CN108277232A - A kind of Se-enriched yeast and preparation method thereof of ease constipation function - Google Patents
A kind of Se-enriched yeast and preparation method thereof of ease constipation function Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A61K36/06—Fungi, e.g. yeasts
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- A61K36/064—Saccharomycetales, e.g. baker's yeast
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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Abstract
The present invention relates to a kind of Se-enriched yeasts and preparation method thereof of ease constipation function.In particular it relates to can be with the engineered Saccharonayces yeast and its construction method of secreting, expressing superoxide dismutase and its purging and intestine lubricating action.The present invention has constructed secretion expression carrier using the secretion signal peptide sequence of saccharomyces cerevisiae mating factor (MF) α with copper-zinc superoxide dismutase (hCu/Zn SOD) cDNA, realize clones and secreting, expressing of the hCu/Zn SOD in saccharomyces cerevisiae, constructed engineering bacteria can within the scope of human temperature the direct active exogenous hCu/Zn SOD of secreting, expressing, expression product may be directly applied to the fields such as medicine, health care of food, play the functions such as its ease constipation.The gene engineering product that the success of Yeast engineering bacterium strain of the present invention is configured to exploitation hCu/Zn SOD is had laid a good foundation, and is had a extensive future.
Description
Technical field
The invention belongs to genetic engineering field, be related to can with the engineered Saccharonayces yeast of secreting, expressing superoxide dismutase and
Its construction method and its application in preparing active ease constipation product.
Background technology
Superoxide dismutase (SOD) is a kind of metalloenzyme being widely present in animal, plant, microorganism, by its knot
The metal ion of conjunction can be divided into tri- kinds of Cu/Zn-SOD, Mn-SOD, Fe-SOD, their major catalytic superoxide anions are free
Disproportionated reaction occurs for base, generates oxygen and hydrogen peroxide.Superoxide dismutase can defend oxygen toxicity, enhancing body radioresistance damage
Hinder ability, can also be pre- anti-aging, there is good curative effect to some diseases such as tumour, inflammation and autoimmune disease etc..Pass through
Content, structure and the function of SOD helps to establish parasite immunity diagnosis index and find special in research parasite and host
The opposite sex inhibits the drug of polypide relevant enzyme.Wherein, Cu/Zn-SOD (SOD1) distributions are most wide, account for 90% or more of total SOD, it is
A kind of enzyme of generally existing, familial amyotrophic lateral sclerosis (FALS), Parkinson's disease (PD), dengue fever, cancer,
There is important physiologic meaning and treatment potentiality in Down ' s syndromes, cataract and a variety of neurological disorders syndromes.
Natural SOD limited sources, and have a foreign protein immunogenicity, external source SOD are not easy to be received by human body etc., make its
Application aspect is very limited, and the SOD prepared with technique for gene engineering is to set up in a wide range enzyme source, reduce cost and obtain no antigen
People source SOD effective way.Currently, external carried out a series of basic research, application and development and clinical test to SOD,
Mainly the research of the purifying including SOD, physicochemical property, clinical efficacy etc. is currently, the SOD of Bacillus coli expression passes through purifying
It is mainly used in some drugs, if applied in health care of food product, SOD products have not easy to maintain, easy inactivation
Or the problem that activity is relatively low.According to retrieval, there are no the reports that the saccharomyces cerevisiae that can express SOD is directly applied to food.
Invention content
The technical problem to be solved in the present invention is to provide a kind of Se-enriched yeast and preparation method thereof with purging and intestine lubricating action, institute
The engineering bacteria of structure can within the scope of human temperature the direct active exogenous hCu/Zn-SOD of secreting, expressing, expression product can
The fields such as medicine, health care of food are directly applied to, the functions such as its ease constipation are played.
Technical scheme is as follows：
One of present invention purpose is to provide a kind of suitable for the secreting, expressing superoxide dismutase in saccharomyces cerevisiae
SOD- saccharomyces cerevisiae recombinant expression carriers.
The recombinant expression carrier is the recombination wine for carrying saccharomyces cerevisiae MF- alpha signals peptide gene and hCu/Zn-SOD genes
Brewer yeast secretion expression carrier.
The carrier that sets out for building recombinant Saccharomyces cerevisiae secretion expression carrier can be that any one can be in saccharomyces cerevisiae
The carrier for expression of eukaryon of secreting expression of exogenous gene, such as pJK3, pMD18-T, pUC19, pET22b (+), preferably pUC19.
