CN107893081A - Gene order, expression vector and the production method of a kind of people source keratin cell growth factor 2 - Google Patents
Gene order, expression vector and the production method of a kind of people source keratin cell growth factor 2 Download PDFInfo
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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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
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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
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
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Abstract
The invention discloses a kind of gene order of people source keratin cell growth factor 2, expression vector and production method, meet the gene order of the people source keratin cell growth factor 2 (KGF 2) of target plant codon preference speciality by design, the suitable plant expression vector for including the gene orders of KGF 2 of structure, plant expression vector is transferred in target plant, through multiplex screening, the transfer-gen plant of high protein expression levels has been obtained, wherein, target plant is alfalfa.Production cost is not only greatly lowered compared with Microbial Expression Systems using alfalfa expression people source KGF 2, and protein product can be ensured correctly to fold and avoid producing inclusion body there is more preferable application prospect.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of gene sequence of people source body keratinized cell growth factor-2
Row, expression vector and production method.
Background technology
Body keratinized cell growth factor-2 (keratinocyte growth factor-2, KGF-2) is fibroblast life
The important member of the long factor (Fibroblast Growth Factor, FGFs) family.1996, Yamasaki etc. took the lead in reporting
It is a kind of to be isolated from rat embryo the novel cell regulatory factor closely related with Keratiocyte growth, it is named as KGF-2.It is secondary
Year, Emoto etc. has cloned people source KGF-2 global cDNA, and the gene code one contains 40 amino acid signal peptides in N-terminal
The single chain polypeptide protein factor of 208 amino acid of total length.
Found by further investigation to KGF-2 mechanism of action, its with 2 kinds of cell membrane surface receptors (FGFR2IIIb and
FGFR1IIIb) interact and then produce adjustment effect, the especially FGFR2IIIb and KGF-2 positioned at surface epithelial cell has
There is higher affinity.After KGF-2 is combined with this receptor, by phosphate acceptor intracellular C-terminal tyrosine residue, and its junket is activated
Propylhomoserin protein kinase activity, and then trigger a series of signal cascade reaction, adjust growth, propagation and the differentiation of epithelial cell.
In the ontogenetic process of whole spinal animals, especially the morphogenesis of the organ such as lung and limb development are required to
KGF-2 regulation；Meanwhile KGF-2 is accelerating Wound healing, treatment enterogastritis and gastrointestinal mucosa breakage etc. field also functions to very
Important effect.Therefore, the focus that KGF-2 has developed as genetically engineered drug.The Human in the U.S. at present
The KGF-2 class medicines of Genome Science companies exploitation have entered II clinical trial phases and have obtained good result.
At present, the KGF-2 in document report is generally prokaryotic expression (such as being realized by traditional escherichia expression system),
But exist more than the destination protein for tending to cause to express due to induced expression condition difference with inclusion bodies, be unfavorable for directly
Separation and Extraction obtains active KGF-2 albumen, and prokaryotes are probably pathogen in itself.Thus, select animal or plant
Thing is as other approach that bioreactor is KGF-2 expression.In view of animal as bioreactor in cell cultivation process
Animal virus in middle may be infected, and will turn into more than using plant by the use of transgenic animals as bioreactor
The public or ethics focus of attention, it is undoubtedly more favorable using plant as bioreactor.
The present inventor is by largely studying, there is provided a kind of to be used as bioreactor production people source by the use of specified plant
The method of KGF-2 albumen, solve using production cost present in the existing prokaryotic expression system technology such as Escherichia coli it is expensive,
Expression product be inclusion body, poor solubility and to host cell it is toxic a series of problems, such as.
The content of the invention
Present inventor has performed studying with keen determination, the people source cutin of alfalfa codon preference speciality is met by design
The gene order of growth factor-2 (KGF-2) simultaneously builds suitable expression vector, and expression vector is transferred into alfalfa
In, the transfer-gen plant of high protein expression levels is obtained, overcomes KGF-2 by prokaryotes and animal as bioreactor table
Up to the shortcomings that, there is great progress meaning, so as to complete the present invention.
It is an object of the invention to provide following aspect：
(1) gene order of a kind of people source body keratinized cell growth factor-2, the gene order are included shown in SEQ ID NO.1
Nucleotide sequence.
(2) a kind of expression vector, wherein, the expression vector includes the nucleotide sequence shown in SEQ ID NO.1.
(3) expression vector according to above-mentioned (2), wherein, the expression vector is plant expression vector.
(4) production method of a kind of people source body keratinized cell growth factor-2, comprises the following steps：
1), construction of expression vector；
2) heredity of the gene pairs plant of people source body keratinized cell growth factor-2, is realized using high flux genetic transfoumation
Conversion；
3), extract people's source body keratinized cell growth factor-2 albumen and purified, obtain the horn cell life of high-purity people source
The long albumen of the factor -2.
(5) production method according to above-mentioned (4), wherein, in step 2), the plant selects legume, preferably
For alfalfa.
(6) production method according to above-mentioned (4) or (5), wherein, in step 3), carried out using base exchange method
The purifying of people source body keratinized cell growth factor-2 albumen；
Preferably, the total soluble protein in plant is extracted, after cation exchange column exchange adsorption, recycling 0.8~
1.0mol/L NaCl carries out gradient elution, collects eluting peak albumen.
