CN103275913A

CN103275913A - Recombinant bacterium with autolysis function as well as preparation method and application thereof

Info

Publication number: CN103275913A
Application number: CN2013101985294A
Authority: CN
Inventors: 周志刚; 杨雅麟; 张美超; 徐俐; 何夙旭; 李青; 余强
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Priority date: 2013-05-24
Filing date: 2013-05-24
Publication date: 2013-09-04
Also published as: WO2014187151A1

Abstract

The invention discloses a recombinant bacterium with an autolysis function as well as a preparation method and an application thereof. The recombinant bacterium is prepared by using the following steps of: introducing an E7 cleavage protein encoding gene and a target encoding gene into a starting bacterium to obtain an engineering bacterium, wherein the E7 cleavage protein is the protein shown in the sequence 2 in a sequence table, and/or the target protein is quenching enzyme which specifically can be the protein shown in the sequence 5 in the sequence table. The recombinant bacterium obtained by using the method has the autolysis function, the wall breaking procedure on the recombinant bacterium in a preparation process of the target protein can be eliminated, and the production cost of the target protein can be reduced.

Description

Reorganization bacterium of a kind of tool self-dissolving function and preparation method thereof and application

Technical field

The present invention relates to a kind of reorganization bacterium and preparation method thereof and application of tool self-dissolving function.

Background technology

Along with pathogenic bacteria resistance multi-drug resistant serious day by day particularly, the discovery of antimicrobial drug effect novel targets and the research and development of novel antibacterial medicine seem particularly important.The Disease-causing gene of numerous pathogenic bacterias is subjected to the regulation and control of quorum sensing (QS), comprises the genes involved of virulence factor, adhesion factor and mediation pathogenic bacteria opposing host immune and medicine.The key of the relevant target gene expression of regulation and control is the concentration of signaling molecule in the QS system in the QS system, as the concentration of effective reduction signaling molecule, just can regulate and control the pathogenic of some pathogenic bacterium.And the cancellation enzyme with degraded signaling molecule function is exactly a kind of important potential source biomolecule means of prevention wherein.

The E7 crack protein is to produce a key component in the SOS responding system in the Colicine cell, and its function is that the output bacteriocin arrives extracellular space under pressure environment.Nearest research shows that the E7 crack protein causes interior membrane damage, may be relevant with the adventitia phospholipase A that activates for film modified outward.

Because many cancellation enzyme sources are in bacterium, therefore most bacterial expression vectors that adopt are studied when carrying out genetically engineered research, and wherein escherichia expression system research is the clearest.Target protein secreting, expressing amount is low, the interior expression of born of the same parents needs broken wall operation increase cost and limit its industrialized this present situation and present colibacillus engineering exists.

Summary of the invention

The preparation method who the purpose of this invention is to provide a kind of bacterium of recombinating, the reorganization bacterium that utilizes this method to obtain has the self-dissolving function, can reduce the broken wall operation of reorganization bacterium, reduces the production cost of target protein.

The preparation method of reorganization bacterium provided by the present invention comprises: the encoding gene of E7 crack protein and the encoding gene of target protein are imported in the bacterium that sets out, obtain the reorganization bacterium.

In aforesaid method, described E7 crack protein can be albumen shown in the sequence table sequence 2; And/or described target protein can be the cancellation enzyme;

Described cancellation enzyme specifically can be the albumen shown in the sequence table sequence 5.

In aforesaid method, the encoding gene of described E7 crack protein can be gene shown in the 52nd to the 195th of sequence table sequence 1;

And/or described target protein encoding gene is gene shown in the 9th to the 818th of sequence table sequence 4.

In aforesaid method, described E7 crack protein encoding gene and target protein encoding gene import in the described bacterium that sets out by recombinant vectors C and obtain engineering bacteria;

Described recombinant vectors C prepares according to the method that comprises the steps:

Insert described E7 crack protein encoding gene expression cassette in plasmid pET-28a (+) T7 promotor upstream and obtain recombinant vectors B; Multiple clone site place in the described T7 promotor downstream of described recombinant vectors B inserts described target protein encoding gene, obtains described recombinant vectors C; The promotor of described E7 crack protein encoding gene expression cassette specifically can be the T7 promotor.

