CN104928307B - A kind of tumorous stem mustard sulphur glycoside enzyme gene MYR1, tumorous stem mustard sulphur glycosides enzyme and its gene engineering expression method - Google Patents
A kind of tumorous stem mustard sulphur glycoside enzyme gene MYR1, tumorous stem mustard sulphur glycosides enzyme and its gene engineering expression method Download PDFInfo
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Abstract
The present invention discloses a kind of tumorous stem mustard sulphur glycoside enzyme gene MYR1, tumorous stem mustard sulphur glycosides enzyme and its gene engineering expression method, and the tumorous stem mustard sulphur glycoside enzyme gene MYR1 nucleotide sequences are as shown in SEQ ID NO.1；The tumorous stem mustard sulphur glycosides enzyme amino acid sequence is shown in SEQ ID NO.2；The gene engineering expression method of tumorous stem mustard sulphur glycosides enzyme, extract the total serum IgE in tumorous stem mustard blade, again using RT PCR methods amplification sulphur glycoside enzyme gene MYR1, and construction of expression vector, expressed using pichia yeast expression system, utilize SalI restriction enzyme site linearized vectors, it is transformed into Pichia pastoris genome, by G418 resistances, geneticin resistant transformant is filtered out, is expressed using methanol induction.Through electrophoretic analysis, recon of the present invention can be expressed, and expressing protein molecular weight is 90KDa, determine the Rate activity of albumen after purification, and the Rate activity of tumorous stem mustard sulphur glycosides enzyme is approximately equal to 0.003U/ μ L.
Description
Technical field
The invention belongs to sulphur glycosides zymotechnic field, and in particular to a kind of tumorous stem mustard sulphur glycoside enzyme gene MYR1, tumorous stem mustard sulphur glycosides
Enzyme and its gene engineering expression method.
Background technology
There is a kind of very important defensive ferment i.e. sulphur glycosides enzyme in crucifer（Myrosinase）, also referred to as meson
Enzyme；Sulphur glycosides enzyme can be degraded sulphur glycosides, and the materials such as glucose, sulfate, isothiocyanate, rhodanate are included in catabolite；
Wherein isothiocyanate has the effect such as anti-inflammatory, pre- anti-cancer；Sulphur glycosides enzyme usually exists in the form of isodynamic enzyme, these same works
Enzyme causes its physical property, chemical property different due to glycosylated degree difference.Have now been found that, sulphur glycosides enzyme at least 3 species
The encoding gene family MA of type（Myr1,TGG1）, MB (Myr2, TGG2), MC (Myr3, TGG3), research show MA gene families
There is 4-5 gene, its sulphur glycosides enzyme encoded is soluble dimer.MB gene families include 10-15 genes, MC gene families by
5 gene compositions, the sulphur glycosides enzyme of MB and MC gene families coding is insoluble compound.
The property to sulphur glycosides enzyme, extracting method have done many researchs in the world at present, first for the Separation Research of sulphur glycosides enzyme
First to mention Bjorkman and be extracted sulphur glycosides enzyme in sinapsis alba seed equal to 1972, and it is purified；And then
Ohtsuru etc. is studied the method for purification of sulphur glycosides enzyme and the property of sulphur glycosides enzyme；Xian Li et al. are also carried among horseradish
Sulphur glycosides enzyme has been got, and has been separated；Bones etc. was in the isolated sulphur glycosides enzyme among Brassica Napus Seedling in 1989；But
For sulphur resources in plant generally than relatively low, directly extraction can not largely obtain sulphur glycosides enzyme.
Preferably to study the property of sulphur glycosides enzyme, Chen and Barbara are by transgenic technology by sulphur glycoside enzyme gene gram
It is grand to be expressed into yeast, but the enzyme expressed is poisonous to yeast, it is impossible to great expression.Domestic scholars are mainly from radish, oil at present
Sulphur glycoside enzyme gene is extracted in dish, arabidopsis, leaf mustard, broccoli and construction of expression vector is expressed；Such as Xiao Haiyan in 2008
Deng building recombinant expression plasmid and the induced expression in Escherichia coli after extracting sulphur glycoside enzyme gene from rape, a 90KDa left sides are obtained
Right protein band；Beam Jin Feng in 2010 etc. extracts sulphur glycoside enzyme gene from broccoli and expressed in Pichia pastoris, obtains
The protein band of 65KDa sizes；Zhang Wei in 2012 et al. extracts sulphur glycoside enzyme gene from cabbage type rape and carrier construction is carried out
Expression；But do not have the extraction sulphur glycoside enzyme gene from tumorous stem mustard at present, and carry out vector construction expression tumorous stem mustard sulphur glycosides enzyme
Correlative study.
The content of the invention
For deficiencies of the prior art, it is an object of the invention to provide a kind of tumorous stem mustard sulphur glycoside enzyme gene
MYR1。
It is a further object of the present invention to provide a kind of tumorous stem mustard sulphur glycosides enzyme.