(7) production method according to one of above-mentioned (4) to (6), wherein, in step 3), obtained people source cutin is thin
The purity of protein of the intracellular growth factor -2 is more than 85 (weight) %.
(8) production method according to above-mentioned (4), wherein, methods described also includes turning to completing heredity in step 2)
The plant of change is identified, filters out the high transfer-gen plant of single copy, expressing quantity.
(9) production method according to above-mentioned (8), wherein, utilize Taqman PCR and western dot blot antagonism
Plant is identified that preliminary screening goes out the transfer-gen plant that copy number is low, expressing quantity is high, then the southern for passing through standard
Blot and western blot are identified it, it is determined that the transfer-gen plant that single copy, expressing quantity are high.
(10) production method according to above-mentioned (8) or (9), wherein, low copy number is carried out using Taqman PCR and turned
When gene plant screens, primer P1 and P2 in reaction system are designed,
Primer P1 includes the nucleotide sequence shown in SEQ ID NO.2；
Primer P2 includes the nucleotide sequence shown in SEQ ID NO.3.
According to gene order, expression vector and the producer of a kind of people source body keratinized cell growth factor-2 provided by the invention
Method, have the advantages that：
(1) production cost is considerably reduced compared with escherichia expression system using alfalfa expression people source KGF-2；
(2) present invention is by building plant expression vector, and is transferred in plant, and the albumen of expression can correctly enter
Modification after row folding and translation, possesses corresponding biological activity, the virus of animal-free cell and pathogen, can direct purification
The destination protein of higher degree is obtained, greatly simplify protein purification technique；
(3) using production method in the present invention, the horizontal transfer-gen plant of high exogenous protein expression, its albumen table be can obtain
Up to horizontal up to 4.76mg/g (being based on plant fresh weight)；
(4) using method for purifying proteins in the present invention, high-purity people source KGF2 albumen is can obtain, purity is in 85 (weights
Amount) more than %.
Brief description of the drawings
Fig. 1 shows expression vector pCAMBIA3301-msKGF2 structure chart；
Fig. 2 shows the western dot blots of alfalfa transgenic plant, wherein, A1:Wild type alfalfa plants；A2-
G12:The clover transfer-gen plant in different explant sources；
Fig. 3 shows the Southern results of hybridization of alfalfa transgenic plant, wherein, M:Nucleic acid molecular weight standard；+:
PCAMBIA3301-msKGF2 plasmids； -:WT lines；1-6：Respectively transformation event msKGF2-102, msKGF2-392,
MsKGF2-831, msKGF2-1562, msKGF2-1567 and msKGF2-2431；
Fig. 4 shows the Western testing results of alfalfa transgenic plant, wherein, M:Protein Marker；wt:
WT lines；1-6：Respectively transformation event msKGF2-102, msKGF2-392, msKGF2-831, msKGF2-1562,
MsKGF2-1567 and msKGF2-2431；
Fig. 5 shows the Tricine-SDS-PAGE electrophoresis detection knots that destination protein purifies in alfalfa transgenic plant
Fruit, wherein, M:Protein Marker；wt:WT lines；1:Transfer-gen plant (transformation event msKGF2-1567)； 2:
Destination protein after purification.
Embodiment
Below by the present invention is described in detail, the features and advantages of the invention will become more with these explanations
To be clear, clear and definite.
Special word " exemplary " is meant " being used as example, embodiment or illustrative " herein.Here as " exemplary "
Illustrated any embodiment should not necessarily be construed as preferred or advantageous over other embodiments.
At present, KGF-2 is prokaryotic expression, and its albumen is expressed with inclusion bodies, and the renaturation of inclusion body has become system
About the bottleneck of bio-pharmaceuticals development, processing procedure include：The broken of thalline, the washing of inclusion body, dissolving, renaturation and purifying,
The contents are multifarious and disorderly, causes purifying process sufficiently complex.Above mentioned problem can not only be overcome by plant-bioreactor, also because transgenosis is planted
Medicine production cost is greatly lowered far below microbial fermentation cost in strain planting cost.
However, floristics is numerous, there are rice, corn and soybean, cotton, tomato, capsicum as bioreactor
Etc. various plants, still there are other plants that there is this potential application.At present it is not yet found that being used for using plant as bioreactor
The report of expressing K GF-2 albumen.Therefore, suitable plant is selected so that target gene successfully obtains expression and needs to solve
One problem.
In the present invention, bioreactor of the legume as KGF-2 is selected, KGF-2 is preferably used as using alfalfa
Bioreactor.Alfalfa, alfalfa, clover are called, the kind more than 50 including mineral nutrient is contained in its cauline leaf
Nutriment and growth factor, its nutritional ingredient many abundanter than other plant.The reason for from alfalfa, mainly includes：1)
Cheap effectively alfalfa is to cultivate most wide, economic value highest leguminous forage, compared with microbial fermentation and transgenic animals more
Be easy to mass produce a variety of foreign proteins, the biochemical characteristic of product is nearly identical with natural products, reduce processing and
The cost isolated and purified；2) purifying is easily collected, clover is a kind of perennial plant, and yield is high, and quality is good, and management is simple, applies
It is less with agriculture chemical, therefore it is easier a large amount of collection purification of target products.
