CN102220256A - Yeast expression system for expressing HAS-Vmip-II fusion protein and construction method thereof - Google Patents

Abstract

The invention discloses an expression system for expressing an HAS-Vmip-II fusion protein, and the expression system is produced by the transformation of plasmid pPICZaA-HSA-vMIP-II into Pichia pastoris. The invention also discloses a construction method of the expression system, comprising the following steps of: extracting recombinant plasmid from escherichia coli containing the plasmid pPICZaA-HSA-vMIP-II, linearizing the recombinant plasmid, transforming Pichia pastoris competent cells by electrotransformation, followed by resistance screening, performing the PCR identification to obtain positive clones, carrying out an inducible expression by the use of a positive clone, followed by the SDS-PAGE analysis of the expression product and Western-Blot identification. According to the invention, the half life of vMIP-II in blood plasma is greatly prolonged without the loss of vMIP-II functions. Therefore, the HAS-vMIP-II fusion protein will reduce the administration frequency and dosage, and exert a drug effect similar to that of vMIP-II.

Description

A kind of yeast expression system and construction process thereof of expressing HSA-vMIP-II fusion rotein

Technical field

The present invention relates to bioengineering field, be specifically related to a kind of yeast expression system and construction process thereof that can efficiently express HSA--vMIP-II fusion rotein.

Background technology

(Kaposi's sarcoma is a kind of blood vessel multiple tumor KS) to Kaposi's sarcoma, is common in the patient of immunodeficient disease.Since AIDS (Acquired Immune Deficiency Syndrome) was popular, oneself became one of African regional most common tumor KS.The high risk population that maybe may suffer from KS to KS carries out biopsy, found Kaposi's sarcoma virus (Kaposi's sarcoma-associate herpsvirus, KSHV) genomic dna, this virus claim human herpes virus 8 (HHV8) again, show that KSHV has participated in the pathology generating process of KS.

Kaposi's sarcoma virus be HIV the infected or transplant patient with immunosuppressor after the pathogenic agent of the normal Kaposi sarcoma that takes place.A gene surplus this virus has 80, the protein product virus macrophage inflammatory protein I(viral macrophage inflammatory protein-I that its K6 and K4 gene are encoded respectively one and be similar to person monocytic cell's inflammatory protein-1 α (MIP-1 α), vMIP-I) and virus macrophage inflammatory protein II (vMIP-II), vMIp-I is by open reading frame (ORF) the K6 genes encoding of 289 bp, the protein molecular size is 10.5 KDa, with the amino acid sequence homology of hMIP-1 β be 37.9%, the vMIP-II is the 10.5KDa albumen by ORF K4 genes encoding, with the amino acid sequence homology of hMIP-1a be 41.1%, the homology of vMIP-I and vMIP-II is 48%, both gene orders have 70% consistence, and another kind of vMIP-III is compared with both genes and had only 37% homology.

94 amino acid of the full genes encoding of vMIP-II, maturation protein is 74 amino acid, with people's MIP homology up to 41%, wherein 8 kinds of nonpolar amino acids account for 40%, have stronger hydrophobicity.VMIP-II albumen has higher homology with the mankind's CC class chemokine on aminoacid sequence.Proteic secondary structure of vMIP-II and folding mode and chemokine have points of resemblance, but also there are differences, even on higher concentration level, also be monomer structure, this just monomer structure makes it can take the receptors bind of any dimeric forms and cell surface, thereby has explained that the cross connection relation is arranged between it and the Chemokine Receptors.The receptor binding site height of vMIP-II N-terminal is unordered, in conservative halfcystine district positively charged amino-acid residue is arranged, and CC class chemokine has hydrophobicity or neutral amino acids residue usually, this zone forms CC class chemokine dimer and plays an important role, thereby explanation vMIP-II lacks the reason of dimeric structure.VMIP-II and Chemokine Receptors calmodulin binding domain CaM mainly are non-representative regions, and this zone comprises that mainly two positions participate in the combination and the activation of acceptor: one is nearly 8～ten amino acid residue of protein N terminal, and another is the N-loop ring.VMIP-II three-dimensional structure is to keep by 2 disulfide linkage and the hydrophobic core group that formed by β lamella, C end superhelix.VMIP-II albumen can wide spectrum ground in conjunction with CC and CXC two class chemokine family receptors, as kind of Chemokine Receptors surplus CCR1, CCR2, CCR3, CCR5, CCR8, CCR10, CXCR4, the CX3CR etc. 10.With the V1 peptide section that derives from vMIP-II N-terminal vMIP-II 26S Proteasome Structure and Function is studied, the result shows that Val-1, Arg-7, Lys-9 residue play an important role in conjunction with the CXCR4 acceptor to the vMIP-II.Therefore vMIP-II albumen not only can play a role by being combined in the immune inflammation pathologic reaction with Chemokine Receptors, but also can act on Chemokine Receptors on the immunocyte, function by excitement or antagonism acceptor are regulated immunocyte has more research using value at aspects such as HIV infection/acquired immune deficiency syndrome (AIDS) and graft-rejections.The vMIP-II can combine with the Chemokine Receptors of the accessory receptor that infects as HIV, and the gp120 of interference HIV can suppress the infection of HIV with combining of accessory receptor.Test confirms that for 89.6 strains (can utilize accessory receptor CCR3, CCR5, CXCR4 to infect widely) of HIV-1 virus, the vMIP-II can have identical with RANTES and SDF-1a or poor slightly blocking-up, and it utilizes the effect of CCR5 and CXCR4 acceptor.Studies confirm that further the vMIP-II is different from RANTES (CCL5) and MIP-1 α in conjunction with combining site and the molecular mechanism of CCR5, and has higher binding constant.Utilize NMR research to think that the vMIP-II plays a role under free state.Existing report, the vMIP-II of clonal expression has the activity of wild-type vMIP-II.

CCR8 also is one of accessory receptor of HIV infection, and especially in cerebral tissue and adenoid course of infection, the vMIP-II also can be by suppressing the cell that HIV infects these tissues in conjunction with CCR8.Accessory receptor role in the HIV infection mechanism is for anti-HIV treatment provides novel targets.In can mediating numerous Chemokine Receptors that external HIV-1 enters cell, CCR5 and CXCR4 have pharmacological significance.The antiviral therapy that with CCR5, CXCR4 is target all has effect in each stage of disease, the R5 strain is the strain of normal propagation, mainly come across the disease asymptomatic stage, therefore the antiviral therapy at CCR5 should carry out in early days, and the X4 strain is mainly in disease progression late period, so the symbol progression of disease is mainly effective in the terminal stage of a disease at the treatment of CXCR4.The major programme that can block CCR5 and CXCR4 that adopts has following three kinds at present: the one, and can not produce enough chemokine persons to those CD8+T cell and give more chemokine artificially; The 2nd, the medicine of auxilliary receptor binding site can be directly blocked in exploitation; The 3rd, disappearance CCR5 gene is used for stem cell transplantation.