Described recombinant vectors C further specifically can prepare according to the method that comprises the steps:

Dna fragmentation shown in the 17th of insertion sequence table sequence 1 to the 195th Nucleotide obtains recombinant vectors A between the Xba of plasmid pET-28a (+) I and Xho I restriction enzyme site, dna fragmentation shown in the 16th of insertion sequence table sequence 3 to the 398th Nucleotide obtains recombinant vectors B between the Sph of described recombinant vectors A I and Bgl II restriction enzyme site, and the dna fragmentation shown in the 9th of insertion sequence table sequence 4 to the 818th Nucleotide obtains described recombinant vectors C between the EcoR of described recombinant vectors B I and Not I restriction enzyme site.

In aforesaid method, the described bacterium that sets out can be intestinal bacteria (Escherichia coli), specifically can be intestinal bacteria (Escherichia coli) BL21 (DE3).

The reorganization bacterium that the present invention protects above-mentioned arbitrary described method to prepare.

The application of reorganization bacterium in the described target protein of preparation that the present invention protects above-mentioned arbitrary described method to prepare.

The present invention also provides a kind of method for preparing target protein, and the reorganization bacterium usefulness fermention medium shaking culture that comprises the steps: above-mentioned arbitrary described method is prepared is to OD ₆₀₀=1.0, add then IPTG to final concentration be 0-1mmol/L(as 0,0.25,0.5 or 1mmol/L), be to continue concussion under the condition of 1.3cm to cultivate 1-24 hours (as 1,2,4,8,12,24 hour) in 30 ℃, 180rpm, rotation radius, obtain described target protein;

When the bacterium that sets out of described reorganization bacterium was intestinal bacteria, the solvent of described fermention medium was water, and solute and final concentration thereof are respectively peptone 12g/L, yeast extract 24g/L, glycerine 4ml/L, KH ₂PO ₄0.231g/L, K ₂HPO ₄1.254g/L and glucose 5g/L.

Described recombinant vectors B also belongs to protection scope of the present invention, and described recombinant vectors B can be used as and makes up the carrier that sets out that target protein is expressed, so that prepare described reorganization bacterium and produce described target protein.

Experimental results show that, recombinant plasmid pET28a (+)/e7t7a thermal shock transformed into escherichia coli (Escherichia coli) BL21 (DE3) that will contain E7 crack protein encoding gene and cancellation enzyme AiiO-AIO6 encoding gene, the reorganization bacterium ETA that obtains, be the IPTG inducing culture of 0.25mmol/L after 8 hours through final concentration, the work of cancellation enzyme enzyme can reach 885.09U/mL in the extracellular protein solution; The present invention has saved needs to collect thalline after intracellular protein is expressed, and a series of processes such as fragmentation have been simplified post-processed greatly, have reduced the loss of enzyme liquid in the treating processes.The present invention has very wide application prospect for the preparation of industrialization target protein provides a method that cost is low, easy and simple to handle.

Description of drawings

Fig. 1 is the structural representation that plasmid pET-28a (+) is transformed part.Last figure is the structural representation of recombinant plasmid pET28a (+)/e7t7, and figure below is the structural representation of recombinant plasmid pET28a (+)/e7t7a, wherein, and P _T7Represent the T7 promotor, E7lysis represents E7 crack protein encoding gene, and MCS represents multiple clone site, and AIO6 represents the encoding gene of cancellation enzyme AiiO-AIO6.

Fig. 2 is the SDS-PAGE figure of different reorganization bacterium protein solution inside and outside acquisition born of the same parents under the different induction times.Wherein, band shown in the arrow is cancellation enzyme AiiO-AIO6, and M is the molecular weight standard of albumen.

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Intestinal bacteria (Escherichia coli) BL21 (DE3) used among the following embodiment are available from Invitrogen company.Plasmid pET-28a (+) is available from Jin Weike (China) life science technique center, and products catalogue is numbered Biovector-182763.

The LB liquid nutrient medium: get peptone 10g, yeast extract 5g and NaCl10g, with after the 0.9L water dissolution, 121 ℃ of autoclaving 20min are cooled to and add 0.1L after 50 ℃ through the D/W of the 50g/L of 108 ℃ of autoclaving 30min.

