CN1924001A - Method of producing human heat shock protein 70 from methyl nutritious yeast - Google Patents

Abstract

The invention discloses a manufacturing, purifying and identifying method of protein for hHSP70, which optimizes to manufacture large scale of industrial ferment material and purify recombined hHSP70.

Description

Method with methylotrophy yeast production HUMAN HEAT SHOCK PROTEINS 70

Invention field

The present invention relates to proteinic production, purifying and authentication method, particularly relate to express recombinant HUMAN HEAT SHOCK PROTEINS 70 (hHSP70) in saccharomyces pastorianus, and the heavy industrialization fermentative production rhHSP70's who optimizes method.

Background of invention

Organism is in the time spent of doing that is subjected to heat or other chemical factors (as high temperature, carbon monoxide, nicotine, anoxic, heavy metal ion, ethanol, uviolizing etc.), can start the expression of one group of specific gene, the protein-heat shock protein(HSP) (HSPs) of synthetic one group of high conservative.Heat shock protein(HSP) (HSP) is also referred to as stress protein, is found to synthetic protein in the reaction of heat-shocked as a kind of cell at first.According to the size of molecular weight, the HSP that has identified so far comprises HSP100, HSP90, HSP70 and small molecules HSP (22KDa, different families such as smHSP).HSP is participation protective cell antagonism unsuitable environmental condition not only, and participates in non-biological chemistry and immunology process that stress cell.HSP can finish different molecular chaperone functions.For example, the member who is positioned the HSP70 family in cytoplasm, nucleus, karyomit(e) or the endoplasmic reticulum not only participates in to immune presented by cells antigen, and participates in proteinic transhipment in the normal cell, folding and assembling.HSP can combine with protein or polypeptide, and has these bonded protein or polypeptide of release down in Triphosaden (ATP), participates in the anti-damage and the cytodifferentiation of cell.Recent study finds that HSPs can will pass to major histocompatibility antigen mixture (MHC) I quasi-molecule by intrinsic pathway with its bonded antigenic peptide or albumen, causes specific cytotoxic T lymphocyte (CTL) reaction.And, also can directly activate gamma delta T cells and NK cell, the immune stimulatory cell discharges various immune-regulating factors, plays a role in the non-specific immunity process.

People such as Srivastava (Immunol.Today, 1988,9:78-83; Proc.Natl.Acad.Sci.USA, 1986, discovering 83:3407-3411), being responsible for the different immunogenic molecules of tumour is that molecular weight is the intracellular protein of glycoprotein and the 84-86KDa (p84/86) of 96KDa (gp96).Can only make the immune response of animal generation with the gp96 or the p84/86 immune mouse that separate from the specific tumors cell at this specific tumors.In addition, shown that HSP70 can cause the immune response of anti-its source tumour rather than other different tumours.Therefore, can think, at this specific immunity of tumor antigen peptide be from the non-covalent complex of HSP and antigen peptide, rather than come from HSP itself.

Antigen peptide in the mixture is transferred to antigen presenting cell, combine with its I of lip-deep main histocompatibility complex (MHC) proteinoid molecule, thereby active cells toxic T lymphocyte (CTL) causes the reaction of CD8+ cytotoxic T cell and kills and wounds target cell.Therefore, might use by the non-covalent complex treatment of the HSP of tumour cell purifying and antigen peptide and prophylaxis of tumours (referring to United States Patent (USP) 5,830,464,6,017,54 and PCT application publication number WO96/10411, WO97/10001).

In addition, also can separate stress protein-peptide complexes in the cell by pathogenic infection, and the infection (PCT 95/24923) of using it for treatment and preventing corresponding pathogenic agent (as virus, bacterium, fungi etc.) to cause.Perhaps, also can use the external sensitization antigen presenting cell of stress protein-peptide complexes, and be used for adoptive immunotherapy (referring to PCT application publication number WO97/10002).

At present, the HSP that is used to prepare stress protein-peptide complexes is HSP70 and tumor antigen peptide particularly, all is mostly to separate from tumour cell and purifying obtains.Yet,, the production of HSP-peptide complex and use are restricted because tumour cell is particularly grown the deficiency in the early stage tumour cell supply.Though someone once utilized the intestinal bacteria system successfully to express HSP70, but because expression product forms inclusion body usually in host cell, so cause the yield of product very low, perhaps purification difficult, be easy to pollute the HSP70 of intracellular toxin and intestinal bacteria itself, often be difficult to adapt to the needs of research and clinical application.Therefore, be necessary to set up and develop recombinant expressed and purifying HSP HSP70 for example, and the method for HSP-antigenic peptide complexes, so that satisfy the demand of laboratory study, and provide this para-immunity therapeutical agent in a large number with prevention for the treatment of tumour and other infectious diseases.

Methylotrophy yeast, particularly pichia pastoris phaff are the eukaryotic expression systems of being furtherd investigate, and have been used to express many useful protein.For example, United States Patent (USP) 5,324,639 disclose the method for particularly producing type-1 insulin like growth factor at the methylotrophy yeast in the pichia pastoris phaff cell; United States Patent (USP) 5,330,901 disclose the method for using pichia pastoris phaff system expression human serum albumin; United States Patent (USP) 5,965,389 provide in the methylotrophy yeast DNA construct of expressing L-L-Glutamic decarboxylase (GAD65) and by the method for the pure GAD65 of preparation; U.S. Patent Publication in pichia pastoris phaff, express the method for platelet-derived cytokine (PDGF); United States Patent (USP) 6,780,615 have described the method for using the reorganization pichia pastoris phaff to produce monellin.Yet, do not see as yet so far about using pichia pastoris phaff to produce the report of HSP 70.

Goal of the invention

An object of the present invention is to provide the method for a kind of use methylotrophy yeast production rhHSP70 (HSP70) polypeptide, it is to cultivate the methylotrophy yeast in the substratum of the carbon source and the energy that this method is included in methyl alcohol, and wherein said methylotrophy yeast is to transform with the DNA construct that comprises the following element that is operably connected: the derivable transcripting promoter of (1) methyl; (2) dna fragmentation of coding heat shock protein 70; (3) transcription terminator and (4) but selection marker, thereby produce the heat shock protein 70 polypeptide with the concentration of 250mg/L substratum at least.

According to a preferred embodiment of the invention, wherein said methylotrophy yeast is a pichia pastoris phaff, and said methyl inducible promoter and transcription terminator are all from pichia pastoris phaff AOX1 gene.

Another object of the present invention provides the methylotrophy yeast that is used for express recombinant HUMAN HEAT SHOCK PROTEINS 70, this yeast can rely on as the growth of the methyl alcohol of the carbon source and the energy, but and is comprised that the DNA construct of dna fragmentation, transcription terminator and the selection marker of the derivable transcripting promoter of methyl, coding heat shock protein 70 transforms.

According to a preferred embodiment of the invention, wherein said methylotrophy yeast is a pichia pastoris phaff, and methyl inducible promoter and transcription terminator are all from pichia pastoris phaff AOX1 gene.

A further object of the present invention provides the DNA construct that is used at pichia pastoris phaff express recombinant HUMAN HEAT SHOCK PROTEINS 70 polypeptide, and this construct comprises the element that is operably connected: but dna fragmentation, transcription terminator and the selection marker of the derivable transcripting promoter of methyl, coding heat shock protein 70.

