CN107641155A - A kind of method of the recombinant Human Serum Albumin Expression in plant - Google Patents
A kind of method of the recombinant Human Serum Albumin Expression in plant Download PDFInfo
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- CN107641155A CN107641155A CN201610576100.8A CN201610576100A CN107641155A CN 107641155 A CN107641155 A CN 107641155A CN 201610576100 A CN201610576100 A CN 201610576100A CN 107641155 A CN107641155 A CN 107641155A
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Abstract
The invention provides a kind of human serum albumin gene of codon optimization, and it being capable of high level expression in plant.Present invention also offers the nucleic acid construct comprising human serum albumin gene, carrier and host cell, and further provide production genetically modified plants and the method using genetically modified plants production recombination human serum albumin.The present invention substantially increases expression of the recombination human serum albumin in plant-bioreactor, so that genetically modified plants low cost production recombination human serum albumin is possibly realized.
Description
Technical field
The present invention relates to plant genetic engineering field, it particularly relates to which efficiently expressing recombinant human serum is white in plant
The method of albumen.
Background technology
The single chain protein matter that human serum albumins is made up of 585 amino acid residues, molecular weight 67kDa, it is people
Main component in blood plasma, secreted after being synthesized in liver and enter blood.Human serum albumins is a kind of medically to make on a large scale
Pharmaceutical protein, annual about 600 tons or so of world market demand, about 3,000,000,000 dollars of market value.Albumin has dimension
Hold colloidal osmotic pressure, Hemorrhagic shock, transport and removing toxic substances, promote the effects such as liver cell reparation and regeneration, be clinically mainly used in：Lose
Suffered a shock caused by blood wound, burn, cranium pressure rise, oedema or abdomen caused by hepatic sclerosis and nephrosis caused by encephaledema and damage
Water, the preventing and treating of Hypoproteinemia, hyperbilirubinemia of newborn, it is saturating to be additionally operable to cardiopulmonary bypass, the auxiliary treatment of burn, blood
The auxiliary treatment and ARDS of analysis.
Current medical human serum albumins is mainly obtained by blood classified filtering, and not only price is higher (about 5 dollars/g),
And production capacity is relatively low, limited blood resource significantly limit the production and use of albumin, and supply far can not meet city
Field needs.
Since clone obtains human serum albumin gene cDNA complete sequences from people's Liver cDNA Library first within 1981,
People are attempting to utilize technique for gene engineering efficiently expressing recombinant human seralbumin in different species always, achieve one
Fixed progress, but it is not high enough still to lock into expression quantity, the highly purified problem such as relatively difficult.
Generally prepared in restructuring bio-pharmaceuticals at present using microbial fermentations such as Escherichia coli, yeast, but the method is present
Shortcomings, such as：Microorganism and human body environment's difference are big, and expression product bioactivity is low, and the microbial toxin of doping is difficult to
Thoroughly remove, complex manufacturing, be hardly formed large-scale production etc..
Also have in recent years using transgenic technology prepare transgenosis animal to prepare human recombination protein product, it is and traditional micro-
Biological expression system is compared, and animal bioreactor has advantages below：(1) proteins biological activity of production is high, can be right
Recombinant protein carries out a variety of post translational processings, very close natural prodcuts；(2) expression quantity is big, cost is low；(3) product easily carries
It is pure, quality is high, avoid chemistry and biotoxin pollution；(4) easy industrialization, external source target gene can be hereditary so that certain
One colony all has same characteristic, can carry out large-scale production by animal reproduction group expanding.But human gene is that radom insertion moves
In the genome of thing, although having great advantage compared with microbial fermentation, there is also many uncontrollable factors and defect：
(1) integration of target gene and expression efficiency are relatively low；(2) it is possible to cause host cell gene to be mutated；(3) transgenic models with
Expected results are not inconsistent, or the disorder of expression individual；(4) the corresponding protein of animal in itself is very difficult to remove in purifying, holds
Easily cause the allergic reaction of the mankind.Therefore, transgenic animals are nor preferable bioreactor.