It is that the set out recombinant Saccharomyces cerevisiae secretion expression carrier of vector construction is named as pUC19-MS with pUC19.Its nucleosides
In acid sequence such as sequence table shown in sequence 4.
The construction method of the recombinant Saccharomyces cerevisiae secretion expression carrier pUC19-MS, it may include following steps：
1) saccharomyces cerevisiae MF- alpha signal peptide genes are expanded：With saccharomyces cerevisiae (Saccharomycescerevisiae) bacterial strain
The cellular lysate liquid of INVSC1 is template, in primer T1：
5'-GGGGTACCATGAGATTTCCTTCTATTTTTACTGC-3'(restriction enzyme sites containing KpnI) and T2：
5'-CGGGATCCTCTTTTATCCAAAGAAACACCT-3'(restriction enzyme sites containing BamHI) guiding under PCR amplification
MF- alpha signal peptide genes, in nucleotide sequence such as sequence table shown in sequence 1；
2) hCu/Zn-SOD genes are expanded：The total mRNA of people's gastric tissue is extracted, then RT-PCR amplifies its total cDNA conduct
PCR templates, in primer I 1：5'-CGGGATCCGCGACGAAGGCCGTGTGCGTGCT-3'(restriction enzyme sites containing BamHI) and
I2：The sites containing XbaI enzyme cutting 5'-GCTCTAGAGAATGTTTATTGGGCGATCC-3'() guiding under PCR amplification hCu/Zn-
Sod gene, in nucleotide sequence such as sequence table shown in sequence 2；
3) structure cloning vector pUC-MS：Saccharomyces cerevisiae MF- alpha signals peptide gene is connected with hCu/Zn-SOD genes into gram
In grand carrier pUC19, the cloning vector pUC-MS for carrying saccharomyces cerevisiae MF- alpha signals peptide gene and hCu/Zn-SOD genes is obtained,
The nucleotide sequence of hCu/Zn-SOD fusions (being made of saccharomyces cerevisiae MF- alpha signals peptide gene and hCu/Zn-SOD genes)
As shown in sequence 3 in sequence table；
3) recombinant Saccharomyces cerevisiae secretion expression carrier is built：By cloning vector pUC-MS and carrier pUC19 through KpnI and
After XbaI carries out double digestion, saccharomyces cerevisiae MF- alpha signals peptide gene is connected with hCu/Zn-SOD genes into saccharomyces cerevisiae secreting type
In expression vector pUC19, the recombinant Saccharomyces cerevisiae secretion expression carrier pUC19- for secreting, expressing hCu/Zn-SOD is obtained
MS, in nucleotide sequence such as sequence table shown in sequence 4.
Another object of the present invention is that providing the SOD- saccharomyces cerevisiaes recombinant expression carrier is preparing active ease constipation food
Or the application in pharmaceutical products.This application for use the SOD- saccharomyces cerevisiaes recombinant expression carrier transformed saccharomyces cerevisiae, and will turn
Change bacterium is added in food or drug as active constituent and active food or pharmaceutical products is prepared.
It is a further object to provide it is a kind of can secreting, expressing superoxide dismutase engineered Saccharonayces yeast, be
Conversion has the SOD recombinant Saccharomyces cerevisiaes of SOD- saccharomyces cerevisiae recombinant expression carriers.
The saccharomyces cerevisiae is the Wine brewing yeast strain suitable for protein expression, such as INVSC1.
Can be common method for transformation in bioengineering field by the method for saccharomyces cerevisiae expression transformed saccharomyces cerevisiae,
The protoplast transformation that such as Li-acetate method, heat shock method, electrotransformation, engagement conversion method PEG are mediated.
Using INVSC1 as starting strain, the conversion of structure has the engineering of recombinant Saccharomyces cerevisiae secretion expression carrier pUC19-MS
Saccharomyces cerevisiae is named as pUC19-MS/INVSC1.
The method of the present invention also provides a kind of in saccharomyces cerevisiae secreting, expressing superoxide dismutase, is included in culture
The above-mentioned conversion of fermentation has recombinant Saccharomyces cerevisiae secretion expression carrier (such as pUC19-MS/ in base (such as YPD culture mediums)
INVSC1), the process of the superoxide dismutase of high activity is obtained.
Ferment recombinant Saccharomyces cerevisiae when need to be added derivant galactolipin, a concentration of 20g/L of derivant galactolipin be added,
Inducing temperature is 30 DEG C, and induction time is 24-48 hours.
Another object of the present invention is to provide engineered Saccharonayces yeast the answering in preparing active food or pharmaceutical products
With.Food or medicine is added as active constituent in the engineered Saccharonayces yeast (also referred to as " SOD recombinant Saccharomyces cerevisiaes ") when the application
Active food or pharmaceutical products are prepared in product product.
