CN106632690A - Recombinant human heat shock protein 10 as well as encoding gene and preparation method thereof - Google Patents
Recombinant human heat shock protein 10 as well as encoding gene and preparation method thereof Download PDFInfo
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Abstract
The invention provides a recombinant human heat shock protein 10. The recombinant human heat shock protein 10 comprises an amino acid sequence of a human heat shock protein 10, an amino acid sequence of a cell penetrating peptide and an amino acid sequence of a flexible connecting peptide, wherein the amino acid sequence of the flexible connecting peptide is used for connecting the amino acid sequence of the human heat shock protein 10 with the amino acid sequence of the cell penetrating peptide. The invention further provides a preparation method of the recombinant human heat shock protein 10. The preparation method comprises the following steps: obtaining genes, transforming, screening a multi-copy recombinant, carrying out fermentation cultivation and inducing expression and purification. The invention further provides an encoding gene of the recombinant human heat shock protein 10, an expression vector containing the encoding gene and a cell line containing the encoding gene.
Description
Technical field
The invention belongs to bioengineering field, and in particular to one kind restructuring human heat shock protein and its encoding gene, this
The bright preparation method for further relating to the restructuring human heat shock protein.
Background technology
Heat shock protein (Heat Shock Proteins, HSPs) is widely present in from bacterium to mammal
One class heat stress proteins matter, will be synthesized this proteinoid to protect organism when organism is exposed to high temperature by thermal excitation
Itself.Heat shock protein-70 (HSP10) is a member in heat-shock protein family, is also called CPN10, GROES or HSPE1, its
It is the highly conserved protein in heredity of a class in being widely present in people, animal, microorganism and plant cell.Human heat shock
Protein 10 gene is located at No. 2 chromosome q33.1 sites, and the mrna length is 306bp, coded product human heat shock protein 10 by
102 amino acid residue compositions, relative molecular mass is about 10KD.
Human heat shock protein 10 is widely present in the various organization of human body, and research shows, HSP10 is in various stressed conditions
Under, such as, great expression under the stimulation of high temperature, ultraviolet, medicine, heavy metal, anoxic etc. protects cell work(as molecular chaperones
Can, the generation of inhibited apoptosis；Type-1 insulin like growth factor receptor pathway can be expanded by posttranslational modification, protect the heart
Myocyte is from ischemia injury；Homoioplastic immunological rejection can be mitigated；Meanwhile, also have research prompting, HSP10 with
Reproduction is infertile relevant.Substantial amounts of research shows that HSP10 has limitless market value, can be not only used for medicine
Exploitation, is also used as the newcomer of biological beauty, in being applied to cosmetics industry.
Although human heat shock protein 10 has wide application, currently also lack high efficient expression human heat shock protein
White 10 method, also, when human heat shock protein 10 is applied in cosmetics, there is a problem of that skin penetration capability is poor.
The content of the invention
The goal of the invention of the present invention is the defect for prior art, there is provided one kind restructuring human heat shock protein, its coding
Gene and preparation method.
On the one hand, the invention provides a kind of recombined human heat shock protein-70, including：
(1) amino acid sequence of human heat shock protein 10；
(2) amino acid sequence of cell-penetrating peptide；With
(3) amino acid sequence of flexible peptide linker, for connecting amino acid sequence and the institute of the human heat shock protein 10
State the amino acid sequence of cell-penetrating peptide.
Aforesaid recombined human heat shock protein-70, the amino acid sequence of the cell-penetrating peptide is SEQ ID in sequence table
No:4 sequence, or, with SEQ ID No in sequence table:The homology of 4 sequence more than 90% (preferably 95%, more preferably
98%) and with the amino acid sequence of identical activity.
Aforesaid recombined human heat shock protein-70, the amino acid sequence of the flexible peptide linker is SEQ ID in sequence table
No:6 sequence.
Aforesaid recombined human heat shock protein-70, the amino acid sequence of the recombined human heat shock protein-70 is in sequence table
SEQ ID No:1 amino acid sequence.
On the other hand, the invention provides the preparation method of recombined human heat shock protein-70, including：
(1) gene is obtained：SEQ ID No in artificial full genome composition sequence table:2 sequence, to pPIC9K carriers and
The SEQ ID No of artificial full genome synthesis:2 sequence carries out Xho I and the double digestions of Not I, reclaims digestion products, is connected with DNA
Enzyme connects, and converts Escherichia coli, extracts plasmid；
(2) convert：Plasmid prepared by step (1) is mixed with competence Pichia pastoris and is converted, bacterium after being converted
Fall；
(3) multicopy recon is screened：Bacterium colony after step (2) is converted further is cultivated, and screens transformant, is obtained
Obtain recombined human heat shock protein-70 engineering bacteria；
(4) fermented and cultured abduction delivering：Fermentation training is carried out to the recombined human heat shock protein-70 engineering bacteria that step (3) is obtained
Support and obtain recombined human heat shock protein-70 zymotic fluid；
(5) purify：To step (4) obtain recombined human heat shock protein-70 zymotic fluid carry out successively separation of solid and liquid, filtration,
Concentration and ion-exchange chromatography obtain product.
Aforesaid preparation method, the purifying of the step (5) is concretely comprised the following steps：The zymotic fluid Jing that step (4) is obtained
Centrifugation carries out separation of solid and liquid, takes supernatant, and micro-filtration is carried out to fermented supernatant fluid, collects filtrate, then carries out desalination and concentration by ultrafiltration, receives
Collection concentrate, then carries out ion-exchange chromatography, you can obtain product.