According to a preferred embodiment of the invention, wherein said methylotrophy yeast is a pichia pastoris phaff, and said methyl inducible promoter and transcription terminator are all from pichia pastoris phaff AOX1 gene.

A further object of the present invention provides the fermentation culture conditions of the optimization of using pichia pastoris phaff scale operation rhHSP70 polypeptide, be characterised in that leavening temperature wherein maintains that 28 ℃～30 ℃, employed pH value are 4.1～5.0, the DO value (dissolved oxygen) of fermented liquid remain on 25%～30% and substratum in be added with the peptone of 0.5% (W/V).

Description of drawings

Fig. 1 shows that the PCR of recombinant plasmid pPICZ alpha/hhsp70 transformed yeast bacterium identifies electrophoretogram.Wherein swimming lane 5 and 11 is dna molecular amount marks; Swimming lane 12 and 13 is PCR products of the yeast genomic dna of empty plasmid carrier conversion; Swimming lane 1～4 and 6～10th, the pcr amplification product of different yeast transformant genomic dnas.

Fig. 2 shows the proteinic sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the rhHSP70 of purifying (SDS-PAGE) result (coomassie brilliant blue staining).Wherein swimming lane 1～4 is respectively the elutriant of fraction collection in the purifying; Swimming lane 5 is hHSP70 standard substance.

Fig. 3 shows the Western engram analysis result of the rhHSP70 of purifying.Wherein swimming lane 3 is hHSP70 standard substance; Swimming lane 1,2 and 4 is that the present invention prepares the also rhHSP70 sample of purifying.

Fig. 4 shows the result who respectively organizes mouse T cell secretion of gamma-IFN level that the enzyme linked immunological spotting method detects.

Fig. 5 shows the killing activity (percentage kill rate) of rhHSP70-antigenic peptide complexes inductive antigen-specific CTL to target cell.

Fig. 6 shows the influence of rhHSP70-antigenic peptide complexes to mouse tumor growth (volume).

Fig. 7 A and 7B show that the rhHSP70-antigenic peptide complexes is to mouse tumor tissue and cyto-architectural influence.Wherein 7A is 400 times of light microscopic photos, and 7B is 200 times of light microscopic photos.

Summary of the invention

The present invention relates to protein production, Purification and Characterization method, particularly relate to the method for in saccharomyces pastorianus, expressing heat shock protein 70.

Pichia pastoris phaff (P.Pastoris) is the yeast expression system that has potentiality that grows up in the 80-90 age in 20th century, owing to it obtains using more and more widely having the incomparable advantage of other system aspect the protein expression.

As eukaryotic expression system, pichia pastoris phaff has the not available advantage of many other protein expression systems: 1. belong to unicellular organism, so kept the characteristics (but high density fermentation cultivate, dry cell weight can reach more than the 130g/L in fermentation tank) of easy operating and fast growth; 2. nutritional requirement is low, and medium component is simple, and production cost is low, is specially adapted to large-scale industrialization production; 3. by homologous recombination, expression vector stably can be incorporated in the host chromosome, be difficult in the continuous passage losing; 4. have strong alcohol oxidase gene (AOX) promoter, can strictly regulate and control the expression of foreign gene; 5. expression is high, and the productive rate of many albumen can reach every liter of above level of gram; The protein of 6. expressing not only can be present in the born of the same parents but also can secrete to born of the same parents, and the secretory volume of pichia pastoris phaff oneself protein matter is very low, extremely is conducive to the purpose product purification; 7. as eukaryotic expression system, correct processing and modification after can translating the albumen of expressing; 8. degree of glycosylation is low.

Pichia yeast expression system is comprised of an exogenous gene expression frame and AOX1 promoter, MCS (MCS) and a terminator sequence that copies from the AOX1 gene (TT). Simultaneously, most of carriers all comprise as the HIS4 gene of selection markers and the sequence (such as ColEI replication origin and anti-ampicillin gene) that exists for copying propagation. In addition, also contain the AOX1 3 ' non-coding area sequence that makes foreign gene can be incorporated into alternative or inserted mode chromosomal AOX1 position. Carrier used in the present invention is to be made of elements such as promoter, terminator, selected marker, reporter gene, origin of replications. Excretion vector also needs to have burst in addition.

The most frequently used promoter is the AOX1 promoter, and its methanol induction is very strong, thereby the foreign gene under its control can obtain higher expression. Most oxidation of ethanol enzyme activities are provided by AOX1 in the cell. When growing take glucose or glycerine as the culture medium of carbon source, AOX1 transcribes and is suppressed; And take methyl alcohol as unique growth carbon source the time, then can induce transcribing and protein expression of AOX1 gene. Selected marker (HIS4) generally is the wild type gene corresponding to the auxotrophy receptor.

The protein of pichia pastoris phaff self secretion seldom, so secreting, expressing is a kind of desirable expression way. Simultaneously, extracellular expression more is conducive to extraction and the purifying of protein. The preferred signal peptide of the present invention is the α factor (α MF). α factor signal sequence is comprised of 87 amino acid, comes from the α sexal maturity factor targeting sequencing of brewer's yeast (S.cerevsia), and the signal peptide of this section sequential coding is inserted the people in the expression vector of pichia pastoris phaff.

Pichia pastoris phaff expression vector of the present invention can be intracellular expression, also can be secreting, expressing. These carriers all comprise an expression cassette that is comprised of 3 ' sequence of the tanscription termination gene of 5 ' AOX1 sequence fragment of 0.9kb and about 0.3kb. Secreted expression carrier can be pPIC9, pPIC9k, pHIL-Sl, pPICZ α, pYAM75P6E6 etc.; The carrier of intracellular expression can be pHIL-D2, pA0815, pPIC3K, pPICZ, pHWO10E121, pGAPZ, pGAPZ α etc. The present invention is pPICZ α preferably.

The pichia pastoris phaff bacterial strain that generally is used for exogenous gene expression comprises Y-11430, M-6100-3, GS115, KM71, SMD1168 etc., and the present invention is pichia pastoris phaff X-33 bacterial strain preferably.

After obtaining carrying the recombinant expression vector of HSP gene, can use methods such as lithium salts method, PEG method, spheroplast method and electroporation to transform the pichia pastoris phaff host cell.Wherein, the preferred method for transformation of the present invention is electroporation ((as referring to Sambrook, ed.Molecular Cloning:A Laboratory Manul (2nd.ed.), Vols.1-3, Cold Spring Harbor Laboratory (1998)).

Import the intravital recombinant expression vector of yeast have only with yeast chromosomal on homologous region recombinate, be incorporated on the karyomit(e), foreign gene can stable existence and is expressed.Among the present invention, in order stably to express goal gene, can be in the single endonuclease digestion site of His or 5 ' AOX1 with plasmid vector linearizing (Sac I), make it to change and be incorporated in the yeast cell karyomit(e) by single cross, be Mut thereby obtain phenotype ⁺And has a transformant that high methanol utilizes ability.

Be used for many posttranslational modification functions that pichia pastoris phaff of the present invention contains typical higher eucaryote.These functions comprise processing, the protein folding of signal peptide, formation, O-and the glycosylation of N-type and the acidylate etc. of disulfide linkage.What wherein, most important and research was maximum is glycosylation.Proteinic glycosylation chain participates in cell recognition, hormone receptor combination, protein positioning and host-microorganism interphase interaction.Glycosylation process is the reaction that also produces the few ribose of being made up of Mans-6-GluNAC2 (high mannose type) through a series of montages.Pichia pastoris phaff can be connected to carbohydrate on the exogenous protein of secreting, expressing.The mean length that pichia pastoris phaff is modified sugar chain is 8～14 seminoses, and outer chain do not contain α-1,3 seminose, so the glycoprotein of its expression is particularly suitable for the treatment application.

