CN1506463A - Method for yeast cell to express human interleukin 10 - Google Patents
Method for yeast cell to express human interleukin 10 Download PDFInfo
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- CN1506463A CN1506463A CNA021280010A CN02128001A CN1506463A CN 1506463 A CN1506463 A CN 1506463A CN A021280010 A CNA021280010 A CN A021280010A CN 02128001 A CN02128001 A CN 02128001A CN 1506463 A CN1506463 A CN 1506463A
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Abstract
The method for yeast cell to express human interleukin 10 (hIL-10) has the technological scheme including artificially synthesizing all-gene sequence of hIL-10 including signal-carrying hIL-10 sequence, named shIL-10, and no-signal hIL-10 sequence, named nshIL-10; adding to the ends with EcorR I enzyme incising sequence and Not I enzyme incising sequence or with EcorR I enzyme incising sequences; double incising with EcorR I and Not I the integral expression vector pPIC3.5K and Ppic9K of destination gene shIL-10, nshIL-10 and Pichia yeast; constituting recombinant plasmid pPIC3.5K/shIL-10 and Ppic9K/nshIL-10; converting Pichia yeast cell via electrical perforation and plasmodic granule process; integrating recombinant plasmid and destination gene to yeast chromosome; and applying methanol to induce Pichia yeast for intracellular soluble expression and extracellular secretion type expression. The present invention is superior to expression in colibacillus.
Description
Technical field
The present invention relates to the manufacture method of human interleukin 10, more specifically say so, belong to biotechnology high-tech research field about using yeast cell expressing human interleukin 10.
Background technology
A kind of material that suppresses Th1 cell generation cytokine (as IFN) of discovery mouse CD4+Th2 emiocytosises such as Fiorentino in 1989 is called cytokine secretion supressor (CSIF), the unified interleukin 10 (IL-10) that is called in back.
IL-10 is normal human's a class important cytokine, and sophisticated IL-10 has 160 amino-acid residues, and the monomer molecule amount is 18.7KD, contain the disulfide linkage that 4 halfcystines form (12-108,62-114).Activity form is to be the few dimer of homology of 39KD by the molecular weight that non covalent bond connects.
People IL-10 is secreted by various kinds of cell, mainly produces by the Th2 cell, and to various kinds of cell and factor performance immunosuppression and regulating effect.But IL-10 suppressor T cell, monocyte, huge cytophilic activity and function are regulated B cell, Tc, Th, mastocyte, granulocyte, DC, keratinocyte and epithelial growth, differentiation.IL-10 suppresses synthetic, the angtigen presentation of proinflammatory cytokine (IL-1, IFN, TNF), chemokine (CC, CXC), PGE2, NO, MHC II and costimulatory molecules (IL-12, CD80/CD86), the expression of downward modulation TLR4, the signal transduction of LPS acceptor, strengthen CD14, CD16, CD64, CD163 and by the clearing factor receptor expression of LPS, IFN-γ and TNF downward modulation, thereby restriction immune response and inflammatory reaction finally finish inflammatory reaction.
When body was in pathological state, IL-10 can suppress pro-inflammatory cytokine and celliferous activity thereof, and the protection body avoids the damage of over-drastic immunopathogenesis.Experimentation on animals and human clinical trial have proved that the restraining effect of IL-10 and anti-inflammatory response bringing into play basic effect in the immunity system of body, point out it having very big application potential as inflammatory colitis, crohn (Crohns disease), atrophic sacroiliitis, multiple sclerosis, psoriatic, chronic hepatitis C aspect organ transplantation and treatment inflammation and the autoimmune disease.IL-10 shows good security and biologic activity aspect clinical treatment, have very important clinical application value, for exploitation rhIL-10 genetically engineered drug has been established solid theory, reliable clinical foundation is provided.
Along with to IL-10 aspect the mechanism of action and the treatment clinical disease deeply and extensive studies, affirmed the biological function of IL-10 on the one hand, expanded its clinical application range on the other hand; Also show simultaneously heavy demand to IL-10, so, become inevitable by the active reorganization of genetic engineering means scale operation high-purity natural hIL-10.
The IL-10 monomer contains the disulfide linkage that 4 halfcystines form, 1 N-glycosylation site, but do not have sugar chain, activity form is the few dimer of homology that is connected by non covalent bond.This constitutional features has determined to express the performance that rhIL-10 just can not influence its natural radioactivity with secreted form.Intestinal bacteria are engineering strains of using always, in intestinal bacteria with inclusion body formal representation hIL-10, it is the metaprotein of non-activity, through purifying and renaturation, still have Partial Protein can not form disulfide linkage accurately and suitable space folding, influenced the biological activity of hIL-10 and complex process, the rate of recovery is low, has increased production cost.
