CN106519023A - Gene cloning method, expressing method and application of psychrophilic yeast metallothionein - Google Patents
Gene cloning method, expressing method and application of psychrophilic yeast metallothionein Download PDFInfo
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Abstract
The invention provides a gene cloning method, expressing method and application of psychrophilic yeast metallothionein. Metallothionein genes are extracted, a primer F1 and a primer R1 are designed, subjected to PCR amplification and then connected with an expression carrier, and a recombinant expression carrier is constructed; the recombinant expression carrier is converted into an expression strain, and a recombination expression transformant is constructed; the expressing and purifying method of recombinant protein is built. The nucleotide sequence and the coded amino acid sequence of the metallothionein genes are shown in the sequence table SEQ ID NO:1 and the sequence table SEQ ID NO:2. The novel metallothionein obtained through cloning comprises 16 pieces of cysteine, multiple metal and heavy metal ions can be chelated through rich sulfydryl, and the novel metallothionein has broad prospects in the medicine field, the food health caring field, the cosmetic additive field, the genetic engineering reagent field, the chemical engineering field, the environmental protection field, the animal field, the agriculture field and the like.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of gene cloning of thermophilic cold yeast metallothionein,
Expression and application.
Background technology
Into a global problem, the minamata disease of Japan and itai-itai, precisely due to hydrargyrum in environment for heavy metal pollution
Cause with the severe overweight of cadmium.Heavy metal element all has stronger bio-toxicity, even in very low concentration range,
Still natural ecosystems may be worked the mischief, and heavy metal pollution has disguised, chronicity and irreversibility.Heavy metal
Can be absorbed by organism, high concentration is concentrated in the enrichment of food chain end, can cause various deformities, poisoning, or even induction cancer
Become etc..As can be seen here, impact of the heavy metal to organism is illustrated, the adaptation mechanism of biological heavy metal is disclosed, so as to effective
Prevention and improvement heavy metal pollution are the task of top priority.
In biological heavy metal coping mechanism, metallothionein plays an important role, can be with heavy metal complexation
And adjust its concentration.Metallothionein (Metallothionein, abbreviation MT) is that a class is widely present in internal low point of biology
Son amount, rich in cysteine, can be by the metal-binding protein of metal inducement, earliest by margoshes and vallee in nineteen fifty-seven
Isolated from horse kidney (Margoshes et al, 1957).It is widely present in almost all of mammal, Fish, amphibious
In class, invertebratess and certain plants and eucaryon, prokaryotic micro-organisms body, the stable state for participating in intracellular necessary metal level is adjusted
Section, inessential heavy metal detoxification, removing free radical, opposing ionizing radiation etc., or even participate in hormone and Growth adjustment, enhancing body
Stress, the depots of zinc element prevent cell carcinogenesis etc., are described as " bodyguard of health " by scientific circles.In medicine, food
The field such as product health care and cosmetics additive, genetic engineering reagent, chemical industry, environmental protection, animal, agricultural has a extensive future.
The South Pole has the extreme environment features such as low temperature, low illumination, oligotrophic, intense radiation, and the South Pole is subjected to heavy metal etc.
The degree of pollution be much higher by it is envisaged that degree, be enriched in heavy metal in organism high compared with other marine organisms.At present,
South Pole heavy metal pollution increasingly causes the concern of people, the metal sulfur of resistance and its induction with regard to Antarctic organism heavy metal
Albumen research is in the starting stage, and great majority research concentrates on the animal bodies such as shellfish and Fish, and also no polar region yeast is related
The report of research.MT produced by Antarctic Yeast has the unrivaled superiority of room temperature yeast, may be a kind of novel metal sulfur
Albumen, special property and composition are adapted with polar region extreme environment, particularly heavy metal stress.
The content of the invention
For current present Research, it is an object of the invention to provide a kind of gene gram of thermophilic cold yeast metallothionein
Grand method, and the recombinant expression protein expression and purification method of the gene.
