CN108250288A - A kind of brain-derived neurotrophic factor hBDNFb albumen of chloroplast expression and preparation method thereof - Google Patents
A kind of brain-derived neurotrophic factor hBDNFb albumen of chloroplast expression and preparation method thereof Download PDFInfo
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- CN108250288A CN108250288A CN201810042307.6A CN201810042307A CN108250288A CN 108250288 A CN108250288 A CN 108250288A CN 201810042307 A CN201810042307 A CN 201810042307A CN 108250288 A CN108250288 A CN 108250288A
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Abstract
Brain-derived neurotrophic factor hBDNFb albumen the invention discloses a kind of chloroplast expression and preparation method thereof.The invention also discloses a kind of brain-derived neurotrophic factor hBDNFb albumen of chloroplast expression, and amino acid sequence is as shown in SEQ ID NO.2.The preparation method of above-mentioned albumen includes the following steps：Build tobacco chloroplast expression vector pWYP23402；Tobacco chloroplast conversion and the homogeneity of hBDNFb genes are screened, and obtain the homogeneous plant that tobacco chloroplast turns hBDNFb genes；HBDNFb expression analysis in chloroplast transgenic tobacco plant；The purifying of brain-derived neurotrophic factor hBDNFb albumen obtains the brain-derived neurotrophic factor hBDNFb albumen of chloroplast expression.Present invention high efficient expression GFP fusion proteins of hBDNFb in tobacco chloroplast, have the condition commercially produced.
Description
Technical field
The invention belongs to genetic engineering field, specifically, be related to a kind of brain-derived neurotrophy of chloroplast expression because
Sub- hBDNFb albumen and preparation method thereof.
Background technology
Growth factor be cell synthesis and secretion a kind of reactive protein, in animal body organize in, cell, growth because
Son and extracellular matrix are co-located among a dynamic environment, and the multiple interaction between three constitutes growth factor in body
The microenvironment inside to play a role.By the research to hematopoietic cell development, it is found that growth factor can be by inducing or adjusting gene
Proliferation and differentiation of the expressional function in cell.As various growth factors and its biological characteristics are constantly disclosed, about life
The long factor is improving various physiology, pathological state, promotes the data of regeneration, wound repair and agglutination etc.
Constantly accumulation, increasingly important role is played in application fields such as medicine.Meanwhile the research of growth factor also has become and makes a living
The research hotspot problem of the subjects such as object chemistry, cell biology, medicine and organizational engineering.
Brain-derived neurotrophic factor b (BDNFb) is one of neurotrophic factor family member, has and promotes neuron life
Physiological action that is long and maintaining neuron normal function, can treat a variety of central nervous system nerve degenerative diseases.However
The natural content of growth factor is extremely low in human body, and every kilogram of tissue is only extractable to obtain the growth factor of microgram level, and so far
It does not find again abundant tissue-derived.Therefore, above-mentioned growth factor is expressed with highly important reason by genetic engineering means
By research significance and market application foreground.
Chloroplaset has the characteristics that semi-autonomous heredity, and chloroplast transformation is that foreign gene is whole by the method for homologous recombination
The operating process closed on Chloroplast gene and expressed in chloroplaset.It is poor that chloroplast transformation method eliminates Plant accepter expression
Different influence, and Chloroplast Inheritance is matrilinear inheritance, is not influenced by karyogene, will not occur that " gene floats as Nuclear transformation
Ecological risk caused by shifting ".In addition, chloroplast expression eliminates the influence of gene silencing, and plant Chloroplast genome
Copy number is very high, and in the chloroplaset being theoretically each converted, the copy number of foreign gene can reach 1-2 ten thousand, because in addition
The expression quantity level of source gene can run up to very high level, compared with traditional microbial fermentation system, production cost
It is cheap, thus as ideal bioreactor.In addition, the most protein of Chloroplast Genetic Engineering production can complete two sulphur
The posttranscriptional modifications such as key crosslinking, correct folding, have correct space conformation and bioactivity, to commercially produce medical protein
It lays a good foundation.(Staub J M, Garcia B, Graves J, the et al.High yield production of such as Staub
a human therapeutic protein in tobacco chloroplast[J].NatBiotechnol,2000,18:
333-338.) for people by auxin channel genes tobacco chloroplast genome, the human growth hormone that transgene tobacco gives expression to compares core
Conversion system is 300 times high, and with normal biological activity.2004, (the Daniell H, Medical such as Daniell
molecular Pharming,Encyclpedia ofplant and crop science.2004:705-710) by insulin
Growth factor I GF21 genes are transferred in tobacco chloroplast, obtain ripe IGF21, expression quantity accounts for the 32% of soluble protein, important
Be that IGF21 can form correct disulfide bond in chloroplaset, and cannot then be formed in Escherichia coli.
Invention content
In view of this, the present invention for it is above-mentioned the problem of, provide a kind of brain-derived neurotrophy of chloroplast expression because
GFP protein tags are merged the N-terminal in BDNFb growth factors by sub- hBDNFb albumen and preparation method thereof, the present invention, to improve
Its expression quantity in tobacco chloroplast.
In order to solve the above-mentioned technical problem, the invention discloses a kind of brain-derived neurotrophic factor of chloroplast expression
HBDNFb albumen, amino acid sequence is as shown in SEQ ID NO.2.