The TB liquid nutrient medium: get peptone 12g, yeast extract 24g and glycerine 4ml, with after the 0.8L water dissolution, 121 ℃ of autoclaving 20min are cooled to add after 60 ℃ 0.1L through autoclaving or with the KH of the membrane filtration degerming of 0.22um ₂PO ₄And K ₂HPO ₄Concentration be respectively the aqueous solution of 2.31g/L and 12.54g/L and 0.1L through the D/W of the 50g/L of 108 ℃ of autoclaving 30min.

Agrobacterium tumefaciens KYC55(Agrobacterium tumefaciens Strain KYC55): document: Cho, K., C.Fuqua, and S.C.Winans.Transcriptional regulation and locations of Agrobacterium tumefaciens genes required for complete catabolism of octopine.J.Bacteriol.1997, the 179:1-8. public can obtain from Institute of Feeds,China Academy of Agriculture Sciences.

Embodiment 1, contain the structure of the double T 7 promoter expression cassettes recombinant vectorss of E7 crack protein

One, the acquisition of E7 crack protein and encoding gene thereof

1, the following three pairs of primers of design:

XE7SF1:5 '-ACAATTCCCC TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAA-3 ' (the underscore base is Xba I restriction endonuclease recognition sequence);

XE7SR1：5′-AAGAAGCAATAAAATAATCCCTGTTATTTTTTTCATGGTATATCTCCTTCTTAAAG-3′；

XE7SF2：5′-AACAGGGATTATTTTATTGCTTCTTGCAGCCATTATTCTTGCTGCATGTCAGGCAA-3′；

XE7SR2：5′-ACTGTCCCGCCCTGAACATCACGGATATAGTTTGCCTGACATGCAGCAAGAATAAT-3′；

XE7SF3：5′-TCCGTGATGTTCAGGGCGGGACAGTATCACCGTCGTCAACTGCTGAACTGACCGGA-3′；

XE7XR3:5 '-GTGGTGGTG CTCGAGTTACTGCGTTTCCACTCCGGTCAGTTCAGCAGTTGACGACG-3 ' (the underscore base is Xho I restriction endonuclease recognition sequence).

2, carry out pcr amplification as template certainly with the primer XE7SF1 in the step 1 and XE7SR1, obtain amplified production E71; Certainly carry out pcr amplification as template with the primer XE7SF2 in the step 1 and XE7SR2, obtain amplified production E72; Certainly carry out pcr amplification as template with the primer XE7SF3 in the step 1 and XE7SR3, obtain amplified production E73; The reaction conditions of described pcr amplification is 95 ℃ of 5min, 94 ℃ of 30sec, 58 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min.

3, E71 and the E72 that obtains with step 2 is template, is that primer carries out pcr amplification with XE7SF1 and XE7SR2; The PCR reaction conditions is 95 ℃ of 5min, 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min; Obtain amplified production E712;

The E73 that obtains with E712 and step 2 is template, is that primer carries out pcr amplification with XE7SF1 and XE7XR3; The PCR reaction conditions is 95 ℃ of 5min, 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min; Obtain amplified production E7123.

4, from the E7123 that step 3 obtains, reclaim the dna fragmentation of purifying 210bp, pEASY-T3 is connected with carrier, the recombinant plasmid that obtains is checked order, the result shows that this recombinant plasmid is the DNA shown in the 1st to the 210th Nucleotide that has inserted sequence table sequence 1 in the TA of carrier pEASY-T3 cloning site place.

E7 crack protein shown in the 52nd to the 195th the nucleotide coding sequence table sequence 2 of sequence table sequence 1, this albumen is made up of 47 amino-acid residues.