Aforesaid preparation method, including：
(1) plasmid is prepared
SEQ ID No in artificial full genome composition sequence table:2 sequence, then to pPIC9K carriers and artificial full base
Because of the SEQ ID No for synthesizing:4 sequence carries out Xho I and the double digestions of Not I, reclaims digestion products, is connected with DNA ligase, and
Conversion Escherichia coli, extract plasmid, are named as pPIC9K-YARA-HSP10；
(2) Pichia pastoris electricity conversion
By the linearizing pPIC9K-YARA-HSP10 plasmids of the restriction endonucleases of Jing Sal I, mix with Pichia pastoris competent cell,
In going to the electricity conversion cup of ice precooling, electric shock, the sorbitol solution for adding ice precooling mixes thalline, and coating MD culture mediums are put down
Plate, is inverted culture, and on MD culture medium flat plates bacterium colony is grown；
(3) multicopy inserts the screening of recon
By the bacterium colony grown on MD culture medium flat plates correspondence be inoculated into G418 concentration be respectively 0.5g/L, 1g/L, 2g/L,
On the YPD flat boards of 3g/L, 4g/L, 5g/L, culture, screening obtains transformant；
(4) ferment
The engineering bacteria for screening is inoculated in BMGY culture mediums, shaken cultivation, is transferred in equipped with FBS as first order seed
In the fermentation tank of culture medium, pH value is transferred into the bulk fermentation equipped with FBS culture mediums 5.5 or so as secondary seed；
(5) purify
The zymotic fluid Jing centrifugations that step (4) is obtained carry out separation of solid and liquid, take supernatant, are 80000D with molecule interception
Hollow Fiber Ultrafiltration system carry out ultrafiltration, collect filtrate, with the rolling ultrafiltration membrane ultrafiltration that molecular weight is 1000D, collect concentration
Liquid, subsequently carries out ion-exchange chromatography, you can obtain product.
On the other hand, the invention provides the encoding gene of recombined human heat shock protein-70.
Aforesaid encoding gene, the encoding gene is SEQ ID No in sequence table:2 nucleotide sequence, or, with
SEQ ID No:2 have at least 90% (preferably 95%, more preferably 98%) homology and encode identical function albumen nucleotides
Sequence.
On the other hand, the invention provides the expression vector containing afore-mentioned code gene.
On the other hand, the invention provides the clone containing afore-mentioned code gene.
The recombined human heat shock protein-70 of the present invention greatly improves human heat shock protein on the premise of activity is not affected
White 10 skin penetration capability.
Description of the drawings
Fig. 1 is the plasmid map of recombinant expression carrier pPIC9K-YARA-HSP10 in embodiment.
Fig. 2 is the PCR primer figure of YARA-HSP10 in embodiment.
Fig. 3 is SDS-PAGE in embodiment.
Fig. 4 is Western-blot results in embodiment.
Fig. 5 is recombined human heat shock protein-70 Immunofluorescence test result figure in embodiment.
Specific embodiment
In order to absolutely prove that the present invention solves the technical scheme that technical problem is implemented to use.With reference to embodiment and attached
Figure elaborates to invention, but the embodiment and protection domain of technical scheme, technical scheme is not merely
It is limited to this.Unless otherwise stated, there are those skilled in the art to be generally understood that for the science and technology for hereinafter occurring and technical term
Implication.
Heat shock protein (Heat Shock Proteins, HSPs) is widely present in from bacterium to mammal
One class heat stress proteins matter, will be synthesized this proteinoid to protect organism when organism is exposed to high temperature by thermal excitation
Itself.Heat shock protein-70 (HSP10) is a member in heat-shock protein family, is also called CPN10, GROES or HSPE1, its
It is the highly conserved protein in heredity of a class in being widely present in people, animal, microorganism and plant cell.Human heat shock
Protein 10 gene is located at No. 2 chromosome q33.1 sites, and the mrna length is 306bp, coded product human heat shock protein 10 by
102 amino acid residue compositions, relative molecular mass is about 10KD, and its amino acid sequence is SEQ ID No in sequence table:3
Sequence.
Cell-penetrating peptide is that a class can carry macromolecular substances into the small peptide of cell, and it is worn film ability and is independent of classics
Encytosis.This kind of material is the polypeptide fragment of the varying length with positive charge, wherein rich in alkali such as arginine, lysines
Acidic amino acid residue, secondary structure all space conformations with alpha-helix.
For currently cannot high efficient expression human heat shock protein 10 and its be used for cosmetics when skin penetration capability it is poor
Problem, the present inventor devises one section of flexible peptide linker, and cell-penetrating peptide is connected into the nitrogen of human heat shock protein 10 (people HSP10)
End, on the premise of activity is not affected, reaches the purpose of the skin penetration capability for improving human heat shock protein 10.
According on one side, the invention provides a kind of recombined human heat shock protein-70, it includes (1) human heat shock protein
10 amino acid sequence；(2) amino acid sequence of cell-penetrating peptide；(3) amino acid sequence of flexible peptide linker.Wherein, it is soft
Property connection peptide be used for connect the amino acid sequence of the human heat shock protein 10 and the amino acid sequence of the cell-penetrating peptide.
In a kind of specific embodiment, the amino acid sequence of the cell-penetrating peptide is YARAAARQARA (i.e. sequence tables
Middle SEQ ID No:4 sequence, abbreviation cell-penetrating peptide YARA), or, with SEQ ID No:4 homology is (preferred more than 90%
More than 95%, more preferably more than 98%) and with the amino acid sequence of identical activity；The amino acid sequence of the flexible peptide linker
It is GGGS.
Inventor's research finds that cell-penetrating peptide YARA can carry surface and corium of the peptide matters into skin, and its turn
The ability of leading is 33 times of cell-penetrating peptide TAT.Cell-penetrating peptide YARA is connected to into human heat shock protein 10 using flexible peptide linker, is weighed
Group human heat shock protein 10, to be not connected with the human heat shock protein 10 of cell-penetrating peptide as control, using immunofluorescence technique to restructuring
Human heat shock protein 10 is tested and analyzed, and is found on the premise of activity is not affected, the cell of recombined human heat shock protein-70
Film penetration capacity has obtained great lifting compared to control.
