CN102021196B - Method for producing recombinant human interleukin-21 by using Pichia pastoris - Google Patents
Method for producing recombinant human interleukin-21 by using Pichia pastoris Download PDFInfo
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Abstract
The invention provides a method for producing recombinant human interleukin-21 by using Pichia pastoris, in particular to a production method of the recombinant human interleukin-21 expressed by the Pichia pastoris, which comprises the following steps: firstly, the reverse transcription PCR (polymerase chain reaction) is carried out in lymphocytes of healthy people to obtain encoding genes of rhIL-21, and the encoding genes are fused in expressional regulatory elements of the Pichia pastoris to construct Pichia pastoris high-level-expression engineering bacteria; and secondly, the Pichia pastoris high-level-expression engineering bacteria are induced to produce a large number of recombinant human interleukins-21 by adding methanol. The Pichia pastoris is very easy to achieve high-density fermentation and has the characteristics of hypersecretion, so that a large number of recombinant human interleukins-21 can be industrially produced easily at low cost.
Description
Technical field
The present invention relates to bioengineering field, relate more specifically to the method for pichia spp Restruction Huamn Interleukin 21 gene.
Background technology
Along with the rise of genetic engineering pharmaceutical, many have the human body native protein factor of pharmaceutical use by recombinant technology scale operation.At present, existing 50 kinds of left and right engineered protein medicines have also been obtained huge economic benefit by various countries' approval listing when having benefited the patient.For example: human growth hormone, insulin human, Epo, G-CSF etc.
(Huamn Interleukin 21 is hIL-21) the I type cytokines that Parrish Novak etc. found in 2000, by the CD4 of activation to Huamn Interleukin 21 gene
+T cell, NKT cell, Tfh cell and Th17 cell produce, and with IL-2, IL-4, IL-15, higher homology are arranged, and belong to γ c family member.Bind interleukin 21 acceptors (IL-21R) that the IL-21 energy is special activate the signaling pathways such as JAK/STAT, show complicated biological effect.IL-21 has immunoloregulation function widely, it is the important cytokine in the immunoregulation network, can be to cell generation effects such as B cell, T cell, NK cells, it can regulate and control differentiation, the apoptosis of B cell and produce the subclass of antibody, promote the cell-mediated acquired immunity of T, strengthen the cytotoxicity of NK cell and the ability of generation IFN-γ, the conversion between mediation active immunity and passive immunization.
HIL-21 is positioned No. 4 karyomit(e)s long-armed upper (4q26-27), transcribe a ripe mRNA who is formed by 642 Nucleotide, the amyloid protein precursor that 162 amino acid of encoding form, wherein front 31 amino acid are signal peptide, and 131 amino acid of back form that to have four spirane structure territory molecular weight be the ripe IL-21 of 15KD.Three t cell activation nfs (NF-AT) binding site is contained in 5 ' the regulation and control zone of IL-21, and the activity of IL-21 promotor acts on cell by Calcium ionophore and produces.IL-21 has the two DNase I of place hypersensitive sites, and all guard in people and mouse in these two sites, and one of them is positioned at the IL-21 promoter region, transcribes relevant with the IL-21 of TCR mediation.HIL-21 can be special in conjunction with Huamn Interleukin 21 gene acceptor (hIL-21R), activate the signaling pathways such as JAK/STAT, show complicated biological effect, it can regulate and control differentiation, the apoptosis of B cell and produce the subclass of antibody, promote the cell-mediated acquired immunity of T, strengthen the cytotoxicity of NK cell and the ability of generation IFN-γ, the conversion between mediation active immunity and passive immunization.The vital role of rhIL-21 in anaphylaxis, inflammatory reaction, autoimmune response and the clinical application such as antitumor.Studies show that in recent years, hIL-21 can effectively be used for the treatment of mammary cancer, renal cell carcinoma and colorectal carcinoma.Drugs approved by FDA IL-21 in 2005 (IL-21, ZymoGenetics produces) is the seldom used medicine for the treatment of of advanced and aggressive melanochrome cancer and tumour.