Two, the acquisition of T7 promotor encoding gene and multiple clone site

Be template with plasmid pET-28a (+), with primer RCE7T7P:5 '-ATGGTACAT GCATGCATGTCGATCCCGCGAAATTAATACGACTC-3 ' (the underscore base is Sph I restriction endonuclease recognition sequence) and E7T7T:5 '-AGTTCGA AGATCTTCAGCAAAAAACCCCTCAAGACCCGTTTA-3 ' (the underscore base is Bgl II restriction endonuclease recognition sequence) carries out pcr amplification; The PCR reaction conditions is 95 ℃ of 5min, 94 ℃ of 30sec, 63 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations, 72 ℃ of 10min; The pcr amplification product that obtains is carried out agarose gel electrophoresis, reclaim the dna fragmentation of purifying 411bp, pEASY-T3 is connected with carrier, the recombinant plasmid that obtains is checked order, the result shows that this recombinant plasmid is the DNA shown in the 1st to the 411st Nucleotide that has inserted sequence table sequence 3 at the TA of carrier pEASY-T3 cloning site place.

The 34th to the 50th coding T7 promotor of sequence table sequence 3, the 217th to the 262nd is multiple clone site.

Three, the structure that contains the double T 7 expression cassette recombinant vectorss of E7 crack protein

1, with the dna fragmentation of 4 210bp that obtain in restriction enzyme Xba I and the Xho I double digestion step 1, reclaims enzyme and cut product.

2, with restriction enzyme Xba I and Xho I double digestion plasmid pET-28a (+), reclaim carrier framework (about 5.4kb).

3, the enzyme of step 1 being cut product is connected with the carrier framework of step 2, obtain recombinant plasmid pET28a (+)/e7, confirm that through order-checking recombinant plasmid pET28a (+)/e7 is the dna fragmentation shown in the 17th to the 195th Nucleotide that has inserted sequence table sequence 1 between the Xba of carrier pET-28a (+) I and Xho I restriction enzyme site.

4, the dna fragmentation of the 411bp that obtains with restriction enzyme Sph I and Bgl II double digestion step 2 reclaims enzyme and cuts product.

5, recombinant vectors pET28a (+)/e7 that obtains with restriction enzyme Sph I and Bgl II double digestion step 3 reclaims carrier framework (about 5.6kb).

6, the enzyme of step 4 being cut product is connected with the carrier framework of step 5, obtain recombinant plasmid pET28a (+)/e7t7, confirm through order-checking, recombinant plasmid pET28a (+)/e7t7 is the dna fragmentation shown in the 16th to the 398th Nucleotide that has inserted sequence table sequence 3 between the Sph of carrier pET28a (+)/e7 I and Bgl II restriction enzyme site, this recombinant plasmid pET28a (+)/e7t7 is the double T 7 expression cassette recombinant vectorss that contain the E7 crack protein, and its structural representation is shown in the last figure among Fig. 1.

The structure of embodiment 2, cancellation enzyme recombinant expression vector

One, the acquisition of cancellation enzyme (AiiO-AIO6) encoding gene

Be template with pale bacillus (Ochrobactrum sp.) M231 genomic dna, design primer A6-F (5 '-CG GAATTCAAATCCCATGAAATCGAGACCAGTC-3 ') (line part be EcoR I restriction enzyme site) and A6-R2 (5 '-AA GCGGCCGCTCAGGCCGTGCAGTCG-3 ') (the line part is Not I restriction enzyme site) carries out pcr amplification; The PCR reaction conditions is 95 ℃ of 5min, 94 ℃ of 30sec, 59 ℃ of 30sec, 72 ℃ of 1min, 30 circulations, 72 ℃ of 10min; To obtain pcr amplification product and carry out agarose gel electrophoresis, reclaim the dna fragmentation of purifying 825bp, pEASY-T3 is connected with carrier, the recombinant plasmid that obtains is checked order, the result shows, this recombinant plasmid is to have inserted the dna fragmentation shown in the 1st to the 825th Nucleotide of sequence table sequence 4 at the TA of carrier pEASY-T3 cloning site place.Cancellation enzyme AiiO-AIO6 shown in the 9th to the 815th the nucleotide coding sequence table sequence 5 of sequence table sequence 4.

Two, the structure of cancellation enzyme recombinant expression vector

1, reclaims the dna fragmentation of the 825bp of purifying with restriction enzyme EcoR I and Not I double digestion step 1, reclaim enzyme and cut product.