Although it is worth noting that, certain progress is obtained using plant bioreactor expression alien gene, outside
The problem of expression of the source gene in plant be not universal high limits it and further developed.
In order to realize expression of the KGF-2 in plant, the present invention devises first meets target plant codon preference
The gene order of the people source body keratinized cell growth factor-2 (KGF-2) of speciality, structure suitably include KGF-2 gene orders
Expression vector, expression vector is transferred in target plant, through multiplex screening, to obtain the plant of the transgenosis of high protein expression levels
Strain.
Selection based on above mentioned problem, the basic conception and bioreactor that solve problem, the first aspect of the present invention, is carried
For a kind of gene order of people source body keratinized cell growth factor-2, it includes the nucleotide sequence shown in SEQ ID NO.1.The base
Because the coded system of sequence has the speciality for meeting alfalfa codon preference, therefore it is named as msKGF2.
In a preferred embodiment, will on the premise of keeping people source KGF-2 amino acid sequences not change
All codons of encoding gene carry out codon replacement using the most suitable coded system of alfalfa codon, and to after modification
Gene order carry out restriction enzyme site information analysis.KGF-2 albumen includes 141 amino acid, by the 2nd ammonia of KGF-2 albumen
Base acid Ser (serine) coding codon is changed to the 2nd most suitable codon TCA to eliminate Psi by the 1st most suitable codon TCT
I restriction enzyme sites；By the 93rd amino acids Gly (glycine) coding codon by the 1st most suitable codon GGT, the 2nd is changed to
Most suitable codon GGA；By the 107th amino acids Arg (arginine) coding codon by the 1st most suitable codon AGA, it is changed to
2nd most suitable codon AGG is to eliminate Acc I restriction enzyme sites.People source keratinocyte growth factor gene order such as SEQ after optimization
Shown in ID NO.1.Codon optimization can be improved to the expression of foreign gene in the present invention, and it is favourable to remove restriction enzyme site
In vector construction.
The second aspect of the present invention, there is provided a kind of to produce people source body keratinized cell growth factor-2 for Genetic Transformation in Higher Plants
Expression vector, the expression vector include the nucleotide sequence shown in SEQ ID NO.1, and the preferably expression vector is plant table
Up to carrier.
Expression vector is to carry target dna fragment to enter the host cell medium that is expanded and expressed, without expression vector,
Target gene cannot be introduced into host cell, is also beyond expression even if into host cell.Table is being built for certain specific plant
Up to during carrier, it is necessary to build specific new support or the existing carrier of transformation so that exogenous gene high-efficient, safely expressed.
In a preferred embodiment, using commercialized pCAMBIA3301 as skeleton, restriction enzyme is utilized
Nco I and BstE II digestion remove gus gene thereon, and using T4 ligases will equally through restriction enzyme Nco I and
The DNA fragmentation of the people source KGF-2 genes for the engineer that BstE II digestion process is crossed is attached, and is built into and is planted suitable for target
The expression vector of strain, i.e. pCAMBIA3301-msKGF2.
Research is found, compared to other plasmid vectors, using pCAMBIA3301-msKGF2 expression vector, can be achieved
High efficient expression of the msKGF2 albumen in target plant.
The third aspect of the present invention, there is provided a kind of production method of people source body keratinized cell growth factor-2, by using purple
Russian fenugreek herb is solved using present in the existing prokaryotic expression system technology such as escherichia expression system as bioreactor
Production cost is expensive and expression product be inclusion body, poor solubility and to host cell it is toxic a series of problems, such as.
In a preferred embodiment, people source KGF-2 production method comprises the following steps：
1) plant expression vector, is built；
2) genetic transformation of msKGF2 gene pairs plant, is realized using high flux genetic transfoumation, obtains a large amount of resistances
Plant；
3), antagonism plant is identified, filters out single copy, expressing quantity high transfer-gen plant (conversion thing
Part)；
4), extract people's source KGF-2 albumen and purified, obtain high-purity people source KGF-2 albumen.
In step 1) of the present invention, on the premise of keeping people source KGF-2 amino acid sequences not change, design including
The gene order of nucleotide sequence shown in SEQ ID NO.1.
In a preferred embodiment, the people source keratinocyte growth factor gene msKGF2 after codon optimization
It is connected into the Restriction enzyme Sma I site of cloning vector pUC19 carriers, is named as after being obtained via chemical synthesis
pUC19-msKGF2.MsKGF2 genes are cut from the carrier using restriction enzyme NcoI and BstEII, through gel electricity
Express and carry with the commercialized plant through identical restriction endonuclease (NcoI and BstEII) digestion removal gus gene after swimming purifying recovery
The large fragment that body pCAMBIA3301 is discharged is connected, and successfully builds the plant expression vector of msKGF2 genes, i.e.,
pCAMBIA3301-msKGF2。
In step 2) of the present invention, the heredity of msKGF2 gene pairs alfalfas is realized using high flux genetic transfoumation
Conversion, obtains a large amount of alfalfa resistant plants.