In the active food or pharmaceutical products, the SOD active matrixes that the SOD recombinant Saccharomyces cerevisiaes secretion generates also are
Active constituent therein.
Engineered Saccharonayces yeast (for example, recombinant Saccharomyces cerevisiae pUC19-MS/INVSC1) described above is preparing for food
Or the application in the SOD active matrixes in pharmaceutical products also belongs to the content of present invention.
The SOD active matrixes are to lure the engineered Saccharonayces yeast (such as pUC-MS/INVSC1) in YPD galactolipins
It leads and carries out 30 DEG C of cultures in culture medium, logarithmic phase (about 16-18h, OD are grown in the saccharomyces cerevisiae:1.3-1.5), warp
Westernblot verifies SOD expression, obtains SOD active matrixes；The YPD galactolipins inducing culture is by following components group
At aqueous solution：1% yeast extract by weight, 2% peptone, 2% glucose；A concentration of 20g/L of galactolipin.
The present invention utilizes the secretion signal peptide sequence of saccharomyces cerevisiae mating factor (MF) α to be disproportionated with copper-zinc superoxide for the first time
Enzyme (hCu/Zn-SOD) cDNA has constructed secretion expression carrier, realize clones of the hCu/Zn-SOD in saccharomyces cerevisiae and
Secreting, expressing proves that saccharomyces cerevisiae engineered yeast strain is expressed with the innate immunity by Western blot analysis
The hCu/Zn-SOD of originality；SOD activity is measured using negative staining positioning mode, is found by comparing activity gel, constructed engineering
Bacterium in addition to expressing the SOD of its own, can also within the scope of human temperature the direct active exogenous hCu/Zn- of secreting, expressing
SOD, expression product may be directly applied to the fields such as food or drug, play the functions such as its ease constipation, Yeast engineering bacterium strain of the present invention
Success be configured to exploitation hCu/Zn-SOD gene engineering product have laid a good foundation, have a extensive future.
Description of the drawings
Fig. 1 is the schematic diagram of carrier pUC19.
Specific implementation mode
The present invention is further elaborated with reference to embodiment.These embodiments be only for illustrative purposes,
And do not limit the scope of the invention and essence.
Method therefor is conventional method unless otherwise instructed in following embodiments, and specific steps can be found in：
《MolecularCloning:ALaboratoryManual》(Sambrook, J., Russell, DavidW.,
MolecularCloning:ALaboratoryManual, 3rdedition, 2001, NY,
ColdSpringHarbor)。
The percent concentration is mass/volume (W/V) percent concentration or volume/volume (V/ unless otherwise instructed
V) percent concentration.
The acquirement approach of various biomaterials described in embodiment be only to provide it is a kind of experiment obtain approach with
Reach specifically disclosed purpose, the limitation to biological material source of the present invention should not be become.In fact, used biomaterial
Source be it is extensive, it is any keep on the right side of the law the biomaterial that can be obtained with moral ethics can be according in embodiment
Prompt is replaced.
The primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Embodiment is being implemented down based on the technical solution of the present invention, gives detailed embodiment and specific
Operating process, embodiment will be helpful to understand the present invention, but protection scope of the present invention is not limited to following embodiments.
The structure of embodiment 1, SOD- saccharomyces cerevisiae recombinant expression carriers
Illustrate that SOD- saccharomyces cerevisiaes recombinant expression carries for building recombinant Saccharomyces cerevisiae secretion expression carrier pUC19-MS
The building process of body.
As shown in Figure 1, being the vector construction recombinant Saccharomyces cerevisiae secretion expression carrier pUC19-MS that sets out with pUC19, specifically
Construction method may include following steps：
1) saccharomyces cerevisiae MF- alpha signal peptide genes are expanded：With saccharomyces cerevisiae (Saccharomycescerevisiae) bacterial strain
The cellular lysate liquid of INVSC1 (being purchased from Invitrogen companies) is template, in primer T1：
5'-GGGGTACCATGAGATTTCCTTCTATTTTTACTGC-3'(restriction enzyme sites containing KpnI) and T2：
5'-CGGGATCCTCTTTTATCCAAAGAAACACCT-3'(restriction enzyme sites containing BamHI) guiding under PCR amplification
MF- alpha signal peptide genes, PCR reaction systems are：Template 1-100ng, 0.5 μM of primer 1 (10 μM), 0.5 μM of primer 2 (10 μM),
DNTP (10mM) 1mM, polymerase 5-10U, 10 times of dilutions of polymerase buffer (10*), 25 microlitres of systems, deficiency supply institute with water
Need volume.PCR reaction conditions are：94 DEG C of pre-degeneration 10min；94 DEG C denaturation 30s, 50 DEG C annealing 30s, 72 DEG C extension 40s, 35
Cycle；Last 72 DEG C of extensions 10min.After reaction, 1% agarose gel electrophoresis detection, warp are carried out to pcr amplification product
PCR amplification obtains the saccharomyces cerevisiae MF- alpha signal peptide genes of 255bp, is consistent with expected results.