The present invention also provides a kind of brain-derived neurotrophic factor hBDNFb albumen for encoding above-mentioned chloroplast expression
Gene, nucleotide sequence is as shown in SEQ ID NO.1.
The invention also discloses a kind of for producing the tobacco chloroplast table of brain-derived neurotrophic factor hBDNFb albumen
Up to carrier pWYP23402, the carrier is using 16S-trnI and trnA-23S as left and right homologous sequence, and insertion chemistry synthesizes therebetween
Prrn-T7g10-GFP＆hBDNFb-aadA-Trps16 expression cassettes；Wherein, Prrn promoters and Trps16 terminators are respectively cigarette
The 16S rRNA promoters of careless Chloroplast gene and rhct1 gene mRNA 3' ends mature sequence (GenBank accession number：NC_
001879)；Contained fusion tag gene GFP, target gene BDNFb and screening-gene aadA using DNA2.0 softwares into
Codon optimization is gone.
The invention also discloses a kind of preparation method of the brain-derived neurotrophic factor hBDNFb albumen of chloroplast expression,
Include the following steps：
1) structure tobacco chloroplast expression vector pWYP23402；
2) the tobacco chloroplast conversion of hBDNFb genes is screened with homogeneity, is obtained tobacco chloroplast and is turned hBDNFb genes
Homogeneous plant；
3) hBDNFb expression analysis in chloroplast transgenic tobacco plant；
4) purifying of brain-derived neurotrophic factor hBDNFb albumen, obtain the brain-derived neurotrophy of chloroplast expression because
Sub- hBDNFb albumen.
Further, the structure tobacco chloroplast expression vector pWYP23402 in the step 1) is as follows：
1.1), optimize brain-derived neurotrophic factor hBDNFb, the brain-derived neurotrophic factor after being optimized
HBDNFb, nucleotide sequence is as shown in SEQ ID NO.1；
1.2), structure tobacco chloroplast expression vector pWYP23402：Expression cassette is synthesized using chemical synthesis full genome,
Expression cassette includes following two parts：The C-terminal addition flexible connection peptide chain of green fluorescence protein gene as fusion protein label
The linker formed with enterokinase recognition site, i.e. amino acid sequence are GSSSGSSSGSSSGSSSDDDDK；And it is used as screening mark
The spectinomycin resistance gene of note gene is placed in after the target gene with green fluorescence protein gene fusion tag, common structure
Into the polycistronic expression frame terminated by tobacco chloroplast promoter Prrn startups, tobacco chloroplast terminator Trps16, wherein,
Tobacco chloroplast promoter Prrn contains ribosome bind site T7g10 sequences；
1.3) artificial synthesized expression cassette carries out digestions processing after the insertion for completing target gene using Nsi I
The segment generated afterwards and " 16Srrn-trnI-trnA-23Srrn in carrying clone's tobacco chloroplast genome of identical enzymatic treatment
Large fragment " cloning vector pEASY-Nt be attached reaction, artificial synthesized expression cassette is made to be inserted into trnI genes and trnA
Between gene, the final tobacco chloroplast expression vector obtained for producing brain-derived neurotrophic factor b is named as
pWYP23402。
Further, the purifying of the brain-derived neurotrophic factor hBDNFb albumen in the step 2) is specially：It will be total
Soluble protein is placed in the ultra-filtration centrifuge tube that molecular weight is 50KD that shuts off, 4000g centrifugations after 0.22 μm of filter membrane filters
30min takes efflux, then is placed in the ultra-filtration centrifuge tube that molecular weight is 30KD that shuts off after 4000g centrifugations 45min, retention part
Liquid be N-terminal fusion GFP labels hBDNFb growth factors preliminary purification product, digested using enterokinase, make growth because
Son separates, then purified with Heparin-Sepharose affinity chromatography column with GFP label proteins, obtains brain-derived neurotrophy
Factor hBDNFb albumen.
Compared with prior art, the present invention can be obtained including following technique effect：
1) present invention high efficient expression GFP fusion proteins of hBDNFb, expression quantity in tobacco chloroplast respectively reaches
The 19.53% of total soluble protein has the condition commercially produced.Headed by high efficient expressions of the hBDNFb in chloroplaset
Secondary report.
2) purifies and separates of the present invention have purified fusion protein, and are cut by enterokinase, the hBDNFb lifes to be dissociated
The Lab Products of the long factor, the purity of product have reached 98%.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaches all the above technique effect.