It is also another object of the present invention to provide the gene engineering expression method of above-mentioned tumorous stem mustard sulphur glycosides enzyme.
Above-mentioned purpose is realized, the present invention uses following technical scheme：A kind of tumorous stem mustard sulphur glycoside enzyme gene MYR1, its nucleotides
Sequence is the sequence shown in SEQ ID NO.1.
A kind of tumorous stem mustard sulphur glycosides enzyme, its amino acid sequence are the sequence shown in SEQ ID NO.2.
A kind of gene engineering expression method of above-mentioned tumorous stem mustard sulphur glycosides enzyme, comprises the following steps：
1）Tumorous stem mustard blade is taken, extracts total serum IgE；
2）With step 1）The total serum IgE of extraction is template, by reverse transcription reaction in PCR instrument, synthesizes the first chain cDNA；
3）Compare the nucleotide sequence of rape and leaf mustard sulphur glycoside enzyme gene, design a pair of forward and reverse primers, the primer is：
Upper primer 1：
5’- GATCCGGTTCTGGCGAATTCGACGACGACGACAAGGATGAAGAAATCAC -3’；
Upper primer 2：
5’- CGACGACGACAAGGATGAAGAAATCACATGTGAAGAAAATAACC-3’；
Lower primer：
5’-GGCGAATTAATTCGCGGCCGCTTATTAAGCATCTGCGAAACGCTTCTTTTG -3’；
4）With step 2）First chain cDNA of synthesis is template, using step 3）Designed primer, enter performing PCR reaction,
Amplification obtains sulphur glycoside enzyme gene MYR1 full length sequence, and the nucleotides sequence of the sulphur glycoside enzyme gene MYR1 is classified as SEQ ID NO.1
It is shown；
5）Extract carrier pPIC9K-S plasmids, and the plasmid described in EcoR I and NotI double digestions；
6）By step 4）The sulphur glycoside enzyme gene MYR1 of acquisition and step 5）Expression vector hybridization after double digestion, is recombinated
Expression vector pPIC9K-S-MYR1；
7）By step 6）Recombinant expression carrier transformed competence colibacillus cell E. coli DH10B, by the cell after conversion containing
Cultivate 18 hours in 37 DEG C on the LB flat boards of ammonia benzyl resistance, through PCR evaluation and screenings positive colony bacterium and be sequenced；
8）By step 7）Correct positive colony culture is sequenced, and extracts plasmid, at Sal I single endonuclease digestion linearisations
Reason, converted for Pichia pastoris SMD1168 electricity；
9）By the recombinant expression carrier pPIC9K-S-MYR1 of Sal I single endonuclease digestion linearization process, electricity is transformed into Pichia pastoris
SMD1168, coating MD flat boards are cultivated 3-6 days in 30 DEG C, and obtain geneticin resistant transformant by G418 resistances, screening；
10）The monoclonal of picking geneticin resistant transformant is to YPD medium cultures, under the conditions of 28 DEG C, with 300
Rpm/h rotating speed shaking between 2-5, centrifuges abandoning supernatant, BMMY fluid nutrient medium weights is added into precipitation to OD600 values
Outstanding cell；Expressed using methanol continuous induction, the final volume concentration for adding methanol to methanol for during which every 24 hours is 0.5% to continue
Induction, after inducing 72 h, the expression of recombinant protein is analyzed and identified using the SDS-PAGE of coomassie brilliant blue staining.
Compared with prior art, the present invention has the advantages that：
1st, content of the tumorous stem mustard sulphur glycosides enzyme in tumorous stem mustard is considerably less, and sulphur glycosides enzyme is directly extracted in tumorous stem mustard to be had necessarily
Difficulty, and natural sulphur glycosides enzyme has a glycosylation modified effect, and prokaryotic expression product lacks sulphur glycosides enzymatic activity, therefore glycosylation is repaiied
Decorations have a significant impact for sulphur glycosides enzyme enzymatic activity；Present invention selection Pichia pastoris is expression system, in processing, outside for expression product
Secretion, translation, modification and glycosylation etc. have very big advantage, and there is Pichia pastoris alcohol oxidase AOX1 genes to open
Mover, can in using methanol as the culture medium of sole carbon source fast-growth, be very beneficial for the purifying of tumorous stem mustard sulphur glycosides enzyme.
2nd, the nucleotide sequence of the present invention relatively plant sulphur glycoside enzyme gene such as rape, leaf mustard, chooses the higher area of homology
Domains, a pair of forward and reverse primers are designed, using the first chain cDNA of tumorous stem mustard as template, expand the sulphur glycoside enzyme gene MYR1 of tumorous stem mustard,
And recombinant expression carrier pPIC9K-S-MYR1 is built, electricity is transformed into Pichia pastoris SMD1168, and sulphur glycoside enzyme gene MYR1 is cloned
Into yeast expression vector, expression obtains tumorous stem mustard sulphur glycosides enzyme, and molecular weight is 90 KDa.