The saccharomyces cerevisiae MF- alpha signal peptide genes of 255bp connect to (structure of carrier refers into pMD18-T carriers《Molecule
Clone》) in, recombinant vector is converted into bacillus coli DH 5 alpha competent cell, sequencing, sequencing knot are carried out to recombinant bacterial strain
Fruit shows to obtain the correct saccharomyces cerevisiae MF- alpha signal peptide genes of sequence through PCR amplification, in nucleotide sequence such as sequence table
Shown in sequence 1.
2) hCu/Zn-SOD genes are expanded：
The total mRNA of people's gastric tissue is extracted with the RneasyMinikit of QIAGEN companies, then uses CASsuperTWO-
STEPRT-PCRkit (being purchased from TIANGEN Biotech (Beijing) Co., Ltd.) amplifies its total cDNA, then using total cDNA as PCR
Template, in primer I 1：
5'-CGGGATCCGCGACGAAGGCCGTGTGCGTGCT-3'(restriction enzyme sites containing BamHI) and primer I 2：
5'-GCTCTAGAGAATGTTTATTGGGCGATCC-3'(III restriction enzyme sites containing Hind) guiding under PCR amplification
HCu/Zn-SOD genes, PCR reaction systems are：Template 1-100ng, 0.5 μM of primer 1 (10 μM), 0.5 μM of primer 2 (10 μM),
DNTP (10mM) 1mM, polymerase 5-10U, 10 times of dilutions of polymerase buffer (10*), 25 microlitres of systems, deficiency supply institute with water
Need volume.PCR reaction conditions are：94 DEG C of pre-degeneration 10min；94 DEG C denaturation 30s, 50 DEG C annealing 30s, 72 DEG C extension 40s, 35
Cycle；Last 72 DEG C of extensions 10min.After reaction, 1% agarose gel electrophoresis detection, warp are carried out to pcr amplification product
PCR amplification obtains the hCu/Zn-SOD genes of 465bp, is consistent with expected results.Again by the hCu/Zn-SOD genes of 465bp
It connects in pMD18-T carriers (being purchased from Invitrogen companies), recombinant vector conversion bacillus coli DH 5 alpha competence is thin
Born of the same parents carry out sequencing to recombinant bacterial strain, and sequencing result shows to obtain the correct hCu/Zn-SOD bases of sequence through PCR amplification
Cause, in nucleotide sequence such as sequence table shown in sequence 2.
3) structure cloning vector pUC-MS：Saccharomyces cerevisiae MF- alpha signals peptide gene is connected with hCu/Zn-SOD genes into gram
In grand carrier pUC19 (being purchased from Invitrogen companies), obtain carrying saccharomyces cerevisiae MF- alpha signals peptide gene and hCu/Zn-SOD
The cloning vector of gene, is named as pUC-MS.Linked system is：Carrier pUC19, MF- alpha signal peptide gene and hCu/Zn-SOD bases
Because of totally 10.0 μ L, ddH2O7.0 μ L, 10 × buffer solution, 2.0 μ L, T4 ligase 1.0 μ L, 20.0 μ L of total volume.Condition of contact is 37
DEG C reaction 2h.
4) structure recombinant Saccharomyces cerevisiae secretion expression carrier pUC19-MS：
Above-mentioned cloned plasmids pUC-MS and plasmid pET22b (+) passes through digestion respectively, and hCu/Zn-SOD cDNA recombinations are arrived
In secreting, expressing type carrier pET22b (+) under the control of T7 promoters, prokaryotic secretion expression plasmid pETSOD is constructed, through digestion
Nucleotide sequence such as sequence table in shown in sequence 3；
By cloning vector pUC-MS and E. coli-yeast shuttle plasmid pYES2.0 (being purchased from Invitrogen companies) warp
After KpnI and XbaI carries out double digestion, eukaryotic expression system plasmid Pyes-MS, such as sequence table of the nucleotide sequence through digestion are built
Shown in middle sequence 4.
