CN104725485B - One kind restructuring active peptide and its synchronic preparation method - Google Patents
One kind restructuring active peptide and its synchronic preparation method Download PDFInfo
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- CN104725485B CN104725485B CN201410665096.3A CN201410665096A CN104725485B CN 104725485 B CN104725485 B CN 104725485B CN 201410665096 A CN201410665096 A CN 201410665096A CN 104725485 B CN104725485 B CN 104725485B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses one kind restructuring active peptide and its synchronic preparation method, the restructuring active peptide is superimposed by active peptide monomer or orienting series form, the active peptide monomer includes blood pressure lowering peptide monomer and anti-oxidation peptide monomer, its amino acid sequence such as sequence table SEQ ID NO：1.The nucleotide sequence such as sequence table SEQ ID NO of restructuring active peptide described in a kind of coding of the present invention：2.Cost of the present invention is low, and the active peptide in the recombinant polypeptide of acquisition can effectively be discharged by pepsin, trypsase and chymotrypsin, and obtained active peptide mixture has high antihypertensive activity and antioxidation activity in vivo.
Description
Technical field
The present invention relates to genetic engineering field, more particularly to a kind of restructuring active peptide and its synchronic preparation method.
Background technology
Biologically active peptide is typically contained in parent protein sequence, not having bioactivity, but by inner or in vitro
Protease or food processing processing can exercise physiological function in vivo after being released.At present, functional activity peptide mainly passes through
It is prepared by zymyhydrolyzed protein raw material.Existing many enzymatic isolation methods prepare the patent report of blood pressure lowering peptide and anti-oxidation peptide at present.
Relative biological extraction method, chemical degradation method and chemical synthesis, enzymatic isolation method have that Product Safety is high, working condition is gentle, right
Protein nutritive value destroys the advantage such as small.But the related activity peptide product occurred on domestic market substantially with
The relatively low complex peptides product form of activity occurs, and such as Soybean Peptide, corn peptide, collagen peptide, function-specific property is not strong, and produces
Product activity needs further to be improved.
The patent report of existing at present a large amount of " purifying highly pure active peptide by conventional separation means ", although researchers reflect
The some peptide fragments made show extremely strong physiologically active, such as IPP, VPP, IKW, LHP blood pressure lowering peptide and YHY, PHH, YKY,
The anti-oxidation peptides such as YPPAK, HDHPVC, but it is low due to containing the effective active peptide fragment ratio in parent protein sequence, cause enzyme
The low-yield in later stage and high separation costs are solved, it is most of high in addition to only a few active peptide segment (IPP, VPP etc.) has product appearance
Active function peptide does not obtain effective exploitation utilization.
With the development of technique for gene engineering, using DNA recombinant techniques, by the gene cloning of expression activity peptide to certain slightly
In biology or animal body, required peptides are directly given expression to by organism, its yield and purity can be greatly increased.At present, it is relevant
The patent report of " preparing active peptide using genetic engineering bacterium " is in the trend increased year by year.Although utilize technique for gene engineering system
Standby high activity small peptide has achieved technical breakthrough, but gene design, expression efficiency and the downstream separation of active peptide multimer
Technique needs further perfect.
In recent years, the research of Celluar and Molecular Biology shows in human body except with hypertensive substance and system
Outside, still with many endogenous depressor substances and system, to maintain the relatively stable of blood pressure.There is research to confirm blood pressure lowering peptide with resisting
Oxidation peptide has different action target spots, different interfering systems and different mechanism of action in vivo, but to hypertension and phase
The prevention and control for being concerned about vascular diseases have positive effect.Many researchs find that multiple efficacies agents are acted on than single
Component has more preferable effect, that is, synergy is presented, its act on may come from the effect of with different mechanism of action the factor it
Between obvious complementation or mutual repair be present.Therefore, if blood pressure lowering peptide and anti-oxidation peptide are united and applied in into the heart
The prevention and control of vascular diseases, it will play good effect.Existing many researchs report synchronously to be prepared by enzymatic isolation method
The relevant report of blood pressure lowering peptide and anti-oxidation peptide.But it there is no at present and " blood pressure lowering peptide synchronously prepared using genetic engineering bacterium and is resisted
The report of oxidation peptide ".