At present, by plant expression system be considered as animal cell culture have prospect substitute be used for mass produce weight
Histone, method for producing protein may refer to for example in genetically modified plants：U.S. Patent No. 5750871, the 5565347th
Number, No. 5464763, No. 5750871 and No. 5565347 etc..Although the raised growth of plant and collection are than protokaryon and very
Nucleus is cheap, but in plant cell alien gene expression quantity it is generally relatively low.
The content of the invention
In order to overcome the shortcomings of the prior art, the present inventor has invented one kind in tobacco plant by studying for a long period of time
The method of middle efficiently expressing recombinant human seralbumin (rHSA).
The first aspect of the present invention provides rHSA amino acid sequence and its coded sequence.
RHSA amino acid sequence such as SEQ ID NO in the application:Shown in 1.
Any nucleic acid that can encode rHSA is used equally for the present invention.Preferably, rHSA coded sequences, which have, is selected from SEQ
ID NO:2 and SEQ ID NO:3 nucleotide sequence.It is highly preferred that rHSA coded sequences are inclined according to the codon of host cell
Good situation has carried out codon optimization.In a most preferred embodiment, rHSA coded sequences such as SEQ ID NO:3 institutes
Show, it is hereinafter referred to as yhsa genes.
Yhsa gene orders be inventor according to tobacco codon preference in natural human serum albumin gene sequence
Engineer obtains on the basis of (hsa genes), and its length is 1830bp, identical with hsa mrna lengths, and encodes identical ammonia
Base acid sequence.
Because plant compares animal, the certain general character of its codon preference, thus the yhsa genes of the present invention can not only
Enough high level expressions in tobacco, can also obtain Gao Shui in other plant, such as arabidopsis, rice, wheat, corn and soybean
Flat expression.Preferably, the plant is plant of Solanaceae.
The second aspect of the present invention provides nucleic acid construct, carrier and the host cell of the nucleic acid including the present invention.
Preferably, the carrier is derived from pCHF3 carriers.
In a specific embodiment, the carrier is pCHF3-yhsa, and it is to carry yhsa genes insertion pCHF3
Structure obtains between KpnI the and SalI restriction enzyme sites of body.
The host cell of the present invention is selected from bacterium, fungi, animal and plant cell.Preferably, the bacterial cell is selected from
Escherichia coli and Agrobacterium, the plant cell are selected from plant of Solanaceae cell.Most preferably, the plant cell is that tobacco is thin
Born of the same parents.
The transgenic bacteria, fungi, animal and plant cell can be directly used in production rHSA as bioreactor, turn
Gene Agrobacterium can be used for mediation and produce genetically modified plants.In the present invention, the zooblast and plant cell do not wrap
Reproduction cell is included, without as propagating materials.
The third aspect of the present invention provides a kind of preparation method for the genetically modified plants for producing recombination human serum albumin,
Including by the present invention nucleic acid, nucleic acid construct or vectors into plant.
In a specific embodiment, methods described includes：
Agrobacterium competent cell is converted with the carrier of the present invention；
Agrobacterium infects vegetable material；And
By vegetable material culture that Agrobacterium is infected, it is regenerated as plant.
The preferred plant of Solanaceae of genetically modified plants, most preferably tobacco.
Preferably, the method for transformation is electroporated.Agrobacterium infects method can be according to vegetable material to be infected
The characteristics of unrestricted choice, for tobacco, preferred leaf disk method.
The fourth aspect of the present invention provide it is a kind of produce rHSA method, including plant and harvest according to the present invention side
Genetically modified plants prepared by method.
According to the difference of genetically modified plants species, its Land use systems also differs, for the plant having, for example (,) it is rice, small
Wheat, corn and soybean etc., rHSA are enriched with its edible portion, therefore directly can be processed the edible portion of these plants
To provide rHSA product.
For the plant that some can not directly eat, such as arabidopsis, tobacco etc., the above method also includes turning from harvest
Separated in gene plant, purify the step of rHSA.