5) induced expressions of the SOD in saccharomyces cerevisiae：
It converts the pETSOD built expression to e. coli bl21 (DE3) competent cell, is induced with IPTG
PETSOD is expressed, and 15%SDS-PAGE electrophoresis, protein immunoblotting and NBT are used also after then taking 1ml bacterium solution ultrasonications
Former method vital staining carries out positioning analysis.
Above-mentioned SOD yeast is cultivated with fermented tea in the culture medium rich in selenium and is co-cultured, finally generates one group
By SOD saccharomycete, thalline that acetic acid bacteria and lactic acid bacteria collectively constitute.This thalline compound is lyophilized, is chilled to minus 20 DEG C,
Then final step ultramicro grinding is carried out, powder is obtained.
The carrier that sets out for building recombinant Saccharomyces cerevisiae secretion expression carrier can be that any one can be in saccharomyces cerevisiae
The carrier for expression of eukaryon of secreting expression of exogenous gene can also be pMD18-T, pJK3, pET22b (+) etc., sheet in addition to pUC19
Field technology personnel can refer to above step process and obtain a variety of SOD- saccharomyces cerevisiaes recombinant expression carriers, not go to live in the household of one's in-laws on getting married one by one herein
It states.
The structure of embodiment 2, SOD recombinant Saccharomyces cerevisiaes
It can illustrate SOD for the engineered Saccharonayces yeast pUC-MS/INVSC1 of secreting, expressing superoxide dismutase by structure
The building process of recombinant Saccharomyces cerevisiae.
With INVSC1 (being purchased from Invitrogen companies) for starting strain, structure conversion has recombinant Saccharomyces cerevisiae secreting, expressing
The engineered Saccharonayces yeast of carrier pUC19-MS, method for transformation are Li-acetate method, and concrete operations can be：
1, a ring saccharomycete is chosen to be inoculated in 5mLYPD culture mediums (by taking INVSC1 as an example) (composed of the following components is water-soluble
Liquid：By weight, 1% yeast extract, 2% peptone, 2% glucose) in, 30 DEG C, 200rpm overnight incubations obtain first order seed；
2,1mL first order seeds are taken to be inoculated in respectively in two bottles of 50mLYPD culture mediums, 30 DEG C, 250-300rpm cultures about 5h
(OD:0.5-0.8), bacterium solution is obtained；
3, two bottles of bacterium solutions are merged, thalline is collected by centrifugation to obtain in 4 DEG C, 5000rpm, 5min；
4, twice, centrifugal condition is identical as step 3 for 25mL ice sterile water washing thalline；
5,1mL0.1M lithium acetates are added into thalline after washing to suspend, then thalline were collected by centrifugation；
6, EP pipes are sub-packed in 50 μ l/ pipes with 0.5mL0.1M lithium acetates suspension thalline (thalline is competent cell) again
In, it is placed in spare on ice；
7,240 μ l of PEG3350 solution, the 1MLiCl solution 36 that 50wt% is added in 50 μ l competent cells are stated upwards in turn
μ l, 50 μ l of conversion DNA (5-10 μ g, that is, the plasmid pUC19-MS built) solution；Violent vortex mixing is until precipitation thalline is complete
Super distributed is uniformly (about 1min)；
8, mixing thalline is incubated 30min in 30 DEG C of water-baths；Again in 42 DEG C of water-bath heat shock 20-25min；
9,13000rpm centrifuges 30s collection precipitations and obtains thalline, is resuspended in 1mlYPD culture mediums, and 30 DEG C of shaking tables are incubated
4h；
10, precipitation thalline is collected by centrifugation again, removes about 800 μ l supernatants, then carries out the coated plate that rises by every 100 μ l/ plates.
Obtained can secreting, expressing superoxide dismutase engineered Saccharonayces yeast, be named as pUC19-MS/INVSC1.
Other Wine brewing yeast strains can be converted with same method can equally obtain it such as yeast strain pYES-MS/INV
Its SOD recombinant Saccharomyces cerevisiae.
Embodiment 3, secreting, expressing superoxide dismutase and its Activity determination in saccharomyces cerevisiae
Engineered Saccharonayces yeast pUC19-MS/INVSC1 is inoculated in two 5mLYPD culture mediums, an addition derivant
A concentration of 20g/L of galactolipin, inducing temperature are 30 DEG C, and induction time is 24-48 hours；Another is not added for negative control
Derivant galactolipin, cultivation temperature are 30 DEG C, and incubation time is 24-48 hours.After culture, each culture supernatant is taken to carry out
15% SDS-PAGE electrophoresis, protein immunoblotting (Western-blot).Culture supernatant protein electrophoresis can see swimming lane 2
There is an apparent band of expression at 19kD；Western blot analysis shows that bacterial strain of the present invention is expressed with day
The hCu/Zn-SOD of right immunogenicity shows successfully to construct biologically active hCu/Zn-SOD expression saccharomyces cerevisiae.