3rd, the present invention uses the tumorous stem mustard sulphur glycosides enzyme after expression and purification, using Sinigrin sinigrins as substrate, measure weight
The activity of histone, the results showed that at 45 DEG C, the albumen expressed by recon can hydrolyze sulphur glycosides, show the life of sulphur glycosides enzyme
Thing activity, Rate activity is about 0.03U/mL.
4th, recon of the present invention can great expression tumorous stem mustard sulphur glycosides enzyme, compared to the method directly extracted, the present invention has more
There is economic value, there is good market promotion prospect.
Brief description of the drawings
Fig. 1 is tumorous stem mustard leaf total serum IgE electrophoresis detection figure；
Fig. 2 is the RT-PCR amplification figures of sulphur glycoside enzyme gene；
Fig. 3 schemes for tumorous stem mustard sulphur glycoside enzyme gene sequence B LASTX；
Fig. 4 schemes for tumorous stem mustard sulphur glycosides enzyme amino acid comparison；
Fig. 5 is that the SDS-PAGE of expression product schemes；
Fig. 6 is fusion protein electrophoretogram after purification；
Fig. 7 is the HPLC figures of detection sulphur resources after determination of activity.
Embodiment
The present invention is described in further detail with reference to specific embodiments and the drawings.
Tumorous stem mustard employed in following embodiments（Brassica juncea (L.) Czern. et Coss. var.
tumida Tsen et Lee）For Yungan xiaoye, storage phase is 3 years, and by Chongqing City Fuling, Lv Yuan developments in agricultural science and technology company carries
For；Escherichia coli DH10B, SMD1168 engineering bacteria, Expression vector pPIC9K are NEB Products.
Without RNase H2O, sterilize paraffin oil, 1 × TAE buffer solutions, 1 × tbe buffer liquid, level pad, elution buffer
Liquid, 6 × loading buffer, GoldviewTM nucleic acid dyes, RNase inhibitor, dNTP Mix, AMV reverse transcriptase, 10
×PCR Buffer、MgCl2, archaeal dna polymerase and agarose be purchased from Shanghai bioengineering Co., Ltd.
Restriction endonuclease EcoR I and Not I are NEB Products.
Other raw materials used are common commercially available unless otherwise specified.
The extraction of the tumorous stem mustard total serum IgE of embodiment 1：
5 mL Trizol reagents are added in centrifuge tube, take 0.5 g tumorous stem mustard blades to be quickly ground to powder in liquid nitrogen
End, it is then transferred into test tube, fully mixes immediately, after room temperature places 10 min, is centrifuged in refrigerated centrifuge, temperature
Degree is arranged to 4 DEG C, and rotating speed is 7200 rpm/min, and supernatant liquid is drawn after centrifuging 25 min；Added into the supernatant liquid
CHCl3The 1 mL and mL of 5 mol/L NaCl solutions 1,3 min acutely are placed after vibration, then at 4 DEG C, 7200 rpm/min are centrifuged
25 min, at this moment sample can be divided into 3 layers, take supernatant；2.5 mL isopropanols are added into supernatant, are centrifuged again after placing 10 min
20 min, abandon supernatant, and centrifuge tube bottom sides form gelatinous precipitate；5 are centrifuged into centrifuge tube plus after 5 mL ethanol washing precipitation
Min, supernatant is abandoned, add the water of 0.25 mL DEPC processing to dissolve precipitation after ethanol volatilization completely, and in -72 DEG C of preservations.
The detected through gel electrophoresis of the total serum IgE of embodiment 2
25 1 × TAE of mL buffer solutions and 0.25 g agaroses are added in conical flask, micro-wave oven is put into and is heated to agarose
Melt, when being cooled to the temperature that the back of the hand can be born, add 2.5 μ L GoldviewTM nucleic acid dyes, system is poured into after mixing
Among offset plate.Take out after the total serum IgE that the extraction of 4 μ L embodiments 1 is dissolved mixes with 1 μ L Loading buffer and be loaded,
After carrying out the min of electrophoresis 30 under 70mA, 100V, observe under ultraviolet light, as a result as shown in figure 1, each swimming lane label divides in Fig. 1
It is not：R1, R2, R3, three swimming lanes are the RNA of extraction, as seen from Figure 1 three bands, and 28S bands and 18S bands
Brightness ratio is about 2:1, it was demonstrated that the total serum IgE of proposition can be used for experiment below.
