CN106282221A - The construction method of a kind of secreting, expressing trehalose synthase gene engineering bacteria and application - Google Patents

The construction method of a kind of secreting, expressing trehalose synthase gene engineering bacteria and application Download PDF

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CN106282221A
CN106282221A CN201610675597.9A CN201610675597A CN106282221A CN 106282221 A CN106282221 A CN 106282221A CN 201610675597 A CN201610675597 A CN 201610675597A CN 106282221 A CN106282221 A CN 106282221A
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npre
tres
trehalose synthase
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苏静
张云霄
王瑞明
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Guangzhou Magigen Biotechnology Corp
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Qilu University of Technology
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Abstract

The present invention relates to construction method and the application of a kind of secreting, expressing trehalose synthase gene engineering bacteria.Construction method step is as follows: (1) PCR amplification NprE signal peptide sequence and trehalose synthase sequence ps TreS；(2) NprE ps TreS fragment is prepared；(3) by NprE ps TreS fragment after double digestion, with as pHT01 plasmid after double digestion connect, proceed in bacillus coli DH 5 alpha, prepare recombiant plasmid pHT01 NprE ps TreS；(4) method that recombiant plasmid is converted by electricity is proceeded in bacillus subtilis, after chloromycetin plate screening, prepare secreting, expressing trehalose synthase gene engineering bacteria.The present invention adds signal peptide fragment NprE first in recombinant vector, and destination protein trehalose synthase can be secreted into the extracellular of recombination engineering and express, thus greatly simplify the step of follow-up enzyme purification by this signal.

Description

The construction method of a kind of secreting, expressing trehalose synthase gene engineering bacteria and application
Technical field
The present invention relates to construction method and the application of a kind of secreting, expressing trehalose synthase gene engineering bacteria, belong to biological skill Art technical field.
Background technology
Trehalose (Trehalose) is a kind of to be connected and composed through α-1,1-glycosidic bond by two pyranoid ring glucose molecules Non-reducing disaccharide, is widely present in the organisms such as antibacterial, yeast, filamentous fungi, plant, insecticide, invertebrates.Grind Study carefully and show, its stable in properties, organism is had very important biological significance.Be mainly manifested in it be the organism energy and The reserve of carbon source, is protein and biomembrane molecule stablizing in the adverse circumstances such as dehydration, high temperature, oxygen-derived free radicals, low temperature Agent and protective agent, be sensing complex and growth regulatory factor, but also one of is the component of some bacteria cell wall, because of This has the good reputation of " sugar of life " at scientific circles' trehalose.Trehalose has the protective effect of mystery to organism, is because sea Algae sugar can form the protection of uniqueness under the severe environmental conditions such as high temperature, high and cold, hyperosmosis and dry dehydration at cell surface Film, effectively protected protein matter molecule invariance inactivation, thus the life process of the body that sustains life and biological characteristic.This is unique Functional characteristic so that trehalose except can as pharmaceutical grade protein, enzyme, vaccine and other biological goods excellent activity protect Protect beyond agent, be also to maintain cytoactive, the important component of moisturizing class cosmetics, more can be as preventing food deterioration, keeping food Product fresh flavor, the particular foodstuff dispensing of lifting food quality.Therefore trehalose can be widely applied to medicine, cosmetic industry with And food service industry, there is tempting development prospect and huge economic benefit.
In view of the using value that trehalose is extensive and important, find that trehalose is efficiently convenient, the grinding of Low-cost production method Study carefully and extensively paid attention to.The production method of trehalose mainly has yeast extraction method, fermentation method, Enzyme optrode at present.Wherein enzyme process is raw Produce trehalose and there is the feature such as higher specificity and quick gentleness, have become as the focus of research and development trehalose industrialized production also As one of feasible way that short-term can take effect.