At present, a kind of high in-vivo activity, the restructuring active peptide and its synchronic preparation method of low cost are lacked.
The content of the invention
It is an object of the invention to provide a kind of high in-vivo activity, the restructuring active peptide and its synchronic preparation method of low cost.
Technical scheme is as follows：One kind restructuring active peptide, the restructuring active peptide is superimposed by active peptide monomer
Or orienting series form, the active peptide monomer includes blood pressure lowering peptide monomer and anti-oxidation peptide monomer, its amino acid sequence is such as
Sequence table SEQ ID NO：1.
The nucleotide sequence such as sequence table SEQ ID NO of restructuring active peptide described in a kind of coding of the present invention：2.
Further, described anti-oxidation peptide monomer have DTHK, YPIL, FLEPDY, YLEPFR, YLEPDY, YDEPEW,
HYRPFW, Y EPDY and IWAPFY；The blood pressure lowering peptide monomer have MRW, WIR, IRA, AMK, MKR, RGY, VAW, DGL, IPP,
IKP, IKPFR, IKPVA, AKF, IW, VAF, VSV, IQY and IVY.
Further, the N-terminal of the active peptide monomer segment peptide fragment is pepsin restriction enzyme site；The active peptide list
The C-terminal of body portion peptide fragment is pepsin or trypsase or chymotrypsin restriction enzyme site；The recombinant angiotensin-converting enzyme inhibitory
And in anti-oxidation peptide between part active peptide monomer by with pepsin or trypsase or chymotrypsin restriction enzyme site
Junction fragment be in series.
The synchronous method for preparing described restructuring active peptide of the invention, comprises the following steps：
(1) artificial synthesized sequence table SEQ ID NO：Gene shown in 2；
(2) structure can high efficient expression and purification of Recombinant blood pressure lowering peptide and the expression vector of anti-oxidation peptide；
(3) with expression vector conversion Host Strains structure genetic engineering bacterium；
(4) engineering bacterium expression restructuring active peptide is utilized；
(5) restructuring active peptide is obtained using label purifying and label removal technology；
(6) blood pressure lowering peptide and anti-oxidant is made using pepsin and/or trypsase and/or chymotryptic
Peptide.
Further, in step (2), the expression vector is pET28a, and Host Strains are E. coli BL21
(DE3)。
Further, in step (2), using DNA recombinant techniques, by sequence table SEQ ID NO：Gene shown in 2 is inserted
In HindIII the and XhoI sites for entering pET28a, and respectively in NcoI the and BamHI sites of the upstream of gene and BamHI and
HindIII sites are cloned into class elastin laminin purification tag ELPs genes and SUMO fusion tag genes, obtain recombination expression and carry
Body pET28a-ESA3.
Further, in step (3), and Transformed E .coli BL21 (DE3), obtain engineering bacteria；
In step (4), using IPTG induce engineering bacterium expression ELPs-SUMO-A3, collect thalline, sonicated cells,
Collect ultrasonic supernatant or inclusion body precipitation；
In step (5), for soluble-expression such as sequence table SEQ ID NO：Target protein shown in 1 directly enters rower
Label technology is purified, and the target protein of inclusion body expression is first carried out being denatured carrying out label technique purifying again with renaturation；Pass through
ELPs label purification techniques fusion proteins and using SUMO protease crackings and add salt centrifugation remove dyad fusion mark
ELPs-SUMO is signed, obtains restructuring active peptide；
In step (6), active peptide is recombinated using pepsin, trypsase and chymotryptic, ultrafiltration obtains
Active peptide is made in small peptide mixture, then desalination impurity elimination, freeze drying example, quality inspection.
Restructuring active peptide of the present invention is preparing the application in treating hypertension agents.
Beneficial effect：Cost of the present invention is low, and the active small peptide in the recombinant polypeptide of acquisition can be by pepsin, trypsase
And chymotrypsin effectively discharges, obtained active peptide mixture has high antihypertensive activity and antioxidation activity in vivo.This hair
The bright small peptide mixer collaboration of finished product can improve blood vessel inner skin cell function while decompression, and multi-angle prevention is cardiovascular with safeguarding
Health；Recombinant bacterium of the present invention can carry out industrial amplification by microbial fermentation, have broad application prospects.The present invention has
Following advantage：
(1) restructuring active small peptide mixture refers to produce using technique for gene engineering and bio-chemistry separation technical research small
Peptide, length have antihypertensive function and anti-oxidant work(inside potent for 2 to more than 10 individual amino acid, the small peptide mixer
Can, there is positive prevention and control effect to angiocardiopathy.