The application conditions optimization of embodiment 4, SOD recombinant Saccharomyces cerevisiae extracellular expression superoxide dismutases
1, the composition for ease constipation food
Culture medium for food is determined as YPD galactolipin inducing cultures, is the aqueous solution containing following composition：It presses
Weight, 1% yeast extract, 2% peptone, 2% glucose；A concentration of 20g/L of galactolipin.
2, the selection for SOD recombinant Saccharomyces cerevisiaes growth time in food
In order to ensure that making full use of for food culture medium nutrition, selection grow to logarithmic phase in saccharomyces cerevisiae,
The making of facial mask is used for after namely about growth 16-18h, saccharomyces cerevisiae growth conditions to be preferable at this time, while also there are enough battalion
Form part, it is ensured that the activity of expressed SOD can be given full play to when for food.
3, the selection for SOD recombinant Saccharomyces cerevisiaes concentration in food
Its OD value can reach 1.3-1.5 after general saccharomyces cerevisiae growth 16-18h, this concentration is determined as being used for food
The concentration of middle SOD recombinant Saccharomyces cerevisiaes, when in use its OD value need to reach 1.3-1.5.
4, the selection of food auxiliary material
After saccharomyces cerevisiae grows to OD values 1.3-1.5 in the medium, become SOD active matrixes, select at this time honey for
Food is made in auxiliary material, and honey dosage is unlimited, and can eat as standard, usual dosage can be 5wt%-15wt%.
Embodiment 5：The application of SOD recombinant Saccharomyces cerevisiaes
1, food：
1) by SOD recombinant Saccharomyces cerevisiaes (such as pUC19-MS/INVSC1) in YPD galactolipin inducing cultures (by following
The aqueous solution that group is grouped as：1% yeast extract by weight, 2% peptone, 2% glucose；A concentration of 20g/L of galactolipin) it is inner into
30 DEG C of cultures of row, growing to logarithmic phase in the saccharomyces cerevisiae, (about 16-18h expresses OD with Westernblot verifications SOD:1.3-
1.5) SOD active matrixes, are obtained；
2) honey is added into the SOD active matrixes makes a concentration of 10wt% of its (honey), -20 DEG C of freezing guarantors after stirring evenly
It deposits, becomes food.
2, drug：Drug can also be made with the method for above-mentioned food.Production method is as above, with above-mentioned steps 1) it is identical
Method obtains SOD active matrixes, is then added into the customary adjuvant of drug, and -20 DEG C of freezen protectives become active drug
Product.
3, nano liposomes
By lecithin (a concentration of 1.0mg/mL), Phospholipid/Cholesterol (mass ratio 1: 5), Tween80/ lecithin (matter
Amount is than being 4: 5), OD recombinant Saccharomyces cerevisiaes thalli powder (25mg), ethyl alcohol (8mL), hydration medium ion (0.05mmol/L) is mixed
Close, hydration time and hydration temperature be respectively 30min, 50 DEG C, ultrasonication post-processes up to liposome, and freeze-drying forms a kind of
The health care probiotic combinations of very useful health.
The present invention is with this embodiment offers the making of a kind of active food or drug and application methods.Production method packet
It includes：So that bioactie agent (such as SOD) is expressed in saccharomyces cerevisiae by way of bioengineering, add it to food or
Active food or drug are formed in drug.