Trehalose synthase (EC5.4.99.16, Trehalose synthase, TreS) is a kind of intramolecular glucoside transfer Enzyme it have only to single step reaction just α-1,4 glycosidic bond of maltose can be converted into α-1,1 glycosidic bond generate trehalose.This enzyme Reaction process is short, easy-regulating, it is not necessary to consume anakinetomer, it is not necessary to phosphate coexists, it is only necessary to a kind of enzyme one-step just reacts Can obtain trehalose, therefore trehalose synthase conversion method is the method for production trehalose suitable for industrialized, has good application Prospect, is paid close attention to widely.Up to the present, the trehalose synthase gene engineering bacteria of the heterogenous expression having been reported that both at home and abroad is all It is the engineering strain built by escherichia coli, and simply intracellular expression.But, compare with escherichia coli, hay bud Spore bacillus is the microorganism of a kind of food safety, and its secretory protein ability is stronger.The present invention by engineered means, Being building up to by trehalose synthase gene on bacillus subtilis secretion expression vector, secreting, expressing is carried by the method utilizing electricity to convert Body is transformed in bacillus subtilis, thus obtain a kind of can be with the genetic engineering bacterium of secreting, expressing trehalose synthase, and by it It is applied in the production of production food pharmaceutical grade trehalose.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that the structure side of a kind of secreting, expressing trehalose synthase gene engineering bacteria Method and application.The present invention utilize that early stage screens can the signal peptide NprE of secreting, expressing trehalose, at commercial vectors Build one on the basis of pHT01 can will be divided by efficient electricity method for transformation by the carrier of trehalose synthase secreting, expressing Secrete expression vector to be transformed in bacillus subtilis, thus obtain a kind of can be with the genetic engineering of secreting, expressing trehalose synthase Bacterium.
Technical solution of the present invention is as follows:
The recombinant expression carrier of bacillus subtilis efficient secretory expression trehalose synthase, nucleotide sequence such as SEQ ID Shown in NO.1.
Above-mentioned recombinant expression carrier, compared with the bacillus subtilis expression vector pHT01 carrier that document is reported, adds Signal peptide fragment NprE of neutral protease, destination protein trehalose synthase can be secreted into extracellular and carry out table by this signal Reach.After trehalose synthase is secreted into extracellular, by obtaining purer liquid of protease after fermentation liquid ammonium sulfate precipitation.
A kind of construction method of secreting, expressing trehalose synthase gene engineering bacteria, step is as follows:
(1) the NprE signal peptide sequence with the genome of bacillus subtilis 168 as template, in PCR amplification Sec approach； It is template PCR amplifications trehalose synthase sequence ps with the genome of Pseudomonas stutzeri (Pseudomonas stutzeri) TreS；
The PCR primer sequence of described NprE signal peptide sequence is as follows:
NprE-F:5 '-AAAGGAGGAAggatccATGGGTTTAGGTAAGAA-3 '；SEQ ID NO.3
NprE-R:5 '-GAATGCTCATactagtAGCCTGAACACC-3 '；SEQ ID NO.4
The PCR primer sequence of described trehalose synthase sequence ps TreS is as follows:
Ps TreS-F:5 '-actagtATGAGCATTCCAGA-3 '；SEQ ID NO.5
Ps TreS-R:5 '-tctagaTTAGATCACCGGGGATGC-3 '；SEQ ID NO.6
(2) use over-lap PCR by signal peptide sequence NprE prepared for step (1) and the Sargassum deriving from Pseudomonas stutzeri Sugar synthase sequences ps TreS merges, and prepares NprE-ps TreS fragment；NprE-ps TreS fusion gene fragment gene sequence is such as Shown in SEQ ID NO.2；
(3) by step (2) prepare NprE-ps TreS fragment after BamH I and Xba I double digestion, with as warp PHT01 plasmid after BamH I and Xba I double digestion connects, and proceeds in bacillus coli DH 5 alpha, prepares recombiant plasmid pHT01-NprE- ps TreS；
(4) method that the recombiant plasmid that step (3) prepares is converted by electricity is proceeded in bacillus subtilis, mould by chlorine After element plate screening, prepare secreting, expressing trehalose synthase gene engineering bacteria.