Summary of the invention
The purpose of this invention is to provide a kind of method of expressing rhIL-10 with secreted form, specifically be that the using yeast cell carries out solubility expression or exocytosis expression in the born of the same parents, this expression improves the correct folding and biologic activity of the space structure of human interleukin 10 effectively.
The yeast of method of the present invention is selected His for use
-Pichia spp GS115 strain and KM71 strain, the conformability expression plasmid is pPIC3.5K, pPIC9K and pAO815.Concrete grammar may further comprise the steps:
1, according to the full gene of codon synthetic hil-10 of pichia spp preference, nucleotide sequence is as follows:
10 20 30 40 50 60
ATGCATTCTT?CTGCTTTGTT?GTGTTGTTTG?GTTTTGTTGA?CTGGTGTTAG?AGCTTCTCCA
TACGTAAGAA?GACGAAACAA?CACAACAAAC?CAAAACAACT?GACCACAATC?TCGAAGAGGT
70 80 90 100 110 120
GGTCAAGGTA?CTCAATCTGA?AAATTCTTGT?ACTCATTTTC?CAGGTAATTT?GCCAAATATG
CCAGTTCCAT?GAGTTAGACT?TTTAAGAACA?TGAGTAAAAG?GTCCATTAAA?CGGTTTATAC
130 140 150 160 170 180
TTGAGAGATT?TGAGAGATGC?TTTTTCTAGA?GTTAAGACTT?TTTTTCAAAT?GAAGGATCAA
AACTCTCTAA?ACTCTCTACG?AAAAAGATCT?CAATTCTGAA?AAAAAGTTTA?CTTCCTAGTT
190 200 210 220 230 240
TTGGATAATT?TGTTGTTGAA?GGAATCTTTG?TTGGAAGATT?TTAAGGGTTA?CTTGGGTTGT
AACCTATTAA?ACAACAACTT?CCTTAGAAAC?AACCTTCTAA?AATTCCCAAT?GAACCCAACA
250 260 270 280 290 300
CAAGCTTTGT?CTGAAATGAT?TCAATTTTAC?TTGGAAGAAG?TTATGCCACA?AGCTGAAAAT
GTTCGAAACA?GACTTTACTA?AGTTAAAATG?AACCTTCTTC?AATACGGTGT?TCGACTTTTA
310 320 330 340 350 360
CAAGATCCAG?ATATTAAGGC?TCATGTTAAT?TCTTTGGGTG?AAAATTTGAA?GACTTTGAGA
GTTCTAGGTC?TATAATTCCG?AGTACAATTA?AGAAACCCAC?TTTTAAACTT?CTGAAACTCT
370 380 390 400 410 420
TTGAGATTGA?GAAGATGTCA?TAGATTTTTG?CCATGTGAAA?ATAAGTCTAA?GGCTGTTGAA
AACTCTAACT?CTTCTACAGT?ATCTAAAAAC?GGTACACTTT?TATTCAGATT?CCGACAACTT
430 440 450 460 470 480
CAAGTTAAGA?ATGCTTTTAA?TAAGTTGCAA?GAAAAGGGTA?TTTACAAGGC?TATGTCTGAA
GTTCAATTCT?TACGAAAATT?ATTCAACGTT?CTTTTCCCAT?AAATGTTCCG?ATACAGACTT
490 500 510 520 530 540
TTTGATATTT?TTATTAATTA?CATTGAGGCT?TACATGACTA?TGAAGATTAG?AAATTAA...
AAACTATAAA?AATAATTAAT?GTAACTCCGA?ATGTACTGAT?ACTTCTAATC?TTTAATT...
2, the external structure recombinant expression vector transforms in the pichia spp, carries out outer secretion expression of born of the same parents and the solubility expression in the born of the same parents:
2.1) external structure recombinant secretor expression vector:
2.1.1) with synthetic not with recombinate the respectively multiple clone site of integrated expression vector pPIC9K and pPIC3.5K of the hIL-10 of original signal sequence and the hIL-10 that has an original signal sequence;
2.1.2) with synthetic not with the recombinate EcoR I site of carrier pAO815 of the fusion gene of the hIL-10 of original signal sequence and α-factor and the hIL-10 that has an original signal sequence, utilize Bgl II and BamH I restriction endonuclease external structure multiple copy expression cassette on pAO815;
2.2) the interior solubility expression carrier of external structure reorganization born of the same parents:
2.2.1) synthetic do not added initiator codon ATG, the multiple clone site of the carrier pPIC3.5K that recombinates with the hIL-10 5 ' end of original signal sequence;
2.2.2) with synthetic do not add initiator codon ATG with the hIL-10 5 ' end of original signal sequence, the EcoR I site of the carrier pAO815 that recombinates utilizes Bgl II and BamH I restriction endonuclease external structure multiple copy expression cassette on pAO815;
3, pichia spp transforms: recombinant plasmid is cut into linear plasmid with restriction enzyme Sal I or Sac I enzyme, transforms pichia spp by electroporation or protoplasm body;
4, Pichia anomala expression hIL-10: in the pichia spp substratum, add methyl alcohol and carry out abduction delivering.