(2) the invention provides the new method of a kind of synchronous production blood pressure lowering peptide and anti-oxidation peptide and thus caused
The small peptide mixer being made up of high activity blood pressure lowering peptide and anti-oxidation peptide.Using DNA recombinant techniques, sent out by genetic engineering bacterium
It is prepared by ferment method.The method for providing the high activity blood pressure lowering peptide for being available for enzymolysis to discharge and anti-oxidation peptide series connection, is provided simultaneously
Designed active peptide Precursor Peptide and the optimization gene that expression can be stablized.Also providing clone has the oligonucleotides of encoding recombinant polypeptide
The expression vector pET28a-ESA3 of sequence and its recombination bacillus coli of conversion.
(3) the inventive method synchronously can effectively be enriched with high activity blood pressure lowering peptide and anti-oxidation peptide, improve active peptide yield, carry
High activity peptide unit mole action intensity；The enzymolysis liquid of recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide of the present invention is without purifying or through simple
Membrane filtration processing after can directly apply；The active peptide monomer in active peptide multimer in the present invention can enter as needed
Row is deleted, and the order of active peptide monomer can also be adjusted as needed.
(4) antihypertensive activity or the higher peptide fragment of antioxidation activity are applied in the present invention as junction fragment, such as this hair
Bright middle N-terminal is aromatic amino acid (F/Y), C-terminal is aromatic amino acid and C-terminal amino acids third from the bottom are Pro
Hexapeptide, such as FLEPDY, YLEPDY, YDEPEW, its C-terminal and N-terminal, which are easy to be cut by pepsin, to be discharged；Such as this hair
IER, VEK, IEK, IQER that bright middle N-terminal is aliphatic amino acid and C-terminal is basic amino acid, the C-terminal of the peptide
It is easy to the release of trypsase, while the N-terminal separate out by downstream close to active peptide.
(5) the part or all of active peptide monomer in recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide of the present invention can by pepsin,
One or more enzyme digestions release in trypsase, chymotrypsin, pancreatin.Finished product small peptide mixing produced by the present invention
Any peptide fragment caused by gastro-intestinal digestion enzyme enzymolysis can be used containing polypeptid acid sequence in thing, preferentially containing with good grounds claim
Active peptide monomer described in 3 and the peptide fragment with notable antihypertensive activity and/or antioxidation activity.The important step of the present invention relates to
And active peptide multimer and its design and optimization of corresponding gene, recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide expression vector and engineering bacteria
Structure, the expression and purifying of recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide, and in recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide activity drop
The enzymolysis of blood pressure peptide and anti-oxidation peptide discharges.
Brief description of the drawings
Fig. 1 is recombinant plasmid schematic diagram constructed by the present invention；
The recombinant plasmid PCR that Fig. 2 is the present invention identifies schematic diagram；
Fig. 3 is that the double digestion of the recombinant plasmid of the present invention identifies schematic diagram；
Fig. 4 is that SDS-PAGE identifies that recombinant protein inclusion body expresses schematic diagram；
Fig. 5 is that SDS-PAGE identifies recombinant protein purification schematic diagram；
Fig. 6 is that SDS-PAGE identifies that recombinant polypeptide A3 purifies schematic diagram；
Fig. 7 is that SDS-PAGE identifies recombinant protein solubility expression schematic diagram.
Embodiment
It will be specifically described, but be not to be construed as to the present invention by the way that specific embodiment is further to the present invention below
The restriction of protection domain.