According to currently preferred, in described step (1), the reaction system of PCR amplification NprE signal peptide is as follows:
2 × Pfu mix 25 μ L, 10 μm ol/L forward primer (NprE-F) 1 μ L, 10 μm ol/L downstream primers (NprE-R) 1 μ L, template 1 μ L, use ddH2O supplies 50 μ L；
PCR response procedures is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 53 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations；72 DEG C of extensions 10min。
According to currently preferred, in described step (1), the reaction system of PCR amplification trehalose synthase sequence ps TreS As follows:
2 × Pfu mix 25 μ L, 10 μm ol/L forward primer (ps TreS-F) 1 μ L, 10 μm ol/L downstream primer (ps TreS-R) 1 μ L, template 1 μ L, use ddH2O supplies 50 μ L；
PCR response procedures is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 4min, 30 circulations；72 DEG C of extensions 10min。
According to currently preferred, in described step (2), the first amplification system of over-lap PCR is 25 μ l:
Trehalose synthase sequence ps TreS 4 μ L；NprE signal peptide fragment 4 μ L；2×Pfu mix 12.5μL；ddH2O 4.5μL；
The first amplification program of described over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 30s, 5 circulations；72 DEG C are prolonged Stretch 10min；
The supplementary amplification system of described over-lap PCR is 25 μ L:
Forward primer (NprE-F) 2 μ L；Downstream primer (ps TreS-R) 2 μ L；2×Pfu mix 12.5μL；ddH2O 8.5μL；
The supplementary amplification program of described over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 5min, 30 circulations；72℃ Extend 10min.
According to currently preferred, the bacillus subtilis in described step (4) is bacillus subtilis WB800N.Hay Bacillus cereus WB800N derives from the biological company limited of Hangzhou treasured match.
According to currently preferred, in described step (4), the condition that electricity converts is as follows:
Competent cell is carried out under the conditions of 2100V electricity and converts 5ms.
The application in preparing trehalose synthase of the above-mentioned secreting, expressing trehalose synthase gene engineering bacteria.
Above-mentioned application, step is as follows:
Secreting, expressing trehalose synthase gene engineering bacteria amplification culture in TB culture medium, to growing logarithmic (log) phase, is trained to TB Support addition IPTG in base, to final concentration 1 μM, 37 DEG C of induction 12h, to be centrifuged, take supernatant, protein concentration, prepare trehalose synthase.
According to currently preferred, described centrifugal condition is 4 DEG C, 10000rpm, 20min.
According to currently preferred, described protein concentration is membrance concentration；It is further preferred that described membrance concentration is for using aperture Size is that 0.2 μm ultrafilter membrane concentrates.
Above-mentioned TB culture medium is this area conventional medium, and every liter of component is as follows:
Peptone 12g, yeast extract 24g, KH2PO42.31g, K2HPO412.54g, glycerol 4ml.
Beneficial effect
1, the present invention adds signal peptide fragment NprE first in recombinant vector, and this signal can be by destination protein trehalose Synthase is secreted into the extracellular of recombination engineering and expresses, thus greatly simplify the step of follow-up enzyme purification；
2, engineering bacteria of the present invention is directly by heterologous protein secretion to culture medium, it is not necessary to cell breakage, it is possible to Obtaining purpose enzyme liquid, and the bacillus subtilis used is food safety bacterial strain, its genetic background understands, genetic operating system is complete Kind and accurate, it is ensured that final expression system is in the status of complete food stage, reach the standard of aliment security level, for downstream Trehalose industrialized production is laid a good foundation.
3, the present invention uses the free expression of recombinant plasmid trehalose synthase gene of high copy can improve genes of interest Sargassum The copy number of sugar synthase, thus improve the expression of exogenous gene, it is achieved trehalose synthase secretion in bacillus subtilis Express.
Accompanying drawing explanation
Fig. 1 is pHT01-NprE-ps TreS vector construction schematic diagram；
Fig. 2 a is the agarose gel electrophoresis figure of NprE signal peptide；
Fig. 2 b is derived from the agarose gel electrophoresis figure of the trehalose synthase sequence ps TreS of Pseudomonas stutzeri；
Fig. 3 is the agarose gel electrophoresis figure that NprE-ps TreS merges fragment；
Fig. 4 is the agarose gel electrophoresis figure after pHT01 plasmid BamH I/Xba I double digestion；
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is further elaborated, but institute of the present invention protection domain is not limited to This.