The present invention provides a kind of cloning process of thermophilic cold yeast metallothionein gene, design and synthesis including primer,
The extraction of thermophilic cold Yeast genome RNA, the PCR amplifications of metallothionein gene and sequencing etc., specifically include following steps：
(1) according to transcription group determination data, design primer and synthesize the pair of primers containing restriction enzyme site, it is thermophilic for expanding
The open reading frame of cold yeast metallothionein gene, introduces the restriction enzyme site of BamH I and Xho two kinds of enzymes of I respectively in primer,
5 ' three protection bases held：
Forward primer F1：5’CGGAATTCATGTCTGCCCCCACCAC3’；
Downstream primer R1：5’CCGCTCGAGAGCCGAGAATCCAGCACT3’；
(2) from polar region, thermophilic cold yeast Rhodotor μ La mucilaginosa AN5 extract total serum IgE, inverse using Takara
Transcript reagent box synthesizes cDNA, and with cDNA as template, using the primer of design, PCR amplifies genes of interest fragment；Reaction interval
Sequence：1. 5min at 94 DEG C, 2. 30s at 94 DEG C, 3. 30s at 56 DEG C, 4. 15s at 72 DEG C, 5. repeat step 2. -4. 30 circulation,
6. 10min at 72 DEG C, 7. 4 DEG C of holdings；
(3) carry out double digestion after PCR primer purification is reclaimed with expression vector respectively, double digestion product purification enters after reclaiming again
Row connection, condition of contact：16 DEG C, 12h.Linked system：10μL.Carrier：2μL；Genes of interest：6μL；10×Ligation
buffer：1μL；T4DNA ligases：1μL；
(4) recombinant expression carrier after connection is transformed into the competent cell of expression strain, picking positive single bacterium colony is entered
Row bacterium solution PCR is simultaneously sequenced.
Such as sequence table SEQ ID NO is obtained according to above-mentioned cloning process：Thermophilic cold yeast metallothionein gene shown in 1 is complete
Long sequence, the aminoacid sequence such as sequence table SEQ ID NO of its coding：Shown in 2.Full length gene 321bp, encodes 106 ammonia
Base acid.
The thermophilic cold yeast metallothionein gene of the present invention can also be coding by SEQ ID NO in sequence table：Ammonia shown in 2
Other any nucleotide sequences of the protein of base acid sequence composition.
The thermophilic cold yeast metallothionein of the present invention is not limited only to SEQ ID NO：Aminoacid sequence shown in 2 sequence tables,
Can also be SEQ ID NO：Through replacing, lacking or add one or several amino acid residues in aminoacid sequence shown in 2
And with identical protein active by SEQ ID NO：The aminoacid sequence of protein derived from sequence shown in 2.
The present invention provides a kind of recombinant expression carrier, and the recombinant expression carrier contains above-mentioned thermophilic cold yeast metallothionein gene
Nucleotide sequence.
The carrier framework of above-mentioned recombiant plasmid is prokaryotic expression carrier pET-28a.
The present invention provides a kind of restructuring table transformant, and the recombination engineering bacteria contains above-mentioned thermophilic cold yeast metallothionein base
Cause or above-mentioned recombinant expression carrier.
Above-mentioned recombinant expression transformants with e. coli bl21 (DH5 α) as Host Strains.
The present invention provides the expression of the thermophilic cold yeast metallothionein gene, including the abduction delivering and egg of gene
White purification, comprises the following steps that：
(1) single bacterium colony of above-mentioned recombinant expression transformants is inoculated in into 50mL's LB fluid mediums (containing kanamycin)
In 250mL conical flasks, 37 DEG C of incubated overnight；
(2) 5mL bacterium solutions overnight are taken in the 500mL conical flasks equipped with 250mL LB fluid mediums (containing kanamycin)
In, 200r/min shaken cultivation to OD600During value about 0.6, the addition 100mM IPTG to final concentration 0.1mM in induction bottle, 37
DEG C shaking table culture abduction delivering 2h；
(3) whole culture fluid are taken, 4 DEG C, 12000r/min is centrifuged 15min；The thalline of centrifugation is added into a small amount of silicon dioxide
With the 50mM phosphate buffers (pH 7.0) of 6mL pre-coolings, being placed in frozen water carries out ultrasonication.Parameter setting：Power 400w, work
Make 2s, be spaced 2s, work 10min.In 4 DEG C, 12000r/min is centrifuged 15min, collects supernatant and precipitates and molten with 8M carbamide
Solution precipitation；
(4) protein purification, 100mM imidazoles eluting foreign proteins, the inspection of Jing SDS-PAGE electrophoresis are carried out by affinity chromatography
Survey and confirm that recombiant protein has higher purity；
According to thermophilic cold yeast metallothionein prepared by said method, its aminoacid sequence such as sequence table SEQ ID NO：2
Shown, containing 106 aminoacid, molecular weight 10.3kDa, isoelectric point, IP 8.5, containing 16 cysteine.