2, carrier pET28a (+)/e7t7 that obtains with restriction enzyme EcoR I and Not I double digestion embodiment 1 step 3 reclaims carrier framework (about 6.0kb).

3, the enzyme of step 1 being cut product is connected with the carrier framework of step 2, obtain recombinant plasmid pET28a (+)/e7t7a, confirm that through order-checking recombinant plasmid pET28a (+)/e7t7a is the dna fragmentation shown in the 9th to the 815th Nucleotide that has inserted sequence table sequence 4 between the EcoR of carrier pET28a (+)/e7t7 I and Not I restriction enzyme site.The structural representation of recombinant plasmid pET28a (+)/e7t7a is shown in the figure below among Fig. 1.

The preparation of embodiment 3, reorganization bacterium

With recombinant plasmid pET28a (+)/e7t7a thermal shock transformed into escherichia coli (Escherichia coli) BL21 (DE3) that embodiment 2 obtains, obtain containing the reorganization bacterium (called after ETA) of recombinant plasmid pET28a (+)/e7t7a.

With recombinant plasmid pET28a (+)/e7t7 thermal shock transformed into escherichia coli (Escherichia coli) BL21 (DE3) that embodiment 1 obtains, obtain containing the contrast bacterium (called after ET) of recombinant plasmid pET28a (+)/e7t7.

With plasmid pET-28a (+) thermal shock transformed into escherichia coli (Escherichia coli) BL21 (DE3), obtain containing the reorganization bacterium (called after 28a) of plasmid pET-28a (+).

With plasmid pET-28a (+)/AiiO-AI06 thermal shock transformed into escherichia coli (Escherichia coli) BL21 (DE3), obtain containing the reorganization bacterium (called after A6) of plasmid pET-28a (+)/AiiO-AI06;

The construction process of described plasmid pET-28a (+)/AiiO-AI06 is as follows:

2, with restriction enzyme EcoR I and Not I double digestion embodiment 1 carrier pET28a (+), reclaim carrier framework.

3, the enzyme of step 1 being cut product is connected with the carrier framework of step 2, obtain recombinant plasmid pET-28a (+)/AiiO-AI06, confirm that through order-checking recombinant plasmid pET28a (+)/AiiO-AI06 is the dna fragmentation shown in the 9th to the 815th Nucleotide that has inserted sequence table sequence 4 between the EcoR of carrier pET-28a (+) I and Not I restriction enzyme site.

Embodiment 4, utilization reorganization bacterium produce target protein-cancellation enzyme (Aii0-AIO6)

One, the growth curve of different reorganization bacterium under different inductive conditions measured

1, four kinds of reorganization bacterium (ETA, ET, 28a, A6) that embodiment 3 is obtained are inoculated in respectively in the 3mL LB liquid nutrient medium (kantlex that contains 50 μ g/mL), and 37 ℃ of shaking culture are spent the night, and obtain incubated overnight liquid.

2, get the incubated overnight liquid 400 μ L that step 1 obtains and be seeded in the 20mL TB liquid nutrient medium (containing 50 μ g/mL kantlex), 37 ℃ of shaking culture 3-3.5h make OD ₆₀₀Reach 1.0, add final concentration then and be 0,0.25,0.5 or the IPTG(inductor of 1.0mmol/L, induce the expression of target protein Aii0-AIO6), after (30 ℃, 180rpm, rotation radius 1.3cm) 0,1,2,4,8,12,24h are cultivated in concussion, get nutrient solution respectively and measure OD ₆₀₀Value,

The IPTG of table 1, different concns induces the OD of different reorganization bacterium nutrient solution behind different induction times ₆₀₀Value

The result of table 1 shows: compare with the reorganization bacterium A6 that expresses in the born of the same parents, the cell density of secreting, expressing reorganization bacterium ETA presents and falls the trend that afterwards rises earlier, because while secreting, expressing E7 crack protein and target protein in the secreting, expressing reorganization bacterium, make that the E7 crack protein is expressed back cracking host when interpolation IPTG induces, discharge the effect that target protein reaches secreting, expressing.Can see in table 1 simultaneously in the IPTG abduction delivering that adds different concns that the cell density of ETA changes little on the whole.But cell density is higher than the IPTG that adds other concentration with the time when ETA interpolation IPTG final concentration 0.5mmol/L induces 4 hours.

