freezing preservation in-70 ℃, took out for second, third batch of abduction delivering afterwards at frozen 2 days and 4 days respectively, measured the relatively inside and outside total enzyme of born of the same parents of each engineering bacteria and lived;

Relatively the enzyme work of different fermentations batch found that, along with the increase of frozen number of days in-70 ℃, e. colibL21 (DE3)/pET-20b (+)- pulenzymatic productivity constantly decline, bacterial classification in-70 ℃ frozen 4 days, in LB substratum, after fermentation, intracellular enzyme work drops to that new conversion bacterial classification fermenting enzyme lives 38%, and this project bacterium unstable properties under frozen state be described, and the ability to express of target protein is decline rapidly; And e. colibL21 (DE3)/pET-22b (+)- puland e. colibL21 (DE3)/pET-28a (+)-PelB- pulenzyme running water flat be 15 U/mL always, keeping 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 under frozen state, can keep producing normally enzyme performance.

4. according to claim 1 based on two methods that check strategy enhancing intestinal bacteria T7 expression system stability, it is characterized in that: the comparison that recombination bacillus coli self-induction is expressed;

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, and pH 7.5 ~ 8.0, distilled water preparation;

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 preparation;

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 ℃, 200 rpm shaking culture, spend the night; Get 2mL nutrient solution and be inoculated in 50mL containing in corresponding antibiotic self-induction substratum, under 37 ℃, 200rpm, cultivate after 2 hours and proceed in 20 ℃ and continue to cultivate 70 hours, measure respectively in born of the same parents exoenzyme running water flat;

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 work be 550 U/mL, show due in carrier lactwo reptation behaviors of operon have been eliminated the background of target protein and have been expressed, and the stability of expression system is improved, and makes bacterial classification also can keep during the fermentation the ability to express of target protein, can not degenerate.