Development of biology has greatly promoted the research and development of polypeptide/protein drug, by in February, 2004, drugs approved by FDA 64 kinds of recombinant polypeptide/protein medicines listings.Pharmacokinetic shows that polypeptide/protein medicaments mainly is eliminated in vivo by effects such as degraded, drainage and receptor-mediated endocytosis.Wherein molecular weight less than the polypeptide factor of 20KDa in metabolic process easily by glomerular filtration, polypeptide factor is partly degraded by proteolytic enzyme wherein again and is discharged from urine during by uriniferous tubules, thereby the transformation period is short.With the interferon-is example, and the interior transformation period of the body of intramuscular injection, generally about 2h-4h, even after six transformation period of heavy dose of administration (24h), its Plasma Concentration can't reach the purpose that suppresses virus replication.For the result of treatment that reaches, need frequent heavy dose of medication, secular frequent injection has not only increased patient's misery and medical expense, and easily causes a series of serious toxic side effect.The exploitation of long-acting polypeptides/protein medicaments has become the important directions of first-generation gene engineering polypeptide/protein drug being carried out secondary development.Prolong at present the protein drug transformation period mainly based on the molecular weight that increases protein drug, reduce glomerular filtration rate(GFR, reduce the immunogenicity of heterologous protein, thereby reduce clearance rate in its body, continue slowly to discharge to keep drug level, three aspects such as prolong drug action time are considered.Common technology has: preparation sustained release preparation, structure mutant, chemically modified and gene fusion etc.

Summary of the invention

The object of the present invention is to provide a kind of yeast expression system that can efficiently express HSA--vMIP-II fusion rotein.

The present invention is achieved through the following technical solutions above-mentioned purpose:

A kind of expression HSA--vMIP-II Expression of Fusion Protein system is converted into pichia pastoris phaff (pichia pastorid) by plasmid pPICZaA-HSA-vMIP-II and obtains.

Described plasmid pPICZaA-HSA-vMIP-II derives from the escherichia coli DH5a that contains plasmid pPICZaA-HSA-vMIP-II.

Described pichia pastoris phaff bacterial strain is preferably yeast X33.

The construction process of above-mentioned expression system, be from the intestinal bacteria that contain plasmid pPICZaA-HSA-vMIP-II, to extract recombinant plasmid, recombinant plasmid is carried out linearizing, utilize electric method for transformation to transform the pichia pastoris phaff bacterium competence cell, behind resistance screening, PCR identifies and obtains positive colony, utilize positive colony to carry out abduction delivering, expression product is carried out SDS-PAGE analyze, Western-Blot identifies, obtains expression system.

The construction process of above-mentioned plasmid is:

Above-mentioned plasmid pPIC ZaA-HSA-vMIP-II derives from the escherichia coli DH5a that contains plasmid pPIC ZaA-HSA-vMIP-II.

In the above-mentioned construction process, PCR is that the genome with recombination yeast is a template, two kinds of different primers increase respectively, first group of upstream primer sequence is shown in SEQ ID NO:1, the downstream primer sequence is shown in SEQ ID NO:2, second group of upstream primer sequence is shown in SEQ ID NO:3, and the downstream primer sequence is shown in SEQ ID NO:4.

Above-mentionedly recombinant plasmid is carried out the restriction enzyme that linearizing uses be PmeI.

The parametric optimization that described electricity transforms is voltage 1.5kV, electric capacity 25 μ F, resistance 200 Ω; The electric shock time is 4～10 seconds, and electric shock once.

The expression product HSA-vMIP-II fusion rotein of described expression system.

The application of HSA-vMIP-II fusion rotein in preparation inhibition peripheral blood mononuclear cell medicine.

Compared with prior art, beneficial effect of the present invention is as follows:

(1) uses pichia spp (pichia pastorid) expression system.Compare with prokaryotic expression system, yeast as unicellular eukaryote has following characteristics: at first, yeast is an eukaryote, can make some proteic glycosylation more stable, carry out posttranslational modification, the formation of for example correct disulfide linkage and the removal of signal peptide, N-combination and O-bonded are glycosylation modified; Secondly, yeast has the unicellular microorganism structure, has the quick growth of bacterial system and is easy to advantage such as genetically engineered operation; Compare with intestinal bacteria, yeast does not have intracellular toxin, does not have lysogenic virus, and yeast and human in close relations, and no pathogenicity is used to bread and alcoholic beverage industry for a long time.Yeast also is an ideal secreting, expressing system.Usually do not have proteinic adding in the yeast culture base, normal yeast secreted protein only is 0. 5% of a yeast cell total protein; It can also be secreted into the exogenous protein that produces in the substratum, is convenient to the product separation purifying, and this can reduce cost to a great extent.Need translate correct folding, the natural excretory protein in back for those, and interior instability of born of the same parents or virose foreign protein, be well suited in the expression system of this kind secretor type, expressing.Bichi yeast system is able to widespread use, reason is that this system has following advantage: the expression output that (1) is high, this system adopts the promotor of methanol induction, single copy alcohol oxidase gene can produce 30% alcohol oxidase of cell total soluble protein amount under this promoters driven, this promotor is introduced expression vector to drive expression of exogenous gene.As expecting, some foreign genes obtain the expression of high level in this system.Reach every liter of 12 gram as the segmental expression output of tetanus toxin C, this result makes genetic engineering technique produce the recombinant protein ability and rises to the gram level more from the milligram level.(2) high-density growth, in the seventies, pichia spp is used to prepare single cell protein, produces the fermentation technique of pichia spp thus, can reach every liter of culture 100 gram stem cells.(3) high-level secretory is in protein-free medium, and this expression vector has yeast saccharomyces cerevisiae a factor signal peptide, expression product can be secreted into outside the born of the same parents.Be used to express substratum and comprise some inorganic salt, trace element, vitamin H and carbon source, and do not have toxin and pyrogen, like this, the excretory expression product is purifying very easily.(4) the glycosylation form of pichia spp approaches Mammals more, and expression product N-bonded seminose generally has only (8～14) individual, and reaches (50～150) in the yeast saccharomyces cerevisiae.And these products do not resemble and comprise terminal a-1 in the yeast saccharomyces cerevisiae, the combination of 3-seminose, and this modification can cause immune genetic.(5) plasmid stability is good, and pichia spp itself and plasmid-free allow to express the assembly series connection and repeat, and be suitable for being incorporated into expression alien gene on the karyomit(e).Report shows, hepatitis B surface antigen still keep single celled productive rate, and its multi-copy integration kept for 150 generations also do not lose after shake bottle and forward fermentor tank to.(6) the secretory product permeability is good, and owing to excessive glycosylation, causing on each N-bonded oligonucleotide chain all has a lot of mannose residues, further has influence on permeability in the yeast saccharomyces cerevisiae.The foreign protein that general molecular weight surpasses 20KDa all can not be secreted in the nutrient solution, and then there is not this phenomenon in pichia spp, has the cover Secretory Pathway extremely similar with eukaryote in its body, is secreted into external from endoplasmic reticulum through golgi body, vesica.Turn to enzyme and human serum protein that molecular weight surpasses 50KDa all can be secreted into outside the born of the same parents, and expression amount is all in the level of 1g/L.Therefore pichia spp is current comparatively ideal heterologous gene expression system, is very suitable for the development and the exploitation of gene engineering product.