Mainly produce hIL-21 by escherichia coli prokaryotic expression system at present, but there is following shortcoming in this system: (1) expression amount is not high, and at intracellular expression, (3) exist with inclusion body bodily form formula (2), needs sex change, renaturation manipulation.U.S. ZymoGenetics company uses this system to produce hIL-21 (application for a patent for invention number: 200380105873.9), the initial expression amount of hIL-21 that obtains is only 2mg/L, rear by methods such as improved construction body and host strains, make overall yield bring up to 50～100mg/L; Make expression level 2～5% bring up to and account for 20% of total protein by what account for total protein by the encoding sequence of optimizing hIL-21.the people such as Japan Asano also produce hIL-21 by escherichia expression system, expression amount can reach 200mg/L (Asano R, Kudo T, Makabe K, et al.Antitumor activity of interleukin-21preparedby novel refolding procedure from inclusion bodies expressed inEscherichia coli.FEBS Lett, 2002, 528 (1-3): 70-76.), but due to the hIL-21 that obtains with the His label, the phenomenon that relative molecular weight increases appears, and be to exist in Escherichia coli cell with inclusion body equally, need broken thalline, the troublesome operation such as renaturation progressively after sex change.At present, domestic hIL-21 by escherichia coli prokaryotic expression system production accounts for the ratio of total protein generally in 10% left and right.The drawback that exists with the inclusion body form in order to overcome e. coli expression product, the people such as Liu Mingjun have given expression to hIL-21 fusion rotein (Liu Mingjun etc. with soluble form in intestinal bacteria, soluble form expression, purifying and the external impact on T cell proliferation of recombinant human il-2's 1 fusion rotein in intestinal bacteria, Shandong medicine, 2008,48 (26): 3-5), but albumen is present in born of the same parents, need equally the cracking thalline, and what express is the GST-IL21 fusion rotein, needs the operations such as cutting GST label.
In sum, this area is in the urgent need to developing the technique for preparing efficiently, easily Huamn Interleukin 21 gene.
Summary of the invention
The purpose of this invention is to provide a kind of efficiently, the method by pichia spp Restruction Huamn Interleukin 21 gene easily.
In a first aspect of the present invention, a kind of expression cassette of Huamn Interleukin 21 gene is provided, described expression cassette from 5 ' to 3 ' comprises following element successively:
(1) the controlling gene pichia pastoris AOX 1 promoter of transcribing;
(2) control the signal peptide sequence of foreign protein secreting, expressing in pichia spp;
(3) encoding sequence of Huamn Interleukin 21 gene; With
(4) terminator codon.
In another preference, described signal peptide sequence is the yeast alpha factor (signal peptide sequence of α-factor).
In another preference, also comprise the AOX1 transcription terminator in described terminator codon downstream.
In another preference, the encoding sequence of described Huamn Interleukin 21 gene is wild-type sequence.
In a second aspect of the present invention, a kind of expression vector is provided, described expression vector carries or contains the expression cassette of the Huamn Interleukin 21 gene described in first aspect present invention.
In another preference, described expression vector is carrier pPIC9K-hIL21.
In a third aspect of the present invention, a kind of Pichia yeast engineering is provided, be integrated with the expression cassette of the Huamn Interleukin 21 gene described in first aspect present invention in the karyomit(e) of described pichia spp, and the secreting, expressing Huamn Interleukin 21 gene.
In another preference, the secreting, expressing amount of the Huamn Interleukin 21 gene of described engineering bacteria is greater than the 200mg/ml fermented liquid.
In another preference, this pichia spp is GS115 (His4
-).
In a fourth aspect of the present invention, a kind of method for preparing Huamn Interleukin 21 gene is provided, comprise step:
(a) under the condition that is fit to fermentation, cultivate the Pichia yeast engineering described in third aspect present invention, and induce pichia pastoris AOX 1 promoter to make engineering bacteria secreting, expressing Huamn Interleukin 21 gene;
(b) isolate the Huamn Interleukin 21 gene of secreting, expressing from fermented liquid.
In another preference, it is the thalli growth stage that step (a) is divided into, the abduction delivering stage.
In another preference, pichia pastoris AOX 1 promoter inductor used is methyl alcohol, and concentration is 0.5～1.0 volume %.