Description of the drawings
Attached drawing described herein is used to provide further understanding of the present invention, and forms the part of the present invention, this hair
Bright illustrative embodiments and their description do not constitute improper limitations of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is vector construction schematic diagram of the present invention；The amino acid sequence of linker is GSSSGSSSGSSSGSSSDDDDK；
When Target gene are BDNFb, chloroplast expression vector pWYP23402；smGFP：Solvable modification type green fluorescent protein base
Cause；aadA：Aminoglycoside -3'- adenylase genes；Prrn、Trps16：Derived from the regulation and control member of tobacco chloroplast genome
Part；T7g10：The boot sequence of T7 phage genes 10；attP、attB：Recombinate enzyme recognition site；P1, P2 are that PCR detections are drawn
Object (P1：5'-GGTCGGAACAAGTTGATAG-3', P2：5'-CAGTAGAGTCTTTCAGTGGC-3'), wild type is estimated expands
It is about 1.2kb to increase segment, and the estimated amplified fragments of transformant are about 3.9kb；BamHI and Kpn I are used for Southern hybridization checks
Endonuclease reaction, between trnI and trnA the segment of about 1.4kb be hybridization probe；
Fig. 2 is homogeneity screening, Molecular Detection and the T1 of chloroplast transformation plant of the present invention for Resistance Identification；Wherein, A：
The chloroplaset that first run screening obtains turns the tobacco resistant plant of BDNFb genes；B：The leaf obtained by continuous 3 wheel high pressure screenings is green
Body turns the Southern results of hybridization of the tobacco resistant plant of BDNFb genes, wherein except chloroplaset turns the tobacco of BDNFb genes
In resistant plant in 2 tobacco resistant plant 3 be non-homogeneous plant, remaining is homogeneous plant；Wherein, M standard molecules
Amount control ,-it is WT lines, 1-4 is transfer-gen plant；
C：The chloroplaset obtained by continuous 3 wheel high pressure screenings turns the SDS-PAGE of the tobacco resistant plant of BDNFb genes
As a result, P is GFP albumen, wt is WT lines, and 1-4 is transfer-gen plant； D：The leaf obtained by continuous 3 wheel high pressure screenings
Green body turns the western results of hybridization of the tobacco resistant plant of BDNFb genes, and P is GFP albumen, and wt is WT lines, 1-4
For transfer-gen plant；
Fig. 3 is the In vivo detection of protein tag green fluorescent protein GFP of the present invention；
Fig. 4 is the growth factor protein sterling and its Activity determination of chloroplast expression of the present invention, wherein, A：Chloroplast expression
Growth factor protein freeze-dried powder；B, C：Three kinds of growth factors promote 3T3 cell proliferation experiments, the tentative confirmation life obtained
The long factor has cell activity；PBS, PBS buffer solution compare；C hBDNFb are the hBDNFb of commercial goods；PD hBDNFb
The respectively hBDNFb in chloroplaset source.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the realization process of technical effect.
1 tobacco chloroplast expression vector pWYP23402 (BDNFb) of embodiment
1) using modern biotechnology informatics technology to the brain-derived neurotrophic factor b in the people source in ncbi database
The nucleotide sequence of (human Brain-derived neurotrophic factorb, BDNFb) gene optimizes.
Under the premise of amino acid sequence is kept not change, all codons of encoding gene are adopted using Gene Designer
Codon replacement is carried out with the most suitable coding mode of tobacco chloroplast codon, the gene order after modification is passed through again
DNAMAN7.0 and 10.0 softwares of VectorNTI analyze restriction enzyme site information therein, and brain-derived neurotrophic factor b is encoded
The coding codon of the 104th amino acids Tyr of gene is changed to the 2nd most suitable codon TCA to disappear by the 1st most suitable codon TCT
Except Nsi I restriction enzyme sites, brain-derived neurotrophic factor b (the human Brain-derived in the people source after modification
Neurotrophic factorb, BDNFb) gene nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence is such as
Shown in SEQ ID NO.2.
2) expression cassette is synthesized using chemical synthesis full genome.Expression cassette includes following two parts：As fusion protein mark
The linker that the C-terminal addition flexible connection peptide chain of the green fluorescence protein gene (GFP) of label is formed with enterokinase recognition site, i.e.,
Amino acid sequence prevents from carrying out space folding initiation in protein synthesis for GSSSGSSSGSSSGSSSDDDDK to facilitate
Excision after problem and separation and Extraction destination protein to GFP labels, target gene pass through reserved Cla I and Xba I digestions position
It puts and is successfully merged with GFP genes；And the spectinomycin resistance gene (aadA) as riddled basins is placed in GFP
After the target gene of fusion tag, collectively form by the tobacco chloroplast promoter Prrn (sequences of T7g10 containing ribosome bind site
Row) start, the polycistronic expression frame that tobacco chloroplast terminator Trps16 is terminated.
3) artificial synthesized expression cassette is after the insertion for completing target gene, after carrying out digestions processing using Nsi I
In carrying clone's tobacco chloroplast genome of the segment of generation and identical enzymatic treatment " 16Srrn-trnI-trnA-23Srrn's
The cloning vector pEASY-Nt of large fragment " is attached reaction, and artificial synthesized expression cassette is made to be inserted into trnI genes and trnA bases
Because between, the final tobacco chloroplast expression vector obtained for brain-derived neurotrophic factor b is named as pWYP23402
(BDNFb)。
According to the method described above, we construct tobacco chloroplast conversion carrier pWYP23402, as shown in Fig. 1.The carrier
Using 16S-trnI and trnA-23S as left and right homologous sequence, the Prrn-T7g10-GFP＆hBDNFb- of insertion chemistry synthesis therebetween
AadA-Trps16 expression cassettes.Wherein, Prrn promoters and Trps16 terminators are respectively the 16S of tobacco chloroplast genome
RRNA promoters and rhct1 gene mRNA 3' ends mature sequence (GenBank accession number：NC_001879)；Three contained bases
Because MSI-99, smgfp and aadA have carried out codon optimization using DNA2.0 softwares.In smgfp upstream region of gene and aadA
Downstream of gene is inserted into the attP sites of homologous recombination enzyme identification and attB sites respectively, it is therefore an objective to for subsequently cutting off selection markers
Gene.