Two, the reorganization bacterium produces the situation of intracellular protein and extracellular protein under different inductive conditions

1, the reorganization bacterium ETA that embodiment 3 is obtained is inoculated in the 3mL LB liquid nutrient medium (kantlex that contains 50 μ g/mL), and 37 ℃ of shaking culture are spent the night, and obtain incubated overnight liquid.

2, get the incubated overnight liquid 400 μ L that step 1 obtains and be seeded in the 20mL TB liquid nutrient medium (containing 50 μ g/mL kantlex), 37 ℃ of shaking culture 3-3.5h make OD ₆₀₀Reach 1.0, adding final concentration then is the IPTG(inductor of 0.5mmol/L, induce the expression of target protein Aii0-AIO6), after (30 ℃, 180rpm, rotation radius 1.3cm) 0,1,2,4,8,12,24h are cultivated in concussion, get nutrient solution 1.6mL respectively, the centrifugal radius 6cm of the centrifugal 5min(of 12,000rpm), collect supernatant liquor and cell precipitation respectively.

3, the cell precipitation that step 2 is obtained is resuspended with 0.1mol/L PBS damping fluid (pH8.0), makes OD ₆₀₀Be 1.0, use The albumen extraction agent (available from Novagen, the products catalogue numbering: 70584-3), and according to operation instruction extracting cell internal secretion expressed proteins, the centrifugal radius 6cm of the centrifugal 10min(of 12,000rpm), collect the solution that supernatant liquor obtains intracellular protein.

4, getting supernatant liquor 1mL that step 2 obtains adds two volumes acetone and is placed on-20 ℃ of precipitation 30min, the centrifugal radius 6cm of the centrifugal 10min(of 12,000rpm), collecting precipitation (being extracellular protein), use 0.1mol/L PBS damping fluid (pH8.0) resuspended then respectively, make its OD ₆₀₀Be 1.0, obtain the solution of extracellular protein.

5, according to the method for step 1-4, obtain the solution of intracellular protein of reorganization bacterium ET and A6 and the solution of extracellular protein respectively.

6, the solution of albumen and the solution of extracellular protein carry out SDS-PAGE in the mycetocyte of respectively recombinating that step 3-5 is obtained, and the result as shown in Figure 2.Wherein, the size of target protein cancellation enzyme (Aii0-AIO6) is 29.47kD.

The result of Fig. 2 shows: compare with expression in the born of the same parents, the outer expression amount of born of the same parents of secreting, expressing reorganization bacterium ETA is far longer than in the born of the same parents, contrast the inside and outside expression of ETA cell simultaneously and can see that the expression amount secreting, expressing amount of ETA contrast intracellular protein has reached 50% of total expression, and induce that expression amount changes little between 1 hour-24 hours.Though with expression amount that abduction delivering in the A6 born of the same parents was compared ETA in 8 hours have only its about 20%, secreting, expressing has saved microorganism collection, processes such as fragmentation.

The result of step 2 proves that E7 crack protein and target protein are expressed simultaneously, target protein can be discharged in the induced liquid under the effect of E7 crack protein.

Three, the cancellation enzyme enzyme activity determination of the extracellular protein that under different induction times, obtains of reorganization bacterium ETA

The extracellular protein solution of the 4 reorganization bacterium ETA that obtain in the step 2 as solution to be measured, is carried out the cancellation enzymic activity respectively and detects, and with the extracellular protein solution of the reorganization bacterium ET that obtains according to 1-4 method in the step 2 in contrast, the result is as shown in table 2.

Table 2, reorganization bacterium extracellular protein solution cancellation enzyme enzyme (U/mL) alive behind 0.5mmol/L IPTG inducing culture different time

Table 2 is the result show, when the IPTG that adds final concentration 0.5mmol/L induced, in the different time of inducing, enzyme was lived and changed not quite.Just can reach the peak value of secretion, and reduce the time of inducing greatly in namely 1 hour.