In a fifth aspect of the present invention, a kind of production method of Pichia anomala expression recombination human IL-21 is provided, it is characterized in that it comprises the following steps: at first, separate from the healthy human body peripheric venous blood and obtain lymphocyte, obtain the encoding gene of rhIL-21 by extracting cell total rna and reverse transcription PCR, be blended in the Pichia anomala expression controlling element, build the pichia spp high expression engineering; Then produce in a large number rhIL-21 by adding the methanol induction of Pichia pastoris high expression engineering.
In another preference, the structure of described rhIL-21 Pichia anomala expression engineering bacteria comprises the following steps: at first, obtain the rhIL-21 encoding gene; Then, build the pichia spp recombinant expression plasmid; At last, expression plasmid transforms pichia spp, recombinant conversion of screening multi-copy integration, then the transformant that screening obtains high expression level is original engineering bacteria.
In another preference, the pichia spp recombinant expression plasmid comprises following element: Pichia anomala expression controlling element, secreting, expressing yeast alpha factor signal peptide encoding sequence, selection markers gene.
In another preference, this Pichia anomala expression controlling element comprises promotor, the transcription terminator of alcohol oxidase 1 gene.
In another preference, this selection markers gene comprises yeast His4 gene, Kanamycin resistant gene, Ampicillin resistant gene.
In another preference, this pichia spp is GS115 (His4
-).
In another preference, the two-step growth method is adopted in described cultivation, i.e. thalli growth stage, abduction delivering stage.
In another preference, described culture condition comprises substratum, temperature, inductor concentration, and wherein substratum refers to semisynthetic medium, and temperature is 28～30 degree Celsius, and inductor is methyl alcohol, and concentration is 0.5%～1.0%.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and in below (eg embodiment) specifically described each technical characterictic can make up mutually, thereby consist of new or preferred technical scheme.As space is limited, this tired stating no longer one by one.
Description of drawings
Fig. 1 is pPIC9K-hIL21 recombinant expression plasmid figure
Fig. 2 is the SDS-PAGE electrophorogram that screens high expression level amount engineering bacteria
Fig. 3 is the SDS-PAGE electrophorogram that in example of the present invention, one week of Pichia yeast engineering is expressed albumen.
Embodiment
The inventor is through extensive and deep research, by a large amount of screenings, be surprised to find that, with the natural encoding sequence of Huamn Interleukin 21 gene, pichia pastoris AOX 1 promoter and the unit construction such as signal peptide sequence (especially α-signal peptide sequence) of controlling foreign protein secreting, expressing in pichia spp together, can realize the high efficient expression of Huamn Interleukin 21 gene, and the rhIL-21 that produces is secreted into outside born of the same parents, need not broken thalline and get final product separation and purification, simple to operate, be convenient to purifying.In addition, the rhIL-21 that produces is not with any label.Completed on this basis the present invention.
Particularly, the inventor has set up a kind of method of pichia spp Restruction Huamn Interleukin 21 gene, wherein use the pichia spp eukaryotic expression system, pichia yeast expression system is a kind of system of effective expression foreign protein, it is except having the simple advantage of intestinal bacteria system operation, also have following principal feature: easily cultivate (1), be particularly suitable for high density fermentation, (2) exogenous gene expression can regulate and control, (3) high expression level, (4) production cost is low, and (5) have certain folding and working ability to recombinant protein.
Find in inventor research and development, if keep the signal peptide sequence of Huamn Interleukin 21 gene self, perhaps with the ORF of Huamn Interleukin 21 gene when other general secreting signal peptides are combined, can not effectively realize secreting, expressing in pichia spp.Yet, with the natural encoding sequence of Huamn Interleukin 21 gene, pichia pastoris AOX 1 promoter and the unit construction such as signal peptide sequence (especially α-signal peptide sequence) of controlling foreign protein secreting, expressing in pichia spp together, but can realize the high efficient expression of Huamn Interleukin 21 gene, its expression amount is far away higher than other combined situation.
In addition, the present invention also unexpectedly finds by analysis, and the CAI value that Huamn Interleukin 21 gene (hIL-21) encoding gene is expressed in pichia spp is 0.74, has the potential (＞0.6) of coding high level expression albumen.Therefore, natural or Huamn Interleukin 21 gene wild-type needn't be optimized with regard to being highly suitable for expression in pichia spp.