The chain cDNA of embodiment 3 first synthesis
Using AMV the first chain cDNA synthetic agent box of Shanghai Sheng Gong bioengineering Co., Ltd, the first chain cDNA is synthesized,
Centrifuge tube is inserted in ice and adds following reaction reagent：
The μ L of total serum IgE 2 of the extraction dissolving of embodiment 1
Oligo dt Primer（0.5 μg/μL） 1 μL
RNase free ddH2O makes final volume be 12 μ L
By the solution that the above-mentioned cumulative volume prepared is 12 μ L, 2 min are centrifuged in 4000 rpm/min, then reaction is mixed
Compound is placed on the min of water-bath 5 in 65 DEG C of water-bath, is inserted into 30 s in ice, centrifuges 2 min, takes supernatant.
Test tube equipped with supernatant is placed on ice, adds following component：
5×Reaction buffer 4 μL
RNase Inhibitor(20M/μL) 1 μL
DNTP Mix(10 mmol/L) 2 μL
AMV Reverse Transcriptase(10Μ/μl) 2 μL
2 min are centrifuged after mixing, carry out reverse transcription reaction in PCR instrument, 42 DEG C of 60 min of processing so as to cDNA synthesis,
Then 85 DEG C of 5 min of processing inactivate enzyme, after processing, are placed in -20 DEG C of refrigerators and preserve.
The amplification of the sulphur glycoside enzyme gene of embodiment 4
Compare the nucleotide sequence of the plant sulphur glycoside enzyme gene such as rape, leaf mustard, choose the higher region of homology, design one
Reverse primer is aligned, enters performing PCR reaction by template of the first chain cDNA that embodiment 3 synthesizes.
Upper primer 1：
5’- GATCCGGTTCTGGCGAATTCGACGACGACGACAAGGATGAAGAAATCAC -3’
Upper primer 2：
5’- CGACGACGACAAGGATGAAGAAATCACATGTGAAGAAAATAACC-3’
Lower primer：
5’- GGCGAATTAATTCGCGGCCGCTTATTAAGCATCTGCGAAACGCTTCTTTTG -3’
Prepare 4 centrifuge tubes, be placed in and add following component successively on ice：
10×PCR Buffer 5 μL（MgCl containing 1mM2）
10×dNTP 5 μL
Upper primer 1（10 μM） 2 μL
Upper primer 2（10 μM） 2 μL
Lower primer（10 μM） 2 μL
The μ L of Pfu archaeal dna polymerases 0.5（5U)
Mixed liquor is mixed with rifle, adds the μ L of template cDNA 2 of the synthesis of embodiment 3, moisturizing makes final volume be 50 μ
L, 2 min are centrifuged, is dispensed into PCR pipe, adds 25 μ L sterilizing paraffin oil.
Reacted as follows in PCR instrument：
94℃ 5min
94℃ 1min
50℃ 30s 35cycle
72℃ 2min
72℃ 10min
4 DEG C of preservations.
PCR primer is detected with agarose gel electrophoresis, and carries out recovery purifying；Then further expand and obtain as template
Obtain the full length sequence of sulphur glycoside enzyme gene.Electrophoretogram is as shown in Fig. 2 swimming lane is respectively designated as in Fig. 2：P1, P2, P3, P4, M；M is
DNA Marker 2000；Remaining four respectively all be represent amplification DNA；As seen from Figure 2, purpose has been amplified
Band.Through sequencing, the full length gene sequence of amplification is as shown in SEQ ID NO.1, the amino acid sequence such as SEQ of the gene code
Shown in ID NO.2.
By the amino acid sequence of the tumorous stem mustard sulphur glycoside enzyme gene full length cDNA sequence of acquisition and its coding on NCBI websites
BLAST, as shown in Figure 3, the results showed that the sequence and cabbage mustard, colea, cabbage heart, Chinese cabbage, radish sulphur glycoside enzyme gene it is same
Source property is 98%, 97%, 96%, 96%, 95% more respectively.
On amino acid levels, amino acid sequence and cabbage mustard, colea, cabbage heart, Chinese cabbage, the trailing plants of tumorous stem mustard sulphur glycosides enzyme
Foretell the similitude for having 97%, 98%, 94%, 93%, 93% respectively, as shown in Figure 4.
Structure, screening and the expression of the recon of embodiment 5
1st, competence Escherichia coli DH10B is prepared：
- 80 DEG C of glycerine frozen strain DH10B are taken out, the ring of picking one is seeded on LB agar plates, and 37 DEG C were cultivated
Night.After bacterium colony is grown, picking monoclonal is into the test tube equipped with 3 mL LB nutrient solutions, 37 DEG C, and 250 rpm/h shakings are overnight.
5 mL bacterium solution is taken to add 500 mL LB nutrient solutions into test tube, again 3 h of shaking culture, when OD600 value is left for 0.3
When right, test tube is taken out into ice bath half an hour.Under the conditions of 4 DEG C, it is dispensed into 50 mL centrifuge tubes and centrifuges, remove supernatant, often pipe is thin
Bacterium 0.1M CaCl2Solution is resuspended, and is repeated once, you can for converting.Unnecessary thalline is dispensed into centrifuge tube, is added sterile
Glycerine is stored under the conditions of -72 DEG C immediately.