In a kind of preferred embodiment, the amino acid sequence of the recombined human heat shock protein-70 of the present invention is sequence
SEQ ID No in list:1 amino acid sequence.
According on the other hand, the invention provides the preparation method of above-mentioned recombined human heat shock protein-70, including：
(1) gene is obtained：SEQ ID No in artificial full genome composition sequence table:2 sequence, to pPIC9K carriers and
The SEQ ID No of artificial full genome synthesis:2 sequence carries out Xho I and the double digestions of Not I, reclaims digestion products, is connected with DNA
Enzyme connects, and converts Escherichia coli, extracts plasmid；
(2) convert：Plasmid prepared by step (1) is mixed with competence Pichia pastoris and is converted, bacterium after being converted
Fall；
(3) multicopy recon is screened：Bacterium colony after step (2) is converted further is cultivated, and screens transformant, is obtained
Obtain recombined human heat shock protein-70 engineering bacteria；
(4) fermented and cultured abduction delivering：Fermentation training is carried out to the recombined human heat shock protein-70 engineering bacteria that step (3) is obtained
Support and obtain recombined human heat shock protein-70 zymotic fluid；
(5) purify：To step (4) obtain recombined human heat shock protein-70 zymotic fluid carry out successively separation of solid and liquid, filtration,
Concentration and ion-exchange chromatography obtain product.
In a kind of preferred embodiment, methods described comprises the steps：
(1) acquisition of gene
One section of flexible peptide linker of design, nucleotides sequence is classified as GGTGGTGGTAGT (SEQ ID No i.e. in sequence table:5 sequence
Row), in the N-terminal of the gene order of human heat shock protein 10, cell-penetrating peptide YARA is connected by flexible peptide linker；Synthesized using full genome
The method synthetic cell cell-penetrating peptide YARA- flexible peptide linker-gene of human heat shock protein 10 (abbreviation YARA-HSP10).The cell-penetrating peptide
It is SEQ ID No in sequence table that the nucleotides sequence of YARA-HSP10 genes is classified as:2 nucleotide sequence, it is specific as follows：
Upstream and downstream introduces respectively the restriction enzyme site of Xho I and Not I, the restricted digestion position of Xho I
Point is CTCGAG, and the restriction enzyme site of Not I is GCGGCCGC..
(2) connection of pPIC9K carriers and recombined human cHSP10 gene
Xho I and Not I double digestions are carried out to pPIC9K, YARA-HSP10, digestion products are reclaimed, is entered with DNA ligase
Row connection, converts e.colistraindh5α, extracts plasmid and is named as pPIC9K-YARA-HSP10, sequencing identification, sequencing result
Show, YARA-HSP10 has correctly been connected to pPIC9K, has illustrated pPIC9K-YARA-HSP10 expression vector establishment successes.
(3) prepared by Pichia pastoris competence
Picking Pichia pastoris single bacterium colony, is seeded to containing in YPD culture medium test tubes, overnight incubation.Culture is forwarded to
In triangular flask containing fresh YPD medium, overnight incubation reaches 1.2～2.0 to OD600 values.Cell culture is centrifuged,
Supernatant is abandoned, ice-cold sterilized water is resuspended by bacterial sediment, be centrifuged, by the same way ice-cold D-glucitol solution
Bacterial sediment is resuspended, and supernatant is abandoned in centrifugation.It is repeated twice.Ice-cold D-glucitol solution is resuspended by bacterial sediment, is placed in
It is stand-by in mixture of ice and water.
(4) Pichia pastoris GS115 electricity conversion
By the linearizing pPIC9K-YARA-HSP10 plasmids of Jing Sal I restriction endonucleases, mix with Pichia pastoris competent cell
It is even, in going to the electric shock cup of ice precooling, shock by electricity 4～10 milliseconds, the D-glucitol aqueous solution for adding ice precooling mixes thalline, applies
Cloth MD culture medium flat plates, are inverted culture until growing bacterium colony on MD culture medium flat plates.
(5) screening of multicopy recon
By the bacterium colony sterile toothpick grown on MD culture medium flat plates correspondence be inoculated into G418 concentration be respectively 0.5g/L,
On the YPD flat boards of 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 30 DEG C of cultures, screening obtains transformant.
(6) the fermented and cultured abduction delivering of recombined human heat shock protein-70 engineering bacteria
The engineering bacteria that screening is obtained is inoculated in BMGY culture mediums, shaken cultivation 24 hours, as first order seed switching
In the fermentation tank equipped with FBS culture mediums, pH value is maintained to cultivate 16～20 hours in 5.5 or so (pH value 5-6), canister culture,
Transfer into big tank as secondary seed, fermented.After fermentation ends, zymotic fluid carries out separation of solid and liquid by centrifugation, in reservation
Clear liquid, destination protein is present in supernatant.
According on the other hand, the invention provides encoding the encoding gene of above-mentioned recombined human heat shock protein-70.It is preferred that
Ground, the encoding gene is SEQ ID No in sequence table:2 nucleotide sequence, or, with SEQ ID No:2 have at least
The nucleotides of 90% homology, the homology of preferably at least 93%, 95%, 97%, 98% or 99% and coding identical function albumen
Sequence.
According on the other hand, present invention also offers the expression vector containing the gene, and the cell containing gene
System.
In a kind of particularly preferred specific embodiment, the present invention is achieved through the following technical solutions：
The present inventor devises one section of flexible peptide linker, and amino acid sequence is GGGS (SEQ ID No i.e. in sequence table:6
Sequence), cell-penetrating peptide YARA is connected with human heat shock protein 10 by flexible peptide linker GGGS, obtain human heat shock protein of recombinating
10, amino acid sequence is SEQ ID No in sequence table:1 sequence.