Embodiment 1
As shown in Figures 1 to 7, Fig. 1 is recombinant plasmid schematic diagram constructed by the present invention；Fig. 2 is the recombinant plasmid of the present invention
PCR identifies schematic diagram, and swimming lane M is DL10000DNA standard specimens, and swimming lane 1,2,3 is same PCR primer (with recombinant plasmid
PET28a-ESA3 is template, and primer 5 '-TTCCTGGAACCGGA-3 ' and 5 '-CCAAGCCACACGA AAC-3 ' draw for upstream and downstream
Thing)；Fig. 3 is that the double digestion of the recombinant plasmid of the present invention identifies schematic diagram, and M is DNA Marker, and swimming lane 1-5 is respectively Nco I/
Bam HI, Bam HI/Hind III, Hind III/Xho I, Bam HI/Xho I, Nco I/Xho I double digestion products, swimming lane
6 be recombinant plasmid；Fig. 4 is that SDS-PAGE identifies that recombinant protein inclusion body expresses schematic diagram, and swimming lane M is standard protein sample, swimming lane 1,2
It is respectively recombinant bacterium ultrasonication stoste, ultrasonic supernatant ultrasound precipitation after inducing with 3；Fig. 5 is SDS-PAGE identification restructuring eggs
White purifying schematic diagram, M protein Marker, the fusion protein after 0 dialysis, swimming lane 1,2,3 be respectively final concentration 1.0,1.5,
The fusion protein of 2.0mol/L sodium chloride after purification；Fig. 6 is that SDS-PAGE identifies that recombinant polypeptide purifies schematic diagram, and swimming lane M is egg
White standard specimen, swimming lane 1 are products of the fusion protein Elps-SUMO-A3 after SUMO protease hydrolyzeds, and swimming lane 2 and 3 is respectively upper
State the centrifugation supernatant precipitation added in enzymolysis product after final concentration 1.0mol/L sodium chloride is saltoutd；Fig. 7 identifies for SDS-PAGE
Recombinant protein solubility expression schematic diagram；Swimming lane M is standard protein sample, and swimming lane 1,2 and 3 is respectively recombinant bacterium ultrasonication after inducing
Stoste, ultrasonic supernatant ultrasound precipitation；
A kind of restructuring active peptide of the present invention, the restructuring active peptide are to be superimposed by active peptide monomer or orienting series form
, the active peptide monomer includes blood pressure lowering peptide monomer and anti-oxidation peptide monomer, its amino acid sequence such as sequence table SEQ ID
NO：1.
The nucleotide sequence such as sequence table SEQ ID NO of restructuring active peptide described in a kind of coding of the present invention：2.
Described anti-oxidation peptide monomer has DTHK, YPIL, FLEPDY, YLEPFR, YLEPDY, YDEPEW, HYRPFW, Y
EPDY and IWAPFY；The blood pressure lowering peptide monomer have MRW, WIR, IRA, AMK, MKR, RGY, VAW, DGL, IPP, IKP, IKPFR,
IKPVA, AKF, IW, VAF, VSV, IQY and IVY.
The N-terminal of the active peptide monomer segment peptide fragment is pepsin restriction enzyme site；The active peptide monomer segment peptide
The C-terminal of section is pepsin or trypsase or chymotrypsin restriction enzyme site；The recombinant angiotensin-converting enzyme inhibitory and anti-oxidant
By the connection sheet with pepsin or trypsase or chymotrypsin restriction enzyme site between part active peptide monomer in peptide
Section is in series.
The synchronous method for preparing described restructuring active peptide of the invention, comprises the following steps：
(1) design and optimization of recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide and its encoding gene；
The artificial synthesized sequence table SEQ ID NO of the present invention：Gene shown in 2；Active peptide monomer in the present invention from
Screened in constructed active peptide database, which part active peptide needs artificially to be changed according to the design of complex function peptide precursor
Make.Total feature of screened active peptide is：Anti- stomach and intestine enzymic digestion；It is easy to the complete release of stomach and intestine enzyme；Lived with high vivo biodistribution
Property.Through preliminary screening, selected ace inhibitory peptide monomer is：MRW、WIR、IRA、AMK、MKR、RGY、VAW、DGL、IPP、IKP、
IKPFR, IKPVA, AKF, IW, VAF, VSV, IQY, IVY etc.；Selecting anti-oxidation peptide monomer is：DTHK、YPIL、FLEPDY、
YLEPFR, YLEPDY, YDEPEW, HYRPFW, Y EPDY, IWAPFY etc..