Since fully optimized dissolved oxygen levels, air flow, fermentation pH value among the present invention, culture condition such as stir speed (S.S.), nutrient, thus can make the yeast high-density growth, thus efficiently expressing of product caused.For example, under the fermentor cultivation condition, reorganization HP70 expression of polypeptides productive rate of the present invention can reach 1.2g/L.

In most cases, integrate the multiple copied foreign gene in the pichia pastoris phaff (P.Pastoris) and can improve expression of recombinant proteins output, so the present invention preferentially selects secretor type, high copy mark and easy-operating shuttle vectors, particularly pPICZ α as HSP70 polypeptide expression carrier.

In addition because when integrating in the His site, the His site of chromosome mutation can and the HIS4 gene locus of expression cassette between the producer exchange can cause losing of expression cassette, so ordinary priority selection AOX1 site.

Among the present invention, the employed substratum of fermentation culture can be substratum such as BMGY/BMMY, BMG/BMM, MGY/MM, but preferably contains the BMGY/BMMY or the BMG/BMM substratum of damping fluid in the composition.

As the unique carbon source and the energy, the add-on of methyl alcohol is generally the 0.5-1.0% of volume of culture (V/V).

In the fermentation culture process, can use known method to detect, and therefrom select to have the cellular segregation thing of high yield, be used for amplifying and produce by the HSP70 content of peptides that is produced by the transformant isolate.

Can use methods such as saturated ammonium sulphate is saltoutd, ion exchange chromatography, affinity chromatography to separate and the required HSP70 polypeptide of purifying.

The present invention first in yeast (pichia spp X-33) efficient secretory expression hHSP70, set up stable rhHSP70 pichia spp and efficiently expressed system.Compare with the expression in intestinal bacteria, method of the present invention has following characteristics: 1. expression system is stable, and the expression cassette that contains goal gene is integrated in the zymic karyomit(e) by homologous recombination, and bacterial classification is stable, does not have the problem of plasmid loss; 2. effective secreting, expressing, the expression of target protein matter is controlled by the strictness of alcohol oxidase promotor, can under methanol induction, start and express and expression product is secreted in the substratum, and the albumen of pichia spp itself seldom is secreted in the substratum, make target protein be easier to purifying; 3. express the output height, bottle scale expression amount that shakes of rhHSP70 in pichia spp can be up to 250mg/L, can reach 1.2g/L during the large scale and high density fermentation culture in fermentor tank; 4. there is not the contaminated with endotoxins problem; 5. the large scale fermentation production cost is low.

Aspect the HSP70 purifying, adopt at present mostly the ConA affinity chromatography in conjunction with DEAE anion-exchange chromatography or ATP/ADP affinity chromatography in conjunction with the DEAE anion-exchange chromatography.As everyone knows, intracellular toxin is made up of lipoprotein and polysaccharide, have negative charge usually, and nucleic acid is also electronegative, therefore uses the anion-exchange chromatography method to cause the pollution of impurity such as intracellular toxin and nucleic acid easily.Someone reported once and pointed out that HSP70 was stable inadequately under higher pH condition, and yield and purity are all lower.For example very fast degraded when 37 ℃ of incubations, 37 ℃ of incubation 2h have obvious degradation when being higher than pH7.7.Because the iso-electric point of hHSP70 is about 5.5, so the present invention adopts and carries out cation-exchange chromatography under the pH4.0 condition and can well avoid nucleic acid and endotoxic pollution, and help keeping the stable of rhHSP70, prevent its degraded.

The present invention has set up the method for efficient secretory expression hHSP70 in yeast, has set up simultaneously and has united the method for using hydrophobic chromatography and cation-exchange chromatography technology purifying hHSP70 product.Use SDS-PAGE, Western engram analysis and protein N-terminal sequencing analysis, the rhHSP70 that confirmation is produced according to the inventive method has sequence and the physico-chemical property identical with natural hHSP70, thereby provides prerequisite for further detecting its inside and outside biologic activity.

Further biological experiment shows, prepare HSP70 according to the inventive method and can be used as molecular chaperones, effectively in conjunction with the lip-deep antigen peptide of target cell, give full-time antigen presenting cell (APC) by receptor-mediated endocytosis with its bonded antigen molecule submission, after APC processing, enter MHC-I quasi-molecule approach, activate specific cytotoxic t lymphocytes (CTL).And HSP70 also can directly activate tumour immunity effector cells such as gamma/delta T cell and NK cell, induces body fluid and cell anti tumor immune response.

The inventor uses synthetic HER2/neu albumen (Hynes NE, Stern DF, Biochem.Biophys.Acta, 1994,198 (1), CTL epitope antigen peptide 165-184) carries out testing in the animal body with the formed mixture of rhHSP70 for preparing according to the inventive method, the result confirms, rhHSP70 is as a kind of non-specific molecules chaperone, it can pass to major histocompatibility antigen mixture (MHC) I quasi-molecule with bonded antigen peptide with it, cause specific cytotoxic T lymphocyte (CTL) reaction, immune stimulatory cell (activatory CTL cell) discharges the active cells factor and promotes the apoptosis of tumour cell.Our a series of experiments show that effectively the rhHSP70 of the present invention's preparation is a kind of effective antigens protein molecular carrier and immunological adjuvant really.

On the basis of above research, the present invention has further inquired into Pichia yeast engineering large scale fermentation (80L fermentor tank) method, optimized the condition of utilizing the yeast large scale fermentation to produce heat shock protein 70, and the product purification method of suitable scale operation.Wherein employed optimal conditions comprises: leavening temperature maintains that 28 ℃～30 ℃, employed pH value are 4.1～5.0, the D0 value (dissolved oxygen) of fermented liquid remain on 25%～30% and substratum in be added with the peptone of 0.5% (W/V).

The result shows, the expression rate that the Pichia yeast engineering large scale fermentation that the present invention sets up is produced the rhHSP70 is about that 1.2g/L fermented liquid, the product rate of recovery are about 80%, degree of purity of production is up to about 95%.These results of study of the present invention will provide necessary base for rhHSP70's suitability for industrialized production and clinical application undoubtedly.

Embodiment

Below by embodiment preferred forms of the present invention is described.These embodiment are intended to further illustrate for example the present invention, rather than limit the await the reply scope of claim of the present invention by any way.

Embodiment 1: the clone and the amplification of people hsp70 gene (hhsp70)

(1) preparation of DNA:

With HeLa cell (2 * 10 ⁶) be seeded in the culture dish of the DMEM nutrient solution that contains 10% new-born calf serum, in 5%CO ₂37 ℃ of cultivations in the incubator.Treat that cell covers with behind the culture dish (about 1 * 10 ⁷Individual cell), discard nutrient solution, wash twice with PBS after, add the ice-cold cell lysis buffer solution of 3ml, room temperature was placed 5 minutes, changed 1.5ml then over to and moved (every pipe 1ml) in liquid (EP) pipe.Add Proteinase K in 5 μ l Proteinase Ks/ml cell lysis buffer solution ratio, mixing places 55 ℃ to place 3～16 hours.After taking out above-mentioned EP pipe and reducing to room temperature, every pipe adds 5 μ l RNA enzymes, places 15～60 minutes for 37 ℃.After sample is reduced to room temperature, add 200 μ l liquor kalii aceticis, concuss made its abundant mixing in 20 seconds.Ice bath is 4 ℃ centrifugal (12000rpm) collection supernatant after 5 minutes.After 70% washing with alcohol, air drying DNA post precipitation carries out quantitatively (OD of agarose gel electrophoresis ₂₆₀/ OD ₂₈₀=1.6～1.8).