The method that above-mentioned cancellation enzymic activity detects is as follows:

1, with a kind of substrate of oxo-C8-HSL(N-acyl homoserine lactones enzyme, it is a kind of gram negative bacterium colony induction signaling molecule acyl homoserine lactones, can make indicator agrobacterium tumefaciens KYC55 fall to being shown as blueness) be dissolved in the dehydrated alcohol, making its final concentration is 10mmol/L, obtains oxo-C8-HSL solution; The ATmm culture medium flat plate is cut into the adhesive tape of 4mm * 60mm size with scalpel.

2, typical curve preparation

In 0.1mol/L PBS damping fluid (pH8.0), add the 10mmol/L oxo-C8-HSL solution that 10 μ L, 5 μ L, 1 μ L, 0.5 μ L, 0.1 μ L, 0.05 μ L, 0.01 μ L, 0.005 μ L and 0.001 μ L step 1 obtain respectively, be settled to 200 μ L respectively with 0.1mol/L PBS damping fluid (pH8.0), shake up, obtain the different standard reaction system of a series of concentration of substrate;

Above-mentioned series of standards reaction system behind 30 ℃ of incubation 30min, is added 50 μ L100g/L SDS aqueous solution termination reactions respectively, obtain series of standards termination reaction liquid;

With toothpick will detect bacterium (agrobacterium tumefaciens KYC55(Agrobacterium tumefaciens KYC55) at interval the 4mm point be connected on the adhesive tape that step 1 makes, get 10 μ L termination reaction drops respectively to the left end of each adhesive tape, counting becomes the Bluepoint number after placing 30 ℃ to cultivate 24h the adhesive tape, and calculating diffusion length (mm), be the longitudinal axis (y) with the oxo-C8-HSL amount that adds in the standard reaction system, be transverse axis (x) with diffusion length (mm), set up regression equation: y=0.00000652 * e ^0.3481x(R ²=0.991); E=2.718281828459045.

3, the enzyme activity determination of solution to be measured

1) reaction system: with the 0.1mol/L PBS damping fluid (pH8.0) of 20 μ L solution to be measured, 179 μ L and the oxo-C8-HSL(substrate of 1 μ L10mmol/L) solution mixes, and shakes up;

2) reaction conditions: 30 ℃ of incubation 30min;

3) stop: in reaction system, add 50 μ L100g/L SDS aqueous solution termination reactions;

4) detect: will detect bacterium (agrobacterium tumefaciens KYC55) interval 4mm point with toothpick and be connected on the adhesive tape that step 1 makes, get the reaction drop of 10 μ L reaction terminatings to the left end of this adhesive tape, counting becomes the Bluepoint number behind 30 ℃ of cultivation 24h, calculate diffusion length (mm), calculate the enzyme of solution to be measured by enzyme work calculation formula and live.

Enzyme (U/mL) unit definition alive: the enzyme amount of decomposing 1nmol oxo-C8-HSL at 30 ℃ of following per minutes is defined as enzyme unit alive.

The regression equation that obtains with step 2 is that following enzyme work calculation formula is set up on the basis:

Enzyme (U/mL)=5 alive * (1.3476 ^Xck-1.3476 ^Xs) * 50/30/10 ⁴Wherein, Xs is that solution to be measured carries out above-mentioned 1)-4) diffusion length after handling; Xck is for replacing solution to be measured to carry out above-mentioned 1 with 0.1mol/L PBS damping fluid (pH8.0))-4) diffusion length after the processing.

Four, different reorganization bacterium are induced the cancellation enzyme enzyme activity determination of the extracellular protein that obtains down in different IP TG concentration

2, get the incubated overnight liquid 400 μ L that step 1 obtains and be seeded in the 20mL TB liquid nutrient medium (containing 50 μ g/mL kantlex), 37 ℃ of shaking culture 3-3.5h make OD ₆₀₀Reach 1.0, add final concentration then and be 0,0.25,0.5 or the IPTG(inductor of 1.0mmol/L, induce the expression of target protein Aii0-AIO6), after (30 ℃, 180rpm, rotation radius 1.3cm) 8h is cultivated in concussion, get nutrient solution 1.6mL respectively, the centrifugal radius 6cm of the centrifugal 5min(of 12,000rpm), collect supernatant liquor respectively.