Inventor has carried out codon optimization to human serum albumin gene sequence, substantially increases it in plant-bioreactor
In expression so that genetically modified plants low cost production rHSA be possibly realized.And due in codon optimisation process
The general character of vegetable codon preference is considered, the yhsa genes of engineer, can also be high in other plant not only in tobacco
Horizontal expression.
Brief description of the drawings
Fig. 1 over-express vector pCHF3 collection of illustrative plates.
Fig. 2 positive plasmid agarose gel electrophoretograms.Wherein, M is DL2000 molecular weight markers, and swimming lane 1~4 is respectively
4 recons, wherein No. 3 recons are the positive.
HSA Western blot identifications in Fig. 3 genetically modified plants.Wherein, swimming lane A is the wild type as negative control
Tobacco, B are the transgene tobacco for being transferred to empty carrier pCHF3 as negative control, and C-F is to be transferred to the table excessively for carrying natural gene
Up to carrier pCHF3-hsa transgene tobacco, G-J is to be transferred to carry the over-express vector pCHF3-yhsa of optimization gene and turn base
Because of tobacco, K is molecular weight marker, and L is the human serum albumins as positive control.
Embodiment
The present invention is further illustrated below by embodiment, but the present invention is not intended to be limited thereto.In the following example not
The experimental method of actual conditions is indicated, generally according to normal condition, or according to the condition proposed by manufacturer.
The human serum albumin gene of embodiment 1 optimizes
Natural human serum albumin gene hsa ORFs length is 1830bp, following (the SEQ ID NO of sequence:2)：
ATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGT
CGAGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGAT
TGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAA
AAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACA
GTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTT
CTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTC
ATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAA
CTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTT
GCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAA
AATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAA
GTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGA
CAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAAC
CTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCT
GATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGA
ATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGA
AGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCT
CAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCG
TTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCA
AATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGT
GTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATG
CTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATA
TATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAG
GCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAA
GGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTATAA
According to the codon preference of tobacco, a kind of optimization human serum albumins base for being capable of high expression in tobacco is devised
Because of ysha, its total length 1830bp, following (the SEQ ID NO of sequence:3)：
ATGAAGTGGGTCACTTTCATTAGTCTCTTGTTTCTGTTCTCTAGTGCATACTCTAGAGGCGTGTTTAGA
AGGGATGCCCATAAATCCGAAGTGGCTCACCGATTTAAAGATTTGGGCGAAGAGAACTTTAAAGCTCTTGTATTAAT
CGCTTTTGCTCAGTATCTTCAACAGTGCCCGTTTGAGGATCATGTGAAGCTGGTAAATGAAGTTACTGAATTTGCTA
AAACTTGCGTCGCTGATGAAAGCGCTGAAAACTGCGACAAATCACTCCATACACTATTTGGTGATAAGTTGTGTACT