(2) clone of hhsp70 gene (PCR method)

Get the people DNA 100ng that as above extracts, set up the PCR reaction system in following ratio:

Human gene group DNA 100ng; Contain Mg ²⁺10 * LA PCR damping fluid, 10 μ l; DNTPs 8 μ l (each 2.5mmol/L); Upstream primer: 5 '-GCATTCGAAACGATGGCCAAAGCCGC-3 ' (SEQ ID NO:1) 1 μ l (20pmol/ μ l); Downstream primer: 5 '-CCGGAATTCGCCCCTAATCTACCTCC-3 ' (SEQ ID NO:2) 1 μ l (20pmol/ μ l); Taq enzyme 1 μ l (5U/ μ l); The sterilization ultrapure water is to final volume 100 μ l.The PCR reaction conditions is: 94 ℃ 4 minutes; 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 2 minutes, totally 28 circulations, then 72 ℃ 8 minutes.Amplified production is carried out 0.8% agarose gel electrophoresis analysis, observe clip size, determine whether to obtain correct goal gene.

(3) structure of cloning vector, conversion and amplification

Reclaim the hhsp70 gene fragment of pcr amplification and carry out 0.8% agarose gel electrophoresis analysis.After the recovery, hhsp70 gene fragment and T carrier are connected 30 minutes for 16 ℃.The ligation system comprises: hhsp70PCR product 0.1～0.3pmol; T carrier 0.03pmol; Solution I (contain dna ligase and be connected damping fluid, see Takara company's T support agent box) 5 μ l; The sterilization ultrapure water is to final volume 10 μ l.

According to ordinary method, from 37 ℃ of XL that cultivate 16 hours ₁(not containing antibiotic LB agar plate) single bacterium colony of picking on the negative culture plate of-Blue (diameter 2～3mm) is inoculated in the 1L culturing bottle that contains 100ml LB substratum, with preparation competence Bacillus coli cells, and frozen standby in-80 ℃.

After getting a frozen competent cell thawing, add above-mentioned connection product, carry out routine and transform.Get competent cell that 200 μ l have transformed then and coat on the LB agar plate that contains X-Gal, IPTG (the two can be before being coated with bacterium half an hour be applied to LB agar plate surface) and penbritin (100 μ g/ml), cultivated 12～16 hours in 37 ℃ of incubators.

(4) evaluation of recombinant vectors

White single bacterium colony (" indigo plant is screened in vain ") after picking transforms at random is inoculated in the LB substratum that contains penbritin (100 μ g/ml), 37 ℃ of strong concussion overnight incubation, the conventional then plasmid DNA of extracting.Get that 1 μ l dissolved DNA throw out carries out the agarose gel electrophoresis quantitative analysis and enzyme is cut evaluation.37 ℃ of PstI digested 2 hours, identified correct clone according to the inscribe zymogram behind 1% agarose gel electrophoresis.Select enzyme and cut the correct clone of evaluation, extract plasmid, be used for dna sequencing.

The structure of embodiment 2:hhsp70 pichia spp secreted expression carrier pPICZ α/hhsp70 and host cell transform

Get above-mentioned order-checking and identify the correct plasmid 50ng that carries the hhsp70 gene, in the 0.2mlEP pipe, set up the PCR reaction system according to the above ratio.Utilize above-mentioned PGR reaction system and condition, introduce the partial sequence and the XhoI site of yeast alpha factor signal peptide by upstream primer 5 '-ATACTCGAGAAGAGAATGGCCAAAGCCGCGGCGATC-3 ' (SEQ ID NO:3), and utilize downstream primer: 5 '-GCTCGATCTAGACCTAATCTACCTCCTCAATGGT-3 ' (SEQ ID NO:4) introduces the XbaI site.After PCR introduces above-mentioned sequence and restriction enzyme site, reclaim the purpose fragment.After cutting with corresponding enzyme, be connected into the plasmid pPICZ α after cutting with same enzyme, make up yeast expression vector pPICZ α/hhsp70, transformed into escherichia coli then extracts plasmid and also carries out enzyme and cut and identify and the determined dna sequence analysis.

Get the correct cultivation bacterium liquid of order-checking, extract plasmid DNA and carry out quantitative analysis with agarose gel electrophoresis method by the plasmid extraction kit specification sheets.Get 20～25 μ g pPICZ α/hhsp70 recombinant plasmids after SacI enzymic digestion (linearizing), with phenol/chloroform extracting and use ethanol sedimentation.Linearizing plasmid is standby on ice with 10 μ l ultrapure waters dissolving postposition.

(not containing antibiotic YPD Agar flat board) single bacterium colony of picking from the negative culture plate of the YPD of pichia spp X-33 is inoculated in the 5ml YPD substratum, 250rpm, and 30 ℃ of concussions were cultivated 8 hours, prepared the yeast competent cell with routine.

Get the above-mentioned competence bacteria of 80 μ l then, mix, move in the 0.2cm electricity conversion cup and carry out the electricity conversion with the linearizing recombinant expression plasmid of 20～25 μ g.The bacterium liquid of getting after 50～100 μ l transform is coated on the YPD flat board that contains Zeocin (100 μ g/ml), and 30 ℃ of incubators were cultivated 2～3 days, observed the upgrowth situation of transformant.

Use PCR method (the primer that uses and reaction conditions the same) screening transformed yeast bacterium then.Behind the centrifugal recovery thalline, extract the pastoris genomic dna performing PCR of going forward side by side with glass bead method and identify that qualification result as shown in Figure 1.

Embodiment 3:HSP70 polypeptide expression and purifying

(1) rhHSP70's (rhHSP70) expression

Get above-mentioned qualification result male clone and be inoculated in 10ml BMGY (pH6.0) substratum, 30 ℃ of concussions were cultivated 24 hours, to OD ₆₀₀Reach 2.0～6.0 o'clock collecting cells.With equal-volume (10ml) BMMY (pH6.0) re-suspended cell precipitation, abduction delivering is cultivated in 30 ℃ of concussions.Induce in the process, replenished a methyl alcohol to final concentration 0.5% in per 24 hours, replenish the sterilization ultrapure water simultaneously, the fermented liquid cumulative volume is remained unchanged.Respectively get the 0.5ml fermented liquid at the 0th, 24,48,72, the 96 and 120 hour equi-time point of cultivating, the centrifuging and taking supernatant is used for protein analysis (SDS-PAGE, Western Blot, n terminal amino acid sequential analysis etc.).