Tobacco chloroplast conversion and the homogeneity of 2 BDNFb genes of embodiment are screened
Biolistic bombardment is carried out with carrier pWYP23402 package bronzes.Concrete operations referring to a kind of people source alkalinity of patent into
Fibroblast growth factor, tobacco chloroplast expression vector and production method (application number：201510310239.3 the applying date：
2015-06-08, publication number：CN104946656A).
A. tobacco explant prepares
Tobacco seed steeps 30sec with 70% ethyl alcohol, during which jiggles；It is careful to discard ethyl alcohol, add in 0.1%HgCl2,
Jiggle 2min；Carefully discard HgCl2Solution, adds rinsed with sterile water 3 times, each 1min.Last time first leaves after rinsing
Less part water, seed suspension is got up, if being poured on dried layer aseptic filter paper, blots water, and carefully seed is inoculated with tweezers
Onto 1/2MSO culture mediums, in 25 DEG C of constant temperature, the photoperiod is to be cultivated in the culturing room of illumination 16h/ dark 8h.
Chloroplast transformation is carried out with PDS 1000/He particle guns (Bio-Rad companies of the U.S.)：Bronze particle diameter is 0.6 μ
M, target distance 9cm can split film using 1100psi, and the micro- bullet of bronze is wrapped up by every milligram of bronze with 0.2 μ g Plasmid DNA.Specifically
It is as follows：
B. the package of the preparation of micro- bullet and bronze
1st, the bronze of a diameter of 0.6 μm of accurate weighing 20mg, is carefully transferred in the import centrifuge tube of 1.5mL.
2nd, 200mL absolute ethyl alcohols, vortex oscillation 3 times, each 2min, interval are added in into the centrifuge tube equipped with bronze
1min。
3rd, after concussion processing, centrifuge tube is placed in and stands 10 minutes under room temperature.
4th, under room temperature, 12,000g centrifuges 1min.
5th, supernatant is carefully siphoned away with pipettor, 200 μ L sterile waters is being added in into the centrifuge tube equipped with bronze, vortex shakes
It swings 30 seconds, bronze is made fully to be resuspended.
6th, under room temperature, 12,000g centrifuges 1min.
7th, step 5,6 are repeated twice.
8th, supernatant is carefully siphoned away with pipettor, 200 μ L sterile waters is being added in into the centrifuge tube equipped with bronze, vortex shakes
It swings 30 seconds, bronze is spare after being fully resuspended.
9th, it is drawn with pipettor and prepares the 100 μ L of bronze suspension Jing Guo above-mentioned processing to the 1.5mL's crossed through high-temperature sterilization
In import centrifuge tube, and the 10 μ L concentration at least tobacco chloroplast expression vector pWX-Nt03 of 1 μ g/ μ L is added in thereto, on ice
Vortex oscillation 30sec.
10th, continue to be added the CaCl of 100 a concentration of 12.5M of μ L into centrifuge tube with pipettor2Solution, and be vortexed on ice
30sec is vibrated to make the abundant mixing of liquid.
11st, slowly the spermidine solution of 40 a concentration of 0.1M of μ L is added in centrifuge tube, while use pipette tips with pipettor
It stirs and evenly mixs.
12nd, oscillation vortex mixer is placed in 4 DEG C of showcase or refrigerator, and centrifuge tube is vibrated into mixing 30min.
13rd, room temperature 12,000g high speed centrifugation 1min, carefully remove supernatant.
14th, 500 μ L absolute ethyl alcohols are added into centrifuge tube with pipettor, and precipitation is gently purged with pipette tips.
15th, in 4 DEG C of showcase or refrigerator, by centrifuge tube vortex oscillation 15sec, precipitation is made thoroughly to be resuspended.
16th, room temperature 12,000g high speed centrifugation 1min, carefully remove supernatant.
17th, 100 μ L absolute ethyl alcohols are added into centrifuge tube with pipettor, vortex oscillation 15sec makes bronze fully suspend standby
With.
18th, by iron hoop, carrier film, film, blocking net etc. can be split after tools soaked in absolute ethyl alcohol 30min, in ultra-clean work
It is air-dried in platform, and carrier film is fitted into iron hoop, 10 μ L points is taken to make its drying of tiling as possible in particle film carrier center with pipettor
It is for use afterwards.
C. biolistic bombardment
1st, the healthy leaves that selection weeds aseptic seedling 3-4 pieces, blade diameter are about 3cm cut back as explant
It is placed on recovery media, is cultivated 4 hours under dark situation up.
2nd, for use 1100psi can be split film and particle slide glass first to impregnate 30 minutes in absolute ethyl alcohol, then sterile
It is air-dried in operation console for use.
3rd, it is cleaned inside and outside the table top, inner wall and particle gun of aseptic operating platform, is opened after wiping purple using 70% alcohol
Outer lamp sterilizing at least more than 30min.
4th, ultraviolet lamp is closed, opens sterile air circulating system, and open the power switch of vacuum pump and particle gun.
5th, the valve of the gas cylinder equipped with helium is unscrewed with spanner, helium pressure valve is adjusted, makes air supply opening helium gas
Pressure is higher than 1300psi.
6th, bombardment exterior door is opened, film door can be split and taken off, can be split in film side bayonet slot disposed with tweezers, then
Again door is installed.