In addition, pichia spp methyl alcohol is unique carbon source, by O ₂Oxidation obtains formaldehyde and hydrogen peroxide, and katalaze enzyme is alcohol oxidase (alcohol oxidase is called for short AOX).Two kinds of alcohol oxidases are arranged, respectively called after AOX1 and AOX2 in the pichia spp body.Alcohol oxidase is to O ₂Affinity very weak, compensate the methyl alcohol metabolism so pichia spp need produce this a large amount of enzymes.When being sole carbon source with methyl alcohol, AOX1 plays a major role, and the alcohol oxidase of generation surpasses total soluble protein of 30%, the phenotype called after Mut of this kind bacterial strain ⁺(Methonal utlization slow).Up to the present existing both at home and abroad Duo Jia laboratory utilizes this system to carry out many successful expression, and the expressed proteins kind relates to enzyme, antigen, antibody, modulin, membranin, proteolytic enzyme and proteinase inhibitor etc.Comprising ox N,O-Diacetylmuramidase (0. 55g/L), human serum protein (3g/L), human epidermal growth factor (0. 4g/L) and M-EGF (0. 45g/L).

Have the cover Secretory Pathway extremely similar in the pichia spp body, be secreted into external through golgi body, vesica from endoplasmic reticulum with eukaryote.The factor that influences secreting, expressing is a lot, as the output of expression vector, host bacterium, gene dosage, Mut phenotype, expression product and quality etc.

The generation of pichia yeast expression system and widespread use are to the crucial influence of oneself generation of development of biotechnology industry.Some products such as cytokine class, vaccine and other biological reagent have entered clinical experimental stage.The Hepatitis B virus vaccine of Pichia anomala expression has also entered market.Further perfect along with aspects such as bacterial strain and carriers, pichia spp becomes main heterologous gene expression system surely, brings into play its advantage.

(2) the present invention uses the pPICZaA plasmid, its signal peptide is from the α-mating factor of yeast saccharomyces cerevisiae (α-factor), and pichia spp secreting, expressing plasmid as a new generation, it also has characteristics is that it has the Zeocin resistant maker gene, and the work of screening transformant to us brings very big facility.The pPICZaA plasmid is that its principal feature is as follows: ⑴ has potent adjustable promotor AOX1(alcohol oxidase, alcohol oxidase as the pichia spp secreting, expressing plasmid of a new generation); ⑵ have Zeocin resistance screening marker gene, and recombinant conversion can directly screen with Zeocin, and promptly in the transformant of growing on the YPDZ flat board, 100% all has the integration of foreign gene, has simplified recombinant conversion zymic screening process greatly.In operating process, Zeocin also can be used to screen the intestinal bacteria transformant that contains expression vector pPICZ α A, needn't use Amp in addition, and is economical and easy; ⑶ have the multiple clone site of inserting for foreign gene in expression vector A0X1 5 ' end promoter sequence downstream, and there is A0X1 3 ' end terminator sequence in the multiple clone site downstream; ⑷ signal peptide α-factor that secernment efficiency is strong.

(3) (human serum albumin HSA) carries out amalgamation and expression with the vMIP-II with human serum albumin in the present invention.HSA is the important component of blood plasma, it also is the carrier of many castle's intrinsic factors and external source medicine, be difficult for seeing through renal glomerulus under the normal circumstances, the protein that it is made up of 585 amino-acid residues, its molecular weight is about 66.5KDa, transformation period in blood plasma is longer, and it is wide and do not have zymetology and an immunologic competence (can resist the effect of organism endoenzyme) to reach in 2 all bodies distributed pole.Simultaneously, HSA is a stable inert protein, can improve stability of drug after protein drug and its fusion, prolongs the protein drug transformation period in vivo.Its expression level is higher, and can improve the expression level of target protein medicine after its fusion, thereby is a kind of ideal biological activity protein carrier.

(4) the HAS-vMIP-II fusion rotein that obtains of expression system of the present invention, the function that has not only kept the vMIP-II, also make obtain significant prolongation its transformation period in blood plasma simultaneously, can reduce administration frequency and dosage, but given play to the drug action similar to the vMIP-II.

Description of drawings

Fig. 1: extracting carrier linearizing electrophorogram, 1.DNA ladder marker, 2.pPICZaA-HSA-vMIP-II;

Fig. 2: the sub-genomic dna PCR of positive colony rear electrophoresis figure, 1 is HSA-vMIP (1.4kb), 2 is recombinant plasmid pPICZaA-HSA-vMIP-II, 3 is plasmid pPICZaA, 4 is DL2000 Marker, 5 is HSA-vMIP (240bp), and 6 is recombinant plasmid pPICZaA-HSA-vMIP-II, and 7 is plasmid pPICZaA;

Fig. 3: the abduction delivering supernatant concentrates rear electrophoresis figure through TCA, 1. expresses supernatant, 2.Marker;

Fig. 4: fusion rotein Western-Blot identifies figure, 1. negative control, 2. target protein band;

Fig. 5: (ammonium sulfate precipitation method concentrates) gradient of saltouing concentrates figure, 1.30% saturation ratio, and 2.40% saturation ratio, 3.50% saturation ratio, 4.60% saturation ratio, 5.70% saturation ratio, 6.80% saturation ratio, 7.Marker 8. expresses supernatant;

Fig. 6: the abduction delivering supernatant is through the record diagram of Ni affinity column purifying, and wherein the small peak on right side is the target protein peak on the ultraviolet absorption curve, and the higher peak of the absorbancy in left side is the absorption peak of other compositions in the supernatant;

Fig. 7: the purified rear electrophoresis figure of target protein, 1.Marker, 2. target protein behind the purifying;

Fig. 8: the Bradford method is surveyed the canonical plotting of protein content;

Fig. 9: the HSA-vMIP-II suppresses graphic representation to the chemotactic of PBMC.

Embodiment

Material that uses in following examples and reagent are: plasmid pPICZa A(is available from Invitrogen); The escherichia coli DH5a that contains plasmid pPICZaA-HSA-vMIP-II; Yeast X33.HMIP-1 β chemokine is available from Peprobiotech; RPMI1640 is available from U.S. GIBCO; HMIP-1 β is available from Britain Peprotech company; Lymphocyte separation medium is available from Tianjin TBD company; EB surrogate, restriction enzyme SacI is available from ancient cooking vessel state biology; Restriction enzyme EcoR I, restriction enzyme XbaI is available from TAKARA BIO INC.