The nucleotide sequence for encoding the gene of above-mentioned recombined human heat shock protein-70 is SEQ ID No in sequence table:2 sequence
Row.
The preparation method of above-mentioned recombined human heat shock protein-70 comprises the steps：
(1) acquisition of gene
One section of flexible peptide linker of design, sequence is GGTGGTGGTAGT (SEQ ID No i.e. in sequence table:5 sequence),
The N-terminal of the gene order of human heat shock protein 10, by flexible peptide linker cell-penetrating peptide YARA is connected；Closed using full genome synthetic method
Into cell-penetrating peptide YARA- flexible peptide linkers-gene of human heat shock protein 10 (abbreviation YARA-HSP10), i.e. SEQ in sequence table
ID No:2 nucleotide sequence.Upstream and downstream introduces respectively the restriction enzyme site of Xho I and Not I.
(2) connection of pPIC9K carriers and recombined human cHSP10 gene
Xho I and Not I double digestions are carried out to pPIC9K, YARA-HSP10, digestion products are reclaimed, is entered with DNA ligase
Row connection, converts e.colistraindh5α, extracts plasmid and is named as pPIC9K-YARA-HSP10, sequencing identification, sequencing result
Show, YARA-HSP10 has correctly been connected to pPIC9K, has illustrated pPIC9K-YARA-HSP10 expression vector establishment successes.
(3) prepared by Pichia pastoris GS115 competence
Picking Pichia pastoris single bacterium colony, is seeded to containing in 5mL YPD culture medium test tubes, 30 DEG C, 225rpm overnight incubations.
The culture for taking 100 μ L is seeded in the 250mL triangular flasks containing 50mL fresh YPD mediums, and 30 DEG C, 225rpm was cultivated
At night, to OD600 values 1.2～2.0 are reached.By cell culture in 4 DEG C, 1500g centrifugation 5min abandon supernatant, and the ice with 50mL is pre-
Cold sterilized water is resuspended by bacterial sediment.Centrifugation, by the same way the D-glucitol with the 1mol/L of the ice precooling of 20mL is molten
Liquid is resuspended by bacterial sediment, and supernatant is abandoned in centrifugation.It is repeated twice.Will with the D-glucitol solution of the 1mol/L of the ice precooling of 0.5mL
Bacterial sediment is resuspended, and its final volume is about 0.7mL.It is placed in stand-by in mixture of ice and water.
(4) Pichia pastoris GS115 electricity conversion
By the linearizing pPIC9K-YARA-HSP10 plasmids of 10uL Jing Sal I restriction endonucleases, with 100uL Pichia pastoris GS115s
Competent cell is mixed, and in going to the electric shock cup of 2mm ice precoolings, shock by electricity 5ms, adds the D- sorbs of the 1mol/L of 1mL ice precoolings
Alcohol solution mixes thalline, is coated with MD culture medium flat plates, and 29 DEG C are inverted 3-4 days, treat to grow bacterium colony on MD culture medium flat plates.
(5) screening of multicopy recon
By the bacterium colony sterile toothpick grown on MD culture medium flat plates correspondence be inoculated into G418 concentration be respectively 0.5g/L,
On the YPD flat boards of 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 29 DEG C of cultures, screening obtains transformant.
(6) the fermented and cultured abduction delivering of recombined human heat shock protein-70 engineering bacteria
The engineering bacteria that obtains of screening is inoculated in 400mL BMGY culture mediums, 30 DEG C of shaken cultivations 24 hours, as
Level seed is transferred in the 5L fermentation tanks equipped with 4L FBS culture mediums, and temperature constant is 30 DEG C, and with ammoniacal liquor pH value is adjusted, and maintains pH value
In 5.5 or so, canister culture 16-20h, transfer into the big tanks of 150L as secondary seed.Inducing temperature is 29 DEG C, and dissolved oxygen control exists
20%～30%, when dissolved oxygen (Dissolved oxygen, DO) suddenly rises, (glycerine in indication basal salt media consumes
To the greatest extent, this stage takes about 20h), (50% glycerine containing 12mL/L PTM1 maintains DO to start flow feeding growth medium
30% or so) ferment.When the weight in wet base of zymotic fluid reaches 300g/L, stop feed supplement, hungry 1h, fed-batch cultivation induction after glycerol depletion
Culture medium (mass concentration is 75% methanol aqueous solution containing 12mL/L PTM1), adds methyl alcohol to maintain 2～3h with low speed stream
Transition stage, after transition stage terminates, improve methanol feeding speed and simultaneously maintain feed rate to carry out in 10.9mL/L/h or so
Fermentation.Dissolved oxygen is made more than 20%, induction fermentation 42h or so by adjusting rotating speed, tank pressure and throughput.After fermentation ends, fermentation
Liquid carries out separation of solid and liquid by centrifugation, retains supernatant, and destination protein is present in supernatant.Jing is determined, and recombined human heat is stopped
The yield of gram protein 10 is up to 450mg/L.