2nd, carrier pPIC9K-S preparation：
Small using Beijing Tiangeng company carries middle amount kit extraction pPIC9K-S plasmids, with the double enzymes of EcoR I and NotI
Cut, digestion system and reaction condition are as follows：
10×NEB Buffer3 5 μL
100×BSA 0.5 μL
EcoR I（20,000 units/mL ） 2 μL
NotI（10,000 units/mL） 2 μL
pPIC9K-S（175 ng/μL） 25 μL
ddH2O 15.5 μL
By above-mentioned well mixed reaction mixture, digestion is stayed overnight under the conditions of 37 DEG C, then enters the fragment after being identified
Row is recycled and is stored under conditions of -20 DEG C.
3rd, the structure of fusion protein expression vector and conversion：
Digestion expression vector hybridizes the sulphur glycoside enzyme gene that embodiment 4 is obtained with this step 2, hybridization system and reaction bar
Part is as follows：
The μ L of 10 × hybrid mixed liquid 5
The sulphur glycoside enzyme gene MYR1 that embodiment 4 obtains（25ng/μL） 6 μL
Step 2 digestion Expression vector pPIC9K-S（20ng/μL） 8 μL
ddH2O 1 μL
Above-mentioned reaction solution is well mixed, after 25 DEG C are reacted 30 minutes；By 10 μ L connection products and 200 μ L this reality
Apply the competent cell DH10B that a step 1 obtains slowly to mix, place half an hour, do not shake on ice；42 DEG C of heat shock 1min,
Plasmid is entered cell, put 2 min on ice immediately；800 μ L LB nutrient solutions are added, half an hour is shaken in 150rpm/h.
The cell of the above-mentioned conversions of 100-150 μ L is taken to be added on the LB flat boards of the resistance of benzyl containing ammonia, cell is uniformly spreadable, directly
To drying.Put upside down flat board, 37 DEG C are cultivated 18 hours；The single bacterium colony grown is cloned into 4 DEG C of preservations, in case examining.Checkout procedure passes through
PCR identifications are crossed, chooses positive colony bacterium, send DNA sequencing identifications.
4th, electricity conversion：
Correct positive colony is sequenced in step 3, the middle amount kit extracting μ g of plasmid 20 is carried with small, with Sal I single endonuclease digestion lines
Propertyization processing.
10×NEB Buffer3.1 50 μL
100×BSA 5 μL
Sal I（20,000 units/mL） 10 μL
pPIC9K-S-MYR1（219ng/μL） 90 μL
ddH2O 345 μL
37 DEG C of digestions are stayed overnight, and 10 μ L digestion products gel analysis are taken, to determine whether that digestion is complete.
Prepare competent cell SMD1168, YPD flat board and draw plate yeast strain SMD1168, culture puts 4 DEG C of refrigerators in 3 days, makes
Standby competent cell；Prepare 1 1.5 mL centrifuge tube ice bath, add the Pichia pastoris competent cell of 100 μ L concentrations
SMD1168,10 μ g DNA to be transformed（PPIC9K-S-MYR1 after Sal I single endonuclease digestion linearization process）（Volume is less than or equal to 10
μL）, mix, stand 5 min on ice.Mixture is transferred to electric shock bottom of a cup portion, need to gently strike to bottom, stand 3 min on ice；
Pulse conversion is done with 1.5kV, 25uF, 200 Ω, cuvette feeding electricity is turned in room, contacted until with two electrodes；Electricity turns one
Secondary, pulse parameter is time 5.5 ms, 1.5kV.Cuvette is removed, adds 1 ice-cold M sorbierites of 1 mL immediately, soft
Cell after dilution is moved into test tube, then gently pressure-vaccum mixes.
Auxotroph screening is carried out, 30 DEG C of culture 1h of cell, then applies MD plates again.150 μ L or so sorb is applied per plate MD
The transformed cells that alcohol is incubated, 30 μ LSMD1168 are taken to apply MD plates, 30 DEG C are cultivated 3-6 days, and incubator need to discharge water anti-dry.Plate puts 4 DEG C
Refrigerator preserves.
5th, hereditary enzyme element resistant transformants are screened：
1）Prepare 2 flat boards, the geneticin concentrations of one are 0.25mg/mL, and another geneticin concentrations are 2.0
Mg/mL, 3 mL aqua sterilisas are drawn in all HIS+On transformant flat board, HIS+ transformants are resuspended with pipette tips, cell suspension is turned
Move on in sterile centrifugation tube, slightly vortex 10S or so.With spectrophotometric determination concentration (1OD600=5 × 107Cell/mL),
10 are applied on every piece of YPD flat board containing Geneticin5Cell, anti-Geneticin, which is cloned 3 days or so, to be occurred.