In a preferred embodiment, alfalfa cultivars of the selection with high frequency regeneration rate are as genetic transformation
Acceptor material.Multiple alfalfa-leaves are cut into two halves, are inoculated on precultivation medium, culturing room's illumination cultivation is used as and lost for 7 days
Pass the explant of conversion.It (is preferably pCAMBIA3301- that explant is placed in and carries plant expression vector by preculture after terminating
MsKGF2 infecting for Agrobacterium) handles 10min in culture medium, discard bacterium solution, is put into and co-cultures on culture medium, light culture 3 days.
After co-cultivation, explant is placed on micro-organisms base, illumination cultivation (the 8 hours dark of illumination in 16 hours) 7 days.Then outer
Implant is transferred on screening/regeneration culture medium and screened, and subculture once, treats that embryoid grows to 1cm or so and (would generally oneself every two weeks
Come off from embryoid clump), it is inoculated into germination medium sprouting.After about 20 days, embryoid, which is sprouted, obtains regrowth.
In further preferred embodiment, the Agrobacterium for carrying plant expression vector is Agrobacterium EHA105.
Suitable Agrobacterium is selected to realize that target gene is vital to the genetic transformation of plant.Research finds, Agrobacterium
EHA105 realizes high genetic transformation of the msKGF2 genes in alfalfa relative to other Agrobacteriums, such as LBA4404
Rate.
In step 3) of the present invention, because the sample size tested is extremely more, High Throughput Screening Assay, i.e. Taqman are utilized
PCR and western dot blots are identified alfalfa resistant plant, and preliminary screening goes out that copy number is low, expressing quantity is high
Transfer-gen plant (transformation event), then it is identified by southern blot and the western blot of standard, really
The high transfer-gen plant of order copy, expressing quantity.
In a preferred embodiment, because the target gene carried by Agrobacterium is radom insertion recipient plant
Genome in, therefore the transfer-gen plant obtained is often containing a foreign gene to multiple copies.The external source of multicopy
There is the phenomenon of gene silencing in gene, cause its unstable expression during transcription and translation.Thus, identify comprise only it is single
The transfer-gen plant of copy foreign gene is very important.
It is of the invention to be judged by the copy number of the high Taqman PCR method antagonism plant of detection accuracy, just
Step filters out the transfer-gen plant of the foreign gene of low-copy.Paramagnetic particle method is used to extract alfalfa STb gene as template, it is determined that
The reaction system and Taqman PCR programs that Taqman PCR are checked, carry out Taqman PCR inspections.Wherein, it is implementation Taqman
PCR method, designs the primer P1 and P2 in reaction system, and primer P1 includes the nucleotide sequence shown in SEQ ID NO.2；Draw
Thing P2 includes the nucleotide sequence shown in SEQ ID NO.3.
Taqman PCR programs are：94 DEG C of pre-degeneration 30sec；94 DEG C of denaturation 5sec, 54 DEG C of annealing 15sec, 72 DEG C are annealed
10sec, 40 circulations；The fluorescent quenching curves of different samples is gone unless turning base between being recycled to the 40th circulation according to the 15th
Because of plant and the transformation event of multicopy.
In a preferred embodiment, the high transfer-gen plant of expression quantity is screened using western dot blots.Will be low
Rotaring gene plant blade grind into powder in liquid nitrogen is copied, ice bath melts to complete after adding PBS, takes a small amount of (several
Microlitre) drop on pvdf membrane or NC films, washed with TBST buffer solutions, add confining liquid closing, sequentially add primary antibody KGF2 works
Make liquid, the secondary antibody with detection mark, after chromogenic reagent, occur hybridization signal on pvdf membrane or NC films, select hybridization signal
Most i.e. several high transformation events of KGF2 expression quantity are further detected by force.
In a preferred embodiment, it is wild-type alfalfa plant and the transgenosis filtered out through dot blot is purple
The blade of the high expression plant of russian fenugreek herb is cut, and extracts STb gene.Carried out according to method routine or that kit provides
Southern blot are analyzed, and hybridization probe is 35S Promoter, size is 1.4kb between smKGF2, NOS terminator
DNA fragmentation, and molecular weight standard is used as using Trans15K DNA Marker.The swimming lane according to where different samples hybridizes
Copy number of the band number analysis foreign gene of signal in genome, selects single copy (the band number of hybridization signal is 1)
Transgenic alfalfa.Wherein, the nucleotide sequence of hybridization probe is as shown in SEQ ID NO.4；Trans15K DNA Marker
For digested plasmid DNA, by 500bp, 1000bp, 1500bp, 3000bp, 5000bp, 7500bp, 10000bp and 15000 bp
8 wire double-stranded DNA band compositions.
In a preferred embodiment, it is wild-type alfalfa plant and the transgenosis filtered out through dot blot is purple
The blade of the high expression plant of russian fenugreek herb is cut, and extracts total soluble protein.Method according to standard carries out blot points of western
Analysis, the strong and weak analysis exogenous protein expression of swimming lane hybridization signal according to where sample are horizontal.