Beneficial effects of the present invention are：
1. the present invention is to the thermophilic cold yeast from the thermophilic cold yeast Rhodotor μ La mucilaginosa AN5 in polar region
Metallothionein gene has carried out successful clone and sequencing.The cloning and expression method of the gene is set up, its whole is determined first
Gene order, and register in Genbank, registration number is KJ866906.Full length gene 321bp, encodes 106 aminoacid, point
Son amount 10.3kDa, isoelectric point, IP 8.5 can recombinate to the gene according to the aminoacid sequence of the sequence or the sequential coding
Expression or gene transfer.
2. the present invention build recombination engineering bacteria containing metal copper ion culture medium in growth rate apparently higher than
Control, heavy metal copper ion have higher resistance, are further to analyze the function of the metallothionein gene engineering bacteria and answer
With laying a good foundation.
3. the novel metal sulfoprotein that present invention clone obtains, containing 16 cysteine, abundant sulfydryl can be chelated
Contents of many kinds of heavy metal ion, it is stronger than the heavy metal binding ability of room temperature yeast metallothionein, in medicine, health care of food and cosmetics
The fields such as additive, genetic engineering reagent, chemical industry, environmental protection, animal, agricultural have a extensive future.
Description of the drawings
Extraction figures of the Fig. 1 for AN5 total serum IgEs；
Fig. 2 is that metallothionein cDNA expands electrophoresis result figure；
Fig. 3 is purpose protein SDS-PAGE electrophoresis result figure；
Fig. 4 is the SDS-PAGE electrophoresis result figures of restructuring metallothionein purification；
Fig. 5 is the growing state figure for turning metallothionein gene escherichia coli in LB culture medium.
Specific embodiment
The specific embodiment of the present invention is described in further detail below by way of specific embodiment.
Embodiment 1：A kind of clone of thermophilic cold yeast metallothionein gene
(1) extraction of thermophilic cold Yeast genome RNA：
1. the Rhodotor μ La mucilaginosa AN5 inoculations of activation are entered into the liquid cultures of YEPD containing 100mL
The 250mL conical flasks of base, while add 8mM CuSO4, induction.10 DEG C of 120rpm are cultivated 3 days.8000rpm centrifugations obtain thalline.
2. by the thalline the being collected by centrifugation distilled water wash of 5 times of volumes, vibrate precipitation and mix, subsequent 4 DEG C, 8000rpm
Centrifugation 8min, abandons supernatant；It is repeated 1 times.
3. anhydrous alcohol is added in mortar, fully burnt, eliminate RNase that may be present in mortar.
4. the thalline for centrifugation being obtained adds the mortar after Liquid nitrogen precooler, is fully ground.
5. thalline after grind 50-100mg add have the 1.5mL RNase-free of 1mL Buffer Rlysis-F from
Heart pipe, fully mixes.
6. 200 μ L chloroforms are added in lysate sample, are fully mixed.4 DEG C of centrifugation 20min of 16000rpm, take supernatant.
7. add the dehydrated alcohol of 1/3 volume, mix, room temperature places 3min, 4 DEG C of 16000rpm centrifugation 5min, remove on
Clearly.
8. precipitated 2 times with 75% washing with alcohol of 700 μ L.
9. room temperature is inverted centrifuge tube 10min, and fully volatilize ethanol.
10. the DEPC-treated ddH of 50 μ L are added2O dissolution precipitations, 1% sepharose electrophoresis identification RNA purity, knot
Really as shown in Figure 1, remaining -80 DEG C of Refrigerator store.