3, getting supernatant liquor 1mL that step 2 obtains adds two volumes acetone respectively and is placed on-20 ℃ of precipitation 30min, the centrifugal radius 6cm of the centrifugal 10min(of 12,000rpm), collecting precipitation (being extracellular protein), use 0.1mol/LPBS damping fluid (pH8.0) resuspended then respectively, make its OD ₆₀₀Be 1.0, obtain the solution of extracellular protein.

4, the extracellular protein solution that step 3 is obtained carries out the cancellation enzymic activity respectively and detects as solution to be measured, and the result is as shown in table 3.

Table 3, the different cancellation enzyme enzyme alive (U/mL) of bacterium through different concns IPTG inducing culture extracellular protein solution after 8 hours of recombinating

Table 3 is the result show, when the IPTG that adds different concns induced, the target protein enzyme work that the ETA exocytosis is expressed will be higher than A6 far away, and ET does not have the expression of target protein simultaneously.The IPTG of different concns induced 8 hours, and enzyme activity is not had obvious variation.Illustrate that the IPTG that uses low concentration (0.25mmol/L) carries out target protein and induces and get final product, so not only can save cost but also can reduce the concentration of IPTG in the product.

Claims

1. the preparation method of the bacterium of recombinating comprises E7 crack protein encoding gene and target protein encoding gene are imported in the bacterium that sets out, and obtains the reorganization bacterium.

2. method according to claim 1, it is characterized in that: described E7 crack protein is albumen shown in the sequence table sequence 2; And/or described target protein is the cancellation enzyme;

3. method according to claim 1 and 2 is characterized in that: described E7 crack protein encoding gene is gene shown in the 52nd to the 195th of sequence table sequence 1;

4. according to arbitrary described method in the claim 1-3, it is characterized in that: described E7 crack protein encoding gene and target protein encoding gene import by recombinant vectors C and obtain the reorganization bacterium in the described bacterium that sets out; Described recombinant vectors C prepares according to the method that comprises the steps:

Insert described E7 crack protein encoding gene expression cassette in plasmid pET-28a (+) T7 promotor upstream and obtain recombinant vectors B; Multiple clone site place in the described T7 promotor downstream of described recombinant vectors B inserts described target protein encoding gene, obtains described recombinant vectors C;

The promotor of described E7 crack protein encoding gene expression cassette specifically can be the T7 promotor;

Described recombinant vector C further specifically can prepare according to the method that comprises the steps: the DNA fragmentation shown in the 17th of insetion sequence table sequence 1 to the 195th nucleotides obtains recombinant vector A between the Xba of plasmid pET-28a (+) I and Xho I restriction enzyme site, DNA fragmentation shown in the 16th of insetion sequence table sequence 3 to the 398th nucleotides obtains described recombinant vector B between the Sph of described recombinant vector A I and Bgl II restriction enzyme site, and the DNA fragmentation shown in the 9th of insetion sequence table sequence 4 to the 815th nucleotides obtains described recombinant vector C between the EcoR of described recombinant vector B I and Not I restriction enzyme site.

5. according to arbitrary described method in the claim 1-4, it is characterized in that: the described bacterium that sets out is intestinal bacteria (Escherichia coli).

6. the reorganization bacterium that arbitrary described method prepares in the claim 1-5.

7. the application of the described reorganization of claim 6 bacterium in the described target protein of preparation.

8. a method for preparing target protein comprises the steps: to use the fermention medium shaking culture to OD the described reorganization of claim 6 bacterium ₆₀₀=1.0, add then IPTG to final concentration be 0-1mmol/L, be to continue concussion under the condition of 1.3cm to cultivate 1-24 hours in 30 ℃, 180rpm, rotation radius, obtain described target protein.

9. method according to claim 8, it is characterized in that: when right requires the bacterium that sets out of 6 described reorganization bacterium to be intestinal bacteria, the solvent of described fermention medium is water, and solute and final concentration thereof are respectively peptone 12g/L, yeast extract 24g/L, glycerine 4ml/L, KH ₂PO ₄0.231g/L, K ₂HPO ₄1.254g/L and glucose 5g/L.

10. the described recombinant vectors B in the described method of claim 4.