GTTGCAACCCTGAGAGAGACCTACGGGGAGATGGCTGACTGCTGTGCCAAGCAGGAACCTGAAAGAAATGAATGCTT
TCTGCAACACAAGGACGATAATCCTAATCTTCCAAGACTCGTGAGACCAGAGGTTGATGTCATGTGCACAGCTTTCC
ATGACAACGAAGAGACATTTCTCAAAAAGTATCTTTATGAGATCGCACGTAGACACCCATATTTTTACGCTCCAGAA
CTACTTTTCTTTGCAAAGCGATACAAGGCCGCTTTTACTGAATGTTGTCAAGCTGCTGATAAAGCAGCTTGCCTGTT
ACCAAAGTTAGATGAACTGAGGGATGAGGGTAAGGCTTCCTCAGCTAAACAGAGACTGAAATGCGCATCACTACAAA
AGTTTGGAGAGAGAGCTTTCAAAGCTTGGGCTGTAGCCCGACTGTCACAAAGGTTCCCTAAGGCCGAATTTGCAGAA
GTTTCAAAGCTAGTTACTGATTTAACAAAGGTACACACCGAATGCTGTCATGGTGATCTATTGGAGTGTGCAGATGA
CCGAGCTGACCTGGCAAAATATATTTGCGAGAACCAAGACAGTATCAGCTCTAAATTAAAAGAGTGCTGCGAGAAAC
CTTTACTAGAAAAAAGCCATTGTATAGCTGAGGTCGAAAACGATGAGATGCCAGCAGATTTGCCATCACTCGCAGCC
GATTTCGTGGAGTCTAAAGACGTGTGCAAGAATTACGCTGAGGCCAAGGACGTATTCTTAGGTATGTTCCTCTATGA
GTACGCCAGAAGGCATCCCGACTACTCTGTGGTACTGTTGCTTAGACTCGCAAAAACCTACGAGACTACCTTGGAGA
AATGTTGTGCTGCAGCTGACCCTCATGAATGCTATGCTAAAGTATTCGATGAATTCAAACCTTTGGTTGAAGAACCC
CAGAACTTGATTAAGCAAAATTGTGAATTGTTCGAACAGTTGGGAGAATACAAGTTTCAAAATGCCCTTCTTGTAAG
GTACACTAAAAAGGTTCCACAGGTTTCTACCCCTACTTTGGTAGAGGTTTCCCGAAATTTGGGGAAGGTTGGTTCCA
AGTGTTGTAAACACCCAGAGGCTAAGAGAATGCCATGTGCTGAGGATTACCTTTCTGTAGTACTCAATCAACTCTGT
GTCCTGCATGAGAAGACACCAGTCTCAGATCGAGTTACTAAATGCTGTACTGAAAGTCTAGTCAATAGAAGGCCATG
CTTCAGCGCTCTTGAAGTTGATGAAACCTACGTTCCTAAGGAATTTAATGCTGAAACTTTTACCTTCCATGCAGACA
TCTGTACTTTAAGTGAAAAAGAGAGGCAAATTAAGAAACAAACTGCTTTAGTTGAGTTAGTAAAACATAAGCCCAAA
GCCACTAAAGAGCAATTGAAGGCTGTAATGGACGACTTTGCCGCCTTTGTTGAAAAATGTTGTAAAGCTGACGATAA
GGAAACTTGCTTTGCAGAGGAAGGGAAAAAGCTTGTTGCAGCAAGTCAAGCAGCTTTGGGTTTGTGA
Hsa genes and yhsa gene code identical human serum albumin HSAs, length 609aa, sequence are as follows
(SEQ ID NO:1)：
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNE
VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV
MCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK
CASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK
ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTY
ETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNL
GKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETF
TFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALG
L
Embodiment 2yhsa gene overexpression carriers pCHF3-yhsa structure
Over-express vector pCHF3 carriers are preserved by this laboratory, and bacterial resistance is Spe+, and plant resistance to environment stress is Kan+, is used for
The overexpression research of gene.
1. selection and the design of primers of restriction enzyme site
Consider over-express vector pCHF3 multiple cloning sites and restriction enzyme site that gene itself contains, be
The sense primer addition restriction enzyme joint KpnI (CGGGGTACC) of yhsa genes, restriction enzyme joint SalI is added for anti-sense primer
(ACGCGTCGAC) primer for, having added joint is yhsa-KpnI-F and yhsa-KpnI-R (being shown in Table 1), and Tm values are 62.9 respectively
℃、66.3℃。
Table 1PCR primers and its sequence
2.PCR amplifying target genes
Enter performing PCR amplification to yhsa gene purpose fragments using KOD-plus-Neo high-fidelities enzyme.
Table 2PCR reaction systems
PCR response procedures：94 DEG C of pre-degeneration 3min；98 DEG C denaturation 15s, 64 DEG C annealing 30s, 68 DEG C extension 45s, totally 32
Circulation；68 DEG C of extension 5min.
The clone of 3.PCR products
(1) electrophoresis
PCR primer electrophoresis in 1.0% Ago-Gel, the DNA fragmentation of expected size is cut under uviol lamp photograph.
(2) DNA fragmentation reclaims
By PCR primer after agarose gel electrophoresis detects, TaKaRa MiniBEST Agarose Gel DNA are used
Extraction Kit carry out gel recovery, the same specification of concrete operation step.
(3) plus polyA is purified
PolyA tails are added to the PCR primer of gel extraction and purified, to carry out TA clones.