(2) PCR reactions amplification metallothionein cDNA
According to transcription group determination data, design primer and synthesize the pair of primers containing restriction enzyme site, it is thermophilic cold for expanding
The open reading frame of yeast metallothionein gene：
Forward primer F1：5’CGGAATTCATGTCTGCCCCCACCAC3’
Downstream primer R1：5’CCGCTCGAGAGCCGAGAATCCAGCACT3’
Primer is respectively provided with the restriction enzyme site (dashed part in primer) of BamH I and Xho two kinds of enzymes of I and 5 ' three for holding
Protection base, primer are synthesized by Shanghai Sheng Gong biological engineering company limited.
PCR reaction systems：Using Takara Reverse Transcriptase kits, according to description method, reversed using the M-MuLV of BBI
By the RNA reverse transcriptions synthesis cDNA for extracting, with this cDNA as template, F1R1 enters performing PCR amplification for primer to record enzyme.
PCR amplification conditions：1. 5min at 94 DEG C, 2. 30s at 94 DEG C, 3. 30s at 56 DEG C, 4. 15s at 72 DEG C, 5. repeatedly walks
Rapid 2. -4. 30 circulation, 6. 10min at 72 DEG C, 7. 4 DEG C of holdings；(M：DL2000；1-5：PET-28a plasmid DNA).
Agarose gel electrophoresiies result finds electrophoresis result and estimated purpose fragment size one as shown in Figure 2, after detection
Cause (320bp or so), band brightness is preferable.
(3) structure of recombinant expression carrier
PCR primer purification carries out double digestion respectively with PET-28a expression vectors after reclaiming, after double digestion product purification is reclaimed
It is attached again, condition of contact：16 DEG C, 12h.Linked system：10μL.Carrier：2μL；Genes of interest：6μL；10×Ligation
buffer：1μL；T4DNA ligases：1μL.
(4) structure of recombinant expression transformants
Recombinant expression carrier after connection is proceeded in bacillus coli DH 5 alpha competent cell, bacterium colony PCR screening positive clones,
Electrophoresis detection result as shown in Figure 3, understands M from the upper result of figure：Marker；1、2、4、5：Bacterium colony PCR positive findingses, size with
Genes of interest is consistent, it was demonstrated that convert successfully.
After positive findingses corresponding bacterium colony amplification culture, extract plasmid and preserve glycerol stock.Plasmid with extraction is as mould
Plate, according to above-mentioned PCR amplification method, enters performing PCR amplification using the primer of design, expands the corresponding glycerol stock of successful plasmid and send
It is sequenced to sequencing company.
Thermophilic cold yeast metallothionein gene total length 321bp in present embodiment, particular sequence are as follows：
In present embodiment, the aminoacid sequence of thermophilic cold yeast metallothionein includes 106 aminoacid, and sequence is as follows：
Embodiment 2：A kind of expression of thermophilic cold yeast metallothionein and purification
(1) abduction delivering of metallothionein gene
1. distinguish single bacterium colony of the picking containing empty expression vector (positive control plasmid) and recombinant expression carrier and be inoculated in 50mL
In the 250mL conical flasks of the LB fluid mediums containing kanamycin, 37 DEG C of incubated overnight；
2. take 5mL bacterium solutions overnight in equipped with 250mL LB fluid mediums (containing kanamycin) 500mL conical flasks in,
200r/min shaken cultivation is to OD600Value about 0.6；
3., in inducing bottle, 100mM IPTG to final concentration 0.1mM, 37 DEG C of shaking table culture abduction delivering 2h are added；
4. 4 DEG C of well-grown recombinant bacterial strain bacterium solution and empty expression vector bacterium solution, 12000r/min, centrifugation is taken respectively
15min.The thalline of centrifugation is added into the 50mM phosphate buffers (pH 7.0) of silicon dioxide and 6mL pre-coolings, is placed in frozen water
Row ultrasonication.Parameter setting：Power 400w, work 2s, is spaced 2s, and work 10min.In 4 DEG C, 12000r/min is centrifuged
15min, collects supernatant and precipitates and with 8M carbamide dissolution precipitations.