(2) purifying of rhHSP70

After with 0.5mol/L NaOH the pH value of fermented supernatant fluid being adjusted to 7.4, centrifugal (12000rpm) 10 minutes is to remove particulate matter.(exchange buffering liquid is solution D: 20mmol/L Tris-Ac, 20mmol/L NaCl, 3mmol/LMgC to carry out sieve chromatography with Sephacryl S-400 resin column then ₁₂, pH7.4).Solution D (20mmol/LTris-Ac, 20mmol/L NaCl, 3mmol/LMgC with 10 times of column volumes ₁₂, pH7.4) balance ATP-Agrose affinity column (Sigma company).Behind the end of the sample, with solution E (solution D that contains 0.5mol/L NaCl) the flushing ATP-Agrose affinity column of about 10 times of column volumes, to OD ₂₈₀Curve is reduced to baseline.Use the solution D and solution F (solution D that contains 3mmol/L ATP) the flushing ATP-Agrose affinity column (flow velocity is the 0.3ml/ branch) of 5 times of column volumes successively, and collect protein peak.Then, with solution B (20mmol/L HAc-NaAc damping fluid (the ie in solution B:NaAc3H of 10 times of column volumes ₂O 4.9g; Glacial acetic acid 9.425ml, ultrapure water is settled to 1L, pH 4.0) balance Mono S resin cation (R.C.), and with the protein sample of above-mentioned collection with 4 times of solution B dilutions, be 4.0 with the vinegar acid for adjusting pH value afterwards, the same centrifugal.To Mono S resin cation (R.C.), application of sample washed to OD with solution B after finishing with 0.5ml/ minute speed application of sample ₂₈₀Value is reduced to baseline.Use solution C (solution B that contains 2mol/L NaCl) gradient elution then, and the fraction collection protein peak.Carry out sieve chromatography with Sephacryl S-400 at last.

The SDS-PAGE analytical results shows, so to reach electrophoresis at least pure for the rhHSP70 of purifying, and product yield and purity reach about 80% and 95% (referring to accompanying drawing 2) respectively.

The physico-chemical property of embodiment 4:HSP70 polypeptide is identified

(1) sds polyacrylamide gel electrophoresis (SDS-PAGE)

Carry out protein molecule flow measurement and preliminary quantitative analysis with the SDS-PAGE method.Method is as follows:

Prepare 10% separation gel 5% and concentrate glue.Get the 48th hour culture supernatant respectively and add 5 * SDS sample buffer, behind the mixing, boiled 3～5 minutes in 4: 1 ratios.Get above-mentioned sample, be cooled to room temperature after, centrifugal (10000rpm) 30 seconds gets the every hole of supernatant application of sample 20 μ l.The 40V electrophoresis is adjusted voltage to 100V to concentrating glue and separation gel intersection, continues the constant voltage electrophoretic separation.After coomassie brilliant blue staining and the decolouring, the observation analysis result.

(2) the Western engram analysis of expression product

According to ordinary method with fermented liquid supernatant behind SDS-PAGE, be transferred on nitrocellulose (NC) film drying at room temperature 30～60 minutes.Above-mentioned NC film is placed plate, add 20ml confining liquid (TTBS that contains 0.2%BSA), room temperature was vibrated 2～3 hours gently or 4 ℃ of sealings are spent the night.Sealing is put into hybridization bag by 0.1ml antibody-solutions/cm with the NC film after finishing ²Film adds the anti-HSP 70 antibody of rabbit, room temperature vibration 1～4 hour.After the film usefulness TTBS rinsing 3～5 times, dilute the goat anti-rabbit antibody to 1 of horseradish peroxidase-labeled with antibody diluent: 200～1: 1000, add in the hybridization bag, with NC film room temperature vibration 2～4 hours.Add 1ml 0.3% (W/V) NiCl or CoCl ₂And 10 μ l 30%H ₂O ₂Solution.After washing film, film is moved in the colour developing liquid, shake and observe color reaction under the room temperature gently.The result as shown in Figure 3.

(3) amino acid sequence analysis of expression product

Proteinic primary structure is the basis of its higher structure, during with the gene recombination technology expression secreted protein, signal peptide mistake cutting phenomenon appears in the heterologous protein of expressing easily in the processing ripening process, influence the higher structure and the bioactive performance of expressing protein then.Therefore, determine under the correct situation of molecular weight, tackle expressed recombinant protein and carry out protein N-terminal amino acids sequence mensuration, to determine the exactness of its primary structure at SDS-PAGE.Protein sequencing working delegation National Center of Blomedical Analysls of this test finishes.Sequencing result shows that the rhHSP70 who prepares according to the inventive method has and the identical aminoacid sequence of wild-type heat shock protein 70.

The biologic activity of embodiment 5:rhHSP70 and application

HSP70 is as a kind of molecular chaperones and molecular vehicle, it can give full-time antigen presenting cell (APC) with its bonded antigen presentation effectively by receptor-mediated endocytosis, after APC processing, enter MHC-I quasi-molecule approach, activate specific cytotoxic t lymphocytes (CTL).In the present embodiment, use the proteic CTL epitope antigen of the HER2/neu peptide of chemosynthesis to combine formed mixture as therapeutic vaccine or experiment medicine with the rhHSP70 that the present invention produces, observe this mixture to the cytotoxic T cell quantity of animal model for tumour and target cell killing activity, inhibition rate of tumor growth, and the influence of the lymphocytic cytokine of T (IFN-) secretion level, so as to judging and the biologic activity of the rhHSP70 that definite the present invention produces.

(1) the breast cancer tumour antigen peptide is synthetic

Application ProPred-I program ( Http:// www.imech.resraghava/propredl/index.htmlWebpage) prediction CTL epi-position is chosen two kinds of sections, i.e. P from antigen predicts the outcome _106-114(Gln-Leu-Phe-Glu-Asp-Asn-Tyr-Ala-Leu) (SEQ ID NO:5) and P _369-377(Lys-Ile-Phe-Gly-Ser-Leu-Ala-Phe-Leu) (SEQ ID NO:6) epi-position restricted peptides is as breast cancer tumour antigen peptide to be synthesized (Kono et al., Inf.j.Cancer, 78 (2): 202-208,1998).

Use ACT90 type Peptide synthesizer (ACT company), with solid phase stepwise coupling method synthetic antigenic peptide.After building-up process finishes, slough the Fmoc protecting group and downcut the synthetic peptide chain, promptly obtain the crude product peptide after the ether washing precipitation from resin.The crude product peptide is with pressing anti-phase liquid chromatography (LC) purifying among the AKT explorer100 (Amersham Phamacia Co.).To combine with the antigen peptide of purifying through the rhHSP70 of processing of ATP affinity chromatography and accurate quantification then, obtain rhHSP70-antigenic peptide complexes (concentration is 0.1 μ g/ μ l).

(2) rhHSP70 of the present invention is to the influence of T cell IFN-(IFN-γ) secretion and specific CTL target cell killing activity

Be in the mammary cancer D2F2 cell (1 * 10 of logarithmic phase with trysinization ⁸/ ml) after, every animal (female 6-8 BALB/c mouse in age in week, mean body weight 18～20g) subcutaneous vaccination D2F2 cell 0.1ml (1 * 10 ⁷Individual cell).Be divided into following six groups (every group of 12 animals) after 7 days at random:

1) blank group: the subcutaneous every side injection PBS 0.1ml of both sides inguinal region weekly, twice totally; 2) rhHSP70 group: both sides inguinal region subcutaneous injection weekly, every side injection rhHSP700.1ml (0.1 μ g/ μ l), twice totally; 3) GM-CSF+P _106-114The polypeptide vaccine group: both sides inguinal region subcutaneous injection, every side injection synthesizes polypeptide P _106-1140.1ml (0.1 μ g/ μ l contains 800ng rhGM-CSF adjuvant) is weekly, twice totally; 4) GM-CSF+P _369-377The polypeptide vaccine group: inguinal region subcutaneous every side injection in both sides synthesizes polypeptide P _369-3770.1ml (0.1 μ g/ μ l contains 800ng rhGM-CSF) is weekly, twice totally; 5) rhHSP70-P _106-114Polypeptide complex group: both sides inguinal region subcutaneous injection weekly, every side injection rhHSP70-P _106-114Polypeptide complex 0.1ml (0.1 μ g/ μ l), twice totally; 6) rhHSP70-P _369-377Polypeptide complex group: both sides inguinal region subcutaneous injection weekly, every side injection rhHSP70-P _369-377Polypeptide complex 0.1ml (0.1 μ g/ μ l), twice totally.