6th, express：
Picking monoclonal to 3 mL YPD medium cultures, parallel two are managed.Under the conditions of 28 DEG C, 300 rpm/h shakings are arrived
OD600 values are between 2-5.Take out 2 mL and centrifuge 10 min (other 4 DEG C of bacterium solutions preserve for protect bacterium), collect cell, in removal
Clear liquid, cell is resuspended to 1 mL with BMMY, carries out induced expression.Every 24 hours, the final volume concentration for adding methanol to methanol was
0.5% to continue to induce（10 μ L or so, because having a small amount of wall built-up）, take 1mL to be centrifuged to centrifuge tube after inducing 72 h, take supernatant
Liquid.These samples are used to analyze expression.
6.1 SDS-PAGE are identified
The Supernatant samples after the 40 above-mentioned centrifugations of μ L are taken, add the μ L of 6 × loading buffer 10, heating of uncapping, concentration
Sample, then carry out 10%Tricine gel electrophoresis, coomassie brilliant blue staining.As a result as shown in figure 5, as shown in Figure 5, by 72
Hour culture after be found that soluble protein and be deposited at 90 KDa and a bright band occur, with the desired value of sulphur glycosides enzyme compared with
It coincide, and 6 clones are positive colony.
6.2 protect bacterium
The bacterial strain for selecting high expression draws plate culture 2-3 days, and picking colony is cultivated in YPD.Cell is collected, sweet containing 10%
The value that OD600 is suspended in the YPD of oil is 50-100, in -80 DEG C of storages.
6.3 extensive expression
PPIC9K-S-Myr1 glycerol stocks draw plate YPD flat boards, and 30 DEG C are cultivated 3-4 days；Picking monoclonal is trained to 3 mL YPD
Support and cultivated in base, parallel two pipe；28 DEG C, shaking culture（300 rpm/h）It is 2-6 or so to OD600 values；It is seeded to and contains 250 mL
In BMGY shaking flask, 28-30 DEG C of acutely vibration, it is 2-6 or so to survey OD600 values to exponential phase, collects cell, and centrifugation removes
Supernatant, cell is resuspended with BMMY and carries out induced expression.400 mL above-mentioned culture is added in each shaking flask, four layers of capping is gone out
Bacterium gauze, is put into shaking table culture.Methanol is added within second day often to induce add within 24 hours methanol to methanol afterwards to methanol final volume 1%
Final volume concentration is 1%, and 72 h collect supernatant.
The purifying of the fusion protein of embodiment 6 and activity identification
1st, purify：
With the level pad of 2 times of column volumes（20 mM TrisCl, 300 Mm NaCl, 20mM imidazoles, pH8.0）With
15 mL/min flow velocity balance Ni2+Post, ready to balance finish, and will mix up the zymotic fluid that pH is 8.0 in advance（Above-mentioned culture simultaneously induces
Purpose enzyme fermentation liquid after expression）With 10 mL/min flow velocity loading.Treat that loading finishes, 5-10 are eluted with level pad
Column volume uses elution buffer until outflow baseline values（20 mM TrisCL, 300mM NaCL, 300mM imidazoles,
pH8.0）Eluted, collect sulphur glycosides enzyme fusion proteins eluting peak, electrophoresis detection.As a result as shown in fig. 6, as seen from Figure 6, passing through
Fusion protein after purification has the band of a 90 KDa sizes on gel, can be further by fusion protein after purification
Measure activity.
2nd, determination of activity：
It is measured by following reaction system, the tumorous stem mustard sulphur glycosides enzyme of the sample enzyme for above-mentioned expression and after purification,
Standard enzyme is Sinigrin/ sinigrins；Measurement result is as shown in table 1：
The reaction reagent table of table 1
Table 1 Reaction reagent table
Above-mentioned reaction system is reacted in 45 DEG C of min of water-bath 10, and reaction immediately boils reaction solution in 100 DEG C after terminating
10 min are inactivated in water-bath, HPLC analyses are carried out after cooling, detect remaining sulphur resources.As a result as shown in fig. 7, by Tu Ke get,
Under the conditions of 45 DEG C, the crude protein expressed by recon can hydrolyze sulphur glycosides, show the bioactivity of sulphur glycosides enzyme, reach
Expected effect, and sample and standard sample are contrasted into discovery, the sulphur glycosides peak height of sample is the same as the sulphur for adding 0.5U enzyme standard items
Glycosides peak height approaches, therefore the sulphur glycosides specific activity of enzyme in sample is about 0.003 U/ μ L.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with
The present invention is described in detail good embodiment, it will be understood by those within the art that, can be to the technology of invention
Scheme is modified or equivalent substitution, and without departing from the objective and scope of technical solution of the present invention, it all should cover in this hair
Among bright right.