Sequence table
<110>Guangxi Tian Chang Investment Co., Ltd
<120>A kind of Se-enriched yeast and preparation method thereof of ease constipation function
<130>Act on behalf of institute's file number
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 255
<212> DNA
<213>Saccharomyces cerevisiae MF- alpha signals peptide gene (Saccharomycescerevisiae)
<400> 1
atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagctgct 60
ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120
tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180
aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240
tctctcgaga aaaga 255
<210> 2
<211> 465
<212> DNA
<213> hCu/Zu-SOD
<400> 2
atggcgacga aggccgtgtg cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 60
ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact 120
gaaggcctgc atggattcca tgttcatgag tttggagata atacagcagg ctgtaccagt 180
gcaggtcctc actttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg 240
catgttggag acttgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt 300
gaagattctg tgatctcact ctcaggagac cattgcatca ttggccgcac actggtggtc 360
catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacaggaaac 420
gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aataa 465
<210> 3
<211> 741
<212> DNA
<213> MF-α and hCu/Zn-SOD
<400> 3
cacttgagcg ataccgataa caccacaagc caaacgactt ccagcgtttc ctgtctttgt 60
actttcttca tttccacctt tgcccaagtc atctgctttt tcatggacca ccagtgtgcg 120
gccaatgatg caatggtctc ctgagagtga gatcacagaa tcttcaatag acacatcggc 180
cacaccatct ttgtcagcag tcacattgcc caagtctcca acatgcctct cttcatcctt 240
tggcccaccg tgttttctgg atagaggatt aaagtgagga cctgcactgg tacagcctgc 300
tgtattatct ccaaactcat gaacatggaa tccatgcagg ccttcagtca gtcctttaat 360
gcttccccac accttcactg gtccattact ttccttctgc tcgaaattga tgatgccctg 420
cactgggccg tcgcccttca gcacgcacac ggccttcgtc gcgaattcta cgtaagcttc 480
agcctctctt ttctcgagag ataccccttc ttctttagca gcaatgctgg caatagtagt 540
atttataaac aataacccgt tatttgtgct gttggaaaat ggcaaaacag caacatcgaa 600
atccccttct aaatctgagt aaccgatgac agcttcagcc ggaatttgtg ccgtttcatc 660
ttctgttgta gtgttgactg gagcagctaa tgcggaggat gctgcgaata aaactgcagt 720
aaaaattgaa ggaaatctca t 741
<210> 4
<211> 2686
<212> DNA
<213> pUC19-MS
<400> 4
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420
cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct 480
gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540
aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600
gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660
agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 780
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1260
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 1320
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680
tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740
ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800
ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860
atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920
cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980
tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040
aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100
tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160
ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220
agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340
agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400
accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460
gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520
cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580
ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 2640
atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 2686
Claims (10)
1. a kind of recombinant expression carrier for the secreting, expressing superoxide dismutase in saccharomyces cerevisiae is to carry saccharomyces cerevisiae
The recombinant Saccharomyces cerevisiae secretion expression carrier of MF- alpha signals peptide gene and hCu/Zn-SOD genes；For building recombinant Saccharomyces cerevisiae
The carrier that sets out of secretion expression carrier, which can be any one, the eukaryotic expression of secreting expression of exogenous gene to be carried in saccharomyces cerevisiae
Body, such as pJK3, pMD18-T, pUC19, pET22b (+), preferably pUC19；With pUC19 be set out vector construction recombination make
Brewer yeast secretion expression carrier is named as pUC19-MS.
2. recombinant Saccharomyces cerevisiae secretion expression carrier according to claim 1, it is characterised in that：The recombinant Saccharomyces cerevisiae
In the nucleotide sequence of secretion expression carrier pUC19-MS such as sequence table shown in sequence 4.
3. recombinant Saccharomyces cerevisiae secretion expression carrier according to claim 2, it is characterised in that：The recombinant Saccharomyces cerevisiae
The construction method of secretion expression carrier pUC19-MS, includes the following steps：
1) saccharomyces cerevisiae MF- alpha signal peptide genes are expanded：With saccharomyces cerevisiae (Saccharomycescerevisiae) bacterial strain
The cellular lysate liquid of INVSC1 is template, in primer T1：5'-GGGGTACCATGAGATTTCCTTCTATTTTTACTGC-3'(contains
KpnI restriction enzyme sites) and T2：5'-CGGGATCCTCTTTTATCCAAAGAAACACCT-3'(restriction enzyme sites containing BamHI) guiding
Lower PCR amplification MF- alpha signal peptide genes, in nucleotide sequence such as sequence table shown in sequence 1；
2) hCu/Zn-SOD genes are expanded：The total mRNA of people's gastric tissue is extracted, then RT-PCR amplifies its total cDNA as PCR moulds
Plate, in primer I 1：5'-CGGGATCCGCGACGAAGGCCGTGTGCGTGCT-3'(restriction enzyme sites containing BamHI) and I2：5'-
The sites containing XbaI enzyme cutting GCTCTAGAGAATGTTTATTGGGCGATCC-3'() guiding under PCR amplification hCu/Zn-SOD genes,
In its nucleotide sequence such as sequence table shown in sequence 2；
3) structure cloning vector pUC-MS：Saccharomyces cerevisiae MF- alpha signals peptide gene is connected with hCu/Zn-SOD genes and is carried into clone
In body pUC19, the cloning vector pUC-MS, hCu/ that carry saccharomyces cerevisiae MF- alpha signals peptide gene and hCu/Zn-SOD genes are obtained
In the nucleotide sequence of Zn-SOD fusions such as sequence table shown in sequence 3；
4) recombinant Saccharomyces cerevisiae secretion expression carrier is built：By cloning vector pUC-MS and carrier pUC19 through KpnI and XbaI into
After row double digestion, saccharomyces cerevisiae MF- alpha signals peptide gene is connected with hCu/Zn-SOD genes into wine yeast secretion type expression carrier
In pUC19, the recombinant Saccharomyces cerevisiae secretion expression carrier pUC19-MS for secreting, expressing hCu/Zn-SOD, nucleotide are obtained
In sequence such as sequence table shown in sequence 4.