Premised on active peptide monomer is easy to the effectively release of gastro-intestinal digestion enzyme in theory, connected using direct head and the tail, or adopt
With suitable connection small peptide (2-3 amino acid residues, there is bioactivity), or by the difference containing part same amino acid residue
Active peptide is overlapped, and the targeted activity peptide orienting series screened are assembled into complex function peptide precursor restructuring active peptide, and
Utilize the design of protein structure prediction software and gene optimization software tip restructuring active peptide and its gene, it is intended to so that restructuring
It can realize that stability and high efficiency is expressed on active peptide gene theory in engineering bacteria cell.
The strategy based on more than, the amino acid sequence of recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide designed by the present invention are as follows：
The nucleotide sequence for encoding recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide is as follows：
(2) structure of recombinant expression carrier and recombinant bacterium；
As shown in figure 1, recombinant plasmid pET28a-ESA3 structure.It is using molecule clone technology, step (1) is designed
And the restructuring active peptide gene cloning optimized enters pET28a HindIII and XhoI sites, and respectively in restructuring active peptide upstream
NcoI and BamHI sites and BamHI and HindIII sites be cloned into class elastin laminin purification tag ELPs genes and SUMO
Fusion tag gene, recombinant expression carrier pET28a-ESA3, and Transformed E .coli BL21 (DE3) are built, obtains engineering bacteria.
As shown in Fig. 2 using recombinant plasmid pET28a-ESA3 as template ,-the TTCCTGGAACCGGA-3 ' and 5 ' of primer 5 '-
CCAAGCCACACGAAAC-3 ' is that upstream and downstream primer enters performing PCR, about 340bp PCR primer is obtained, with expected purpose fragment
The size for recombinating active peptide is close.
As a result recombinant plasmid confirms construction of recombinant plasmid success through further double digestion and sequencing identification.Structure can be high
Imitate the expression vector of expression and purification recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide；The expression vector is pET28a, and Host Strains are big
Enterobacteria E.coli BL21 (DE3).Using DNA recombinant techniques, by sequence table SEQ ID NO：Gene insertion shown in 2
In pET28a HindIII and XhoI sites, and respectively in NcoI the and BamHI sites of the upstream of gene and BamHI and
HindIII sites are cloned into class elastin laminin purification tag ELPs genes and SUMO fusion tag genes, obtain recombination expression and carry
Body pET28a-ESA3.
(3) with expression vector conversion Host Strains structure genetic engineering bacterium；And Transformed E .coli BL21 (DE3), obtain
Engineering bacteria；Induced expressions of the recombinant protein Elps-SUMO-A3 in E.coli BL21 (DE3)
E.coli BL21 (DE3) single bacterium for choosing the pET28a-ESA3 containing recombinant plasmid falls within the Kana that 20ml contains 50 μ g/ml
LB culture mediums (1%Tryptone, 0.5%Yeast extract, 1%NaCl, pH 7.0) in, 37 DEG C, 200rpm shaking tables training
Support overnight, after will be incubated overnight liquid and transferred by 2% inoculum concentration into the TB nutrient solutions (1.2% of the Kana containing 50 μ g/ml
Tryptone, 2.4%Yeast extract, 0.4%glycerol, 0.2%KH2PO4,1.6%K2HPO4) in, 37 DEG C,
200rpm, Shaking culture to thalline OD values reach 0.6-0.8, add IPTG to final concentration of 0.2mM, 37 DEG C, Fiber differentiation 5h
Afterwards, SDS-PAGE analyses are sampled, as Fig. 2 is learnt, engineering bacteria sample before relative induction, has a molecular weight about after Fiber differentiation 5h
45kDa protein band occurs, and recombinant protein Elps-SUMO-A3 indicated above successfully obtains in E.coli BL21 (DE3) to be melted
Close expression.Analyzed through gray scale scanning, the expression quantity of recombinant protein accounts for more than the 65% of total protein of cell.
(4) engineering bacterium expression restructuring active peptide is utilized；Elps-SUMO-A3 inclusion bodys prepare and renaturation
Culture gained bacterium solution is dispensed, 4000rpm centrifugations 5min obtains bacterial sediment, and 20ml is separately added into precipitation
PH 8.0PBS buffer solutions are resuspended, the ultrasonication 50min under 300W, work 2s, interval 8s, condition of ice bath of the bacterium solution after resuspension.