Biological material source:
Bacillus subtilis 168 is purchased from the biological company limited of Hangzhou treasured match；
Bacillus subtilis WB800N is purchased from the biological company limited of Hangzhou treasured match；
Shuttle plasmid pHT01 is purchased from the biological company limited of Hangzhou treasured match；
Pseudomonas stutzeri (Pseudomonas stutzeri) is purchased from Chinese Academy of Sciences's Culture Collection (CGMCC)；
Embodiment 1: construction recombination plasmid
(1) clone obtains signal peptide gene fragment
With the genome of hay 168 as template, design primer carries out PCR amplification, obtains NprE signal peptide fragment.
Described PCR primer sequence is as follows:
NprE-F:5 '-AAAGGAGGAAggatccATGGGTTTAGGTAAGAA-3 '
NprE-R:5 '-GAATGCTCATactagtAGCCTGAACACC-3 '
Described PCR reaction system is as follows:
2 × Pfu mix 25 μ L, 10 μm ol/L forward primer (NprE-F) 1 μ L, 10 μm ol/L downstream primers (NprE-R) 1 μ L, template 1 μ L, use ddH2O supplies 50 μ L；
Above-mentioned PCR reaction is carried out according to following program:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 53 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations；72 DEG C of extensions 10min。
PCR passes through 1% agarose gel electrophoresis analytic plate segment length after terminating is short, cuts purpose bar according to clip size Band, uses the raw work glue in Shanghai to reclaim test kit recovery and cuts glue product.
(2) clone obtains Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3) trehalose synthase gene
According to Pseudomonas stutzeri trehalose synthase full length nucleotide primers disclosed on NCBI, primer sequence As follows:
Ps TreS-F:5 '-actagtATGAGCATTCCAGA-3 '
Ps TreS-R:5 '-tctagaTTAGATCACCGGGGATGC-3 '
With the genome of Pseudomonas stutzeri (Pseudomonas stutzeri) as template, above-mentioned primer is utilized to carry out PCR expands, and PCR reaction system is as follows:
2 × Pfu mix 25 μ L, 10 μm ol/L forward primer (ps TreS-F) 1 μ L, 10 μm ol/L downstream primer (ps TreS-R) 1 μ L, template 1 μ L, use ddH2O supplies 50 μ L；
Above-mentioned PCR reaction is carried out according to following program:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 4min, 30 circulations；72 DEG C of extensions 10min。
PCR passes through 1% agarose gel electrophoresis analytic plate segment length after terminating is short, cuts purpose bar according to clip size Band, uses the raw work glue in Shanghai to reclaim test kit recovery and cuts glue product.
(3) the Pseudomonas stutzeri trehalose synthase that NprE signal peptide fragment step (1) prepared prepares with step (2) Genetic fragment carries out over-lap PCR, prepares NprE-ps TreS fragment；
The first amplification system of described over-lap PCR is 25 μ l:
Trehalose synthase sequence ps TreS 4 μ L；NprE signal peptide fragment 4 μ L；2×Pfu mix 12.5μL；ddH2O 4.5μL；
The first amplification program of described over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 30s, 5 circulations；72 DEG C are prolonged Stretch 10min；
The supplementary amplification system of described over-lap PCR is 25 μ L:
Forward primer 2 μ L；Downstream primer 2 μ L；2×Pfu mix 12.5μL；ddH2O 8.5μL；
The supplementary amplification program of described over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 5min, 30 circulations；72℃ Extend 10min.-20 DEG C of preservations；
(4) by the NprE-ps TreS fragment for preparing in step (3) through BamH I/Xba I double digestion with as after double digestion PHT01 plasmid connect, proceed in bacillus coli DH 5 alpha；After identifying successfully and checking order correctly, by prepared recombinant vector name For pHT01-NprE-ps TreS.
Described PCR primer enzyme action system is:
PCR primer 50 μ L；BamHⅠ2.0μL；XbaⅠ2.0μL；10×buffer K 6.0μL；
Reaction condition: 37 DEG C of reaction 4h.