A) Ex Taq enzymes normal work system：μ L of gel recovery product 15.75, the μ L of 10 × Buffer 5, the μ of 2mM dNTP 4
L, the μ L of 0.25 μ L and DEPC water of Ex Taq 25,72 DEG C of reaction 30min after being well mixed.
B) mixing of 1/10 volume 3M NaAc solution is added in above-mentioned 50 μ L systems, adds 2.5 times of anhydrous second of cumulative volume
Alcohol mixes, and is put in -70 DEG C of refrigerator 20min, or -20 DEG C of at least 30min.
C) 12,000rpm centrifugations 15min under the conditions of 4 DEG C, abandons supernatant.
D) rinsed with 500 μ L75% ethanol, 12,000rpm centrifugations 5min, abandons supernatant under the conditions of 4 DEG C.
E) repeat step e).
F) normal temperature is dried to not having ethanol taste, and 10~20 μ L DEPC water dissolve.
(4) connection conversion
A) purified product and PMD-19T carriers are attached using DNA Ligation Kit (Solution I).Press
According to carrier:Fragmental volumes ratio is 1:3~1:10, carrier and fragmental volumes sum are equal to the ratio and dosage of Solution I volumes
Carrier, purified product and Solution I is mixed.
B) above-mentioned mixed liquor is connected overnight in 16 DEG C of water-baths.
C) take 50 μ L bacillus coli DH 5 alpha competent cells to be placed on ice to melt, add 5 μ L connection products, with pipette tips by its
Mix.
D) after standing 30min on ice, 42 DEG C of heat shock 90s.After heat shock, then 2~3min is being placed on ice, then add 500 μ
L nonreactive LB fluid nutrient mediums, in 37 DEG C of incubator 150rpm shaken cultivations 1h.
E) prepare the LB flat boards containing Amp, take 100 μ L cultures to be uniformly coated on above, after planar surface drying, in
37 DEG C are inverted 12~16h of culture.
(5) screening of recombinant clone
A) single bacterium colony of random picking independence, is cultivated in the LB fluid nutrient mediums (100 μ g/mL) containing Amp, 37 DEG C of trainings
Support about 12h.
B) a small amount of bacterium solution is taken to add in 1.5mL centrifuge tubes, boiling water bath 10min, 12000rpm centrifugation 3min.
C) 1 μ L of supernatant is taken as template, and 20 μ L systems enter performing PCR amplification.
D) size of Insert Fragment is detected with 1% agarose gel electrophoresis.If stripe size meets, you can tentatively recognizes
It is positive colony for the bacterial plaque.Positive colony is delivered into Beijing six directions Huada gene company sequencing, confirmation whether there is base mutation etc..
(6) positive plasmid DNA is extracted
Recombinant plasmid dna, the same specification of concrete operation step are extracted using TIANpure Midi Plasmid Kit.
5. double digestion step
(1) KpnI and SalI double digestions, 37 DEG C of incubation 2h while to positive plasmid and over-express vector pCHF3 are carried out.
The double digestion system of table 3
(2) by digestion products after agarose gel electrophoresis detects, target gene fragment and over-express vector fragment are reclaimed,
And conversion is connected, method is same as above.
(3) screening and identification of recon：There is the clone of Insert Fragment by the screening of bacterium solution PCR methods first, choosing to expand
Increase the bacterium colony for expected size DNA fragmentation.After positive colony expands culture, extraction plasmid carries out double digestion checking, and can detection
Obtain being expected the purpose fragment of size, method is same as above.
6. electrophoresis detection
1 × TBE is prepared as electrophoretic buffer using DEPC processing water, and 1% Ago-Gel is prepared with it.Will be double
Digestion products enter row agarose gel electrophoresis, and the parameter of electrophoresis apparatus is arranged to voltage 220V, 20~30min of time.After electrophoresis
Imaging is (referring to Fig. 2) in gel imaging system.