Embodiment 1 HAS-vMIP-II yeast expression system makes up

1. alkaline lysis extracts the plasmid that contains goal gene from intestinal bacteria

(1) cultivate the escherichia coli DH5a that recombinant plasmid pPICZ aA-HSA-vMIP-II transforms, the results of thalline: 1.5ml bacterium liquid is poured in the Eppendorf tube, and centrifugal 30 seconds of 12000g inhales and removes nutrient solution.Supernatant will be drawn totally, otherwise can influence the quality of plasmid.In case of necessity can be centrifugal 2 times.

(2) bacterial sediment is resuspended in the ice-cold solution I of 100 μ l (the solution I preparation: 50 mM glucose/25 mM Tris-HCl/10 mM EDTA, pH 8.0; Behind the autoclaving in 4 ℃ of storages), add 1/50 volume RNase A stock solution, thermal agitation makes it to disperse fully.Glucose can be replaced by NaCl, is beneficial to store; Extraction is greater than the plasmid of 15kb, bacterium should be suspended from etc. to ooze in the sucrose solution, and handle with N,O-Diacetylmuramidase; Bacterial precipitation is disperseed fully, and it is abundant to be beneficial to the alkali lye effect; Can in centrifuge tube, add toothpick or rifle head during concussion, raise the efficiency; During preparation RNase A stock solution, boiling time is unsuitable long or too short; Solution I was prepared 1 time in per 2 months again.

(3) add 200 μ l solution II (solution II preparation: 0.2M NaOH/1% SDS, fresh preparation), slightly put upside down mixing 5 times, place 5min on ice.If cellular lysate gets fully, the solution very thickness that can become then; Do not want concuss, otherwise can sneak into genomic dna; Again prepared 1 time in per 2 months.

(4) add the ice-cold solution III of 150 μ l (solution III preparation: 5mol/L potassium acetate 60ml, glacial acetic acid 11.5ml, water 28.5ml), slightly shake mixing 10 times.The solution that is made into is 3mol/L to potassium, is 5mol/L to acetate moiety, about pH4.8; The bulk precipitation of trying not big, it is centrifugal to be beneficial to down the step; Again prepared 1 time in per 1 month, and packing is stored in the narrow-mouth bottle.

(5) centrifugal 5 minutes kinds of 12000g are transferred to supernatant in another centrifuge tube.

(6) in supernatant, add isopyknic chloroform (precooling), put upside down mixing after, room temperature leaves standstill 5min.Centrifugal 5 ~ the 10min of 12000g carefully draws supernatant liquor.

(7) add 2 times of volume dehydrated alcohols (precooling) at supernatant, the centrifugal 5 ~ 10min of 12000g removes supernatant.This moment, precipitation was plasmid DNA.

(8) add 1ml 70% ethanol (precooling) washing DNA precipitation, centrifugal, the careful suction removed supernatant.For washing fully, can wash 2 times.

(9) in air, leave standstill 3 ~ 10min, after waiting to precipitate drying, be dissolved among the 50 μ l TE.

2. the preparation of pichia spp bacterium competence cell

(1) at first carry out the flat board cultivation to protecting the yeast X33 that plants, wait to grow single bacterium colony, picking yeast list bacterium colony is seeded in the 50ml triangular flask that contains 10ml YPD liquid nutrient medium 30 ℃, 200r/min overnight incubation;

(2) culture of getting 100-500 μ l is seeded to the triangle that contains the 100ml fresh culture and shakes in the bottle, and 28 ~ 30 ℃, 200r/min overnight incubation reach 1.3 ~ 1.5 to OD600;

(3) with cell culture in 4 ℃, the centrifugal 5min of 1500g, with the sterilized water of the ice precooling of 100ml that bacterial sediment is resuspended;

(4) 3 centrifugal set by step, with the sterilized water of the ice precooling of 100ml that bacterial sediment is resuspended;

(5) 3 centrifugal set by step, with the Sorbitol Solution USP of the 1mol of the ice precooling of 20ml that bacterial sediment is resuspended;

(6) 3 centrifugal set by step, with the Sorbitol Solution USP of the 1mol of the ice precooling of 0.4ml that bacterial sediment is resuspended, its final volume is about 0.6ml, and it is packed as the packing of 150 μ l portions.

3. linearizing DNA electricity transforms

At first cultivate the intestinal bacteria that recombinant plasmid pPICZ aA-HSA-vMIP-II transforms, therefrom extract recombinant plasmid (method of extraction plasmid as previously mentioned), and utilize restriction enzyme PmeI is carried out enzyme and is cut 1h and make its linearizing.Enzyme is cut system: pPICZaA-HSA-vMIP-II (200ng/ μ l) 10 μ l, restriction enzyme PmeI (10 unit/ μ l) 2 μ l, L Buffer (10 *) 4 μ l, dd H ₂O 14 μ l.

Linearizing DNA is dissolved in the TE solution, thalline mixing with above-mentioned steps 6 gained, the electricity that goes to the precooling of 0.2cm ice transforms in the cup, and electricity transforms a cup ice bath 5min, the data that provides according to electric conversion instrument, determine that suitable electricity transforms parameter, be preferably: voltage 1.5kV, electric capacity 25 μ F, resistance 200 Ω, the electric shock time is 4～10msec, and electric shock once.After electric shock finished, the Sorbitol Solution USP that adds the precooling of 1ml ice went to the thalline mixing in the EP pipe of 1.5ml.With the expressive host X33 competent cell after the above-mentioned electric shock as for 1h under 30 ℃ of environment, then suspension is coated ((YPD plate culture medium preparation: peptone 20g on the YPD flat board that contains Zeocin, yeast extract 10g, glucose 20g, agar powder 20g, adding distil water is to 1000ml, 121 ℃ of autoclaving 20min).Place 30 ℃ to cultivate 2～3 days flat board, occur until single bacterium colony.

4. the screening of positive colony

Expressive host X33 competent cell after electricity changes is cultivated on the YPD flat board through the Zeocin resistance screening and is grown about 10 clones, and these clones are exactly pichia pastoris phaff X33 transformant.Picking contains in the YPD liquid nutrient medium of Zeocin in 3ml at the single colony inoculation that grows on the YPDZ, 30 ℃, 180～200rpm/min shaking table cultivation (YPDZ substratum preparation: YPD plate culture medium adding Zeocin 100ug/ml) in 2～3 days.