7th, it takes out corpuscular emission device and screws off outer cover, be sequentially placed into blocking net and the iron equipped with the carrier film for carrying bronze
Ring makes the one side that carrier film speckles with bronze downward (vegetable material side i.e. to be bombarded), screws on outer cover again and will be micro-
Grain emitter is pushed into bombardment room along card slot.
The 8th, culture dish equipped with vegetable material to be bombarded is placed on to the appropriate card slot that bombardment room is inserted into sample disc
Position, distance is 3cm between each card slot, and several card slots are spaced between corpuscular emission device and sample disc and show target distance
For 3 × n cm, whether closing is bombarded the external door of room and gone through seals.
9th, it according to particle gun operation rules, proceeds as follows successively, opens VAC switches (upper gear) and start to vacuumize, directly
When not being further added by for desirable value (27~28niHg) to vacuum pressure meter reading, switch is adjusted at HOLD switches rapidly
(lower block), then long-press FIRE, which is switched, injects helium into particle gun, can split film when the helium pressure of injection reaches and can bear
Maximum pressure when, can split film rupture helium by particle gun room enter and the bronze on carrier film is squeezed into vegetable material,
FIRE switches are unclamped at this time.
10th, switch is moved at VENT (middle apron), the atmospheric pressure of release bombardment indoor and outdoor, when pressure vacuum gauge is read
When number is restored to 0, you can open bombardment exterior door.
11st, bombardment room door is opened, takes out in sample disc equipped with the culture dish of vegetable material crossed through biolistic bombardment, covers
Culture dish lid is simultaneously sealed with sealed membrane.
12nd, it takes out corpuscular emission device and the iron hoop being packed into before and blocking net is taken out and be soaked in absolute ethyl alcohol again
In.
13rd, film door can be split to remove, is taken out damaged splitting film or film fragment can be split with tweezers.
14th, step 6-13 is repeated, until the biolistic bombardment process of all material terminates.
15th, the main valve of helium tank is closed, it is gentle to live in the gas-guide tube that FIRE switches are bled off between particle gun room and helium tank for long
Helium in body accelerating tube when pressure gauge reading is 0, cuts off the power supply of particle gun, vacuum pump, superclean bench successively.
D. homogeneity screening
The first round screens：The vacuum side of blade bombarded is restoring to support 25 DEG C of light cultures 3 days on base upward.It, will after light culture
Blade is cut into the small pieces of 5mm × 5mm, and the back side is placed on downward on screening and culturing medium I (spectinomycin containing 500mg/L), with 16h light
According to 25 DEG C of cultures of photoperiod of, 8h dark, culture medium is replaced within every 20 days.After generating adventitious bud, detected with 365nm ultraviolet lamps, it will
The adventitious bud for generating green fluorescence is cut, and is placed on root media I (spectinomycin containing 500mg/L).PCR detecting and selecting property marks
Remember gene GFP, aadA and target gene hBDNFb.
Second wheel screening：The stronger plant of transfer-gen plant Green fluorescence is chosen, its leaf is cut into 5mm × 5mm's
Small pieces, the back side are placed on downward on screening and culturing medium II (spectinomycin containing 625mg/L), with 16h illumination, the photoperiod 25 of 8h dark
DEG C culture, every 20 days replace culture medium.After generating adventitious bud, it is (grand mould containing 625mg/L in root media II to cut adventitious bud
Element) on take root.PCR detects its degree of homogenization.
Third round is screened：The plant through PCR detection high conversion rates is chosen, its leaf is cut into the small pieces of 5mm × 5mm, the back of the body
It is placed on screening and culturing medium III (spectinomycin containing 750mg/L) down, as the above method is screened；This screening go out it is indefinite
It is repeated once after bud, the adventitious bud finally obtained is taken root on root media III (spectinomycin containing 750mg/L).PCR is detected
Its degree of homogenization.
Fourth round is screened：The plant through PCR detection high conversion rates is chosen, its leaf is cut into the small pieces of 5mm × 5mm, the back of the body
It is placed on screening and culturing medium IV (spectinomycin containing 1000mg/L) down, as the above method is screened；This screening is going out not
It is repeated once after normal bud, the adventitious bud finally obtained is taken root on root media IV (spectinomycin containing 1000mg/L).PCR is examined
Its degree of homogenization is surveyed, and passes through southern hybridization verification homogeneity testing results.
PCR detection method is as follows：PCR reactions are shown in Fig. 1 using positions of the primer P1 and P2 on tobacco chloroplast genome
It is shown.PCR programs are：95 DEG C of pre-degeneration 5min；95 DEG C denaturation 20sec, 56 DEG C annealing 20sec, 72 DEG C extension 2min, 30
Cycle；Extend 5min after 72 DEG C.
Southern blot analysis methods are as follows：Take 4 μ g chloroplast DNA BamHI and Kpn I digestions, 0.8% agar
Gel 80V electrophoresis is transferred to after 3 hours on nitrocellulose filter, and UV crosslinking twice, 30 seconds every time, is spaced 2 minutes.According to
The method that Roche companies kit provides carries out Southern hybridization analysis, and probe is trnI-trnA sections BamHI and Kpn
The segment of about 1.4kb between I restriction endonucleases.Concrete operations are referring to a kind of people source basic fibroblast growth factor of patent, tobacco
Chloroplast expression vector and production method (application number：201510310239.3 the applying date：2015-06-08, publication number：
CN104946656A)。
Exogenous protein expression is analyzed as follows：By a kind of people source basic fibroblast growth factor of patent, tobacco chloroplast
Expression vector and production method (application number：201510310239.3 the applying date：2015-06-08, publication number：
CN104946656A method extraction total soluble protein).