The genetic transformation of the over-express vector of embodiment 3
1. the preparation of Agrobacterium competence
(1) agrobacterium strains (EHA105 and GV3101) for being stored in -80 DEG C are taken out, the oese to be sterilized with calcination is stained with
A small amount of bacterium solution is taken, in the flat lining out of YEB solid mediums of the Rif containing 25mg/L, 28 DEG C are cultivated 18h or so.
(2) picking single bacterium is fallen within the 5mL YEB fluid nutrient mediums containing 25mg/L Rif, 28 DEG C of 250rpm shaken cultivations
16~24h.
(3) by volume 1:50 contain the bacterium solution access 50mL after activation in 25mg/L Rif YEB fluid nutrient mediums,
28 DEG C of 250rpm are cultivated to OD600=0.5 or so.
(4) bacterium solution for cultivating end is transferred in the 50mL centrifuge tubes of precooling, 4 DEG C of 5000rpm centrifuge 10min, abandon
Clearly.
(5) the sterile ultra-pure water for the thalline precooling collected is resuspended, repeat step (4).
(6) thalline is resuspended with the glycerine of 1mL 10%, often the μ L of pipe 100 are dispensed into sterile 1.5mL centrifuge tubes, in -80 DEG C
Preserve.
2. electric shocking method converts the screening of Agrobacterium and positive colony
(1) Agrobacterium competent cell is taken out from -80 DEG C to be placed on ice, after its thawing, adds what 2 μ L were prepared
PCHF3-yhsa expression vector DNAs, inhaled with liquid-transfering gun and play mixing.
(2) bacterium solution drawn with liquid-transfering gun after mixing is squeezed into the electric shock cup of precooling, and electric shock cup is inserted in electric shock tank,
2500V voltages, shock by electricity 5ms.
(3) the nonreactive YEB fluid nutrient mediums of 500 μ L precoolings are added after shocking by electricity into electric shock cup in superclean bench, then will
It is transferred in 1.5mL centrifuge tubes, 28 DEG C, 250rpm 2~3h of shaken cultivation, foreign gene is given full expression in Agrobacterium.
(4) 100 μ L bacterium solutions are drawn, are coated on Rif containing 25mg/L and the YEB flat boards of expression vector corresponding resistant, 28
DEG C be inverted culture 24~48h.
(5) picking monoclonal cultivates 18h or so in the YEB fluid nutrient mediums containing same concentrations antibiotic, and temperature is set
28 DEG C are set to, rotating speed 250rpm.Enter performing PCR with the corresponding primer of expression vector to identify.Final concentration is added in positive bacterium solution
20% glycerine, in -80 DEG C of preservations.
3. agriculture bacillus mediated tobacco genetic transformation
(1) Aseptic seedling culture
Appropriate primary tobacco seed is taken, it is sterile with 75% alcohol surface sterilization 1min after soaking 24h in sterilized water
Water is cleaned, then with 10% 10~15min of hydrogen peroxide dipping, sterile water wash 3~4 times.Sterile-processed seed point is sowed at
On MS plating mediums, seedling is transferred in the tissue culture bottle containing MS culture mediums when 3~4 leaflets are grown, in artificial gas
Wait room culture 45d or so.Growth selection is healthy and strong, leaf color is dark green, vein clearly infect by blade progress Agrobacterium.
(2) infect and subculture
A) Agrobacterium activation is with infecting.The identified correct rear Agrobacterium original bacteria liquid preserved is taken out, after it melts completely
500 μ L are drawn in the 50mL YEB fluid nutrient mediums containing corresponding resistant, in 28 DEG C of 250rpm shaken cultivations to OD600It is close
0.6.Bacterium solution is transferred in the 50mL centrifuge tubes for the precooling that sterilizes in advance, 4000rpm centrifugation 10min, abandons supernatant and collect thalline.With
Thalline is resuspended in the MS0 fluid nutrient mediums of precooling, and 4000rpm centrifuges 10min again, abandons supernatant, collects thalline and with the MS0 of precooling
Fluid nutrient medium is resuspended to OD600=0.6 or so.Then final concentration of 20mg/L AS is added, prepares to be used to infect.Use scissors
The edge of aseptic blade is cut, the leaf dish that blade is cut into 1.0 × 1.0cm along master pulse is put into Agrobacterium and infected in liquid and invaded
Dye, time of infection is 5~6min.