<110>Yangtze Normal University；
<120>A kind of tumorous stem mustard sulphur glycoside enzyme gene MYR1, tumorous stem mustard sulphur glycosides enzyme and its gene engineering expression method；
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1591
<212> DNA
<213>Artificial sequence
<220>
<223>SEQ ID NO.1 nucleotide sequence
<400> 1
gatgaagaaa tcacatgtga agaaaataac cctttcacat gttctaacac tgatattttg 60
tcctctaaaa acttcggtaa agattttatc ttcggagtcg cttcatccgc ataccaaatt 120
gaaggtggta gaggtcgtgg agttaatgtc tgggatggtt tctcccatcg gtatccagaa 180
aaggccggtt ccgatctgaa gaacggcgac acaacctgtg aatcttatac tcgttggcaa 240
aaggatgtcg atgttatggg tgaacttaac gccaccggat ataggttctc cttcgcctgg 300
tcccgtatca tccctaaggg taaagtcagc cgtggagtca accaaggtgg acttgattac 360
taccataaac ttatcgatgc actgctggaa aagaacatca ctccattcgt caccttattc 420
cattgggatc tgccacaaac actgcaagat gaatacgaag gtttccttga taggcaaatt 480
attcaagatt ttaaggatta cgctgatctg tgttttaagg aatttggagg taaagttaaa 540
cattggatca ctattaatca attgtacacc gtccctacga ggggatatgc tattgggacc 600
gatgccccag gtaggtgttc cccaatggtc gataccaagc atcgttgtta cggtggaaac 660
tcatccaccg aaccttatat tgttgcacat aaccaacttt tagctcatgc tacagttgtc 720
gatctgtata ggacaaagta caagttccaa aagggcaaaa tcggaccagt tatgatcaca 780
cgttggttct tgccatttga tgaatcagat ccagcctcta tcgaagccgc tgaacgtatg 840
aaccaattct ttcatggttg gtatatggaa cctttgacta agggacgtta cccagatatt 900
atgagacaaa tcgtcggcag ccgtctgcct aacttcaccg aagaagaagc agaattagtt 960
gccggttcct acgatttctt aggactgaac tactatgtca ctcaatacgc acaacctaag 1020
cctaacccat acccttcaga aacacataca gccatgatgg atgccggtgt caagctgacc 1080
tacgataact cccgcggaga atttctggga cctctgttcg tcgaagataa agtcaacgga 1140
aactcatact attaccctaa gggtatctac tatgtcatgg attactttaa gaccaagtac 1200
ggtgacccac tgatctacgt caccgaaaac ggtttctcca ccccatcctc cgaaaataga 1260
gaacaagcta tcgccgatta taagcgtatc gattacttgt gttcacattt gtgtttcctg 1320
cgtaaagtca tcaaggaaaa gggagttaat gtcaggggtt actttgcttg ggctctggga 1380
gataactacg agttttgtaa gggttttaca gttagattcg gactgtctta cgttaattgg 1440
gaggatctgg atgatcgtaa tctcaaggaa tccgggaaat ggtatcaacg tttcattaat 1500
ggtacagtca agaacgccgt caagcaagat ttcttgaggt cctccctgtc ctctcaatct 1560
caaaagaagc gtttcgcaga tgcttaagtt a 1591
<210> 2
<211> 528
<212> PRT
<213>Artificial sequence
<220>
<223>SEQ ID NO.2 amino acid sequence
<400> 2
DEEITCEENN PFTCSNTDIL SSKNFGKDFI FGVASSAYQI EGGRGRGVNV WDGFSHRYPE 60
KAGSDLKNGD TTCESYTRWQ KDVDVMGELN ATGYRFSFAW SRIIPKGKVS RGVNQGGLDY 120
YHKLIDALLE KNITPFVTLF HWDLPQTLQD EYEGFLHRQI IQDFKDYADL CFKEFGGKVL 180
HWITINQLYT VPTRGYAIGT DAPGRCSPMV DTKHRCYGGN SSTEPYIVAH NYLLAHATVV 240
DLYRTKYKFQ KGKIGPVMIT RWFLPEDESD PASIEAAERM NQFFHGWYME PLTKGRYPDI 300
MRQIVGSRLP NFTEEEAELV AGSYDFLGLN YYMTQYAQPK PNPYPSETHT AMMDAGVKLT 360
YDNSRGEFLG PLFVEDKVNG NSYYYPKGIY YVLDYFKTKY GDPLIYVTEN GFSTPSDENR 420
EQAIADYKRI DYLCSHLCFL RKVIKEKGVN VRGYFAWALG DNYEFCKGFT VRFGLSYVNW 480
EDLDDRNLKE SGKWYQRFIN GLVKNAVKQD FLRSSLSSQS QHKRFADA 528
<210> 3
<211> 49
<212> DNA
<213>Artificial sequence
<220>
<223>The nucleotide sequence of upper primer 1
<400> 3
gatccggttc tggcgaattc gacgacgacg acaaggatga agaaatcac 49
<210> 4
<211> 44
<212> DNA
<213>Artificial sequence
<220>
<223>The nucleotide sequence of upper primer 2
<400> 4
cgacgacgac aaggatgaag aaatcacatg tgaagaaaat aacc 44
<210> 5
<211> 51
<212> DNA
<213>Artificial sequence
<220>
<223>The nucleotide sequence of lower primer
<400> 5
ggcgaattaa ttcgcggccg cttattaagc atctgcgaaa cgcttctttt g 51
Claims (2)
1. a kind of tumorous stem mustard sulphur glycoside enzyme gene MYR1, it is characterised in that its amino acid sequence is the sequence shown in SEQ ID NO.2
Row, its nucleotides sequence are classified as the sequence shown in SEQ ID NO.1.