5. yeast genes group PCR identifies positive colony

(1) extracts pastoris genomic dna

Contain Zeocin with becoming muddy YPD() liquid medium, the centrifugal collection thalline of room temperature is in 1.5mL Ep pipe, with the SCE solution suspension thalline of using 0.5mL after the aqua sterilisa washing once again, and add helicase and the 10 μ l mercaptoethanols that 1 μ l concentration is 200mg/mL, the centrifugal supernatant that goes behind 37 ℃ of 200r/min oscillation treatment 1h.Sedimentary thalline suspends again with 0.5mLTris/EDTA (containing 50mmol/L Tris-HCL, 20mmol/L EDTA) and adds 50 μ l 10%SDS, handles 20min for 65 ℃.The KAc that adds 200 μ l 5mol/L is put 30-60min on ice behind the inversion mixing.Room temperature is centrifugal and supernatant is transferred in the new centrifuge tube, adds the dehydrated alcohol of about 1mL precooling, and centrifugal 5 min of room temperature go to be inverted behind the supernatant and volatilize the DNA precipitation.The DNA of gained dissolves again with the TE of 30 μ l and adds an amount of RNase digestion.37 ℃ of insulation 1h degradation of rna.

(2) 0.8% agarose gel electrophoresis

The preparation of sepharose: take by weighing agarose 0.16g, add 10 times of TAE electrophoretic buffer 20ml, heating for dissolving on electric furnace is mixed with 0.8% sepharose, and cool slightly back adds EB surrogate 1 μ l.Pour sepharose solution into the electrophoresis template, insert comb.After the condensation comb is transferred to, running gel is put into electrophoresis chamber.Extracting sample solution 20 μ l are added to solution in the sample well, add standard molecular weight DNA simultaneously in another sample well.The general dna sample preferably is controlled between 0.5～1.0 μ g.Add the TAE damping fluid, voltage 100V is kept in energising, and electrophoresis stops electrophoresis during to the glue middle and lower part.Electrophoresis finishes, and observes down in ultraviolet lamp, and the position that DNA exists presents fluorescence.

5. PCR identifies transformant

Genome with recombination yeast is a template, and two kinds of different primers increase respectively.

First group of primer: upstream primer sequence FP is shown in SEQ ID NO:1, and downstream primer sequence RP predicts amplification segment 1.4kb shown in SEQ ID NO:2.

Second group of primer: upstream primer sequence FP is shown in SEQ ID NO:3, and downstream primer sequence RP predicts amplification segment 240bp shown in SEQ ID NO:4.

The PCR reaction system: in ice bath, according to the form below 1 order adds an aseptic 0.5ml centrifuge tube with each composition.

Table 1

10×PCRbuffer	2 μl
		dNTP mix （2mM）	2 μl
Primers F P (10pM)	0.5 μl
		Primer RP (10pM)	0.5 μl
Taq enzyme (2U/ μ l)	0.5 μl
		Dna profiling	0.5 μl
Add ddH 2O extremely	20 μl

The PCR condition: initial 94 ℃, pre-sex change 5min, 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 1.5min, and after totally 31 circulations, 72 ℃ are extended 5min again.Obtain the PCR product by 0.8% gel electrophoresis analysis, it the results are shown in Figure 2.As can be seen from Figure 2, amplified the band that conforms to expection size (1.4kb) at first swimming lane, the 5th swimming lane has amplified the band that conforms to expection size (240bp).The result prove absolutely goal gene HSA-vMIP-II homologous recombination be incorporated on the saccharomycetic genomic dna of engineering bacteria.

6. the methanol induction of the positive yeast strain of pPICZaA-HSA-vMIP-II is expressed

The preparation of BMGY substratum: peptone 20g, yeast extract 10g, yeast nitrogen (not sulfur acid ammonia) 3.4g, sulfate of ammoniac 10 g, D-vitamin H 0.04g, glycerine 10ml, phosphoric acid buffer pH 6.0 100ml of 1M, distilled water adds to 1000ml.Changing 10ml/L glycerine into 5ml/L methyl alcohol when preparation BMMY gets.

The positive single colony inoculation of picking yeast in the 5m1YPD substratum, 30 ℃ of 180rpm shaken overnight.By 1% volume YPD bacterium liquid is inoculated in 40m1 BMGY substratum, 30 ℃ of 180rpm shake-flask culture spend the night.Centrifugal extraction bacterial sediment is transferred among the 200m1BMMY, and 30 ℃ of 180rpm continued shaking culture 3 days, and after abduction delivering is initial, added 100% methyl alcohol every 24h, and volume is 1% of a substratum cumulative volume.Yeast fermentation broth is through 9000rpm, and the centrifugal removal yeast of 10min carries out the SDS-PAGE electrophoretic analysis to expressing supernatant.

Electrophoresis result is seen Fig. 3.The protein band that conforms to theoretical value 79.4KDa size has appearred between the first swimming lane 66KDa and 97KDa.The result shows that tentatively positive yeast has given expression to HSA-vMIP-II fusion rotein behind methanol induction thus.

7. Western-Blot identifies the methanol induction expressing protein

(1) preparation of SDS-polyacrylamide gel

Install electrophoresis chamber and sheet glass by BIO-RAD company protein electrophorese operation instructions.Dispose 6 mL, 10% SDS-PAGE and concentrate glue 12%SDS-PAGE separation gel, add to mounted joining in the glue sheet glass rapidly, reach level altitude after, add water to the edge of glass, room temperature is vertically placed.After glue to be separated is gathered fully, with the water on filter paper exhaustion upper strata.Dispose spacer gel (concentrating glue), rapid mixing behind the adding TEMED pouring into concentrated glue on the polymeric separation gel, inserts clean comb rapidly, avoids entering bubble, and room temperature is vertically placed.

(2) SDS-PAGE electrophoresis

After treating that concentrated glue solidifies fully, carefully take out comb, and remove aerial oddments, and be fixed on the electrophoresis chamber, respectively add 1 * Tris-glycine electrophoretic buffer up and down in the groove.By predetermined order application of sample, the application of sample amount guarantees that every porin content is identical, connects power supply, and carries out electrophoresis by the constant voltage mode.Deposition condition: sample is when concentrating glue, and voltage is the 60V constant voltage; When sample entered separation gel, voltage changed the 120V constant voltage into, and near separation gel when bottom, powered-down stops electrophoresis until the sample dyestuff.Unload the lower-glass clamping plate from electrophoresis chamber, carefully take off the glass plate, downcut one jiao of gel, mark gel direction is carried out immunoblotting and is detected.