(7) purifying of recombined human heat shock protein-70
After fermentation ends, zymotic fluid centrifugation takes supernatant 80L, with the doughnut that molecule interception is 80000D
Ultrafiltration system carries out ultrafiltration, collects filtered solution, with the rolling ultrafiltration membrane ultrafiltration that molecular weight is 1000D, concentrate is collected, by upper
State two step ultrafiltrations and can remove pigment and some foreign proteins that Pichia pastoris fermentation is produced, obtain recombined human heat shock protein-70
Crude protein concentrate.Recombined human heat shock protein-70 crude protein liquid is carried out into cation-exchange chromatography, filler selects CM
Sepharose FF, are purchased from GE companies of the U.S., and the pH with 3 times of column volumes is 7.5 20mmol/L phosphate buffers balance, on
Sample, the pH value containing 1.0mol/L NaCl with 10 times of column volumes is 7.5 20mmol/L phosphate buffers wash-out, collects weight
The group protein liquid of human heat shock protein 10, with the rolling ultrafiltration membrane ultrafiltration that molecular weight is 1000D, desalination, collects concentrate, with freezing
Drying machine is freezed, and obtains recombined human heat shock protein-70, and fermented supernatant fluid expression reaches 450mg/L.
Embodiment
The assay method and material source in the embodiment of the present invention is illustrated below.
PPIC9K is purchased from Invitrogen companies
Xho I and Not I are purchased from Thermo Fisher companies
117 amino acid of recombined human heat shock protein-70 total length of the present embodiment, the protein amino acid sequence is sequence
SEQ ID No in table:1 sequence, it is specific as follows：
Above-mentioned gene recombinant human heat shock protein-70 preparation method is as follows：
(1) acquisition of gene
One section of flexible peptide linker of design, sequence is GGTGGTGGTAGT (SEQ ID No i.e. in sequence table:5 sequence),
The N-terminal of the gene order of human heat shock protein 10, by flexible peptide linker cell-penetrating peptide YARA is connected；Closed using full genome synthetic method
Into YARA cell-penetrating peptides-gene of flexible peptide linker-human heat shock protein 10 (abbreviation YARA-HSP10).Cell-penetrating peptide YARA-HSP10
It is SEQ ID No in sequence table that the nucleotides sequence of gene is classified as:2 nucleotide sequence, it is specific as follows：
Restriction enzyme site Xho I and Not I are introduced in upstream and downstream.The electrophoresis knot of recombined human cHSP10 gene
Fruit sees Fig. 1.
(2) double digestion of pPIC9K carriers and recombined human cHSP10 gene
Xho I and Not I double digestions are carried out to PPIC9K, YARA-HSP10, digestion system is as follows：
After mentioned reagent adds well, 37 DEG C, DNA purification kits are used in PCR instrument temperature control digestion 1 hour, to specifications, from
Specific band is reclaimed on 1.2% Ago-Gel.
(3) connection of pPIC9K carriers and recombined human cHSP10 gene (i.e. YARA-HSP10)
PPIC9K and YARA-HSP10 fragments are attached using T4DNA ligases, reaction system is as follows：
After above-mentioned system is mixed, in 22 DEG C, connect 4 hours, be named as pPIC9K-YARA-HSP10.
(4) conversion of connection product
The pipe 40uL of DH5a competent cells 1 of freezing is taken, is placed in and is thawed on ice, add above-mentioned connection product pPIC9K-
YARA-HSP10, mixes, and puts 30min on ice, 42 DEG C of water-bath heat shock 90s, and quickly pipe is transferred in ice bath, makes cell cool down 2-
3min, adds the LB fluid nutrient mediums of 300uL sterilizings, mixes, and 37 DEG C incubate 1 hour, draw 200uL bacterium solutions, are applied to containing ammonia
On the solid LB flat boards of parasiticin (50ug/mL), 37 DEG C, culture 12-18 hours are inverted, choose single bacterium colony and be inoculated in containing ammonia
In the 5mL LB liquid mediums of parasiticin (50ug/mL), 37 DEG C, 225rpm, shaking table culture 14 hours enters performing PCR to bacterium solution
Identification (Fig. 2), identifies that positive bacterium solution sends sequencing outside, and sequencing result shows, cell-penetrating peptide YARA-HSP10 is correctly connected to
PPIC9K, illustrates pPIC9K-YARA-HSP10 expression vector establishment successes.
(5) prepared by Pichia pastoris GS115 competence
Picking Pichia pastoris single bacterium colony, is seeded to containing in 5mL YPD culture medium test tubes, 30 DEG C, 225rpm overnight incubations.
The culture for taking 100 μ L is seeded in the 250mL triangular flasks containing 50mL fresh YPD mediums, and 30 DEG C, 225rpm was cultivated
At night, to OD600 values 1.2～2.0 are reached.By cell culture in 4 DEG C, 1500g centrifugation 5min abandon supernatant, and the ice with 50mL is pre-
Cold sterilized water is resuspended by bacterial sediment.Centrifugation, by the same way the D-glucitol with the 1mol/L of the ice precooling of 20mL is molten
Liquid is resuspended by bacterial sediment, and supernatant is abandoned in centrifugation.It is repeated twice.Will with the D-glucitol solution of the 1mol/L of the ice precooling of 0.5mL
Bacterial sediment is resuspended, and its final volume is about 0.7mL.It is placed in stand-by in mixture of ice and water.
(6) Pichia pastoris GS115 electricity conversion
By the linearizing pPIC9K-YARA-HSP10 plasmids of 10uL Jing Sal I restriction endonucleases, with 100uL Pichia pastoris GS115s
Competent cell is mixed, and in going to the electric shock cup of 2mm ice precoolings, shock by electricity 5ms, adds the D- sorbs of the 1mol/L of 1mL ice precoolings
Alcohol solution mixes thalline, is coated with MD culture medium flat plates, and 29 DEG C are inverted 3-4 days, treat to grow bacterium colony on MD culture medium flat plates.
(7) screening of multicopy recon
By the bacterium colony sterile toothpick grown on MD culture medium flat plates correspondence be inoculated into G418 concentration be respectively 0.5g/L,
On the YPD flat boards of 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 29 DEG C of cultures, screening obtains transformant.