A kind of 2. gene engineering expression method of tumorous stem mustard sulphur glycosides enzyme, it is characterised in that comprise the following steps：
1）Tumorous stem mustard blade is taken, extracts total serum IgE；
2）With step 1）The total serum IgE of extraction is template, by reverse transcription reaction in PCR instrument, synthesizes the first chain cDNA；
3）Compare the nucleotide sequence of rape and leaf mustard sulphur glycoside enzyme gene, design a pair of forward and reverse primers, the primer is：
Upper primer 1：
5’- GATCCGGTTCTGGCGAATTCGACGACGACGACAAGGATGAAGAAATCAC -3’；
Upper primer 2：
5’- CGACGACGACAAGGATGAAGAAATCACATGTGAAGAAAATAACC-3’；
Lower primer：
5’-GGCGAATTAATTCGCGGCCGCTTATTAAGCATCTGCGAAACGCTTCTTTTG -3’；
4）With step 2）First chain cDNA of synthesis is template, using step 3）Designed primer, enter performing PCR reaction, amplification
Sulphur glycoside enzyme gene MYR1 full length sequence is obtained, the nucleotides sequence of the sulphur glycoside enzyme gene MYR1 is classified as shown in SEQ ID NO.1；
5）Extract carrier pPIC9K-S plasmids, and the plasmid described in EcoR I and NotI double digestions；
6）By step 4）The sulphur glycoside enzyme gene MYR1 of acquisition and step 5）Expression vector hybridization after double digestion, is recombinantly expressed
Carrier pPIC9K-S-MYR1；
7）By step 6）Recombinant expression carrier transformed competence colibacillus cell E. coli DH10B, by the cell after conversion in benzyl containing ammonia
Cultivate 18 hours in 37 DEG C on the LB flat boards of resistance, through PCR evaluation and screenings positive colony bacterium and be sequenced；
8）By step 7）Correct positive colony culture is sequenced, and extracts plasmid, using Sal I single endonuclease digestion linearization process, uses
Converted in Pichia pastoris SMD1168 electricity；
9）By the recombinant expression carrier pPIC9K-S-MYR1 of Sal I single endonuclease digestion linearization process, electricity is transformed into Pichia pastoris
SMD1168, coating MD flat boards are cultivated 3-6 days in 30 DEG C, and obtain geneticin resistant transformant by G418 resistances, screening；
10）The monoclonal of picking geneticin resistant transformant is to YPD medium cultures, under the conditions of 28 DEG C, with 300 rpm/h
Rotating speed shaking to OD600 values for 2-5 between, centrifuge abandoning supernatant, the resuspension of BMMY fluid nutrient mediums added into precipitation carefully
Born of the same parents；Expressed using methanol continuous induction, the final volume concentration of during which every 24 hours plus methanol to methanol is for 0.5% to continue to lure
Lead, after inducing 72 h, the expression of recombinant protein is analyzed and identified using the SDS-PAGE of coomassie brilliant blue staining.
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| WO2009106985A3 (en) * | 2008-02-27 | 2010-04-01 | University Of Copenhagen | Biosynthetic engineering of glucosinolates |
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| WO2009106985A3 (en) * | 2008-02-27 | 2010-04-01 | University Of Copenhagen | Biosynthetic engineering of glucosinolates |
Non-Patent Citations (3)
| Title |
|---|
| characterization of a brassica napus myrosinase pseudogene: myrosinases are members of the bga family of beta-glycosidases;lenman M. et al.;《plant mol biol.》;19930228;第21卷(第3期);463-474 * |
| uniprotkb-q00326(MYRO_BRANA);UNIPROT;《UNIPROT》;19921201;序列21-548 * |
| 西兰花硫代葡萄糖苷酶基因BoMyr2在毕赤酵母中的表达;梁锦锋等;《中国食品学报》;20100228;第68页左栏1.3-右栏1.7节 * |
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