(2) purification of restructuring metallothionein
Protein purification is carried out using the mode of affinity chromatograph, with Ni-NTA as medium, imidazoles final concentration be set to 50mM,
100mM, 200mM carry out the eluting of foreign protein, and as a result as shown in Figure 4 Jing PAGE gel electrophoresis, in imidazole concentration is
Under conditions of 100mM, obtaining a specific band and molecular size range, identical with destination protein (this albumen Jing is commonly formed dimerization
Body, molecular weight about 20kDa), it was demonstrated that thermophilic cold yeast metallothionein gene has obtained correct expression in restructuring thalline.In Fig. 4
Each marker annotations are as follows：M：Albumen marker；1：Recombiant plasmid is not induced；2：Recombinant plasmid I PTG final concentration 0.1mM induces 2h；
3：Albumen obtained by 50mM imidazoles eluting；4：100mM imidazoles eluting；5：Albumen obtained by 200mM imidazoles eluting.
Embodiment 3：Recombiant protein is analyzed to the resistance test of metal ion
The e. coli bl21 for proceeding to recombiant plasmid is inoculated in the LB fluid mediums containing kanamycin and is cultivated,
OD680For 0.42 when, add the Cu of final concentration of 1mM2+, continue culture 2 days, detect the OD of thalline680, as a result as shown in Figure 5,
cK：Control, i.e., do not turn the thalline of metallothionein gene；mt-BL21：Turn the thalline of metallothionein gene.Turn metal sulfur
The escherichia coli of protein gene containing metal copper ion culture medium in growth rate apparently higher than control, heavy metal copper ion
There is higher resistance.
The novel metal sulfoprotein that present invention clone obtains, containing 16 cysteine, abundant sulfydryl can chelate many
Heavy metal species ion, it is stronger than the heavy metal binding ability of room temperature yeast metallothionein, add in medicine, health care of food and cosmetics
Plus the field such as agent, genetic engineering reagent, chemical industry, environmental protection, animal, agricultural has a extensive future.
Here description of the invention and application are illustrative, are not wishing to limit the scope of the invention to above-described embodiment
In, therefore, the present invention is not limited by the present embodiment, and the technical scheme that any employing equivalence replacement is obtained is in present invention protection
In the range of.
Claims (10)
1. a kind of cloning process of thermophilic cold yeast metallothionein gene, it is characterised in that comprise the steps：
(1) from polar region, thermophilic cold yeast Rhodotor μ La mucilaginosa AN5 extract total serum IgE, using Takara reverse transcriptions
Test kit synthesizes cDNA；
(2) with cDNA as template, using forward primer F1 (5 ' CGGAATTCATGTCTGCCCCCACCAC3 ') and downstream primer R1
(5 ' CCGCTCGAGAGCCGAGAATCCAGCACT3 ') enters performing PCR amplification；Response procedures：1. 5min at 94 DEG C, 2. at 94 DEG C
30s, 3. 30s at 56 DEG C, 4. 15s at 72 DEG C, 5. repeat step 2. -4. 30 circulation, 6. 10min at 72 DEG C, 7. 4 DEG C of holdings；
(3) carry out double digestion after PCR primer purification is reclaimed with expression vector respectively, double digestion product purification is connected after reclaiming again
Connect, condition of contact：16 DEG C, 12h；Linked system：10μL；Carrier：2μL；Genes of interest：6μL；10×Ligation buffer：
1μL；T4DNA ligases：1μL；
(4) recombinant expression carrier after connection is transformed into the competent cell of expression strain, picking positive single bacterium colony carries out bacterium
Liquid PCR is simultaneously sequenced.
2. the cloning process of thermophilic cold yeast metallothionein gene according to claim 1, it is characterised in that utilize the party
The thermophilic cold yeast metallothionein gene sequence that method is obtained is SEQ ID NO：1, the aminoacid sequence of its coding is SEQ ID
NO：2.
3. a kind of thermophilic cold yeast metallothionein, it is characterised in that its aminoacid sequence such as sequence table SEQ ID NO：Shown in 2.
4. a kind of recombinant expression carrier, it is characterised in that comprising the thermophilic cold yeast metallothionein gene described in claim 2
Nucleotide sequence.
5. recombinant expression carrier according to claim 4, it is characterised in that the carrier framework of the recombinant expression carrier is original
Nuclear expression carrier pET-28a.