B) co-culture.Blade after infecting is pulled out, Agrobacterium bacterium solution is blotted on aseptic filter paper, is tiled downwards with blade face
Co-cultured to G on culture medium, be put into phjytotron (temperature is 26 DEG C, humidity 40%) dark culturing 3 days.
C) S1 subcultures.Blade and blade after co-culturing 3 days is transferred on S1 differential mediums up.If explant band
There is more Agrobacterium, then with the sterilizing water washing of the Cef containing 500mg/L, S1 differentiation trainings are transferred to after being blotted with sterilizing filter paper
Support base.5~8 are placed per ware, middle dark culturing is placed under illumination and continues to cultivate for 1 week or so in the controlled environment chamber, to blade edge
Edge grows clump bud, bud length to 0.5cm or so.
D) S2 subcultures.Clump bud long on S1 differential mediums is transferred on S2 differential mediums with tweezers.Clump bud can be broken
What is taken is directly inoculated with clump bud, it is impossible to directly breaks being inoculated with together with related blade of taking, and removes the blade part of raw clump bud
Point.Optical culture treats that clump bud grows up to seedling in 1~2 week.
E) S3 subcultures.Seedling on S2 differential mediums is transferred on S3 differential mediums, optical culture 1~2 week.At this
During one, seedling growth is more healthy and stronger.
F) culture of rootage.Bottom enlargement and the leaf of bottom jaundice are removed to the healthy and strong seedling on S3 differential mediums
Piece, inoculate in the tissue culture bottle containing R root medias, optical culture 1~2 week, so that seedling is taken root as early as possible.
(3) acquisition of transgene tobacco
Treat small seedling rooting 3~10, during 2~3cm of root long or so, the lid of blake bottle is opened and carries out hardening.About 3d is left
Behind the right side, by seedling replanting into the nutritive cube for filling sterile soil, and plastic covering film carries out moisturizing.Can be according to children after about one week
The upgrowth situation of seedling throws off preservative film, makes its fast-growth under field conditions (factors).
4. it is overexpressed yhsa genes T0For the positive identification of tobacco
(1) DNA is extracted
Transgenic tobacco leaf about 100mg is gathered in 2.0mL centrifuge tubes, is said according to Plant Genomic DNA kit
Bright book extracts genomic DNA.
(2) PCR is identified
Because transgenic positive plant contains the 35S promoter on pCHF3 carriers, and nontransgenic plants do not contain.Cause
This, according to the sequences Design forward primer pCHF3-35S-F of 35S promoter, the reverse primer yhsa- with expanding yhsa genes
SalI-R together, to T0In generation, is overexpressed plant and carries out positive identification, and amplified fragments size is about 1850bp.
Enter performing PCR reaction by template of transgenic tobacco leaf DNA, the DNA by the use of over-express vector is right as the positive
According to by the use of non-transgenic tobacco leaf DNA and sterilized water as negative control, passing through agarose gel electrophoresis and detect transgenosis and plant
Whether strain is positive strain.PCR reaction systems and reaction condition are same as above.
Human serum albumin HSA expression quantity is identified in the transgene tobacco of embodiment 4
Prepared respectively with the method for previous embodiment and be transferred to the over-express vector pCHF3-hsa for carrying natural gene and optimization
Each 4 plants of the over-express vector pCHF3-yhsa of gene transgene tobacco, albumen table is carried out using Western blot method
Identified up to amount.