Broken liquid obtains the isometric 6mol/l urea resuspension of inclusion body precipitation, ice bath 10h in 4 DEG C, 8000rpm centrifugation 10min.4
DEG C, 8000rpm centrifugation 10min, retain supernatant.The supernatant after inclusion body redissolution is taken respectively at dialyzate 1, dialyzate 2, dialyzate
Ice bath gradient dialysis 6h (dialyzate Tris-HCl containing 10mmol/l pH 8.0,1mmol/l EDTA, 150mmol/l in 3
NaCl, 10% glycerine).The urea concentration of dialyzate 1 is 4mol/l, and the urea concentration of dialyzate 2 is 2mol/l, and the urea of dialyzate 3 is dense
Spend for 1mol/l.Dialysis terminates, and sample is centrifuged into 10min under the conditions of 4 DEG C, 8000rpm, reservation supernatant, i.e. renaturation obtain can
The fusion protein Elps-SUMO-A3 of dissolubility.
(5) recombinant protein Elps-SUMO-A3 is isolated and purified
Restructuring active peptide is obtained using label purifying and label removal technology；After dialysis, the restructuring egg after folding again
Also contain a small amount of foreign protein in white solution, need to be purified.The recombinant protein of above-mentioned preparation is due to containing Elps labels, therefore
It can use and add salt centrifugal purification.Dialysis gained protein solution is taken, the NaCl for adding final concentration of 1.5mol/l is well mixed after 30
Water-bath 10min under the conditions of DEG C, normal temperature centrifugation, retains precipitation.For soluble-expression such as sequence table SEQ ID NO：Mesh shown in 1
Mark albumen directly carries out label technique purifying, and the target protein of inclusion body expression is first carried out being denatured entering row label again with renaturation
Technology purifies；By ELPs label purification techniques fusion proteins and using SUMO protease crackings and salt centrifugation is added to remove
Dyad fusion tag ELPs-SUMO, obtain restructuring active peptide；
(6) active peptide is made using pepsin and/or trypsase and/or chymotryptic
Obtained using pepsin, trypsase and chymotryptic recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide, ultrafiltration
Blood pressure lowering peptide and anti-oxidation peptide is made in small peptide mixture, then desalination impurity elimination, freeze drying example, quality inspection.Added into precipitation etc.
4 DEG C of pH 8.0PBS buffer solutions of volume precooling are resuspended, and are centrifuged after ice bath 6h, and supernatant is purifying gained protein solution, is passed through
Centrifuge cycle can obtain the target protein of purity more than 95% twice.
A kind of coli strain for preparing bioactive peptide of the present invention, the bacterial strain is Escherichia coli；Preservation is entitled big
Intestines Escherichia Escherichia coli；It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center
(CGMCC), preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Preservation date：On October 24th, 2014；Preservation is compiled
Number：CGMCC No.9842.
Escherichia coli microbial inoculum prepared by Escherichia coli of the present invention.
Escherichia coli microbial inoculum of the present invention, its active component are at least one of following (a) (b) (c)：
(a) fermentation culture medium of the Escherichia coli described in claim 1；
(b) the ultrasonic degradation supernatant of claim 1 gained Bacillus coli cells；
(c) the ultrasonic degradation precipitation of claim 1 gained Bacillus coli cells.
The method that the present invention prepares the Escherichia coli microbial inoculum, comprises the following steps：
(1) bacterial strain single bacterium is fallen within Kana of the 20ml containing 50 μ g/ml LB culture mediums, temperature is 37 DEG C, stir speed (S.S.)
For 200rpm shaking table cultures 12-14h；The LB culture mediums are by mass percentage by 1%Tryptone, 0.5%Yeast
Extract, 1%NaCl, pH 7.0 component composition.
(2) nutrient solution to be transferred by 2% inoculum concentration in the TB nutrient solutions of the Kana containing 50 μ g/ml, temperature is 37 DEG C,
Stir speed (S.S.) is 200rpm；The TB nutrient solutions by mass percentage by 1.2%Tryptone, 2.4%Yeast extract,
0.4%glycerol, 0.2%KH2PO4And 1.6%K2HPO4Component composition.
(3) strain Escherichia coli culture to thalline OD values is reached 0.6-0.8 by shaking flask, and addition IPTG is to final concentration of
0.02-0.2mM, temperature are after 20-37 DEG C of Fiber differentiation time is 5-20h, sampling SDS-PAGE analyses, Escherichia coli bacterium are made
Agent.