In an example of the present invention, the invention provides a kind of production method of Pichia anomala expression recombination human IL-21, it comprises the following steps: at first, separate from the healthy human body peripheric venous blood and obtain lymphocyte, obtain the encoding gene of rhIL-21 by extracting cell total rna and reverse transcription PCR, be blended in the Pichia anomala expression controlling element, build the pichia spp high expression engineering, then produce in a large number rhIL-21 by adding the methanol induction of Pichia pastoris high expression engineering.
Separate from the healthy human body peripheric venous blood and obtain lymphocyte, extract cell total rna and reverse transcription PCR, obtain the sequence of coding hIL-21, this sequence does not comprise the encoding sequence of hIL-21 self signal peptide, after cutting the generation sticky end with MunI, Not I enzyme, insertion EcoR I, Not I enzyme are cut on the commercially available yeast expression vector pPIC9K of processing, are built into recombinant expression plasmid pPIC9K-hIL21.
The yeast expression vector of indication of the present invention refers to the fabric shuttle-type plasmid pPIC9K, and it comprises following various element:
(1) the plasmid replication element in intestinal bacteria source.
(2) intestinal bacteria selection markers is as Ampicillin resistant gene (Ampr).
(3) Pichia anomala expression controlling element is as AOX1 gene promoter 5 ' AOX1, transcription terminator (TT) 3 ' AOX1.
(4) pichia spp secreting, expressing signal peptide sequence.As the yeast alpha factor (signal peptide of α-factor).
(5) multiple clone site
(6) pichia spp selection markers: as yeast His4 gene, Kanamycin resistant gene.
(7) can be on pichia spp karyomit(e) specific site the distinguished sequence of recombination and integration, as 5 ', the 3 ' sequence of AOX1.
Expression plasmid pPIC9K-hIL21 transforms bacillus coli DH 5 alpha, screening positive clone.One side is used for preserving, the amplification plasmid, is used on the other hand the analyses such as enzyme is cut, order-checking and detects.The recombinant expression plasmid pPIC9K-hIL21 that therefrom Screening and Identification is correct uses Sac I with the pPIC9K-hIL21 linearizing, adopts electric method for transformation to transform pichia spp.
Have the Kanamycin resistance marker according to the expression plasmid that changes over to, with G418 concentration gradient 1,2,3,4mg/mL screening multi-copy integration recon; At last express with shaking flask the recombinant bacterial strain of further verifying and screening high expression level amount.
The production rhIL-21 of shake-flask culture recombinant bacterial strain.Pichia spp is methylotrophic yeasts, and it can utilize methyl alcohol to be sole carbon source.The AOX1 promotor is methanol inducible promoters, can be suppressed by the carbon source of glycerine, the utilization of glucose isopreference.The present invention adopts two-step growth method shake-flask culture to produce hIL-21.Fs, engineering bacteria is inoculated in the semisynthetic medium BMGY that glycerine is carbon source (concrete formula is seen example) and cultivates and reached 4 left and right to nectar degree OD600 in 22～26 hours.Remove substratum after centrifugal, again add the semisynthetic medium BMMY (concrete formula is seen example) take methyl alcohol as carbon source, added methyl alcohol in every 24 hours to 0.5%～1.0% of final concentration, induce engineering bacterium expression hIL-21, the methyl alcohol final concentration all can be induced the expression of hIL-21 between 0.5%～1.0%, protein expression difference is not obvious.Cultivate and finish the rear centrifugal thalline of removing, collect culture supernatant, use polyacrylate hydrogel electrophoresis (SDS-PAGE) and enzyme linked immunosorbent assay (ELISA) to detect the expression of hIL-21 in supernatant.
ABIPROCISE is used in front 15 the amino acid whose detections of the N end of target protein
TM492cLC (GC320078) instrument is completed, and detection method is according to N-terminal sequencing standard method (SCI-S-006).
The invention provides a kind of great commercial value, scale operation prepares the method for rhIL-21, and its main outstanding advantages is:
1) built pichia spp high expression level rhIL-21 production engineering bacterium, shake-flask culture can make expression amount up to arriving approximately 230mg/L.
2) rhIL-21 that produces is secreted into outside born of the same parents, need not broken thalline, and rhIL-21 is present in substratum, and is simple to operate, is convenient to purifying.