The tobacco leaf of quality is taken etc. with Eppendorf pipes, is ground in liquid nitrogen, the protein loading for adding 1 times of volume is delayed
Fliud flushing, vibration, boiling water bath 10min, 2min on ice is immediately placed on, 5000rpm centrifugation 10min, takes supernatant.15 μ l samples are through PAGE
Glue separates.Point sample order be：Swimming lane A is the wild-type tobacco as negative control, and B is to be transferred to empty carrier as negative control
PCHF3 transgene tobacco, C-F be transferred to carry natural gene over-express vector pCHF3-hsa transgene tobacco, G-J
To be transferred to the transgene tobacco for the over-express vector pCHF3-yhsa for carrying optimization gene, K is molecular weight marker, and L is as sun
Property control human serum albumins (Sigma A9511).After coomassie brilliant blue R250 dyes and decolourizes, adjustment applied sample amount is to equal
It is even consistent；Electrophoresis again.PAGE gel is fine to nitric acid by Protein transfer with the method for electrotransfer in transfering buffering liquid
Tie up on plain film.Shift the nitrocellulose filter finished to rinse in TBST buffer solutions, be transferred to (5% degreasing of 10ml confining liquids
Milk powder) in 37 DEG C closing 2h.After the completion of closing, specific antisera (Sigma A2672) (1 is added directly in confining liquid:
10000), 37 DEG C of reaction 2h.Nitrocellulose filter is rinsed 3 times in TBST buffer solutions, adds and dilutes (1 with TBST:
5000) the goat-anti Mouse Antisera (Sigma A3562) of alkaline phosphatase mark, 37 DEG C of reaction 1h.Again film 3 is washed with TBST
It is secondary.Under the conditions of lucifuge, nitrocellulose filter is placed in the alkaline phosphatase containing 330 μ g/ml NBT and 165 μ g/ml BCIP and delayed
It is clear to band to be developed the color in fliud flushing.
From figure 3, it can be seen that the expression quantity for being transferred to rHSA in pCHF3-yhsa transgene tobacco is significantly larger than transferred to
PCHF3-hsa transgene tobacco.
The present invention is not limited to above-mentioned preferred forms, and anyone can draw other various under the enlightenment of the present invention
The product of form, however, make any change in its shape or structure, it is every that there is skill identical or similar to the present application
Art scheme, is within the scope of the present invention.
Claims (21)
1. a kind of protein of separation, its amino acid sequence such as SEQ ID NO:Shown in 1.
2. a kind of nucleic acid of separation, it encodes the protein described in claim 1.
3. nucleic acid according to claim 2, it, which has, is selected from SEQ ID NO:2 and SEQ ID NO:3 nucleotide sequence.
4. nucleic acid according to claim 3, its nucleotide sequence such as SEQ ID NO:Shown in 3.
5. a kind of nucleic acid construct, it includes the nucleic acid described in claim any one of 2-4.
6. a kind of carrier, it includes the nucleic acid described in claim any one of 2-4.
7. carrier according to claim 6, it is derived from pCHF3 carriers.
8. a kind of host cell, it includes the carrier described in claim 6 or 7.
9. host cell according to claim 8, it is selected from bacterium, fungi, animal and plant cell.
10. host cell according to claim 9, it is selected from Escherichia coli and agrobatcerium cell.
11. host cell according to claim 9, it is plant of Solanaceae cell.
12. host cell according to claim 11, it is tobacco cell.
13. a kind of preparation method for the genetically modified plants for producing recombination human serum albumin, including by any one of claim 2-4
Nucleic acid construct described in described nucleic acid, claim 5 or the vectors into plant described in claim 6 or 7.
14. the method according to claim 11, including：
(1) with the carrier conversion Agrobacterium competent cell described in claim 6 or 7；
(2) Agrobacterium obtained with step (1) infects vegetable material；
(3) by vegetable material culture that Agrobacterium is infected, be regenerated as plant.
15. according to the method for claim 14, wherein the method for transformation of step (1) is electroporated.
16. according to the method for claim 14, wherein the vegetable material of step (2) is leaf dish.
17. according to the method described in claim any one of 13-16, wherein the plant is plant of Solanaceae.
18. according to the method for claim 17, wherein the plant is tobacco.
19. the method for recombination human serum albumin is produced a kind of, including plants and harvests described in claim any one of 13-18
Genetically modified plants prepared by method.
20. according to the method for claim 19, in addition to from the genetically modified plants of harvest separate, purification of Recombinant human serum
Albumin.
21. according to the method for claim 20, directly contain recombinant human serum albumin egg with the genetically modified plants production of harvest
White product.
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