4. it is a kind of can secreting, expressing superoxide dismutase engineered Saccharonayces yeast, be to be had the right requirement 1- with Saccharomyces cerevisiae transformant
Any one of 3 recombinant Saccharomyces cerevisiae secretion expression carriers, preferably pUC19-MS, obtained recombinant Saccharomyces cerevisiae；The wine
Brewer yeast is the Wine brewing yeast strain suitable for protein expression, such as INVSC1；The method for transformation is Li-acetate method, heat shock method, electricity
The protoplast transformation etc. that conversion method, engagement conversion method PEG are mediated；Using INVSC1 as starting strain, the conversion of structure has weight
The engineered Saccharonayces yeast of group saccharomyces cerevisiae secretion expression carrier pUC19-MS is pUC19-MS/INVSC1.
5. a kind of method of the secreting, expressing superoxide dismutase in saccharomyces cerevisiae, including claim of fermenting in the medium
The 4 engineered Saccharonayces yeast, obtain the process of the superoxide dismutase of high activity.
6. secretory expression method according to claim 5, it is characterised in that：The culture medium is the induction training of YPD galactolipins
Base is supported, is aqueous solution composed of the following components：1% yeast extract by weight, 2% peptone, 2% glucose；Galactolipin it is dense
Degree is 20g/L；The fermentation temperature is 30 DEG C, and induction time is 24-48 hours.
7. application of the engineered Saccharonayces yeast described in claim 4 in the SOD active matrixes in preparing for ease constipation product.
8. a kind of SOD active matrixes, by engineered Saccharonayces yeast (such as pUC19-MS/INVSC1) described in claim 4 in YPD half
30 DEG C of cultures are carried out in lactose inducing culture, and logarithmic phase (about 16-18h, OD are grown in the saccharomyces cerevisiae:1.3-1.5), warp
Westernblot verifies SOD expression, obtains SOD active matrixes；The YPD galactolipins inducing culture is by following components group
At aqueous solution：1% yeast extract by weight, 2% peptone, 2% glucose；A concentration of 20g/L of galactolipin.
9. a kind of pharmaceutical composition, described pharmaceutical composition by described in claim 4 engineered Saccharonayces yeast with it is one or more
Auxiliary material forms.
10. a kind of nano liposomes contain lecithin, cholesterol, which is characterized in that the nano liposomes further include that right is wanted
Seek the engineered Saccharonayces yeast described in 4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109456989A (en) * | 2018-10-31 | 2019-03-12 | 陕西慧康生物科技有限责任公司 | A kind of raising Pichia pastoris secretion expression carrier construction method |
| CN115161209A (en) * | 2022-05-13 | 2022-10-11 | 江苏农林职业技术学院 | Saccharomyces cerevisiae engineering strain capable of effectively improving organism immunity of aquatic animals, construction method and application |
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| CN102177247A (en) * | 2008-08-12 | 2011-09-07 | 格利科菲公司 | Improved vectors and yeast strains for protein production: CA2+ ATPase overexpression |
| CN103333913A (en) * | 2013-07-08 | 2013-10-02 | 中国人民解放军疾病预防控制所 | Engineering saccharomyces cerevisiae being capable of secreting and expressing superoxide dismutase, and construction method thereof and applications of same in preparation of active beauty products |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109456989A (en) * | 2018-10-31 | 2019-03-12 | 陕西慧康生物科技有限责任公司 | A kind of raising Pichia pastoris secretion expression carrier construction method |
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| CN115161209A (en) * | 2022-05-13 | 2022-10-11 | 江苏农林职业技术学院 | Saccharomyces cerevisiae engineering strain capable of effectively improving organism immunity of aquatic animals, construction method and application |
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Application publication date: 20180713 |