Coli strain of the present invention is preparing the application in treating hypertension agents.
Embodiment 2
The difference of embodiment 2 and embodiment 1 is：According to step (1) in embodiment 1 and step (2) structure engineering bacteria
pET28a-ESA3/E.coli BL21(DE3).In the LB culture mediums that engineering bacteria single bacterium is fallen within to Kana of the 20ml containing 50 μ g/ml,
37 DEG C, 200rpm shaking table cultures are stayed overnight, after will be incubated overnight liquid and transferred by 2% inoculum concentration into the TB of the Kana containing 50 μ g/ml
In nutrient solution, 37 DEG C, 200rpm, Shaking culture to thalline OD values reaches 0.6-0.8, adds IPTG to final concentration of 0.02mM,
After 20 DEG C of Fiber differentiation 20h, sampling SDS-PAGE analyses.
As Fig. 4 is learnt, engineering bacteria sample before relative induction, has after Fiber differentiation 20h under the conditions of the low inducer concentrations of low temperature
One molecular weight about 45kDa protein band occurs；After sonicated cells, the Soluble expression levels of target protein reach 60%
More than.Ultrasonication supernatant is taken, the NaCl for adding final concentration of 1.5mol/l is well mixed the water-bath under the conditions of 30 DEG C
10min, normal temperature centrifugation, retains precipitation.4 DEG C of pH 8.0PBS buffer solutions that isometric precooling is added into precipitation are resuspended, ice bath 6h
After centrifuge, supernatant is purifying gained protein solution, and the target of purity more than 95% can be obtained by centrifuge cycle twice
Albumen.
Embodiment 3
The difference of embodiment 3 and embodiment 1 is：Recombinate preparation and its activity identification of small peptide mixer.Take appropriate system
The ELPs-SUMO-A3 of standby gained, SUMO protease, 30 DEG C of enzymolysis 5h are added according to 3% addition.After enzymolysis terminates, to enzyme
Solve the sodium chloride that final concentration 1.5mol/l is added in liquid, after 30 DEG C of water-bath 10min, 8000r/min centrifugation 10min, retain supernatant
Liquid, as recombinate active peptide.In-vitro simulated human body natural's physiology digestion process, by 2mg/ml restructuring activity peptide solution hydrochloric acid
PH to 2.0 is adjusted, adds 2% pepsin, 4h is hydrolyzed at 37 DEG C, boiling water bath terminating reaction, pH to 7.0 is adjusted, takes partial hydrolysis
Liquid determines peptide activity.The pancreatin (or trypsase and chymotrypsin protein enzymatic mixture) of addition 2% in remaining hydrolyzate, 37
DEG C hydrolysis 4h, boiling water bath terminating reaction.Pepsin hydrolysis liquid (H1)；Pancreatin hydrolysis liquid (H2)；Complex enzyme for hydrolyzing liquid (H3).Through
External ACE inhibitory activity and antioxidation activity detection, as a result show polymer recombinate active peptide gastro-intestinal digestion enzyme hydrolyzate H1,
H2 and H3 shows extremely strong ACE inhibitory activity and antioxidation activity, wherein, hydrolyzate H2 ACE suppresses IC50In 1.0 μ
Below g/ml, SHR zoopery also confirm that stomach and intestine enzyme hydrolyzate H1, H2 and H3 of restructuring active peptide are respectively provided with significant decompression effect
Should.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, the present invention
Claimed scope is by appended claims, specification and its equivalent thereof.
Claims (9)
1. one kind restructuring active peptide, it is characterised in that：It is described restructuring active peptide be superimposed by active peptide monomer or orienting series and
Into, the active peptide monomer includes blood pressure lowering peptide monomer and anti-oxidation peptide monomer, its amino acid sequence such as sequence table SEQ ID
NO ：1.
A kind of 2. nucleotide sequence such as sequence table SEQ ID NO of the restructuring active peptide encoded described in claim 1：2.