(3) western blotting

Wear gloves and cut 6 filter paper and 1 pvdf membrane, size is in full accord with gel, and places transfering buffering liquid to soak 30min.The order of 3 metafiltration paper, pvdf membrane, gel, 3 metafiltration paper is sequenced, make it complete matching, accurately overlapping, interlayer does not stay bubble, and makes marks at one jiao with pencil.The fixed order of pressing black plate holder → sponge pad → 3 metafiltration paper → gel → pvdf membranes → 3 metafiltration paper → sponge pad → transparent clips is good, be positioned in the transfer groove by red-black direction, in groove, fill it up with transfering buffering liquid (2.9g glycine, 5.8gTris alkali, 0.37gSDS, add 200ml methyl alcohol, constant volume 1000ml), connect power supply, 4 ℃ of 300mA, 99V shifts 90min, end power supply then, take out pvdf membrane, the band in the clip polyacrylamide gel electrophoresis between 66KDa and the 97KDa is hybridized, and gel gone in the Coomassie brilliant blue dyestuff dye, detect albumen and whether shift fully.Transfer printing finishes pvdf membrane is washed film 3 times with PBS, each 5min.Powered-down, transfer film is taken out in stripping assembly.The sealing damping fluid that adds 100mL is placed 16h for 4 ℃.The mice Anti-His of 5 μ l is added in the sealing damping fluid of 20ml.The damping fluid adding for preparing is contained in the little lunch box of transfer film incubated at room 1h, vibration gently.Take out transfer film with tweezers, put into the cleaning buffer solution incubated at room of 20-30ml, gently vibration.Repeat 2 times.HRP-Goat-Anti-mice IgG of 5 μ l is added in the sealing damping fluid of 20mL.The damping fluid adding for preparing is contained in the little lunch box of transfer film incubated at room 1h, vibration gently.Take out transfer film with tweezers, put into the cleaning buffer solution incubated at room of 20-30ml, gently vibration.Repeat 2 times.The film of handling well is put into freshly prepared colour developing liquid, and the 5-10min colour developing is waited in vibration gently.Observe the colour developing result.

The result as shown in Figure 4.Find protein band can with mouse-anti His antiserum(antisera) generation immune response.The result verification of immunoblotting the existence of fusion rotein, prove that this band is a target protein HSA-vMIP-II fusion rotein band.HAS-vMIP-II yeast expression system successfully constructs.

The purifying of II fusion rotein

Yeast fermentation broth is through 3500rpm, the centrifugal removal yeast of 20min thalline, to express supernatant with phosphoric acid buffer dialysis 24h(phosphoric acid buffer 20mmol/L pH7.0) change liquid three times, filter the solution of dialysing with 0.45 μ m mocromembrane then, the membrane filtration of then again filtrate being used again 0.22 μ m once removes macromolecular substance and lipid.

1. (ammonium sulfate precipitation method concentrates) gradient of saltouing concentrates saturation ratio and explores

The beaker that will fill protein soln (expression supernatant) places on the magnetic stirring apparatus, adds the 16.4g/100ml ammonium sulfate solids under 0 ℃ of condition while stirring slowly in solution, forms 30% saturation ratio.This process is finished at 5-10min, and continues to stir 10-30min.The centrifugal 10min of 10000rpm, collecting precipitation is used for the SDS-PAGE electrophoresis.

In protein solution, add 5.6g/100ml, 5.8g/100ml, 6.0g/100ml, 6.2g/100ml, 6.5g/100ml ammonium sulfate solids respectively by above-mentioned steps, form 40%, 50%, 60%, 70%, 80% saturation ratio solution.Be used for electrophoresis behind the centrifugal collecting precipitation.Electrophoresis result is seen Fig. 5, and the target protein concentrated effect is best when showing 60% saturation ratio.

2. the purifying of abduction delivering product

(1) saltouts (ammonium sulfate precipitation method)

Place on the magnetic stirring apparatus 4 ℃ to stir in the beaker that fills protein soln, add the saturation ratio of 29.1g/100ml ammonium sulfate solids while stirring slowly to solution 50%.Finish at 5-10min, and continue to stir 10-30min.The centrifugal 10min of 10000g gets supernatant, adds the saturation ratio of 6.0g/100ml ammonium sulfate solids to solution 60%.Finish at 5-10min, and continue to stir 10-30min.The centrifugal 10min of 10000g removes supernatant, and precipitation is used for dialysis.

(2) dialysis

With 10m mol/L NaHCO ₃, 1m mol/L EDTA solution boils dialysis tubing 30min.After boiling, the distilled water thorough washing.With transfer pipet or funnel protein is moved into dialysis tubing, dialysis tubing is answered reserving space in order to avoid burst.Put 10 times of protein solns with the upper volume damping fluid, magnetic stirring apparatus slowly stirs.Change dialysis buffer liquid after a few hours.Behind the exchange buffering liquid 4 times, the protein soln in the dialysis tubing is used for purifying.

(3) purifying of target protein

Abduction delivering supernatant application Ni affinity column and dialysis method are carried out purifying, and the used level pad of Ni affinity chromatography column purification is 50mmol NaH ₂PO ₄, 300mmol NaCl, 10mmol imidazoles pH 8.0; Lavation buffer solution is 50mmol NaH ₂PO ₄, 300mmol NaCl, the 20mmol imidazoles, pH 8.0; Elution buffer is 50mmol NaH ₂PO ₄, 300mmol NaCl, the 250mmol imidazoles, pH 8.0, through wash-out, collect the eluted protein peak, the protein solution that obtains is carried out SDS-PAGE determine target protein, dialyse with deionized water then, and dialysis 24h changes liquid 4 times.

After saltouing, dialysing, abduction delivering supernatant application Ni affinity column and dialysis method are carried out purifying, collect second peak of purifying record diagram (Fig. 6), the solution that obtains is carried out the SDS-PAGE electrophoresis determine.Electrophoresis result is seen Fig. 7.The protein band that conforms to theoretical value 79.4KDa size has appearred between the second swimming lane 66KDa and the 97KDa in the electrophoresis result.Electrophoresis result shows and obtains purer HSA-vMIP-II fusion rotein.

(4) the Bradford colorimetry is surveyed protein content

Coomassie brilliant blue method (Bradford method), according to protein and the design of dyestuff principle of combining, Coomassie brilliant blue G-250 dyestuff, in acidic solution with protein bound, make the position (lmax) of the maximum absorption band of dyestuff, become 595nm by 465nm, the color of solution also becomes blueness by brownish black.The absorbance A that under 595nm, measures ₅₉₅, be directly proportional with protein concn.

Method: the BSA solution that disposes 0.01mg/ml, 0.02mg/ml, 0.05mg/ml, 0.1mg/ml respectively, get 6 test tubes and add Bradford working fluid 1mL respectively, in No. 1 pipe, add the NaCl solution 0.1mL of 0.15mmol/L as blank, each 0.1ml of BSA solution that in 2～No. 5 pipes, adds different concns respectively, add sample solution 0.1ml in No. 6 pipes, with the wavelength measurement absorbancy of 595nm, do typical curve.Calculate protein concentration in the sample according to typical curve then, see Fig. 8.Calculate from the typical curve of Fig. 8, the content of purified back target protein is: 24.5ug/ml.