Secreting, expressing human activated protein IL-10 has good advantage in the eukaryotic cell yeast: it not only can prevent the host bacterium to the degraded of expression product, alleviate the metabolic load of host cell and expression product toxic action to the host, and can promote secretory protein to fold, recover its native conformation and activity by suitable mode.Especially the activity form of hIL-10 is the few dimer of homology that connects with non covalent bond, and secreting, expressing hIL-10 just might keep its biologic activity.Utilize yeast cell to carry out secreting, expressing rhIL-10, target protein will be secreted in the nutrient solution in a large number, can form disulfide linkage and correct space structure accurately, has kept the hIL-10 natural radioactivity; Owing to be not subjected to the influence of host bacterial intracellular protein, reduced purifying process, improved the rate of recovery; Simple in structure, the growth of yeast cell simultaneously fast, be easy to cultivation and fermentation, low, the target protein output height of production cost.
Description of drawings
Fig. 1 makes up the secretion expression's carrier p3K/shIL that has original signal sequence
Fig. 2 makes up the secretion expression's carrier p9K/nshIL that has the alpha factor signal sequence
Fig. 3 makes up single copy non-secretory expression vector pAO/nshIL
Fig. 4 makes up secretion expression's carrier pAO/shIL that single copy has original signal sequence
Fig. 5 makes up secretion expression's carrier pAO/ahIL that single copy has the alpha factor signal sequence
Fig. 6 makes up the multiple copied expression vector
Embodiment
1. the full gene of synthetic hIL-10, nucleotide sequence is as follows:
10 20 30 40 50 60
ATGCATTCTT?CTGCTTTGTT?GTGTTGTTTG?GTTTTGTTGA?CTGGTGTTAG?AGCTTCTCCA
TACGTAAGAA?GACGAAACAA?CACAACAAAC?CAAAACAACT?GACCACAATC?TCGAAGAGGT
70 80 90 100 110 120
GGTCAAGGTA?CTCAATCTGA?AAATTCTTGT?ACTCATTTTC?CAGGTAATTT?GCCAAATATG
CCAGTTCCAT?GAGTTAGACT?TTTAAGAACA?TGAGTAAAAG?GTCCATTAAA?CGGTTTATAC
130 140 150 160 170 180
TTGAGAGATT?TGAGAGATGC?TTTTTCTAGA?GTTAAGACTT?TTTTTCAAAT?GAAGGATCAA
AACTCTCTAA?ACTCTCTACG?AAAAAGATCT?CAATTCTGAA?AAAAAGTTTA?CTTCCTAGTT
190 200 210 220 230 240
TTGGATAATT?TGTTGTTGAA?GGAATCTTTG?TTGGAAGATT?TTAAGGGTTA?CTTGGGTTGT
AACCTATTAA?ACAACAACTT?CCTTAGAAAC?AACCTTCTAA?AATTCCCAAT?GAACCCAACA
250 260 270 280 290 300
CAAGCTTTGT?CTGAAATGAT?TCAATTTTAC?TTGGAAGAAG?TTATGCCACA?AGCTGAAAAT
GTTCGAAACA?GACTTTACTA?AGTTAAAATG?AACCTTCTTC?AATACGGTGT?TCGACTTTTA
310 320 330 340 350 360
CAAGATCCAG?ATATTAAGGC?TCATGTTAAT?TCTTTGGGTG?AAAATTTGAA?GACTTTGAGA
GTTCTAGGTC?TATAATTCCG?AGTACAATTA?AGAAACCCAC?TTTTAAACTT?CTGAAACTCT
370 380 390 400 410 420
TTGAGATTGA?GAAGATGTCA?TAGATTTTTG?CCATGTGAAA?ATAAGTCTAA?GGCTGTTGAA
AACTCTAACT?CTTCTACAGT?ATCTAAAAAC?GGTACACTTT?TATTCAGATT?CCGACAACTT
430 440 450 460 470 480
CAAGTTAAGA?ATGCTTTTAA?TAAGTTGCAA?GAAAAGGGTA?TTTACAAGGC?TATGTCTGAA
GTTCAATTCT?TACGAAAATT?ATTCAACGTT?CTTTTCCCAT?AAATGTTCCG?ATACAGACTT
490 500 510 520 530 540
TTTGATATTT?TTATTAATTA?CATTGAGGCT?TACATGACTA?TGAAGATTAG?AAATTAA...