SDS PAGE and Western blot are analyzed as follows：The albumen for extracting transgenic tobacco plant and WT lines is thick
Extract takes 10 μ l of protein solution to be mixed with 2 × sample-loading buffer of equivalent respectively, and boiling water warm bath 5min is taken on 10 μ l mixed liquors
Sample is separated by electrophoresis in 12% PAGE gel.80V voltages prerunning 1h during electrophoresis, later 160V voltages to electrophoresis knot
Beam.Two pieces of glue, one of coomassie brilliant blue staining, another piece of half-dried electric rotary device using BIO-RAD companies are run simultaneously
It is transferred on PVDF films, by a kind of people source basic fibroblast growth factor of patent, tobacco chloroplast expression vector and production
Method (application number：201510310239.3 the applying date：2015-06-08, publication number：CN104946656A method) is done
WesternBlot is analyzed.
Elisa assay is as follows：The concentration of protein crude extract is measured with Branford methods, is then resisted respectively with anti-GFP
Body carries out elisa assay, and concrete operations are expressed referring to a kind of people source basic fibroblast growth factor of patent, tobacco chloroplast
Carrier and production method (application number：201510310239.3 the applying date：2015-06-08, publication number：CN104946656A).
Expression vector is imported in tobacco chloroplast genome by particle bombardment, is sieved by the spectinomycin of 500mg/L
Choosing obtains resistance regeneration bud point (Fig. 2A) respectively.The resistant plant that further screening obtains is removed through Southern hybridization analysis
It is non-homogeneous plant that chloroplaset, which turns in the tobacco resistant plants of BDNFb genes 2, remaining is homogeneous plant.Chloroplaset base
Because group with BamHI and Kpn I digestion rear electrophoresis transferring films.We are it is expected that Wild type control plants should be in the position of about 2.4kb
It puts and hybridization signal occurs, and if transfer-gen plant can only detect hybridization in homogeneous homozygotic state at about 5.2kb
Signal, and two hybridization signals of 5.2kb and 2.4kb can then occur simultaneously in heterogeneousization plant.As shown in Figure 2 B, Southern is miscellaneous
It hands over and shows that chloroplaset turns in the tobacco resistant plants of BDNFb genes 2 and two hybridization signals of 5.2kb and 2.4kb occurs, be heterogeneous
Change plant.Remaining is homogeneous plant.
For the expression quantity of testing goal gene, we have also carried out SDS-PAGE and Western hybridization, have as a result seen Fig. 2 C
With Fig. 2 D.The chloroplast transgenic plant of different growth factors is regardless of whether hybridization signal, and its hybridization letter, occurs in homogeneity
Number molecular size is all higher than GFP albumen and is consistent with expected size.
The In vivo detection of 3 protein tag green fluorescent protein GFP of embodiment
According to a kind of people source basic fibroblast growth factor of patent, tobacco chloroplast expression vector and production method
(application number：201510310239.3 the applying date：2015-06-08, publication number：CN104946656A method), to transgenosis
Material carries out whole strain fluorescence analysis.
Fluoroscopic examination is carried out to the GFP labels of the destination protein N-terminal of transfer-gen plant expression.In the ultraviolet light of 365nm
Under, transfer-gen plant sends out strong green fluorescence in darkroom, shows that green fluorescent protein obtains great expression, and compares strain
It then shows as from red-emitting fluorescent (Fig. 3).
Separation, extraction and the removal of GFP labels of the growth factor protein of 4 chloroplast expression of embodiment extract non-turn respectively
Genetic contrast and the mature leaf of transgenosis homogeneity plant, the grind into powder in liquid nitrogen, every gram of blade add in the PBS of 5mL
Total soluble protein in buffer solution extraction plant leaf, after 0.22 μm of filter membrane filters, it is the super of 50KD to be placed in the molecular weight that shuts off
It filters in centrifuge tube, 4000g centrifugation 30min take efflux, then are placed in 4000g in the ultra-filtration centrifuge tube that molecular weight is 30KD that shuts off
After centrifuging 45min, retention part liquid is the preliminary purification product of the hBDNFb growth factors of N-terminal fusion GFP labels.
It is digested using enterokinase, growth factor is made to be separated with GFP label proteins, then with the affine layers of heparin-Sepharose
Analysis column is purified, and albumen after purification carries out cell proliferation experiment using Balb/c 3T3 cells to above-mentioned growth factor, with
Whether active detect it.
The mature leaf of transgenosis homogeneity plant, the grind into powder in liquid nitrogen are extracted, every gram of blade adds in 5mL's
PBS buffer solution extracts total soluble protein in blade, and after 0.22 μm of filter membrane filters, it is the super of 50KD to be placed in the molecular weight that shuts off
It filters in centrifuge tube, 4000g centrifugation 30min take efflux, then are placed in 4000g in the ultra-filtration centrifuge tube that molecular weight is 30KD that shuts off
After centrifuging 45min, retention part liquid is the preliminary purification product of the BDNFb growth factors of N-terminal fusion GFP labels.Purifying
Anteroposterior diameter ELISA is detected, and loss of proteins ratio is controlled within 30%, and the recombinant protein of the label containing GFP accounts for about albumen after purification
More than 73%.