The biological activity assay of embodiment 3 purifying proteins (chemotactic suppresses experiment)

The preparation of PBMC: get about 4ml lymphocyte separation medium in a clean centrifuge tube, with 4ml fresh and healthy people's peripheral blood with isopyknic RPMI1640 liquid dilution after, careful is added on the lymphocyte separation medium, room temperature 1300 rpm, 12 min; Solution will be divided into 3 layers, and the cotton-shaped peripheral blood mononuclear cell that is of middle one deck is got buffy coat in the clean centrifuge tube of another, add the washing of 4～5ml RPMI1640 liquid, 1200 rpm, 8 min; Outwell supernatant, use 4ml RPMI1640 liquid (containing 10% calf serum) resuspended again, calculate cell density in cell counting count board.

Calculate the amount of diluent by the cell density of adjusting, make cell density transfer to 1 * 10 ⁶Individual/ml, and be resuspended in RPMI1640 nutrient solution (containing 10% calf serum).

Above-mentioned cell suspension is handled 30min with the HSA-vMIP-II (0.5ng/ml, 5ng/ml, 50ng/ml, 500ng/ml) of different concns under 4 ℃ of conditions, the cell of handling without HSA-vMIP-II is as blank, as the positive control hole, other is provided with a hMIP negative control hole of handling without hMIP-1 β with the cultivation datum hole of the hMIP-1 β that contains 10ng/ml.

Lower floor's cell of 24 hole chemotactic cells is added 250 μ l chemotactic damping fluids (containing 10ng/ml hMIP-1 β, 0.5%BSA, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates and RPMI-1640 nutrient solution) attention: need carefully allow film contact with following indoor chemotactic damping fluid, bubble can not be arranged, can not allow the liquid phase in each hole mix.

The upper strata cell adds 1 * 10 of 100 μ l ⁶The cell suspension of individual/ml density, wherein No. 1 is to handle without HSA-vMIP-II, same chamber does not at present add the blank group of chemokine, No. 2 is to handle without HSA-vMIP-II, but positive controls with the chemokine chemotactic, be respectively the HSA-vMIP-II treatment group of 0.5ng/ml, 5ng/ml, 50ng/ml, 500ng/ml for 3～No. 6, all experimental ports all repeat to do 3 parallel holes, and the chemotactic plate is at 37 ℃, 5%CO ₂Hatch 2h in the incubator, allow cell from face down chemotactic motion of film.

The concentration (ng/ml) of HSA-vMIP-II/hMIP
	0/0 0/10 0.5/10 5/10 50/10 500/10

The careful cell that takes out will descend the ventricular cell mixing.

Light microscopic counting: with hemocyte technology plate counting cells density

Number of cells=cell density * volume 250 μ l

Chemotactic inhibiting rate=(1-experimental group chemotactic arrives down, and the cell count/control group chemotactic of chamber arrives the cell count of chamber down) * 100%

Data processing: adopt SPSS10.0 software to analyze.

Table 3:vMIP-II suppresses experiment to the chemotactic of PBMC

* compare P＜0.05 with positive control (HSA-vMIP-II/hMIP-1 β, 0/10) group

The result calculates: go out EC50 by the SPSS10.0 computed in software: the A value that suppresses probability 50% place under the effect of HSA-vMIP-II is 0.67734, and its antilogarithm is 4.76, and promptly the effective concentration of EC50 is 4.76ng/ml.

The average chemotactic inhibiting rate of four concentration treatment group is respectively: 37.52%, 48.52%, 59.64%, 70.03%.

Discuss

Pichia spp has been widely used since being developed to the exogenous protein expression system beginning of the eighties always, and some recombinant proteins have become formal product and entered market.So far existing hundreds of kind foreign protein is in this system's successful expression, and the high expression level amount of having reported is respectively 22g/L (in the born of the same parents) and 14.8g/L (secretion).Find that pichia yeast expression system can be also to find in the born of the same parents and secretion mode expression alien gene encoded protein the time, secreting, expressing is a kind of ideal protein production mode, the foreign protein of expressing in the secretion mode can account for 80% of culture supernatant total protein, and this will help being further purified of target protein.Can be divided into two classes for the signal peptide that pichia spp is selected: i.e. the signal peptide of foreign protein self and derive from the zymic signal peptide.

Human serum albumin itself just belongs to secretory protein, with the signal peptide sequence of self can be in pichia spp successful expression, and very high expression level is arranged.Clinical practice shows that prolonging the protein drug transformation period is to increase the most effectively means of its interior curative effect.The exploitation of long-acting protein drug has become an important directions of first-generation gene engineering product being carried out secondary development.

Different protein drugs have different internal metabolism mechanism, and the strategy of therefore developing depot drug product is also inequality.Less than for the protein drug of 70KDa, the filtration of renal glomerulus is a total mechanism for molecular weight, and increasing its molecular weight then is a general strategy, and the method for the molecular weight of increase protein drug commonly used has protein-crosslinking, PEG to modify and albumen merges.Compare with other method, the human serum albumin integration technology has the following advantages: (1) HSA is connected by peptide bond through the protein translation system in born of the same parents with target protein, does not need extra extracorporeal treatment; (2) expression level of HSA is higher, with the expression level that can improve target protein after its fusion, thus the output of raising target protein; (3) HSA is a stable 'inertia' albumen, with the stability that can improve target protein after its fusion; (4) the HSA fusion rotein has the longer transformation period of protein drug of modifying than PEG.The clinical effectiveness that HGS (Human Genome Sciences) company announces shows that the transformation period of HSA-IFN fusion rotein Albuferon reaches 145h, and the transformation period of the Interferon, rabbit that PEG modifies has only 40～80h.HGS company has albumin fusion growth hormone (Albutropin) at present, albumin merges granulocyte somatomedin (Albugranin) and albumin fused interferon products such as (Albuferon) has successively entered I/II phase clinical study, and has demonstrated transformation period and security preferably and interior curative effect in the long body.

This research and utilization alkaline lysis is extracting secretor type recombinant expression vector pPICZaA-HSA--vMIP-II from intestinal bacteria, by electric transformed yeast bacterium X33, resistance screening, PCR identifies, we have obtained stable transformant, and utilize this project bacterium to carry out abduction delivering in a small amount.By a small amount of abduction delivering, purifying has obtained purer target protein.

We must make the recombinant expression plasmid DNA linearizing of extraction before recombinant expression plasmid being carried out the electricity conversion, because have only linearizing DNA could successfully be integrated in the yeast genes group, simultaneously, the restriction enzyme selected to linearizing also has strict demand: at first, whole recombinant expression plasmid preferably only contains this enzyme restriction enzyme site; Secondly, this restriction enzyme site can not occur in goal gene.Therefore, we analyze all restriction enzyme sites of the gene of constructed recombinant expression plasmid according to utilizing computer software, and we have selected for use PmeI that recombinant expression plasmid has been carried out linearization for enzyme restriction.