AAACTATAAA?AATAATTAAT?GTAACTCCGA?ATGTACTGAT?ACTTCTAATC?TTTAATT...
2. it is as follows to make up the integrated expression vector mode of pichia spp:
2.1) Pichi strain GS115 (HIS used in the present invention
-) and expression vector pPIC3.5K, pPIC9K, pAO815 all come from Invitrogen company.
2.2) method of construction expression recombinant plasmid is as follows:
2.2.1) using the hIL-10 gene of primer PCR amplification synthetic, upstream primer 5 ' end contains EcoR I enzyme and cuts sequence, and downstream primer 3 ' end contains Not I enzyme and cuts sequence.
The PCR reaction is allocated by following reagent:
Upstream primer 0.5 μ l
Downstream primer 0.5 μ l
10×reaction?buffer 5μl
MgCl
2 3μl
dNTP 3μl
Archaeal dna polymerase Taq 2U
Sterilized water up to 50 μ l
Place on the DNA cloning instrument by 94 ℃ 4min; 94 ℃, 30sec, 55 ℃, 20sec, 72 ℃, 20sec circulates 35 times; 72 ℃, 10min.Get 5 μ l amplified productions at 1% agarose electrophoresis, 90V, 15min.Under ultraviolet lamp, observe and take pictures.
2.2.2) when the dna fragmentation size of above-mentioned pcr amplification meets with the size of expection, use OMEGA GEL EXTRACT KIT to reclaim fragment, concrete operations are undertaken by the test kit specification sheets.
2.2.3) enzyme cuts PCR product and plasmid: reaction solution is composed as follows,
PCR product plasmid (pPIC3.5K or pPIC9K)
Substrate 50 μ l 50 μ l
10×H?buffer 10μl 10μl
BSA 10μl 10μl
Not?I 10U 10U
EcoR?I 10U 10U
Sterilized water up to 100 μ l up to 100 μ l
37 ℃, after 3 hours, according to 2.2.2) method carries out fragment and reclaims.
2.2.4) ligation:
Enzyme is cut PCR product 10 μ l
Digested plasmid 5 μ l
10×ligase?buffer 2μl
T4?ligase 1μl
Sterilized water up to 20 μ l
16 ℃, spend the night.
2.2.5) transformed competence colibacillus bacterium DH5 α: the preparation of intestinal bacteria competence is carried out according to a conventional method.Get above-mentioned connection product 10 μ l and carefully mix with competence bacterium 100 μ l, 4 ℃, 30min, 42 ℃, 2min adds 500 μ l LB nutrient solutions then, 37 ℃, 30min, 6000rpm, the centrifugal 1min of room temperature, its supernatant 550 μ l, surplus solution suspension bacterial precipitation, all bacterium liquid is coated with the Amp-LB flat board, cultivates 14 hours for 37 ℃.
2.2.6) enzyme cuts evaluation: 4 single colony inoculations of picking are in 5ml Amp-LB nutrient solution at random, and 37 ℃ of shaking culture 12 hours are used OMEGA MINIPREP PLASIMAD KIT extracting plasmid, press the test kit specification sheets and operate.It is composed as follows that enzyme is cut system:
10×H?buffer 2μl
BSA 10μl
Not?I 10U
Sterilized water up to 20 μ l
2.3) have the reorganization (showing) of the shIL-10 and the integrated expression vector pPIC3.5K of original signal sequence as Fig. 1
Primer amplification shIL-10 gene (total length 537bp), and add respectively that at 5 ends and 3 ends the enzyme of EcoR I and Not I cuts sequence, with EcoR I and Not I difference double digestion shIL-10 and expression vector pPIC3.5K, shIL-10 is inserted on the pPlC3.5K, obtain the p3K/shIL recombinant plasmid.
2.4) nshIL-10 and the pPIC9K construction of recombinant plasmid (showing as Fig. 2) of no signal sequence
Primer amplification nshIL-10 gene (total length 480bp), and add respectively that at 5 ends and 3 ends the enzyme of EcoR I and Not I cuts sequence, with EcoR 1 and Not I difference double digestion nshIL-10 and expression vector pPIC9K, the nshIL-10 place is inserted on the pPIC9K, obtain the p9K/nshIL recombinant plasmid.