3. the restructuring active peptide according to claim 1, it is characterised in that：Described anti-oxidation peptide monomer have DTHK,
YPIL, FLEPDY, YLEPFR, YLEPDY, YDEPEW, HYRPFW, YEPDY and IWAPFY；The blood pressure lowering peptide monomer has
MRW, WIR, IRA, AMK, MKR, RGY, VAW, DGL, IPP, IKP, IKPFR, IKPVA, AKF, IW, VAF, VSV, IQY and
IVY。
4. the restructuring active peptide according to claim 1, it is characterised in that：The N of the active peptide monomer segment peptide fragment
End is pepsin restriction enzyme site；The C ends of the active peptide monomer segment peptide fragment be pepsin or trypsase or
Chymotrypsin restriction enzyme site；By with stomach egg between the active peptide monomer of part in the recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide
White enzyme or the junction fragment of trypsase or chymotrypsin restriction enzyme site are in series.
5. the preparation method of a kind of recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide, its feature exists, in comprising the following steps：
（1）Artificial synthesized sequence table SEQ ID NO：Gene shown in 2；
（2）Structure can high efficient expression and purification of Recombinant blood pressure lowering peptide and the expression vector of anti-oxidation peptide；
（3）With expression vector conversion Host Strains structure genetic engineering bacterium；
（4）Active peptide is recombinated using the engineering bacterium expression；
（5）Restructuring active peptide is obtained using label purifying and label removal technology；
（6）Blood pressure lowering peptide and anti-oxidant is made using pepsin and/or trypsase and/or chymotryptic
Peptide.
6. the preparation method of the recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide according to claim 5, it is characterised in that：In step
（3）In, the expression vector is pET28a, and Host Strains are E. coli BL21 (DE3).
7. the preparation method of the recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide according to claim 6, it is characterised in that：In step
(2)
In, using DNA recombinant techniques, by sequence table SEQ ID NO：Gene shown in 2 inserts pET28a'sHindIII
WithXhoIn I sites, and respectively in the upstream of geneNcoI andBamHI sites andBamHI andHinDIII sites gram
It is grand enter class elastin laminin purification tag ELPs genes and SUMO fusion tag genes, obtain recombinant expression carrier pET28a-
ESA3。
8. the preparation method of the recombinant angiotensin-converting enzyme inhibitory and anti-oxidation peptide according to claim 6, it is characterised in that：
In step (3), and Transformed E .coli BL21 (DE3), obtain engineering bacteria；
In step (4), engineering bacterium expression ELPS-SUMO-A3 is induced using IPTG, collects thalline, ultrasonication is thin
Born of the same parents, collect ultrasonic supernatant or inclusion body precipitation；
In step (5), for soluble-expression such as sequence table SEQ ID NO：Target protein shown in 1 is directly carried out
Label technique is purified, and the target protein of inclusion body expression is first carried out being denatured carrying out label technique purifying again with renaturation；Pass through
ELPs label purification techniques fusion proteins and using SUMO protease crackings and add salt centrifugation remove dyad fusion
Label
ELPs-SUMO, obtain restructuring active peptide；
In step (6), active peptide is recombinated using pepsin, trypsase and chymotryptic, ultrafiltration obtains small
Active peptide is made in peptide mixture, then desalination impurity elimination, freeze drying example, quality inspection.
9. according to claim 1, the restructuring active peptide described in 3 any one is preparing the application in treating hypertension agents.
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| WO1999016461A1 (en) * | 1997-09-26 | 1999-04-08 | Calpis Co., Ltd. | Antistress agents and functional foods |
| CN103709253A (en) * | 2013-12-20 | 2014-04-09 | 南开大学 | Method for preparing GLP-1 (Glucose-Like Peptide) and analogue thereof by biological synthesis |
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| WO1999016461A1 (en) * | 1997-09-26 | 1999-04-08 | Calpis Co., Ltd. | Antistress agents and functional foods |
| CN1279614A (en) * | 1997-09-26 | 2001-01-10 | 卡尔皮斯株式会社 | Antistress agents and functional foods |
| CN103709253A (en) * | 2013-12-20 | 2014-04-09 | 南开大学 | Method for preparing GLP-1 (Glucose-Like Peptide) and analogue thereof by biological synthesis |
| CN103804466A (en) * | 2013-12-20 | 2014-05-21 | 广西大学 | Decolorizing method of silkworm chrysalis proteolysis antioxidative peptide |
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