Expression vector is by after the linearization for enzyme restriction, can produce stable recombinant conversion with P.Pastoris genomic homologous sequence generation homologous recombination, at present the linearizing DNA of carrier is transferred to the host bacterium and mainly contain 4 kinds of methods: be i.e. electroporation, protoplasm body, PEG method and lithium salts method.Wherein the electroporation transformation efficiency is the highest and can obtain containing the transformant of multiple copied foreign gene sometimes, it mainly utilizes the effect of fast-changing high field and makes cell absorb exogenous dna fragment, because electroporation is easy, quick, efficient, so be the method for present optimal conversion P.Pastoris.The electricity transformation efficiency is subjected to all multifactor influences, mainly contains the following aspects: the cell that 1. is in logarithmic phase is very responsive to electric shock, is to be suitable for most carrying out the growth phase that electricity transforms, so the pichia methanolica bacterium is cultivated mid-log phase and carries out electricity and be converted into suitable; 2. foreign DNA concentration can only could obtain maximum transformation efficiency within the specific limits, and the foreign DNA excessive concentration is crossed the low transformation efficiency that all influences, and this may be the reason that an optimum matching is arranged between the amount of foreign DNA and the recipient bacterium; 3. working as 2 centimetres of conversions of fixing electricity conversion cup width and take the lead in raising along with the increasing of voltage, descend on the contrary but work as the too high transformation efficiency of voltage, may be the mass mortality that too high voltage has caused competent cell.But because the experiment condition restriction, the electric conversion instrument parameter that this experiment electricity uses when changeing is non-adjustable, can only carry out electricity according to the parameter of having set and change.Because parameter is non-adjustable, makes electric transfer efficient not high, it is a lot of to expend time in.Based on taking all factors into consideration of above factor, the present invention's suggestion is: approximate between 1.0 to 1.3 to OD600 at thalli growth, electricity transforms parameter and is: voltage 1.5kV, electric capacity 25 μ F, resistance 200 Ω.The electric shock time is 4～10 seconds, and electric shock once.

This experiment utilizes the anti-Zeocin characteristic of recombinant yeast pichia pastoris to carry out positive-selecting.By add Zeocin preparation YPDZ solid medium in YPD, if bacterium colony normal growth on the YPDZ flat board, then positive clone is sub.Because the methanol yeast expression vector does not contain the yeast replication origin, is incorporated in the host cell chromosome so have only by homologous recombination, foreign gene can stable existence.During reorganization with restriction enzyme with the carrier linearizing, make it to be incorporated into AOXI gene location or His4 site in the yeast genes group.Foreign gene after the integration is with the zymic growth existence of can stably going down to posterity.Following 2 kinds of integration modes are arranged usually: a kind of situation is that single cross is changed, and promptly inserts, and foreign gene is inserted on the yeast chromosomal by reorganization, and the AOXI gene still keeps, and the transformant phenotype that obtains like this is Mut ⁺Another kind of situation is double exchange, promptly replaces, and foreign gene has been replaced the AOXl gene on the karyomit(e) by reorganization, causes the disappearance of AOXI gene, and the transformant phenotype that obtains like this is Mut ^sHomologous recombination produces the integration of multiple copied sometimes, generally accounts for the 1%-10% of transformant.But no matter be to adopt single cross to change or double exchange, the transformant that obtains a wherein part is a false positive.So only, be necessary to use PCR method that a small amount of transformant is carried out multiple sieve, therefore need carry out PCR and identify to positive colony by being far from being enough in the substratum screening that contains Zeocin.This experiment takes the extracting pichia spp to come positive colony is carried out multiple sieve because of the method that group DNA carries out PCR.

Utilize affinity column to carry out protein purification, it is concentrated to saltout earlier before the purifying, and the dialysis desalination can improve purity.According to the actually operating experience, the best salting point of different batches expression supernatant is also inequality, therefore saltouts to concentrate and need grope best salting point before different batches is expressed supernatant liquor.

This experiment is not optimized expression condition, but there are some researches show, the expression of exogenous gene amount can reach more than 10 times of test tube in the suitable fermentation condition fermentor tank if provide, but according to the optimization step in the real attenuation work, the relevant report of method and documents and materials and experience, we can inquire into from following several respects: 1. induction time is to the influence of Pichia anomala expression HSA-vMIP-II, 2. medium pH is to the influence of Pichia anomala expression HSA-vMIP-II, 3. methanol concentration is to the influence of Pichia anomala expression HSA-vMIP-II, 4. inoculum size is to the influence of Pichia anomala expression HSA-vMIP-II, 5. influence of leavening temperature Pichia anomala expression HSA-vMIP-II etc.

Suppress in the experiment at chemotactic, find through the pretreated PBMC cell of HSA--vMIP-II be with respect to untreated fish group dose-dependently reduction to the chemotactic migration of hMIP-1 β.Proved and utilized pichia yeast expression system to express the reorganization HSA--vMIP-II that obtains that more obvious to the chemotactic inhibition effect of PBMC, protein-active is comparatively remarkable.

SEQUENCE?LISTING

 

＜110〉Ji'nan University

 

＜120〉a kind of yeast expression system and construction process thereof of expressing HSA-vMIP-II fusion rotein

 

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Claims (9)

1. express HSA--vMIP-II Expression of Fusion Protein system for one kind, be converted into pichia pastoris phaff by plasmid pPICZaA-HSA-vMIP-II and obtain.

2. the described expression system of claim 1 is characterized in that described plasmid pPICZaA-HSA-vMIP-II derives from the escherichia coli DH5a that contains plasmid pPICZaA-HSA-vMIP-II.

3. the described expression system of claim 1 is characterized in that described pichia pastoris phaff bacterial strain is yeast X33.

4. the construction process of the described expression system of claim 1, be from the intestinal bacteria that contain plasmid pPICZaA-HSA-vMIP-II, to extract recombinant plasmid, recombinant plasmid is carried out linearizing, utilize electric method for transformation to transform the pichia pastoris phaff bacterium competence cell, obtain recombination yeast, behind resistance screening, PCR identifies and obtains positive colony, utilizes positive colony to carry out abduction delivering, expression product is carried out SDS-PAGE analyze, Western-Blot identifies, obtains expression system.

5. construction process according to claim 4, it is characterized in that described PCR is that genome with recombination yeast is a template, two kinds of different primers increase respectively, first group of upstream primer sequence is shown in SEQ ID NO:1, the downstream primer sequence is shown in SEQ ID NO:2, second group of upstream primer sequence is shown in SEQ ID NO:3, and the downstream primer sequence is shown in SEQ ID NO:4.

6. construction process according to claim 4 is characterized in that describedly recombinant plasmid is carried out the restriction enzyme that linearizing uses being PmeI.

7. construction process according to claim 4 is characterized in that the parameter that described electricity transforms is voltage 1.5kV, electric capacity 25 μ F, resistance 200 Ω; The electric shock time is 4～10 seconds, and electric shock once.

8. according to the expression product HSA-vMIP-II fusion rotein of the described expression system of claim 1.

9. the application of HSA-vMIP-II fusion rotein according to claim 6 in preparation inhibition peripheral blood mononuclear cell medicine.

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