2.5) external structure multiple copied hIL-10 expression cassette
2.5.1) construction process: have primer difference pcr amplification gene nshIL-10, shIL-10, α hIL-10 that EcoR I enzyme is cut sequence, reclaim the PCR product and also cut with EcoR I enzyme.Cut the pA0815 plasmid with EcoR I enzyme simultaneously, 37 ℃, 2 hours; Add isopyknic phenol/chloroform, vibration, the centrifugal 5min of 13200rpm; Get supernatant and add isopyknic chloroform again, vibration, the centrifugal 5min of 13200rpm; Get supernatant, add 1/10 3M NaAc, the dehydrated alcohol of 2 times of volumes ,-30 ℃ of 30min; 4 ℃, the centrifugal 10min of 13200rpm, its supernatant precipitates with 70% cold ethanol rinsing twice, drying at room temperature; The sterilized water that adds pH8.0 suspends and precipitates.
EcoR I enzyme is cut the dephosphorylation of pAO815 plasmid:
pAO815/EcoR?I 20μl
10×CIAP?buffer 5μl
CIAP 3μl
Sterilized water up to 50 μ l
37℃，30min。After phenol/chloroform extracting, be connected with nshIL-10, shIL-10, α hIL-10 that EcoR I enzyme is cut, the transformed competence colibacillus intestinal bacteria, be coated with dull and stereotyped the cultivation, choose single colony inoculation Amp-LB liquid shaking culture, extracting plasmid, EcoR I enzyme are cut and are identified goal gene whether recombinate (method is the same) on the pAO815 plasmid; And then identify the goal gene both forward and reverse directions of recombinant plasmid with HindIII and BamH I:
Recombinant plasmid (pAO/nshIL, 10 μ l
pAO/shIL、pAO/ahIL)
10×K?buffer 2μl
BamH?I 5U
HindIII 5U
Sterilized water up to 20 μ l
37 ℃, 3 hours, get 10 μ l and carry out 1% agarose electrophoresis, under ultraviolet lamp, observe and take pictures.If except the 7.7kb that has an appointment, also have the fragment of 640bp, the illustration purpose fragment is a forward on the pAO815 carrier, if there is not the 640bp band, is that other bands are then for reverse.
2.5.2) nshIL-10 of no signal sequence 5 ' end adds initiator codon ATG, hold at 5 ' end and 3 ' again to add that the enzyme of EcoRI cuts sequence; Be connected to after EcoR I enzyme is cut through EcoR I enzyme cut with the dephosphorylized plasmid pAO815 of alkaline phosphatase on, screening forward recombinant plasmid pAO/nhIL (showing) as Fig. 3.
2.5.3) shIL-10 that has an original signal sequence holds at 5 ' end and 3 ' and add that the enzyme of EcoR I cuts sequence; Be connected to after EcoR I enzyme is cut through EcoR I enzyme cut with the dephosphorylized plasmid pAO815 of alkaline phosphatase on, screening forward recombinant plasmid pAO/shIL (showing) as Fig. 4.
2.5.4) zymic secretory signal sequence α-factor is connected the 5 ' end of hIL-10, form fusion gene α hIL-10, hold at the 5 ' end and 3 ' of this fusion gene again to add that the enzyme of EcoR I cuts sequence; Be connected to after EcoR I enzyme is cut through the EcoRI enzyme cut with the dephosphorylized plasmid pAO815 of alkaline phosphatase on, screening forward recombinant plasmid pAO/ahIL (showing) as Fig. 5.
2.5.5) the recombinant plasmid pAO/nshIL, the phO/shIL that make up of aforesaid method, pAO/ahIL cut with Bgl I and BamH I pair respectively, reclaim the fragment of about 2200bp, be single copy expression cassette, refined solution adds T4 ligase enzyme and damping fluid thereof, totally 20 μ l, 16 ℃ of ligations of spending the night; Add Bgl II 2U, BamH I 2U, 10 * K buffer, 10 μ l, sterilized water to 100 μ l then, 37 ℃ 2 hours, purpose is that enzyme cuts the expression cassette that elimination head-head, tail-tail connects; It is corresponding with it and be connected through the recombinant plasmid (containing corresponding single expression cassette that copies) that BamH I enzyme is cut and dephosphorylation is handled behind phenol/chloroform extracting and ethanol sedimentation (method is the same) that enzyme is cut product, Transformed E .coli DH5 α, choose single bacterium colony, the extracting plasmid, carry out enzyme with Bgl II and BamH I again and cut evaluation and screening, electrophoresis observation is except having 4028bp and 2393bp, also have 2039bp (pAO/ahIL) or 1829bp (pAO/shIL) or 1769bp (pAO/hIL), 4076 (2 * 2039) bp (pAO/ahIL) or 3658 (2 * 1829) bp (pAO/shIL) or 3538 (2 * 1769) bp (pAO/hIL), 6117 (3 * 2039) bp (pAO/ahIL) or 5487 (3 * 1829) bp (pAO/shIL) or 5307 (3 * 1769) bp (pAO/hIL) ... then expression is 1,2,3 ... Deng the band and the plasmid band of multiple copy expression cassette, select the recombinant plasmid that contains maximum copy numbers and further transform pichia spp (showing) as Fig. 6.