Described pHT01 plasmid enzyme restriction system is:
PHT01 plasmid 50 μ L；BamHⅠ2.0μL；XbaⅠ2.0μL；10×buffer K 6.0μL
Reaction condition: 37 DEG C of reaction 4h.
Product after PCR primer and carrier double digestion is through 1% agarose gel electrophoresis, and uses DNA gel to reclaim, then Reclaim test kit with DNA glue and carry out glue recovery.
Described linked system is:
PHT01 digested plasmid 5.0 μ L；PCR primer 3.0 μ L after enzyme action；10×T4buffer 1.0μL；T4-DNA Ligase1.0μL
Reaction condition: 16 DEG C of reaction 10h.
The NprE-ps TreS gene connected and pHT01 are connected product conversion importing bacillus coli DH 5 alpha competence thin In born of the same parents.
Embodiment 2: bacillus subtilis WB800N electricity turns the preparation of competent cell
The mono-bacterium colony of bacillus subtilis WB800N of picking fresh LB solid culture primary surface in 5ml LB culture medium, mistake Night cultivates.Taking 2.5mL overnight culture to access in 40mL growing microorganism culture medium (LB+0.5M sorbitol), 37 DEG C, 200rpm shakes Swinging cultivation to OD600 is between 0.85～0.95.By bacterium solution ice-water bath 10min, then 5000g, 4 DEG C of centrifugal 5min, collect bacterium Body.Culture medium (0.5M sorbitol, 0.5M mannitol, 10% glucose) resuspended thalline is turned with the electricity of 50mL pre-cooling, 5000g, 4 DEG C Centrifugal 5min, removes supernatant, so rinsing 4 times.Thalline after washing is resuspended in 1ml electricity and turns in culture medium, be sub-packed in EP pipe, often Pipe subpackage 60 μ L.
Embodiment 3: recombiant plasmid is proceeded in bacillus subtilis WB800N
50ng plasmid DNA (1～8 μ L) is joined in 60 μ l competent cells, hatches 2min on ice, add the electricity of pre-cooling In revolving cup (2mm), under the conditions of 2100V, carry out electricity convert 5ms.After electric shock, take out cup and add 1mL thalline immediately again Soviet Union's culture medium (LB+0.5M sorbitol+0.38M mannitol), after 3h is cultivated in 200rpm vibration recovery, coats chloride mould by 37 DEG C On the LB flat board of element.37 DEG C of incubator overnight incubation.The bacterial strain of screening chloramphenicol resistance.
Embodiment 4: the cultivation of positive recombinant bacterium and qualification
By above-mentioned positive restructuring bacterium colony, it is inoculated in LB fluid medium the overnight incubation (containing chloromycetin), draws 1mL bacterium Liquid, utilizes the test kit that Shanghai biological engineering company limited provides to extract genomic DNA, with the genomic DNA that obtains as template, It is that primer carries out PCR amplification with ps TreS-F, ps TreS-R and NprE-F, NprE-R respectively.
The first amplification system of described bacterium colony PCR is 20 μ L:
Forward primer 1 μ L；Downstream primer 1 μ L；Template 2 μ l；2×Pfu mix 10μL；ddH2O 6μL；
The first amplification program of described bacterium colony PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 2min 30s, 30 circulations； 72 DEG C extend 10min；
Agarose gel electrophoresis proves that exogenous sequences ps TreS and NprE signal peptide have been connected respectively to pHT01 plasmid the most On.
Embodiment 5: the fermentation of positive recombinant bacterium
Recombinant bacterium pHT01-NprE-ps TreS embodiment 4 built is inoculated in 50mL LB fluid medium and (contains The chloromycetin of concentration 25 μ g/mL), 37 DEG C, 200rpm overnight incubation, transfer in TB culture medium by 1% inoculum concentration next day, cultivate 7h, to growing logarithmic (log) phase, adds IPTG to final concentration of 1 μM, 37 DEG C of induction 12h, takes fermentation liquid at 4 DEG C, under the conditions of 10000rpm Centrifugal 20min, supernatant is the outer crude enzyme liquid of born of the same parents.Mensuration for enzyme activity.