In step 4) of the present invention, purification of recombinant human source body keratinized cell growth factor-2, the people source of alfalfa expression is obtained
KGF-2。
In a preferred embodiment, the total soluble protein of alfalfa blade will be extracted from through cation exchange
Method is purified.Total soluble protein recycles the NaCl of various concentrations to carry out gradient after cation exchange column exchange adsorption
Elution, each eluting peak albumen is collected respectively and carries out polyacrylamide gel electrophoresis detection, analysis recombination human source KGF-2 elutions institute
Need NaCl concentration.People source KGF-2 is eluted completely when NaCl concentration reaches 0.8~1.0mol/L, and preferably NaCl concentration is
0.9mol/L.Albumen after purification is placed in after polyacrylamide gel electrophoresis the observation purifying effect after coomassie brilliant blue staining
Fruit, and pass through thin layer scanning analyzing proteins purity.
Embodiment
In embodiment, agents useful for same source is as follows：
msKGF2：Oneself design and transfer to Shanghai Sheng Gong Bioisystech Co., Ltd to synthesize；PUC19 carriers：Purchased from Dalian
Precious biotech firm；PCAMBIA3301 carriers：Preserved by Jilin Academy of Agricultural Science；Restriction enzyme NcoI and BstEII：Purchase
From NEB companies；Alfalfa (imperial lucerne 803M26)：Preserved by Jilin Academy of Agricultural Science；Agrobacterium EHA105：By Jilin Province
Academy of Agricultural Sciences preserves；Primer P1：Oneself design and transfer to Shanghai Sheng Gong Bioisystech Co., Ltd to synthesize；Primer P2：Oneself
Design and transfer to Shanghai Sheng Gong Bioisystech Co., Ltd to synthesize；The anti-KGF2 of primary antibody mouse：Have purchased from adopted Beijing Yi Qiao Divine Land science and technology
Limit company；The sheep anti mouse of secondary antibody alkali phosphatase enzyme mark：Purchased from adopted Beijing Yi Qiao Divine Land Science and Technology Ltd.；Restriction enzyme
PsiI：Purchased from NEB companies；DIG High Prime DNA Labeling and Detection Starter Kit I reagents
Box：Purchased from product (Shanghai) Co., Ltd. of Roche Diagnistics；Trans15K DNA Marker：It is limited purchased from the full formula gold biology in Beijing
Company；SP Sepharose Fastaflow cation exchange columns：Purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd.
A kind of production method of people source body keratinized cell growth factor-2 of embodiment 1
The people source KGF-2 of high-purity is obtained using following production method：
1) plant expression vector pCAMBIA3301-msKGF2 is built
All codons of encoding gene 1a) are used into the most suitable coding of alfalfa codon using Gene Designer
Mode carries out codon replacement, passes through the softwares point of DNAMAN7.0 and Vector NTI 10.0 again to the gene order after modification
Restriction enzyme site information therein is analysed, by the 2nd amino acids Ser (serine) coding codon by the 1st most suitable codon TCT,
The 2nd most suitable codon TCA is changed to eliminate Psi I restriction enzyme sites；By the 93rd amino acids Gly (glycine) coding password
Son is changed to the 2nd most suitable codon GGA by the 1st most suitable codon GGT；By the 107th amino acids Arg (arginine) coding
Codon is changed to the 2nd most suitable codon AGG to eliminate Acc I restriction enzyme sites, the people after optimization by the 1st most suitable codon AGA
Shown in source KGF-2 gene orders as SEQ ID NO.1；
After 1b) the people source keratinocyte growth factor gene msKGF2 after codon optimization obtains via chemical synthesis
It is connected into the Restriction enzyme Sma I site of cloning vector pUC19 carriers, is named as pUC19-msKGF2.Using restricted interior
Enzyme cutting NcoI and BstEII cuts msKGF2 genes from the carrier, after gel purified recovery, and through identical inscribe
The commercialized plant expression vector pCAMBIA3301 that enzyme (NcoI and BstEII) processing removes gus gene thereon is discharged
Large fragment be connected, successfully build msKGF2 genes plant expression vector, be named as pCAMBIA3301-msKGF2, such as scheme
Shown in 1.
2) the high flux genetic transformation of msKGF2 gene pairs alfalfa
" acceptor materials of the imperial lucerne 803M26 " as genetic transformation, will for alfalfa cultivars of the selection with high frequency regeneration rate
At least 3000 alfalfa-leaves scalpels or operation shearing are cut into two halves, are inoculated on precultivation medium, culturing room's light
According to 7 days explants as genetic transformation of culture.After preculture terminates, explant is placed in carrying plant expression vector
PCAMBIA3301-msKGF2 infecting for Agrobacterium EHA105 handles 10min in culture medium, discard bacterium solution, is put into co-cultivation training
Support on base, light culture 3 days.After co-cultivation, explant is placed on micro-organisms base, illumination cultivation (illumination in 16 hours 8 hours
It is dark) 7 days.Then explant is transferred on screening/regeneration culture medium and screened, subculture once, treats that embryoid is grown to every two weeks
1cm or so, germination medium sprouting is inoculated into, after about 20 days, embryoid, which is sprouted, obtains regrowth, is obtained through counting final
3534 plants of resistance regeneration plants.