3. recombinant vectors transforms the yeast transformant of screening multiple copy expression cassette in pichia spp and the body:
3.1) recombinant plasmid that makes up by aforesaid way behind Sal I linearization for enzyme restriction, use electroporation or protoplasm body recombinant plasmid be incorporated on the pichia spp karyomit(e).
3.1.1) electroporation:
Inoculation His
-The single bacterium colony of pichia spp GS115 is in 500ml YPD nutrient solution, and incubated overnight is to OD
600Be about 1.3-1.5.4 ℃, 3000rpm, centrifugal 5min, collecting cell, the freezing sterilized water of 250ml suspends and precipitates; Wash again once with freezing sterilized water by this method, after freezing 1M sorbyl alcohol is washed twice, yeast cell is suspended in the 1ml sorbyl alcohol.Get the DNA of 5-20 μ g, change competent cell with 80 μ l electricity and be mixed in the freezing 0.2cm electricity revolving cup, hatched on ice 5 minutes through Sal I linearization for enzyme restriction.
Use electroporation BIO-PAD Gene Pulse Xcell
TMElectroporation System carries out electroporation with voltage 2000V, electric capacity 25 μ F, resistance 200 Ω, adds the freezing 1M sorbyl alcohol of 1ml then, gets 200-600 μ l solution and is coated with the MD flat board.Single bacterium colony is selected in 30 ℃ of cultivations.
3.1.2) protoplasm body:
His
-Pichia spp GS115 is through being cultured to OD
600Be 0.2-0.3,3000rpm, the centrifugal 5min of room temperature, collecting cell with 20ml sterilized water, 20mlSED, 20ml 1M sorbyl alcohol, 20mlSCE washing, is resuspended in the 1mlSCE liquid successively at last.The Lyticase (Sigma product) that adds 1 μ l 1mg/ml, 30 ℃ of about 15min of peptic cell wall, the centrifugal 5min of 2500rpm obtains protoplastis, respectively washes 1 time with 20ml 1M sorbyl alcohol, 20ml CaS again, is suspended at last among the CaS of 0.6ml.Get protoplastis 100 μ l, add the recombinant plasmid incubated at room 10min of 10 μ g through Sal I linearization for enzyme restriction, add 1mlPEG/CaT incubated at room 10min again, through the centrifugal 10min of 2000rpm room temperature, precipitation suspends with 150 μ lSOS liquid, room temperature is slowly mixed with 850 μ l 1M sorbyl alcohols after placing 20min, gets 100 μ l and mixes back coating RD flat board with 2ml RD soft agar upper strata substratum, cultivates 6 days for 30 ℃.Select single bacterium colony.
3.2) yeast transformant of G418 screening multiple copy expression cassette:
Selecting 50 recons from RD flat board or MD flat board is seeded on the MD flat board, so being seeded in 96 orifice plates that contain 200 μ l YPD nutrient solutions goes down to posterity three times, make the concentration basically identical of each recon, respectively getting 10 μ l is seeded in and contains on different concns G418 (0.25,0.5,1.0,2.0,3.0,4.0mg/ml) the YPD flat board 30 ℃ of cultivations.Select the recon (transformant that promptly contains multiple copy expression cassette) of anti-high density and do abduction delivering.
4. pichia spp transformant abduction delivering hIL-10 albumen:
The transformant list colony inoculation that will contain multiple copy expression cassette is in 20ml BMGY, and 30 ℃ of shaking culture 48 hours are collected thalline and also are suspended among the 10ml BMMY, and 30 ℃ of shaking culture are every 12 hours sampling 1ml and added 0.5% methyl alcohol in per 24 hours.Separate 5 days sampling supernatants, carry out SDS-PAGE and Western Blotting according to a conventional method and identify and detect, the high recon of screening expression amount carries out a large amount of inducing culture.