Above-mentioned TB culture medium is this area conventional medium, and every liter of component is as follows:
Peptone 12g, yeast extract 24g, KH2PO42.31g, K2HPO412.54g, glycerol 4ml.
Above-mentioned LB fluid medium, every liter of component is as follows:
Tryptone 10g, yeast extract 5g, NaCl 10g, pH 7.0.
Embodiment 6: the detection that trehalose synthase enzyme is lived
The maltose solution of 500 μ L 60% is added, the outer crude enzyme liquid of 500 μ L born of the same parents in reaction system.React at 37 DEG C 12h.Measured the conversion ratio of trehalose by the method for high-pressure liquid phase, during mensuration, use nh 2 column；Column temperature is 40 DEG C of flowings Using the mixed solution of acetonitrile and water mutually, the two ratio is 3:1；Flow velocity is 1mL/min；Detector is Composition distribution；Detection Time is 25min.Conversion ratio is calculated according to equation below.
Software matching is utilized according to maltose peak area, trehalose peak area and glucose peaks area in efficient liquid phase result Obtaining curve, calculate the quality of three, wherein m3 is the quality being converted into trehalose, and m2 is the quality being converted into glucose, m1 For remaining the quality of maltose.
Result shows, the trehalose synthase gene engineering bacteria constructed by the present invention is capable of trehalose synthase at hay bud Secreting, expressing in spore bacillus.Maltose in the outer crude enzyme liquid of born of the same parents is converted into the conversion ratio of trehalose up to 72%.Have extensively Prospects for commercial application.
Comparative example 1
The trehalose synthase deriving from Pseudomonas stutzeri (Pseudomonas stutzeri) is cloned into shuttle plasmid In pHT43, this plasmid contains AmyQ signal peptide, by inducing it to express with IPTG as derivant, is successfully realized it withered Intracellular expression in grass bacillus cereus, but be the failure to realize its extracellular expression.
Comparative example 2
According to the content disclosed in Chinese patent literature CN103215300A (application number 201310174692.7), by Sargassum Sugar synthase expression element is incorporated in bacillus subtilis chromosome and builds integrated recombined bacillus subtilis.Result shows, Total maltose is converted into the conversion ratio of trehalose and reaches 63.1%.
The present invention is to be cloned into by trehalose synthase gene in shuttle plasmid pHT01, and the method converted by electricity will restructuring Plastid transformation in bacillus subtilis WB800N, the heredity that this plasmid not only can be stable in bacillus subtilis, and Because its copy number is higher, the target gene dosage that it carries increases the most accordingly, therefore, and the trehalose synthase base constructed by the present invention Because engineering bacteria is capable of trehalose synthase secreting, expressing in bacillus subtilis, its maltose is converted into turning of trehalose Rate is up to 72%.For comparing more integrated recombined bacillus subtilis production trehalose synthetase, use the side of the present invention Method production trehalose synthase seems and more has superiority.

Claims (10)

1. the recombinant expression carrier of bacillus subtilis efficient secretory expression trehalose synthase, nucleotide sequence such as SEQ ID Shown in NO.1.
2. the construction method of a secreting, expressing trehalose synthase gene engineering bacteria, it is characterised in that step is as follows:
(1) the NprE signal peptide sequence with the genome of bacillus subtilis 168 as template, in PCR amplification Sec approach；To execute The genome of family name pseudomonas (Pseudomonas stutzeri) is template PCR amplifications trehalose synthase sequence ps TreS；
The PCR primer sequence of described NprE signal peptide sequence is as follows:
NprE-F:5 '-AAAGGAGGAAggatccATGGGTTTAGGTAAGAA-3 '；
NprE-R:5 '-GAATGCTCATactagtAGCCTGAACACC-3 '；
The PCR primer sequence of described trehalose synthase sequence ps TreS is as follows:
Ps TreS-F:5 '-actagtATGAGCATTCCAGA-3 '；
Ps TreS-R:5 '-tctagaTTAGATCACCGGGGATGC-3 '；
(2) the signal peptide sequence NprE and the trehalose deriving from Pseudomonas stutzeri that use over-lap PCR step (1) to be prepared close Enzyme sequence ps TreS merges, and prepares NprE-ps TreS fragment；NprE-ps TreS fusion gene fragment gene sequence such as SEQ Shown in ID NO.2；
(3) by step (2) prepare NprE-ps TreS fragment after BamH I and Xba I double digestion, with as through BamH PHT01 plasmid after I and Xba I double digestion connects, and proceeds in bacillus coli DH 5 alpha, prepares recombiant plasmid pHT01-NprE-ps TreS；
(4) method that the recombiant plasmid that step (3) prepares is converted by electricity is proceeded in bacillus subtilis, put down by chloromycetin After screen choosing, prepare secreting, expressing trehalose synthase gene engineering bacteria.