(8) the fermented and cultured abduction delivering of recombined human heat shock protein-70 engineering bacteria
The engineering bacteria for screening is inoculated in 400mL BMGY culture mediums, 30 DEG C of shaken cultivations to OD600 up to 10 or so,
Transfer in equipped with 4L FBS (40g containing glycerine, K in 1L2SO4 18.2g、H3PO426.7mL、CaSO4.2H2O 0.93g、MgSO4
14.9g, KOH 4.13g is mixed) in the 5L fermentation tanks of culture medium, temperature constant is 30 DEG C, and with ammoniacal liquor pH value is adjusted, and maintains pH
5.5 or so, dissolved oxygen is controlled in 20%～30%, canister culture 16-20h value, is transferred into the big tanks of 150L.In sweat, when
(the sweet Fuel Exhausted in indication basal salt media, this stage takes when dissolved oxygen (Dissolved oxygen, DO) suddenly rises
20h), the glycerine for flowing plus 18.15mL/h/L mass percentage concentrations are 50% is started, in the glycerine that mass percentage concentration is 50%
(contain CuSO in 1L containing 12mL/L trace element PTM14.5H2O 6g、NaI 0.08g、MnSO4.H2O 3g、Na2MoO4.H2O
0.2g、H3BO3 0.02g、H2SO4 5mL、CoCl2.6H2O 0.5g、ZnCl2 20g、FeSO4.7H2O 75g, biotin 0.2g,
It is mixed).When the weight in wet base of zymotic fluid reaches 300mg, stop feed supplement, hungry 1h, fed-batch cultivation Fiber differentiation after glycerol depletion
Base (methyl alcohol containing 12mL/L PTM1), adds methyl alcohol to maintain the transition stage of 2～3h with low speed stream, and temperature setting is 28 DEG C.
After transition stage terminates, improve methanol feeding speed and maintain feed rate to be fermented in 10.9mL/L/h or so.By adjusting
Section rotating speed, tank pressure and throughput make dissolved oxygen more than 20%, induction fermentation 42h or so.After fermentation ends, zymotic fluid by be centrifuged into
Row separation of solid and liquid, retains supernatant, and destination protein is present in supernatant.HSP10 yield is up to 450mg/L.
(9) purifying of recombined human heat shock protein-70 albumen
After fermentation ends, zymotic fluid centrifugation takes supernatant 80L, with the doughnut that molecule interception is 80000D
Ultrafiltration system carries out ultrafiltration, collects filtered solution, with the rolling ultrafiltration membrane ultrafiltration that molecular weight is 1000D, concentrate is collected, by upper
State two step ultrafiltrations and can remove pigment and some foreign proteins that Pichia pastoris fermentation is produced, obtain recombined human heat shock protein-70
Crude protein concentrate.Recombined human heat shock protein-70 crude protein liquid is carried out into cation-exchange chromatography, filler selects CM
Sepharose FF, are purchased from GE companies of the U.S., and the pH with 3 times of column volumes is 7.5 20mmol/L phosphate buffers balance, on
Sample, the pH value containing 1.0mol/L NaCl with 10 times of column volumes is 7.5 20mmol/L phosphate buffers wash-out, collects weight
The group protein liquid of human heat shock protein 10, with the rolling ultrafiltration membrane ultrafiltration that molecular weight is 1000D, desalination, collects concentrate, with freezing
Drying machine is freezed, and obtains recombined human heat shock protein-70, and fermented supernatant fluid expression reaches 450mg/L.
(10) detection of gene recombinant human heat shock protein-70
(10.1) reagent preparation
Low bisacrylamide mother liquor：9.6g acrylamides, 0.3g methylene diacrylamides are dissolved in 20mL water；
High bisacrylamide mother liquor：9.3g acrylamides, 0.6g methylene diacrylamides are dissolved in 20mL water；
Gel buffer liquid：2.422g trishydroxymethylaminomethanes, 0.02g lauryl sodium sulfate (SDS), are dissolved in 20mL
Water, HCl adjusts pH to 8.45；
Anode buffer liquid：12.11g trishydroxymethylaminomethanes are dissolved in water, and are settled to 500mL, and with HCl pH to 8.9 is adjusted；
Cathode buffer：6.06g trishydroxymethylaminomethanes, 8.89g trimethylglycines (Tricine), 0.5g 12
Sodium alkyl sulfate (SDS), adds water to 500mL；
Fixer：30mL ethanol, 0.5mL glutaraldehydes, adds water and is settled to 100mL；
Dyeing liquor：0.125g coomassie brilliant blue R250s, 10mL glacial acetic acid, 45mL methyl alcohol adds water to 100mL；
Destainer：10mL glacial acetic acid, 45mL methyl alcohol, adds water to 100mL；
(10.2) preparation of glue
The separation gel that mass percentage concentration is 16% is prepared by such as lower volume：
To the separation gel that 6mL is irrigated between glass plate, one layer of redistilled water, polyase 13 0min are covered immediately.
As lower volume prepares the spacing glue that mass percentage concentration is 10%：
Separation gel upper strata redistilled water is gone, filter paper is blotted, irrigate 1mL spacing glues, add water water seal, polyase 13 0min.
Prepare the concentration glue that mass percentage concentration is 4% by such as lower volume again：
After spacing glue polymerization, the concentration glue of 2mL is irrigated, plug sample comb, polyase 13 0min.
(10.3) electrophoresis
40uL protein samples are mixed with 10uL sample-loading buffers, 100 DEG C of heating 5min of water-bath is put into, at electrophoresis tank bottom
Portion adds anode buffer liquid, and the glue for making is put into into electrophoresis tank together with its device, and between two pieces of glue Cathode buffer is injected,
30V electrophoresis prerunning 10min, then loading.When dyestuff enters separation gel from spacing glue, high voltage to 90V continues electrophoresis
Until terminating.Electrophoresis result such as Fig. 3.