Wherein：Precultivation medium (common 1L)：MS culture medium 4.74g, sucrose 30g, caseinhydrolysate 2g/L, 2,4-D (2,
4- dichlorphenoxyacetic acids) 2.00mg/L, KT (kinetin) 0.25mg/L and 8g/L agar, pH5.8；
Infect culture medium (1L)：MS culture medium 4.74g, sucrose 30g, MES (morpholinoethyl sulfonic acid) 2g/L, 2,4-D
2.00mg/L KT 0.25mg/L and 8g/L agar, pH5.8；
Co-culture culture medium (1L)：MS culture medium 4.74g, sucrose 30g, caseinhydrolysate 2g/L, 2,4-D 2.00mg/L,
KT 0.25mg/L and 8g/L agar, pH5.8；
Micro-organisms base (1L)：MS culture medium 4.74g, sucrose 30g, caseinhydrolysate 2g/L, 2,4-D 2.00mg/L, KT
0.25mg/L, 250mg/L cef (cephalosporin), 250mg/L carb (carbenicillin) and 8g/L agar, pH5.8；
Screening/regeneration culture medium (1L)：MS culture medium 4.74g, sucrose 30g, caseinhydrolysate 2g/L, 2,4-D
2.00mg/L, KT 0.25mg/L, 125mg/L cef, 125mg/L carb, 1.5mg/L basta (nonselective herbicide),
Agar powder 8g/L, pH=5.8；
Germination medium (1L)：MS culture medium 4.74g, sucrose 30g, 125mg/L cef, 125mg/L carb, 1.5mg/
L basta, agar powder 8g/L, pH=5.8.
3) to the screening of alfalfa
3a) low-copy clover transformed plant is filtered out using Taqman round pcrs
The STb gene of 3534 plants of alfalfa resistance regeneration plants is all obtained using paramagnetic particle method extraction, because magnetic bead is inhaled
Attached DNA maximum bearing capacity is certain, therefore the DNA concentration after extraction is basically identical, thus can extract in this way pale reddish brown
The STb gene of clover is that template carries out Taqman PCR inspections.Taqman PCR check that the primer used is：Primer P1, base sequence
Row are as shown in SEQ ID NO.2；Primer P2, base sequence is as shown in SEQ ID NO.3.
The reaction system that Taqman PCR are checked：The μ L of 2 × TransStart Green qPCR SuperMix 10, primer
6.8 μ of μ L, ddH2O of P1 0.4 μ L, primer P2 0.4 μ L, 2 μ L, Passive Reference Dye (50 ×) of template DNA 0.4
L。
Taqman PCR programs are：94 DEG C of pre-degeneration 30sec；94 DEG C of denaturation 5sec, 54 DEG C of annealing 15sec, 72 DEG C are annealed
10sec, 40 circulations；The curve that is quenched of different fluorescents between the 40th circulation is recycled to according to the 15th, is gone unless turning
The transformation event of gene plant and multicopy.
318 plants of low-copy clovers are tentatively filtered out from the 3534 plants of Alfalfa regeneration plants obtained after testing to turn
Gene plant.
Expression analysis 3b) is carried out to transfer-gen plant by western dot blots
By obtained 318 plants of low-copy rotaring gene plant blade grind into powders in liquid nitrogen, by 1：2 (W/V) ratios
PBS is added, ice bath melts to complete；Vortex 30sec；15000g centrifuges 20min under the conditions of 4 DEG C, takes 1 μ L of supernatant liquid
Drop on pvdf membrane, washed 6 times, each 5min with TBST buffer solutions, add confining liquid 100mL, 37 DEG C of closing 2h；Outwell envelope
Liquid is closed, the anti-KGF2 working solutions of primary antibody mouse that 100 mL are diluted with confining liquid, 37 DEG C of coating 1h are added after washing as stated above；
Fall primary antibody working solution, such as the goat-anti for the secondary antibody alkali phosphatase enzyme mark that preceding method washing, addition 100mL confining liquids dilute
Mouse, it is coated with 1h；Secondary antibody working solution is outwelled, such as preceding method is washed, and adds 10mL developer BCIP/NBT, the reaction of room temperature half-light is until go out
After being now expected hybridization signal, film 5min is washed with 50ml distilled waters, with terminating reaction.
For dot blot result as shown in Fig. 2 point A1 is WT lines, remaining point is the hybridization spot of transgenic alfalfa.Through
The strong and weak comparative analysis of hybridization signal finally determines that most the i.e. high alfalfa of KGF2 expression quantity turns base to 6 plants of hybridization signals by force
Because of plant, respectively F5 (transformation event msKGF2-102), A7 (transformation event msKGF2-392), B8 (transformation events
MsKGF2-831), D9 (transformation event msKGF2-1562), E9 (transformation event msKGF2-1567) and D10 (transformation events
msKGF2-2431)。
Wherein, PBS NaCl containing 137mmol/L, 2.7mmol/L KCl, 10mmol/L Na2HPO4, 2mmol/L
KH2PO4；TBST buffer solutions contain 20 mmol/L Tris-HCl, 150mmol/L NaCl, 0.05%Tween 20；Confining liquid is
5% skimmed milk power/TBST buffer solutions.