Attached solution formula:
1.LB substratum (pH to 7.0):
Tryptones 10g
Yeast extract 5g
NaCl 10g
The LB solid medium adds 2% agar again
2.YPD substratum:
Peptone 2%
Glucose 2%
The YPD solid medium adds 2% agar again.
3.MD solid medium:
Yeast nitrogenous base 1.34%
Vitamin H 4 * 10
-5%
Glucose 2%
Agar 2%
4.RD solid medium:
Yeast nitrogenous base 1.34%
Vitamin H 4 * 10
-5%
Glucose 2%
Sorbyl alcohol 1M
The kilnitamin 0.005% of no Histidine
Upper strata soft agar 0.8%
Lower floor's agar 2%
5.SCE：
Sorbyl alcohol 1M
EDTA 1mM
Citrate buffer solution (pH5.8) 10mM
6.SED
1ml DTT+19ml SE (1M sorbyl alcohol, 25mM EDTA (pH8.0)
7.CaS
Sorbyl alcohol 1M
Tris-HCl(pH7.5) 10mM
CaCl
2 10mM
8.SOS
Sorbyl alcohol 1M
0.3 times of YPD
CaCl
2 10mM
9.CaT
Tris?pH7.5 20mM
CaCl
2 20mM
10.BMGY
Peptone 2%
Yeast nitrogenous base 1.34%
Vitamin H 4 * 10
-5%
Glucose 2%
Potassium phosphate buffer pH6.0 100mM
11.BMMY
Peptone 2%
Yeast nitrogenous base 1.34%
Vitamin H 4 * 10
-5%
Glucose 2%
Potassium phosphate buffer pH6.0 100mM
Methyl alcohol 0.5%
Claims (3)
1. a method of making recombination human interleukins-11 0 is characterized in that: using yeast expressing human interleukin 10.
2. according to the method for right 1 described manufacturing recombination human interleukins-11 0, it is characterized in that: yeast is selected His for use
-Pichia spp GS115 strain and KM71 strain.
3. according to the method for right 1 described manufacturing recombination human interleukins-11 0, it is characterized in that may further comprise the steps:
1) according to the full gene of codon synthetic hIL-10 of pichia spp preference, nucleotide sequence is as follows:
10 20 30 40 50 60
ATGCATTCTT?CTGCTTTGTT?GTGTTGTTTG?GTTTTGTTGA?CTGGTGTTAG?AGCTTCTCCA
TACGTAAGAA?GACGAAACAA?CACAACAAAC?CAAAACAACT?GACCACAATC?TCGAAGAGGT
70 80 90 100 110 120
GGTCAAGGTA?CTCAATCTGA?AAATTCTTGT?ACTCATTTTC?CAGGTAATTT?GCCAAATATG
CCAGTTCCAT?GAGTTAGACT?TTTAAGAACA?TGAGTAAAAG?GTCCATTAAA?CGGTTTATAC
130 140 150 160 170 180
TTGAGAGATT?TGAGAGATGC?TTTTTCTAGA?GTTAAGACTT?TTTTTCAAAT?GAAGGATCAA
AACTCTCTAA?ACTCTCTACG?AAAAAGATCT?CAATTCTGAA?AAAAAGTTTA?CTTCCTAGTT
190 200 210 220 230 240
TTGGATAATT?TGTTGTTGAA?GGAATCTTTG?TTGGAAGATT?TTAAGGGTTA?CTTGGGTTGT
AACCTATTAA?ACAACAACTT?CCTTAGAAAC?AACCTTCTAA?AATTCCCAAT?GAACCCAACA
250 260 270 280 290 300
CAAGCTTTGT?CTGAAATGAT?TCAATTTTAC?TTGGAAGAAG?TTATGCCACA?AGCTGAAAAT
GTTCGAAACA?GACTTTACTA?AGTTAAAATG?AACCTTCTTC?AATACGGTGT?TCGACTTTTA
310 320 330 340 350 360
CAAGATCCAG?ATATTAAGGC?TCATGTTAAT?TCTTTGGGTG?AAAATTTGAA?GACTTTGAGA
GTTCTAGGTC?TATAATTCCG?AGTACAATTA?AGAAACCCAC?TTTTAAACTT?CTGAAACTCT
370 380 390 400 410 420
TTGAGATTGA?GAAGATGTCA?TAGATTTTTG?CCATCTGAAA?ATAAGTCTAA?GGCTGTTGAA
AACTCTAACT?CTTCTACAGT?ATCTAAAAAC?GGTACACTTT?TATTCAGATT?CCGACAACTT
430 440 450 460 470 480
CAAGTTAAGA?ATGCTTTTAA?TAAGTTGCAA?GAAAAGGGTA?TTTACAAGGC?TATGTCTGAA
GTTCAATTCT?TACGAAAATT?ATTCAACGTT?CTTTTCCCAT?AAATGTTCCG?ATACAGACTT
490 500 510 520 530 540
TTTGATATTT?TTATTAATTA?CATTGAGGCT?TACATGACTA?TGAAGATTAG?AAATTAA...