3. construction method as claimed in claim 2, it is characterised in that in described step (1), PCR amplification NprE signal peptide Reaction system is as follows:
2 × Pfu mix 25 μ L, 10 μm ol/L forward primer NprE-F 1 μ L, 10 μm ol/L downstream primer NprE-R 1 μ L, mould Plate 1 μ L, uses ddH2O supplies 50 μ L；
PCR response procedures is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 53 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations；72 DEG C of extensions 10min。
4. construction method as claimed in claim 2, it is characterised in that in described step (1), PCR expands trehalose synthase sequence The reaction system of row ps TreS is as follows:
2 × Pfu mix 25 μ L, 10 μm ol/L forward primer ps TreS-F 1 μ L, 10 μm ol/L downstream primer ps TreS-R1 μ L, template 1 μ L, use ddH2O supplies 50 μ L；
PCR response procedures is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C extend 4min, 30 circulations；72 DEG C of extensions 10min。
5. construction method as claimed in claim 2, it is characterised in that in described step (2), over-lap PCR expand body for the first time System is 25 μ l:
Trehalose synthase sequence ps TreS 4 μ L；NprE signal peptide fragment 4 μ L；2×Pfu mix 12.5μL；ddH2O4.5μL；
The first amplification program of described over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 30s, 5 circulations；72 DEG C of extensions 10min；
The supplementary amplification system of described over-lap PCR is 25 μ L:
Forward primer NprE-F 2 μ L；Downstream primer ps TreS-R 2 μ L；2×Pfu mix 12.5μL；ddH2O 8.5μL；
The supplementary amplification program of described over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 5min, 30 circulations；72 DEG C of extensions 10min。
6. construction method as claimed in claim 2, it is characterised in that the bacillus subtilis in described step (4) is hay Bacillus cereus WB800N.
7. construction method as claimed in claim 2, it is characterised in that in described step (4), the condition that electricity converts is as follows:
Competent cell is carried out under the conditions of 2100V electricity and converts 5ms.
8. the secreting, expressing trehalose synthase gene engineering bacteria that claim 2 builds application in preparing trehalose synthase.
Apply the most as claimed in claim 8, it is characterised in that step is as follows:
By secreting, expressing trehalose synthase gene engineering bacteria, in TB culture medium, amplification culture is to growing logarithmic (log) phase, to TB culture medium Middle addition IPTG, to final concentration 1 μM, 37 DEG C of induction 12h, is centrifuged, takes supernatant, protein concentration, prepare trehalose synthase.
Apply the most as claimed in claim 9, it is characterised in that described centrifugal condition is 4 DEG C, 10000rpm, 20min；
Preferably, described protein concentration is membrance concentration；It is further preferred that described membrance concentration for pore size be 0.2 μm surpass Filter membrane concentrates.
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CN107698666A (en) * 2017-10-23 2018-02-16 华南理工大学 A kind of signal peptide for effectively improving secretion and its application
CN107857801A (en) * 2017-10-23 2018-03-30 华南理工大学 A kind of signal peptide and its application that can be used for improving secernment efficiency
CN114015678A (en) * 2021-09-30 2022-02-08 中南民族大学 Aminopeptidase Amp0279 derived from Bacillus sphaericus C3-41 as well as recombinant strain and application thereof

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