Immune back 7 days for the second time, 4 animals of every group of picked at random, disconnected neck is put to death, and aseptic separation splenic lymphocyte is carried out conventional T cell IFN-γ secretion and is measured (enzyme linked immunological spotting method) and specific CTL detection (cytotoxic T cell granzyme method for releasing).Wherein calculate the CTL killing activity by following formula.

CTL killing activity (%)=100% * (detecting hole OD value-blank hole OD value)

/ (enzyme release aperture OD value-blank hole OD value)

But since activatory CTL cell external be subjected to specific antigen and stimulate after secretion of gamma-IFN, the ability that therefore detects the post-stimulatory T emiocytosis of specific antigen IFN-γ (adopts the enzyme linked immunological spotting method, ELISPORT), can infer the number of Specific CTL Cells.

T cell IFN-γ secretion measurement result shows that (PBS) compares with control group, rhHSP70 group, GM-CSF+P _106-114Group, GM-CSF+P _369-377Group, rhHSP70-P _106-114Group and rhHSP70-P _369-377The equal showed increased of mouse boosting cell quantity (P＜0.01) of group secretion of gamma-IFN.And, rhHSP70-P _106-114Group and GM-CSF+P _106-114Group, rhHSP70-P _369-377Group and GM-CSF+P _369-377Group, rhHSP70-P _106-114Group and rhHSP70 group, and rhHSP70-P _369-377Group is compared with the rhHSP70 group, and the mouse boosting cell number of secretion of gamma-IFN is showed increased (P＜0.01) also.In addition, rhHSP70-P _106-114Group and rhHSP70-P _369-377Group has been compared significant difference (P＜0.05), GM-CSF+P _106-114Group and GM-CSF+P _369-377Group is compared difference not statistically significant (P＞0.1).The result referring under tabulate 1 and accompanying drawing 4.

Table 1rhHSP70-antigenic peptide complexes to the influence of mouse T lymphocyte IFN-γ excretory (x ± S, n=4)

	The PBS group	The rhHSP70 group	GM-CSF+ P 106-114Group	GM-CSF+ P 369-377Group	rhHSP70- P 106-114Group	rhHSP70- P 369-377Group
							The spot number	9.75± 3.79	22.83 ± 3.71	67.42 ± 6.57	65.08 ± 5.32	89.17 ± 3.46	94.50 ± 6.42

GM-CSF: granulocyte-huge is had a liking for colony-stimulating factor.

The result that specific CTL detects shows that (PBS) compares with control group, GM-CSF+P _106-114Group, GM-CSF+P _369-377Group, rhHSP70-P _106-114Group and rhHSP70-P _369-377The Specific CTL Cells that the group immunostimulation produces obviously strengthens (P＜0.01) to the lethal effect of specific target cell.And, rhHSP70-P _106-114Group and GM-CSF+P _106-114Group, rhHSP70-P _369-377Group and GM-CSF+P _369-377Group, rhHSP70-P _106-114Group and rhHSP70 group, and rhHSP70-P _369-377Group is compared with the rhHSP70 group, and the CTL cell that the former produces also strengthens (P＜0.01) to the lethality of target cell.In addition, rhHSP70-P _106-114Group and rhHSP70-P _369-377Group, GM-CSF+P _106-114Group and GM-CSF+P _369-377Group is compared, and all has significant difference (P＜0.05).Control group is compared then difference not statistically significant (P＞0.1) with the rhHSP70 group.The results are shown in down tabulation 2 and accompanying drawing 5.

Table 2 experiment mice specific CTL to the kill rate (%) of target cell (x ± S, n=4)

	The PBS group	The rhHSP70 group	GM-CSF+ P 106-114Group	GM-CSF+ P 369-377Group	rhHSP70- P 106-114Group	rhHSP70- P 369-377Group
							The target cell kill rate	9.24 ± 0.51	10.88 ± 2.14	24.71 ± 2.83	27.28 ± 3.04	34.76 ± 3.63	37.26 ± 3.28

(3) rhHSP70 of the present invention is to the upgrowth situation of mouse tumor tissue and the influence of tumour cell structure

Put to death animal on the 30th day behind the second immunisation mouse, the weighing knurl is heavy and calculate tumour inhibiting rate, to analyze the restraining effect of various vaccines to tumor growth.The result as seen, GM-CSF+P _106-114Group, GM-CSF+P _369-377Group, rhHSP70-P _106-114Group and rhHSP70-P _369-377The tumor weight and the volume of group are starkly lower than control group (P＜0.01, P＜0.01, P＜0.01, P＜0.01).And, rhHSP70-P _106-114Group and rhHSP70-P _369-377The tumor weight and the volume of group are significantly less than GM-CSF+P _106-114Group and GM-CSF+P _369-377Group (P＜0.01, P＜0.01, P＜0.01, P＜0.01).RhHSP70 group compares gross tumor volume with control group and weight reduces to some extent, but significant difference not remarkable (P＜0.05).The result referring under tabulate 3 and accompanying drawing 6.

The restraining effect that table 3rhHSP70-antigenic peptide complexes vaccine is grown to mouse D2F2 breast tumor (x ± S, n=8)

The experiment group	Average knurl heavy (g)	Tumour inhibiting rate (%)	Mouse mean body weight (g)
					Before the inoculation	After the inoculation
			PBS control group rhHSP70 organizes GM-CSF+P 106-114Group GM-CSF+P 369-377Group rhHSP70-P 106-114Group rhHSP70-P 369-377Group	2.83±0.83 2.57±0.72 1.81±0.56 1.78±0.43 1.24±0.49 1.08±0.35			0 9.2 36.2 37.1 56.2 61.8	18.85±0.72 19.37±0.81 18.98±0.77 19.21±0.76 19.27±0.83 19.17±0.77	28.13±3.09 31.26±2.93 33.32±2.76 34.43±2.65 33.78±2.53 34.66±2.39

Tumor tissue section's light microscopic after HE dyeing is observed down and is found GM-CSF+P _106-114Group and GM-CSF+P _369-377Group, rhHSP70-P _106-114Group and rhHSP70-P _369-377The visible big area necrosis region of tumor tissues of group, in the necrosis region or periphery as seen leukocyte infiltration is arranged.The cell cytosol at necrosis region place is red to be dyed, and cellularstructure disappears; The karyon major part presents cracked, and the nuclear chromatin disintegration is a small shreds; Nuclear membrane breaks, and the chromatin fragment is scattered in the endochylema.The part cell is karyolysis, and nuclear staining is lighter, only sees the profile of nuclear, even karyolysis disappears.Be worth should be mentioned that a large amount of necrosis regions are also arranged at the tumor tissues periphery, and at rhHSP70-P _106-114Group and rhHSP70-P _36-377Tumor tissues fibrosis phenomenon has appearred in group.Then do not see big area necrosis (referring to accompanying drawing 7A and 7B) in PBS group and the rhHSP70 group mouse tumor tissue.