It is digested using enterokinase, growth factor is made to be separated with GFP label proteins, then is affine with heparin-Sepharose
Chromatographic column is purified, and gropes to confirm through condition, the buffer solution formed with the PB of the NaCl containing 0.7mol/L and 10mmol/L
Growth factor is eluted, best purification effect and high organic efficiency can be obtained, obtains the BDNFb that purity is 98%, freeze-dried processing
Primary Lab Products (Fig. 4 A) are obtained afterwards.Albumen after purification carries out cell Proliferation using 3T3 cells to above-mentioned growth factor
Experiment, as a result as shown in Figure 4 B, compared with negative control, growth factor is respectively provided with apparent promotion 3T3 cell Proliferation effects, takes off
Its potentiality as pharmaceutical protein raw material is shown.Meanwhile the bioactivity of this growth factor in chloroplaset source and commercially available quotient
Activity difference comparative experiments between the albumen of product as a result, it has been found that, the former outline is higher than the latter, but difference is not significantly only compared with the latter
8.7% is improved, it is consistent (Fig. 4 C) to be accordingly regarded as effect.Present invention GFP of high efficient expression hBDNFb in tobacco chloroplast melts
Hop protein, expression quantity have respectively reached the 19.53% of total soluble protein, have the condition commercially produced.The present invention is pure
Change has isolated and purified fusion protein, and is cut by enterokinase, the Lab Products of hBDNFb growth factors to be dissociated,
The purity of product has reached 98%.
Several preferred embodiments of invention have shown and described in above description, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, is not to be taken as the exclusion to other embodiment, and available for various other combinations, modification
And environment, and can be carried out in the scope of the invention is set forth herein by the above teachings or related fields of technology or knowledge
Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in power appended by invention
In the protection domain of profit requirement.
Sequence table
<110>Jilin Academy of Agricultural Science
<120>A kind of brain-derived neurotrophic factor hBDNFb albumen of chloroplast expression and preparation method thereof
<130> 2017
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 396
<212> DNA
<213>Brain-derived neurotrophic factor b (the human Brain-derived neurotrophic factor in people source
b)
<400> 1
atgggagtat ctgaaactgc tcctgcttct agaagaggag aattagctgt atgtgatgct 60
gtatctggat gggtaactga tagaagaact gctgtagatt taagaggaag agaagtagaa 120
gtattaggag aagtacctgc tgctggagga tctcctttaa gacaatattt ttttgaaact 180
agatgtaaag ctgataatgc tgaagaagga ggacctggag ctggaggagg aggatgtaga 240
ggagtagata gaagacattg ggtatctgaa tgtaaagcta aacaatctta tgtaagagct 300
ttaactgctg atgctcaagg aagagtagga tggagatgga ttagaattga tactgcttgt 360
gtatgtactt tattatctag aactggaaga gcttaa 396
<210> 2
<211> 131
<212> PRT
<213>Brain-derived neurotrophic factor b (the human Brain-derived neurotrophic factor in people source
b)
<400> 2
Met Gly Val Ser Glu Thr Ala Pro Ala Ser Arg Arg Gly Glu Leu Ala
1 5 10 15
Val Cys Asp Ala Val Ser Gly Trp Val Thr Asp Arg Arg Thr Ala Val
20 25 30
Asp Leu Arg Gly Arg Glu Val Glu Val Leu Gly Glu Val Pro Ala Ala
35 40 45
Gly Gly Ser Pro Leu Arg Gln Tyr Phe Phe Glu Thr Arg Cys Lys Ala
50 55 60
Asp Asn Ala Glu Glu Gly Gly Pro Gly Ala Gly Gly Gly Gly Cys Arg
65 70 75 80
Gly Val Asp Arg Arg His Trp Val Ser Glu Cys Lys Ala Lys Gln Ser
85 90 95
Tyr Val Arg Ala Leu Thr Ala Asp Ala Gln Gly Arg Val Gly Trp Arg
100 105 110
Trp Ile Arg Ile Asp Thr Ala Cys Val Cys Thr Leu Leu Ser Arg Thr
115 120 125
Gly Arg Ala
130
Claims (6)
1. the brain-derived neurotrophic factor hBDNFb albumen of a kind of chloroplast expression, which is characterized in that its amino acid sequence is such as
Shown in SEQ ID NO.2.
2. the gene of the brain-derived neurotrophic factor hBDNFb albumen of coding chloroplast expression described in claim 1, special
Sign is that nucleotide sequence is as shown in SEQ ID NO.1.
3. it is a kind of for producing the tobacco chloroplast expression vector pWYP23402 of brain-derived neurotrophic factor hBDNFb albumen,
It is characterized in that, the carrier is using 16S-trnI and trnA-23S as left and right homologous sequence, the Prrn- of insertion chemistry synthesis therebetween
T7g10-GFP＆hBDNFb-aadA-Trps16 expression cassettes；Wherein, Prrn promoters and Trps16 terminators are respectively Tobacco Leaf
The 16S rRNA promoters of green body genome and rhct1 gene mRNA 3' ends mature sequence (GenBank accession number：NC_
001879)；Contained fusion tag gene GFP, target gene BDNFb and screening-gene aadA is carried out using DNA2.0 softwares
Codon optimization.