3c) hybridized by southern blot and copy number analysis is carried out to transfer-gen plant
By wild type alfalfa plants and the leaf of the high expression plant of the transgenic alfalfa filtered out through western dot blots
Piece is cut, and grind into powder extracts STb gene with CTAB methods in liquid nitrogen.10 μ g DNA PsiI digestions are taken, 0.8% agar coagulates
Glue 80V electrophoresis is transferred on nitrocellulose filter after 3 hours, it is UV-crosslinked twice, 30 seconds every time, be spaced 2 minutes.According to
The method that DIG High Prime DNA Labeling and Detection Starter Kit I kits provide is carried out
Southern blot are analyzed, and hybridization probe is 35S Promoter, size is 1.4kb between smKGF2, NOS terminator
DNA fragmentation (as shown in SEQ ID NO.4), using Trans15K DNA Marker as molecular weight standard.
Results of hybridization as shown in figure 3, except the transformation event msKGF2-102 of the 1st swimming lane is multicopy transformation event, remaining
Transfer-gen plant is single copy transfer-gen plant.
3d) hybridized by western blot and expression analysis is carried out to transfer-gen plant
The blade of wild type alfalfa plants and the high expression plant of the transgenic alfalfa filtered out through dot blot is cut, in liquid
Grind into powder in nitrogen, by 1：2 (W/V) ratios add PBS, and ice bath melts to complete；Vortex 30sec；In 4 DEG C of conditions
Lower 15000g centrifuges 20min, and it is total soluble protein to take supernatant；
Take 10 μ L protein solutions to be mixed with 2 × sample-loading buffer of equivalent, the min of boiling water warm bath 5, then carry out 16.5%
Tricine-SDS-PAGE electrophoresis detections, and dyed with coomassie brilliant blue R250；It will not carry out examining horse after electrophoresis under the same terms
The gel of this light blue R250 dyeing, 0.2 μm of nylon membrane is transferred to by half-dried transferring film instrument under 200mA constant current conditions
On, washed 6 times with TBST buffer solutions, each 5min；Add confining liquid 100mL, 37 DEG C of closing 2h；Confining liquid is outwelled, by above-mentioned
The anti-KGF2 working solutions of primary antibody mouse that 100mL is diluted with confining liquid, 37 DEG C of coating 1h are added after method washing；Outwell primary antibody work
Liquid, such as preceding method washing, the sheep anti mouse for the secondary antibody alkali phosphatase enzyme mark that 100mL confining liquids dilute is added, is coated with 1h；
Fall secondary antibody working solution, such as preceding method is washed, and adds 10mL developer BCIP/NBT, and the reaction of room temperature half-light is until there is expected hybridization letter
After number, film 5min is washed with 50mL distilled waters, with terminating reaction.
As shown in figure 4, the strong and weak analysis result of swimming lane hybridization signal shows according to where sample, positioned at turning for the 5th swimming lane
Foreign protein expression highest in change event msKGF2-1567, its protein expression level are (fresh based on plant up to 4.76mg/g
Weight).
4) people source KGF2 purifying
The total soluble protein of transformation event msKGF2-1567 blades is filtered with 0.22 μm of filter membrane, through SP
After Sepharose Fastaflow cation exchange column exchange adsorptions, recycle 0.9mol/L NaCl to carry out gradient elution, receive
Collect eluting peak albumen and carry out 16.5%Tricine-SDS-PAGE electrophoresis detections.Albumen after purification is placed in 16.5%
Observed after Tricine-SDS-PAGE electrophoresis through coomassie brilliant blue staining.
As a result as shown in figure 5, destination protein electrophoresis is only single band, then albumen is shown by thin layer scanning analysis result
Purity is in 85 (weight) more than %.Understand, high-purity people source KGF2 albumen can obtain by production method of the present invention.
The present invention is described in detail above in association with embodiment and exemplary example, but these explanations are simultaneously
It is not considered as limiting the invention.It will be appreciated by those skilled in the art that without departing from the spirit and scope of the invention,
A variety of equivalencing, modification or improvement can be carried out to technical solution of the present invention and embodiments thereof, these each fall within the present invention
In the range of.Protection scope of the present invention is determined by the appended claims.