Large scale fermentation preparation and the purifying of embodiment 6:rhHSP70

(1) optimal ph determines

Choose the higher rhHSP70 Pichia yeast engineering of expression amount, 30 ℃, 250rpm concussion were cultivated about 24～36 hours in 10ml YPD substratum.Get above-mentioned BMGY 8ml respectively, press the amount of showing and add 1mol/L Na with damping fluid ₂HPO ₄With the 0.5mol/L citric acid, to be mixed with the BMGY of different pH values.Respectively add the 1ml Pichia yeast engineering behind the mixing, 30 ℃, 250rpm concussion were cultivated about 30 hours, made its OD ₆₀₀≈ 5.

The pH value	1mol·L -1Na 2HPO 4(μl)	0.5mol·L -1Citric acid (μ l)
			2.2 2.8 3.4 4.0 4.6 5.2	20 158.5 285 385.5 467.5 536	980 841.5 715 614.5 532.5 464

Room temperature centrifugal (3000rpm) 5 minutes, supernatant discarded, and in the bacterial sediment thing, add the BMMY 8ml that does not contain damping fluid.Add 1mol/L Na by amount as implied above then ₂HPO ₄With the 0.5mol/L citric acid, being mixed with the BMMY of different pH values, 30 ℃, 250rpm shake cultivation.In the abduction delivering process, replenished a methyl alcohol to final concentration 0.5% in per 24 hours, replenish the sterilization deionized water simultaneously, the fermented liquid cumulative volume is remained unchanged.Respectively get the 0.5ml fermented liquid at the 0th, 24,48,72, the 96 and 120 hour equi-time point of cultivating, centrifugal collection supernatant carries out quantification of protein analysis (SDS-PAGE method).

(2) other Optimizing Conditions of Fermentation

A) with frozen engineering bacteria on YPD Agar (containing Zeocin 100 μ g/ml) plate 30 ℃ streak culture.Reach about 2mm to colony diameter, picking mono-clonal bacterium colony joins in the 10ml YPD nutrient solution (seed culture medium), and 30 ℃, 250rpm concussion were cultivated 16～24 hours.Then above-mentioned culture is joined in the YPD nutrient solution (2L), 30 ℃, 250rpm concussion were cultivated OD about 24 hours ₆₀₀Reach about 10.

B) accumulation of biomass

Preparation 30L FM21 substratum adds in the 80L fermentor tank autoclaving (121 ℃, 30 minutes).When treating in the fermentor tank substratum cool to room temperature, the pH that regulates the FM21 substratum with ammoniacal liquor then adds 36.75ml PTM1 trace element mixture and 13.5ml vitamin H stock solution to required numerical value.In fermentor tank, add 2 and go up the bacterium liquid of stating cultivation, begin to carry out fs fermentor cultivation (being that glycerine is cultivated the amplification thalline stage).This stage culture temperature is 30 ℃, stirring velocity 500rpm, jar internal pressure 10psi, and DO value (dissolved oxygen) maintains more than 20%, feeds pure oxygen in case of necessity.In this stage, take a sample at least every day 2 times, survey OD ₆₀₀And wet cell weight, analyze the yeast growth conditions, naked eyes and mirror are observed bacterium liquid down.Get rid of living contaminants, and leave and take supernatant and be used for analysis of protein.The DO value rises near 100% after about 24 hours, shows in the substratum glycerine approach exhaustion.Promptly change over to this moment and replenish the glycerine stage, with further increase cell density.According to adding the PTM1 trace element in 50% glycerine of ratio behind autoclaving of every liter of 12mlPTM1 trace element.Behind the mixing, the speed of this mixture with the initial fermented liquid of 18.2ml/h/L (being 546ml/h) is joined in the fermentor tank, reach 180～220g/L to the thalline weight in wet base.The DO value rises near after 100%, continues to keep " glycerine hunger " state 30 minutes, enters the methanol induction expression phase then.

C) methanol induction rhHSP70's is synthetic

According to the ratio of 12ml/L, in methyl alcohol, add the PTM1 trace element, behind the mixing, join abduction delivering in the fermentor tank with the speed of the initial fermented liquid of 3.6ml/h/L (being 108ml/ hour).Keeping this low rate 2～3 hours, is the growth environment of sole carbon source so that yeast adapts to gradually with methyl alcohol.The DO value by the fluctuation become more greatly relatively stable after, continue to keep this low rate and added methyl alcohol 1 hour.

Strengthen to replenish the speed (7.2ml/h/L) of methyl alcohol then, and this speed kept speed is continued to increase to 11ml/h/L after 2 hours.Simultaneously, monitoring DO value and fermented liquid temperature and judge whether methyl alcohol is excessive.If the DO value ascensional range in 1 minute that stops behind the additional methyl alcohol illustrates that greater than 10% carbon source is limited, otherwise explanation methyl alcohol is excessive.Under the limited situation of carbon source, must accelerate to replenish the speed of methyl alcohol; Should transfer the speed of mending methyl alcohol slowly if methyl alcohol is excessive.

Behind the beginning abduction delivering,, detect OD every sampling in 6 hours ₆₀₀And wet cell weight, so as to analyzing saccharomycetic growth conditions.Leave and take the culture supernatant during this, be used for the protein quantification analysis.The continuous induction fermentation finished fermentation after 72 hours.Get the fermented liquid supernatant of each time point, carry out SDS-PAGE and analyze.

Experimental result:

1) fermented liquid pH value is to the influence of rhHSP70 fermentation yield:

Observed under extensive condition pH value to the influence of rhHSP70 productive rate, discovery almost detects less than rhHSP70 in the pH value is lower than 4.0 fermented liquid supernatant; Under pH value 4.1～5.0 conditions, the 18th hour expression amount of fermentation promptly reaches the climax.Continue to prolong fermentation time, the expression amount of target protein reduces gradually, and the foreign protein band increases.During pH value 4.8, at the 48th～54 hour of methanol induction, the rhHSP70 expression amount was the highest, can reach 1.2g/L.Continue to prolong fermentation time, the expression amount of rhHSP70 reduces on the contrary.

2) dissolved oxygen is to the influence of rhHSP70 fermentation expression output:

During the fermentation, the DO value (dissolved oxygen) of fermented liquid should remain between 25%～30%.The too high expression to rhHSP70 of dissolved oxygen can increase production cost on the contrary without any favorable influence.

3) medium component is to the influence of rhHSP70 fermentation expression output:

BSM substratum and FM21 substratum have been compared in this experiment, found that FM21 is more suitable for fermentative production rhHSP70 than BSM substratum.And the peptone that adds 0.5% (W/V) in the substratum can make the expression amount of rhHSP70 slightly increase, and particularly can make to express the 30th～36 hour after being advanced to methanol induction and expressing, peak, and foreign protein content reduces.

4) temperature is to the influence of rhHSP70 fermentation expression output:

The optimum growth temp of pichia spp is 30 ℃, and it is fatal to pichia spp that the temperature rising reaches 32 ℃.Sometimes reducing leavening temperature can increase the output of target protein.Report that in the methanol induction stage temperature is reduced to 25 ℃ from 30 ℃, can make the output of being cloned into the galactose oxidase in the pichia pastoris increase by 4 times.For rhHSP70, obtained good effect at 28 ℃～30 ℃ temperature bottom fermentation.