(11) the Western-blot protein immunoblots identification of recombined human heat shock protein-70
Tricine-SDS polyacrylamide gel electrophoresises, electrophoresis knot are carried out to recombined human heat shock protein-70 after purification
Shu Hou, is put in electrotransfer buffer solution and soaks 20min, while soaking 6 filter paper, 1 polyvinylidene fluoride film.From negative pole to positive pole
Sequentially it is：Three metafiltration paper-gel-polyvinylidene fluoride film-tri- metafiltration paper, constant current 300mA, electrotransfer 1 hour.Electricity turns to terminate
Afterwards, it is 5% polyvinylidene fluoride film deionized water rinsed clean to be soaked in polyvinylidene fluoride film containing mass percentage concentration
(1.21g trishydroxymethylaminomethanes, 8.8g sodium chloride adjust pH to the TBS of bovine serum albumin(BSA) after the 800mL that adds water dissolvings with hydrochloric acid
To 7.5, add water and be settled to 1L) in buffer solution, room temperature is closed 1 hour, add from rabbit polyclonal antibody (one resists, 1：
500), 4 DEG C overnight, film 4 times (10min/ time), addition horseradish peroxidating are washed with TBST (in 1L TBS add 0.5mL polysorbas20s)
Thing enzyme mark goat anti-rabbit igg (two resist, and 1:1000), 37 DEG C are incubated 1 hour, and with TBST film 4 times, substrate diaminobenzidine are washed
(DAB) develop the color.Western-blot qualification results such as Fig. 4.There is chromogenic reaction at 12KD, specific band occur, explanation is obtained
Obtained correct recombined human heat shock protein-70.
(12) immunofluorescence technique detects the cell membrane penetration capacity of recombined human heat shock protein-70
(12.1) cell culture
Fresh F12 complete culture solutions (are purchased from into Chinese medicines group, containing 10% hyclone, the benzyl mould of ammonia containing 100ug/ml
Element) to take out from 4 DEG C of refrigerators, room temperature is placed 1 hour, and is placed in super-clean bench together with the experiment appliance such as centrifuge tube, culture dish,
Ultraviolet sterilization 30min, quickly takes out frozen l cell (l929) from liquid nitrogen container, and 37 DEG C of water-baths are put at once
In, quick-thawing；After thawing, in being sucked 10ml centrifuge tubes, the complete culture solution for adding 2ml fresh, to reduce DMSO pair
The injury of cell, is centrifuged 5min in low speed centrifuge with 1000rpm afterwards；Supernatant is sucked, then adds 4ml new in centrifuge tube
Fresh nutrient solution, cell is blown and suck in culture dish after hanging, and supplementing culture medium is put into CO to 8ml2(CO2Concentration 5%, O2Concentration
15.5%) cultivate in incubator.
(12.2) Immunofluorescence test
Cultivate to the l cell of certain density and digested with the pancreatin of 3ml 0.25%, postdigestive cell difference
With the human heat shock protein 10 (1mg/ml) containing not connected cell-penetrating peptide and the cell of recombined human heat shock protein-70 (1mg/ml)
Nutrient solution blows after hanging and is inoculated in 24 orifice plates, cultivates 48h, cell culture fluid is discarded, with 3 (5min/ of cell PBS
It is secondary), cell is fixed with 4% methyl alcohol, room temperature fixes 30min, then with PBS 3 times (5min/ time), with 1%
The agent of Triton permeable membranes carries out permeable membrane to cell and processes 30min, then with PBS 3 times (5min/ time), lowlenthal serum closes fluid-tight
Close 45min, PBS 3 times (5min/ time), add the heat shock protein-70 from rabbit polyclonal antibody (one resists, 1：
500), 4 DEG C of night incubations, next day places at room temperature 30min, then with PBS 3 times (5min/ time), uses (different with FITC
Thiocyanic acid fluorescein) and fluorescently-labeled two anti-lucifuges incubation 45min (two resist, and 1:1000), then with 3 (5min/ of PBS
It is secondary), taken a picture with Laser Scanning Confocal Microscope, observe albumen distribution situation in the cell.Immunofluorescence test result such as Fig. 5, restructuring
Human heat shock protein 10 (Y-HSP10) is relatively not connected with the heat shock protein-70 (HSP10) of cell-penetrating peptide, and cell-penetrating ability is obtained
Greatly improve.
Sequence table
<110>Shaanxi Hui Kang biotechnologies Co., Ltd
<120>A kind of recombined human heat shock protein-70 and its encoding gene and preparation method
<160> 6
<210> 1
<211> 117
<212> PRT
<213>Recombined human heat shock protein-70
<400> 1
YARAAARQAR AGGGSMAGQA FRKFLPLFDR VLVERSAAET VTKGGIMLPE KSQGKVLQAT 60
VVAVGSGSKG KGGEIQPVSV KVGDKVLLPE YGGTKVVLDD KDYFLFRDGD ILGKYVD 117
<210> 2
<211> 354
<212> DNA
<213>Artificial sequence
<400> 2
tacgctagag ctgctgctag acaagctaga gctggtggtg gtagtatggc aggacaagcg 60
tttagaaagt ttcttccact ctttgaccga gtattggttg aaaggagtgc tgctgaaact 120
gtaaccaaag gaggcattat gcttccagaa aaatctcaag gaaaagtatt gcaagcaaca 180
gtagtcgctg ttggatcggg ttctaaagga aagggtggag agattcaacc agttagcgtg 240
aaagttggag ataaagttct tctcccagaa tatggaggca ccaaagtagt tctagatgac 300
aaggattatt tcctatttag agatggtgac attcttggaa agtacgtaga ctga 354
<210> 3
<211> 102
<212> PRT
<213>Human heat shock protein 10
<400> 3
MAGQAFRKFL PLFDRVLVER SAAETVTKGG IMLPEKSQGK VLQATVVAVG SGSKGKGGEI 60
QPVSVKVGDK VLLPEYGGTK VVLDDKDYFL FRDGDILGKY VD 117
<210> 4
<211> 11
<212> PRT
<213>Cell-penetrating peptide YARA
<400> 4
YARAAARQAR A 60
<210> 5
<211> 12
<212> DNA
<213>Artificial sequence
<400> 5
GGTGGTGGTA GT 60
<210> 6
<211> 4
<212> PRT
<213>Connection peptide
<400> 6
GGGS 60
Claims (11)
1. a kind of recombined human heat shock protein-70, it is characterised in that include：
(1) amino acid sequence of human heat shock protein 10；
(2) amino acid sequence of cell-penetrating peptide；With
(3) amino acid sequence of flexible peptide linker, for connecting the amino acid sequence of the human heat shock protein 10 and described thin
The amino acid sequence of born of the same parents' cell-penetrating peptide.