Sequence table
<110>Wenzhou University
<120>Gene order, expression vector and the production method of a kind of people source body keratinized cell growth factor-2
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 426
<212> DNA
<213>People source KGF-2 (Homo sapiens)
<400> 1
atgtcatata atcatcttca aggtgatgtt agatggagaa aacttttttc ttttactaaa 60
tattttctta aaattgaaaa aaatggtaaa gtttctggta ctaaaaaaga aaattgtcct 120
tattctattc ttgaaattac ttctgttgaa attggtgttg ttgctgttaa agctattaat 180
tctaattatt atcttgctat gaataaaaaa ggtaaacttt atggttctaa agaatttaat 240
aatgattgta aacttaaaga aagaattgaa gaaaatggat ataatactta tgcttctttt 300
aattggcaac ataatggtag gcaaatgtat gttgctctta atggtgaagg tgctcctaga 360
agaggtcaaa aaactagaag aaaaaatact tctgctcatt ttcttcctat ggttgttcat 420
tcttaa 426
<210> 2
<211> 16
<212> DNA
<213>Primer P1 (Homo sapiens)
<400> 2
gatgttagat ggagaa 16
<210> 3
<211> 17
<212> DNA
<213>Primer P2 (Homo sapiens)
<400> 3
aagaatagaa taaggac 17
<210> 4
<211> 1444
<212> DNA
<213>Hybridization probe (Homo sapiens)
<400> 4
gatgttagat ggagaaccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 60
gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 120
aatgctagag cagcttgagc ttggatcaga ttgtcgtttc ccgccttcag tttagcttca 180
tggagtcaaa gattcaaata gaggacctaa cagaactcgc cgtaaagact ggcgaacagt 240
tcatacagag tctcttacga ctcaatgaca agaagaaaat cttcgtcaac atggtggagc 300
acgacacact tgtctactcc aaaaatatca aagatacagt ctcagaagac caaagggcaa 360
ttgagacttt tcaacaaagg gtaatatccg gaaacctcct cggattccat tgcccagcta 420
tctgtcactt tattgtgaag atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt 480
gcgataaagg aaaggccatc gttgaagatg cctctgccga cagtggtccc aaagatggac 540
ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag 600
tggattgatg tgatatctcc actgacgtaa gggatgacgc acaatcccac tatccttcgc 660
aagacccttc ctctatataa ggaagttcat ttcatttgga gagaacacgg gggactcttg 720
accatgtcat ataatcatct tcaaggtgat gttagatgga gaaaactttt ttcttttact 780
aaatattttc ttaaaattga aaaaaatggt aaagtttctg gtactaaaaa agaaaattgt 840
ccttattcta ttcttgaaat tacttctgtt gaaattggtg ttgttgctgt taaagctatt 900
aattctaatt attatcttgc tatgaataaa aaaggtaaac tttatggttc taaagaattt 960
aataatgatt gtaaacttaa agaaagaatt gaagaaaatg gatataatac ttatgcttct 1020
tttaattggc aacataatgg taggcaaatg tatgttgctc ttaatggtga aggtgctcct 1080
agaagaggtc aaaaaactag aagaaaaaat acttctgctc attttcttcc tatggttgtt 1140
cattcttaag gtgaccagct cgaatttccc cgatcgttca aacatttggc aataaagttt 1200
cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta 1260
cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat 1320
gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa 1380
ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcggggtc cttattctat 1440
tctt 1444
Claims (10)
1. the gene order of a kind of people source body keratinized cell growth factor-2, it is characterised in that the gene order includes SEQ ID
Nucleotide sequence shown in NO.1.
2. a kind of expression vector, it is characterised in that the expression vector includes the nucleotide sequence shown in SEQ ID NO.1.
3. expression vector according to claim 2, it is characterised in that the expression vector is plant expression vector.
4. the production method of a kind of people source body keratinized cell growth factor-2, it is characterised in that comprise the following steps：
1), construction of expression vector；
2), realize that the heredity of the gene pairs plant of people source body keratinized cell growth factor-2 turns using high flux genetic transfoumation
Change；
3), extract people's source body keratinized cell growth factor-2 albumen and purified, obtain high-purity people source Keratiocyte growth factor 1
Sub -2 albumen.
5. production method according to claim 4, it is characterised in that in step 2), the plant selects legume, excellent
Elect alfalfa as.
6. the production method according to claim 4 or 5, it is characterised in that in step 3), carried out using base exchange method
The purifying of people source body keratinized cell growth factor-2 albumen；
Preferably, the total soluble protein in plant is extracted, after cation exchange column exchange adsorption, recycling 0.8~
1.0mol/L NaCl carries out gradient elution, collects eluting peak albumen.
7. the production method according to one of claim 4 to 6, it is characterised in that in step 3), obtained people source cutin is thin
The purity of protein of the intracellular growth factor -2 is more than 85 (weight) %.
8. production method according to claim 4, it is characterised in that methods described is also included to completing heredity in step 2)
The plant of conversion is identified, filters out the high transfer-gen plant of single copy, expressing quantity.
9. production method according to claim 8, it is characterised in that utilize Taqman PCR and western dot blots pair
Resistant plant is identified that preliminary screening goes out the transfer-gen plant that copy number is low, expressing quantity is high, then passes through southern
Blot and western blot determine the high transfer-gen plant of single copy, expressing quantity.
10. production method according to claim 8 or claim 9, it is characterised in that low copy number is carried out using Taqman PCR and turned
When gene plant screens, primer P1 and P2 in reaction system are designed,
Primer P1 includes the nucleotide sequence shown in SEQ ID NO.2；
Primer P2 includes the nucleotide sequence shown in SEQ ID NO.3.
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| CN110551200A (en) * | 2019-07-26 | 2019-12-10 | 温州大学 | Production method of fibroblast growth factor-18 and application of fibroblast growth factor-18 in preparation of hair promoting preparation |
| CN110656128A (en) * | 2019-10-18 | 2020-01-07 | 周口师范学院 | Recombinant construct containing human keratinocyte growth factor gene expression and application thereof |
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