5) the methyl alcohol flow acceleration is to the influence of rhHSP70 fermentation expression:

Though pichia spp can be the sole carbon source growing multiplication with methyl alcohol, methyl alcohol is still a kind of toxic substance to Pichia yeast, so its concentration in substratum generally must not be higher than 2%.Originally discover that when stream added methyl alcohol and induces, although the expression amount of target protein matter and methanol consumption amount are proportionate within the specific limits, the flow acceleration of methyl alcohol was not that The faster the better.With regard to fermentation expression rhHSP70, be that the initial fermentating liquid volume of 8.5ml/h/L is advisable with the final flow acceleration of methyl alcohol, the methyl alcohol flow acceleration is crossed and lowly can be prolonged induction time, increases the pollution of foreign protein; The methyl alcohol flow acceleration is too high also can to cause expression amount to descend and the foreign protein increase.

(3) large scale purification system and purification process

Continuous flow centrifugation is handled and is drawn the rhHSP70 fermented liquid that content has been determined in the mark analysis through SDS-PAGE and Western.Get supernatant, the pH value is adjusted to 6.0, and add the 1mol/L phosphate buffered saline buffer (pH6.0) of 1/20 volume.Get the 500ml fermented supernatant fluid, slowly add (NH under the stirring at room respectively ₄) ₂SO ₄27.5g, 56.5g, 88g, 121g, 157g, 195g, 236g and 280.5g, make (NH ₄) ₂SO ₄Saturation ratio reach 10%, 20%, 30%, 40%, 50%, 60%, 70% and 80% respectively.With the fermented liquid supernatant of different ammonium sulfate saturation ratios centrifugal (10000rpm) 10 minutes, the precipitation of getting after centrifugal was carried out SDS-PAGE and Western engram analysis.Found that (the NH of 50% above saturation ratio ₄) ₂SO ₄Contain rhHSP70 in the precipitation.Getting the 500mlpH value again is adjusted to 6.0 fermented supernatant fluid room temperature and adds (NH respectively ₄) ₂SO ₄42g, 72g, 104g, 136g make (NH ₄) ₂SO ₄Saturation ratio be respectively 15%, 25%, 35%, 45%.Behind about 2 hours of the precipitation at room temperature, the same centrifugal.Use (the NH of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% saturation ratio respectively ₄) ₂SO ₄Solution flushing Phenyl Sepharose 6 Fast Flow hydrophobic chromatography resins (10 times of gel volumes) are with balanced gel.Add identical (NH then respectively ₄) ₂SO ₄The fermented supernatant fluid of saturation ratio carries out drainage column and separates, and eluate (is used 20mmolL ^-1Phosphate buffered saline buffer (pH 6.0) wash-out) carrying out SDS-PAGE analyzes.Determine the best (NH of rhHSP70 hydrophobic chromatography thus ₄) ₂SO ₄Saturation ratio is 40%.

With 10 times of column volume solution B balance SP Sepharose XL resin cation (R.C.)s, be used to be further purified the protein soln (pH4.0) of above-mentioned hydrophobic chromatography wash-out, and with above-mentioned elute soln (20mmol/L HAc-NaAc damping fluid, 0.5molL ^-1NaCl, pH 4.0) wash-out.RhHSP70 with ultra-filtration equipment after concentrated and purified, and with the 10mmol/L phosphate buffered saline buffer (wherein contain 138mmol/L NaCl, 2.7mmol/L KCl, 1mmol/LKCl) doubling dilution 3～5 times is with desalination.SDS-PAGE analyze to determine proteinic purity and the concentration behind the purifying, and with Bradford method accurate quantification.The result shows that behind hydrophobic chromatography and cation-exchange chromatography purifying, the proteinic yield of rhHSP70 is about 80%, and purity can reach more than 95%.

Sequence table

＜110〉the holy first Science and Technology Ltd. in Jilin

＜120〉use the methylotrophy yeast to produce rhHSP70's method

<140>

<141>

<160>6

<210>1

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as the structure cloning vector.

<400>1

GCATTCGAAA CGATGGCCAA AGCCGC

<210>2

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as the structure cloning vector.

<400>2

CCGGAATTCG CCCCTAATCT ACCTCC

<210>3

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as construction of expression vector.

<400>3

ATACTCGAGA AGAGAATGGC CAAAGCCGCG GCGATC

<210>4

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as construction of expression vector.

<400>4

GCTCGATCTA GACCTAATCT ACCTCCTCAA TGGT

<210>5

<211>9

＜212〉amino acid

＜213〉artificial sequence

<220>

＜223〉the proteic CTL epitope antigen of the HER2/neu of chemosynthesis peptide.

<400>4

GlnLeuPheGluAspAsnTyrAlaLeu

<210>6

<211>9

＜212〉amino acid

＜213〉artificial sequence

<220>

＜223〉the proteic CTL epitope antigen of the HER2/neu of chemosynthesis peptide.

<400>4

LysIlePheGlySerLeuAlaPheLeu

Claims (7)

1, a kind of method of using methylotrophy yeast production rhHSP70 polypeptide, this method comprises: cultivate the methylotrophy yeast in the substratum that with methyl alcohol is the carbon source and the energy, wherein said methylotrophy yeast is to transform with the DNA construct that comprises the following element that is operably connected: the derivable transcripting promoter of (1) methyl; (2) dna fragmentation of coding HUMAN HEAT SHOCK PROTEINS 70; (3) transcription terminator and (4) but selection marker, thereby obtain rhHSP70's polypeptide with the concentration of 250mg/L substratum at least.

2, according to the process of claim 1 wherein that said methylotrophy yeast is a pichia pastoris phaff, and said methyl inducible promoter and transcription terminator are all from pichia pastoris phaff AOX1 gene.

3, the methylotrophy yeast culture that is used for express recombinant HUMAN HEAT SHOCK PROTEINS 70, this yeast can rely on as the growth of the methyl alcohol of the carbon source and the energy, but and this yeast comprised what the DNA construct of dna fragmentation, transcription terminator and the selection marker of the derivable transcripting promoter of methyl, coding HUMAN HEAT SHOCK PROTEINS 70 transformed.

4, be used for DNA construct at pichia pastoris phaff express recombinant HUMAN HEAT SHOCK PROTEINS 70 polypeptide, this construct comprises the element that is operably connected: but dna fragmentation, transcription terminator and the selection marker of the derivable transcripting promoter of methyl, coding HUMAN HEAT SHOCK PROTEINS 70.

5, according to the construct of claim 4, wherein said methylotrophy yeast is a pichia pastoris phaff, and methyl inducible promoter and transcription terminator are all from pichia pastoris phaff AOX1 gene.

6, use the fermentation culture conditions of the optimization of pichia pastoris phaff scale operation rhHSP70 polypeptide, be characterised in that leavening temperature wherein maintains that 28 ℃～30 ℃, employed pH value are 4.1～5.0, the DO value (dissolved oxygen) of fermented liquid remain on 25%～30% and substratum in be added with the peptone of 0.5% (W/V).

7, the method for purifying heat shock protein 70, be characterised in that use successively hydrophobic chromatography and cation-exchange chromatography technical point from.

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