2. recombined human heat shock protein-70 according to claim 1, it is characterised in that the amino acid of the cell-penetrating peptide
Sequence is SEQ ID No in sequence table:4 sequence, or, with SEQ ID No in sequence table:The homology of 4 sequence exists
More than 90% (preferably 95%, more preferably 98%) and with the amino acid sequence of identical activity.
3. recombined human heat shock protein-70 according to claim 1, it is characterised in that the amino acid of the flexible peptide linker
Sequence is SEQ ID No in sequence table:6 sequence.
4. the recombined human heat shock protein-70 according to any one of claim 1-3, it is characterised in that the recombined human heat is stopped
The amino acid sequence of gram protein 10 is SEQ ID No in sequence table:1 amino acid sequence.
5. the preparation method of the recombined human heat shock protein-70 described in any one of claim 1-4, it is characterised in that include：
(1) gene is obtained：SEQ ID No in artificial full genome composition sequence table:2 sequence, to pPIC9K carriers and manually
The SEQ ID No of full genome synthesis:2 sequence carries out Xho I and the double digestions of Not I, reclaims digestion products, is connected with DNA ligase
Connect, and convert Escherichia coli, extract plasmid；
(2) convert：Plasmid prepared by step (1) is mixed with competence Pichia pastoris and is converted, bacterium colony after being converted；
(3) multicopy recon is screened：Bacterium colony after step (2) is converted further is cultivated, and screens transformant, is weighed
The group engineering bacteria of human heat shock protein 10；
(4) fermented and cultured abduction delivering：Fermented and cultured is carried out to the recombined human heat shock protein-70 engineering bacteria that step (3) is obtained to obtain
Obtain recombined human heat shock protein-70 zymotic fluid；
(5) purify：Carry out separation of solid and liquid, filtration, concentration successively to the recombined human heat shock protein-70 zymotic fluid that step (4) is obtained
Product is obtained with ion-exchange chromatography.
6. preparation method according to claim 5, it is characterised in that the purifying of the step (5) is concretely comprised the following steps：Will
The zymotic fluid Jing centrifugations that step (4) is obtained carry out separation of solid and liquid, take supernatant, and micro-filtration is carried out to fermented supernatant fluid, collect filtrate,
Desalination and concentration by ultrafiltration is carried out again, concentrate is collected, and then carries out ion-exchange chromatography, you can obtain product.
7. the preparation method according to claim 5 or 6, it is characterised in that include：
(1) plasmid is prepared
SEQ ID No in artificial full genome composition sequence table:2 sequence, then closes to pPIC9K carriers and artificial full genome
Into SEQ ID No:4 sequence carries out Xho I and the double digestions of Not I, reclaims digestion products, is connected with DNA ligase, and converts
Escherichia coli, extract plasmid, are named as pPIC9K-YARA-HSP10；
(2) Pichia pastoris electricity conversion
By the linearizing pPIC9K-YARA-HSP10 plasmids of the restriction endonucleases of Jing Sal I, mix with Pichia pastoris competent cell, go to
In the electricity conversion cup of ice precooling, electric shock, the sorbitol solution for adding ice precooling mixes thalline, is coated with MD culture medium flat plates,
Culture is put, on MD culture medium flat plates bacterium colony is grown；
(3) multicopy inserts the screening of recon
By the bacterium colony grown on MD culture medium flat plates correspondence be inoculated into G418 concentration be respectively 0.5g/L, 1g/L, 2g/L, 3g/L,
On the YPD flat boards of 4g/L, 5g/L, culture, screening obtains transformant；
(4) ferment
The engineering bacteria for screening is inoculated in BMGY culture mediums, shaken cultivation, is transferred in cultivating equipped with FBS as first order seed
In the fermentation tank of base, pH value is transferred into the bulk fermentation equipped with FBS culture mediums 5.5 or so as secondary seed；
(5) purify
The zymotic fluid Jing centrifugations that step (4) is obtained carry out separation of solid and liquid, take supernatant, are in 80000D with molecule interception
Fibre ultrafiltration system carries out ultrafiltration, collects filtrate, with the rolling ultrafiltration membrane ultrafiltration that molecular weight is 1000D, collects concentrate, with
After carry out ion-exchange chromatography, you can obtain product.
8. the encoding gene of recombined human heat shock protein-70 described in any one of claim 1-4.
9. encoding gene according to claim 8, it is characterised in that the encoding gene is SEQ ID No in sequence table:
2 nucleotide sequence, or, with SEQ ID No:2 have at least 90% (preferably 95%, more preferably 98%) homology and coding
The nucleotide sequence of identical function albumen.
10. containing the expression vector of encoding gene described in claim 8 or 9.
11. clones containing encoding gene described in claim 8 or 9.
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