AAACTATAAA?AATAATTAAT?GTAACTCCGA?ATGTACTGAT?ACTTCTAATC?TTTAATT...
2) the outer recombinant expression vector that makes up transforms in the pichia spp, carries out outer secretion expression of born of the same parents and the solubility expression in the born of the same parents:
(1) external structure recombinant secretor expression vector:
A, synthetic not with recombinate the respectively multiple clone site of integrated expression vector pPIC9K and pPIC3.5K of the hIL-10 of original signal sequence and the hIL-10 that has an original signal sequence;
B, with the recombinate EcoR I site of carrier pAO815 of the fusion gene that is not formed by connecting with the hIL-10 and the α-factor of original signal sequence of synthetic and the hIL-10 that has original signal sequence, utilize Bgl II and BamH I restriction endonuclease external structure multiple copy expression cassette on pAO815;
(2) solubility expression carrier in the external structure reorganization born of the same parents:
A, synthetic do not added initiator codon ATG, the multiple clone site of the carrier pPIC3.5K that recombinates with the hIL-10 5 ' end of original signal sequence;
B, with synthetic do not add initiator codon ATG with the hIL-10 5 ' end of original signal sequence, the EcoR I site of the carrier pA0815 that recombinates utilizes Bgl II and BamH I restriction endonuclease external structure multiple copy expression cassette on pA0815;
3) conversion of pichia spp: recombinant plasmid is cut into linear plasmid with restriction enzyme Sal I or Sac I enzyme, transforms pichia spp by electroporation or protoplasm body;
4) Pichia anomala expression hIL-10: in the pichia spp substratum, add methyl alcohol and carry out abduction delivering hIL-10.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100383249C (en) * | 2005-04-28 | 2008-04-23 | 中国人民解放军第三军医大学 | Expression of interleukin 24 from yeast cell |
| CN100591754C (en) * | 2005-10-14 | 2010-02-24 | 中国科学院大连化学物理研究所 | Yeast system capable of coexpressing CYP and CPR |
| CN101892168A (en) * | 2010-07-06 | 2010-11-24 | 集美大学 | Pichiapastoris expression strain for recombinant duck interleukin 2 and construction method and application thereof |
| CN102392043A (en) * | 2011-12-13 | 2012-03-28 | 江南大学 | Method for preparing human interleukin 32beta (hIL-32beta) from Pichia pastoris |
| CN102533840A (en) * | 2011-12-13 | 2012-07-04 | 江南大学 | Method for preparing human interleukin 29 (hIL-29) mature peptide by using Pichia pastoris |
| CN102021196B (en) * | 2009-11-20 | 2013-06-26 | 上海杰隆生物工程股份有限公司 | Method for producing recombinant human interleukin-21 by using Pichia pastoris |
| CN105603027A (en) * | 2016-01-21 | 2016-05-25 | 遵义医学院 | Preparation process and application of interleukin-10 |
-
2002
- 2002-12-07 CN CNB021280010A patent/CN100494386C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100383249C (en) * | 2005-04-28 | 2008-04-23 | 中国人民解放军第三军医大学 | Expression of interleukin 24 from yeast cell |
| CN100591754C (en) * | 2005-10-14 | 2010-02-24 | 中国科学院大连化学物理研究所 | Yeast system capable of coexpressing CYP and CPR |
| CN102021196B (en) * | 2009-11-20 | 2013-06-26 | 上海杰隆生物工程股份有限公司 | Method for producing recombinant human interleukin-21 by using Pichia pastoris |
| CN101892168A (en) * | 2010-07-06 | 2010-11-24 | 集美大学 | Pichiapastoris expression strain for recombinant duck interleukin 2 and construction method and application thereof |
| CN102392043A (en) * | 2011-12-13 | 2012-03-28 | 江南大学 | Method for preparing human interleukin 32beta (hIL-32beta) from Pichia pastoris |
| CN102533840A (en) * | 2011-12-13 | 2012-07-04 | 江南大学 | Method for preparing human interleukin 29 (hIL-29) mature peptide by using Pichia pastoris |
| CN105603027A (en) * | 2016-01-21 | 2016-05-25 | 遵义医学院 | Preparation process and application of interleukin-10 |
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|---|---|
| CN100494386C (en) | 2009-06-03 |
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