6. a kind of recombinant expression transformants, it is characterised in that comprising thermophilic cold yeast metallothionein gene described in claim 2
Recombinant expression carrier described in nucleotide sequence or any one of claim 4～5.
7. recombinant expression transformants according to claim 6, it is characterised in that the recombinant expression transformants are with escherichia coli
BL21 (DH5 α) is Host Strains.
8. a kind of expression of thermophilic cold yeast metallothionein gene, cultivates recombinant expressed turn described in induction claim 6
Change body, obtain the thermophilic cold yeast metallothionein of restructuring, it is characterised in that comprise the steps：
(1) the plasmid conversion single bacterium colony of extraction is inoculated in into the 250mL conical flasks of the 50mL LB fluid mediums containing kanamycin
In, 37 DEG C of incubated overnight；
(2) take 5mL bacterium solutions overnight in equipped with containing kanamycin 250mL LB fluid mediums 500mL conical flasks in,
200r/min shaken cultivation is to OD600During value about 0.6, the addition 100mM IPTG to final concentration 0.1mM in induction bottle, 37 DEG C
Shaking table culture abduction delivering 2h；
(3) whole culture fluid are taken, 4 DEG C, 12000r/min is centrifuged；The thalline of centrifugation is added into a small amount of silicon dioxide and 6mL pre-coolings
50mM pH 7.0 phosphate buffer, being placed in frozen water carries out ultrasonication, collects and supernatant and precipitates and molten with 8M carbamide
Solution precipitation；
(4) protein purification, 100mM imidazoles eluting foreign proteins, Jing SDS-PAGE electrophoresis detection card are carried out by affinity chromatography
The real destination protein for obtaining purification.
9. one kind recombinates thermophilic cold yeast metallothionein as claimed in claim 3 in the addition of medicine, health care of food and cosmetics
Application in agent field.
10. one kind is recombinated thermophilic cold yeast metallothionein as claimed in claim 3 in prevention and administers heavy metal pollution field
In application.
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| CN107699578A (en) * | 2017-09-30 | 2018-02-16 | 中国农业科学院麻类研究所 | A kind of ramie metallothionein gene and its recombinant protein and application |
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| CN110105446A (en) * | 2019-04-11 | 2019-08-09 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | The metallothionein and its expression and application that a kind of metallothionein gene MT27, its coding obtain |
| CN116410943A (en) * | 2023-05-26 | 2023-07-11 | 维塔探索(广东)科技有限公司 | Alternaria aequorea thioredoxin, and preparation method and application thereof |
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| GENBANK: KJ866906.1: "Rhodotorula mucilaginosa isolate AN5 metallothionein mRNA, complete cds", 《GENBANK》 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699578A (en) * | 2017-09-30 | 2018-02-16 | 中国农业科学院麻类研究所 | A kind of ramie metallothionein gene and its recombinant protein and application |
| CN107699578B (en) * | 2017-09-30 | 2019-12-20 | 中国农业科学院麻类研究所 | Ramie metallothionein gene, recombinant protein thereof and application thereof |
| CN109851670A (en) * | 2019-04-11 | 2019-06-07 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | The metallothionein and its expression and application that a kind of metallothionein gene MT18, its coding obtain |
| CN110105446A (en) * | 2019-04-11 | 2019-08-09 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | The metallothionein and its expression and application that a kind of metallothionein gene MT27, its coding obtain |
| CN109851670B (en) * | 2019-04-11 | 2021-12-31 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Metallothionein gene MT18, metallothionein obtained by encoding metallothionein gene MT18, expression and application of metallothionein |
| CN110105446B (en) * | 2019-04-11 | 2022-02-18 | 中国科学院遗传与发育生物学研究所农业资源研究中心 | Metallothionein gene MT27, metallothionein obtained by encoding metallothionein gene MT27, expression and application of metallothionein |
| CN116410943A (en) * | 2023-05-26 | 2023-07-11 | 维塔探索(广东)科技有限公司 | Alternaria aequorea thioredoxin, and preparation method and application thereof |
| CN116410943B (en) * | 2023-05-26 | 2023-09-29 | 维塔探索(广东)科技有限公司 | Alternaria aequorea thioredoxin, and preparation method and application thereof |
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