4. a kind of preparation method of the brain-derived neurotrophic factor hBDNFb albumen of chloroplast expression, which is characterized in that including
Following steps：
1) structure tobacco chloroplast expression vector pWYP23402；
2) the tobacco chloroplast conversion of hBDNFb genes is screened with homogeneity, obtains the homogeneity that tobacco chloroplast turns hBDNFb genes
Change plant；
3) hBDNFb expression analysis in chloroplast transgenic tobacco plant；
4) purifying of brain-derived neurotrophic factor hBDNFb albumen obtains the brain-derived neurotrophic factor of chloroplast expression
HBDNFb albumen.
5. the preparation method of the brain-derived neurotrophic factor hBDNFb albumen of chloroplast expression according to claim 4,
It is characterized in that, the structure tobacco chloroplast expression vector pWYP23402 in the step 1) is as follows：
1.1), optimize brain-derived neurotrophic factor hBDNFb, the brain-derived neurotrophic factor hBDNFb after being optimized,
Nucleotide sequence is as shown in SEQ ID NO.1；
1.2), structure tobacco chloroplast expression vector pWYP23402：Expression cassette, expression are synthesized using chemical synthesis full genome
Frame includes following two parts：The C-terminal addition flexible connection peptide chain and intestines of green fluorescence protein gene as fusion protein label
The linker that Kinase recognition site is formed, i.e. amino acid sequence are GSSSGSSSGSSSGSSSDDDDK；And it is used as selection markers base
The spectinomycin resistance gene of cause is placed in after the target gene with green fluorescence protein gene fusion tag, collectively form by
The polycistronic expression frame that tobacco chloroplast promoter Prrn starts, tobacco chloroplast terminator Trps16 is terminated, wherein, tobacco
Chloroplast promoter Prrn contains ribosome bind site T7g10 sequences；
1.3) artificial synthesized expression cassette produces after the insertion for completing target gene after carrying out digestions processing using Nsi I
" 16Srrn-trnI-trnA-23Srrn's is big in carrying clone's tobacco chloroplast genome of raw segment and identical enzymatic treatment
The cloning vector pEASY-Nt of segment " is attached reaction, and artificial synthesized expression cassette is made to be inserted into trnI genes and trnA genes
Between, the final tobacco chloroplast expression vector obtained for producing brain-derived neurotrophic factor b is named as pWYP23402.
6. the preparation method of the brain-derived neurotrophic factor hBDNFb albumen of chloroplast expression according to claim 3,
It is characterized in that, the purifying of the brain-derived neurotrophic factor hBDNFb albumen in the step 2) is specially：It will be total soluble
Albumen is placed in after 0.22 μm of filter membrane filters in the ultra-filtration centrifuge tube that molecular weight is 50KD that shuts off, and 4000g centrifugation 30min take stream
Go out liquid, then be placed in the ultra-filtration centrifuge tube that molecular weight is 30KD that shuts off after 4000g centrifugations 45min, retention part liquid is N-terminal
The preliminary purification product of the hBDNFb growth factors of GFP labels is merged, is digested using enterokinase, makes growth factor and GFP labels
Protein Separation is opened, then is purified with Heparin-Sepharose affinity chromatography column, obtains brain-derived neurotrophic factor hBDNFb
Albumen.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109411014A (en) * | 2018-10-09 | 2019-03-01 | 中国科学院昆明植物研究所 | A kind of cyclic method of plant chloroplast full-length genome assembling based on the sequencing of two generations |
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| CN1793377A (en) * | 2005-12-08 | 2006-06-28 | 上海交通大学 | Process for structuring carrier of expressing seven exogenous geng at same time in chloroplast |
| CN1978466A (en) * | 2005-12-06 | 2007-06-13 | 中国人民解放军军事医学科学院毒物药物研究所 | Transduction peptide-human brain-derived neurotrophic factor fusion protein and its use |
| CN104946656A (en) * | 2015-06-08 | 2015-09-30 | 吉林省农业科学院 | Human-derived bFGF (Basic Fibroblast Growth Factor), tobacco chloroplast expression vector and production method |
| WO2017072498A1 (en) * | 2015-10-26 | 2017-05-04 | Quethera Limited | Genetic construct |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1978466A (en) * | 2005-12-06 | 2007-06-13 | 中国人民解放军军事医学科学院毒物药物研究所 | Transduction peptide-human brain-derived neurotrophic factor fusion protein and its use |
| CN1793377A (en) * | 2005-12-08 | 2006-06-28 | 上海交通大学 | Process for structuring carrier of expressing seven exogenous geng at same time in chloroplast |
| CN104946656A (en) * | 2015-06-08 | 2015-09-30 | 吉林省农业科学院 | Human-derived bFGF (Basic Fibroblast Growth Factor), tobacco chloroplast expression vector and production method |
| WO2017072498A1 (en) * | 2015-10-26 | 2017-05-04 | Quethera Limited | Genetic construct |
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| CN109411014A (en) * | 2018-10-09 | 2019-03-01 | 中国科学院昆明植物研究所 | A kind of cyclic method of plant chloroplast full-length genome assembling based on the sequencing of two generations |
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