3) rhIL-21 that produces is not with any label, as His label, C-myc epi-position, closer to the native protein of hIL-21.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The structure of rhIL-21 engineering bacteria
1.hIL-21 the acquisition of gene fragment order and analysis
At first, analyzed the encoding gene of hIL-21, result shows, do not contain the sequence of the effect substrate of this activator lytic enzyme of coding Pro-Glu-Ser-Thr in this gene, do not contain the sequence that coding is subject to X-Phe-X-Arg-Gln, the Gln-Arg-X-Phe-X (X=arbitrary amino acid) of lysosome cutting yet.Consider simultaneously the codon preference characteristic different from the people of yeast, having analyzed the CAI value that the hIL-21 encoding gene expresses in pichia spp is 0.74, potential (＞0.6) with coding high level expression albumen, the CAI value is that a statistics of synonym preference is measured, and can be used for the level that predicted protein matter is expressed.Therefore, directly separate from the healthy human body peripheric venous blood and obtain lymphocyte, extract cell total rna, and obtain the complete encoding gene of hIL-21 by reverse transcription PCR, this encoding gene does not comprise the encoding sequence of hIL-21 self signal peptide, introduces respectively Mun I, Not I restriction enzyme site at the two ends of this gene.
Method is as follows:
The design primer amplification is removed the hIL21 reading frame of endogenous signal peptide, introduces respectively Mun I and Not I restriction enzyme site in the upstream and downstream primer, and primer sequence is as follows:
IL21F：5’GCT
CAATTGCAAGATCGCCACATGATTAG-3’；(SEQ ID NO：2)
IL21R：5’-AAT
GCGGCCGCTCAGGAATCTTCACTTCCGT-3’(SEQ ID NO：3)。
94 ℃ of 5min, 94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 30s (30 circulations), 72 ℃ of 10min, amplified production are connected with Expression vector pPIC9K through EcoR I, Not I double digestion after Mun I, Not I double digestion, are built into recombinant expression vector.
2. the structure of recombinant expression plasmid
After the hIL-21 encoding gene that obtains is cut the generation sticky end with Mun I, Not I enzyme, be connected to the yeast expression vector pPIC9K upper (Mun I and EcoR I are as isocaudarner) that cuts processing take EcoR I, Not I enzyme, build recombinant expression plasmid pPIC9K-hIL21.The recombinant plasmid transformed bacillus coli DH 5 alpha, through enzyme cut, the proof procedure such as DNA sequencing, therefrom filter out the right-on transformant of structure, amplification and a large amount of recombinant plasmid pPIC9K-hIL21 that extract see Fig. 1.
Wherein, in described plasmid the partial sequence of the expression cassette of Huamn Interleukin 21 gene as shown in SEQID NO:1.
ATGAGATTTCCTTCA ATTTTTACTGCAGTTTTATTCGCAGCATCCTCC GCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAA TTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGAT GTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATA AATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGA GAAAAGAGAGGCTGAAGCTTACGTAGAATTGCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTGA(SEQ ID NO：1)
Wherein underscore is the base sequence of α-factor, and what follow later is the coding base sequence of ripe IL-21.
The aminoacid sequence of the IL-21 that gives expression to is as shown in SEQ ID NO:4
YVELQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO：4)
Wherein, the aminoacid sequence of ripe IL-21 is compared with natural IL-21 and will be had more four of fronts amino acid YVEL since the 5th at last, does not generally affect function.
3. screening multiple copied recombinant conversion is sub
After recombinant plasmid pPIC9K-hIL21 and empty carrier pPIC9K use SacI linearization for enzyme restriction with 5～10 μ g, adopt electric method for transformation to transform Pichia yeast GS115 (electric Transformation Parameters: 1500V, 25 μ F and 200 Ω).Add 1mL precooling 1mol/L sorbyl alcohol immediately in electric revolving cup after electricity transforms, 30 ℃ of incubations 1 hour are coated on the YPD flat board that contains 0.5mg/mL G418 30 ℃ of constant temperature culture.The transformant that grows is inoculated into respectively contains that G418 content is 1,2,3, on the YPD flat board of 4mg/mL, obtain recombinant conversion of multi-copy integration on the YPD flat board that contains 4mg/mLG418.
Shaking flask abduction delivering hIL-21
1. screen high expression level amount engineering bacteria
The employing two-step approach is cultivated, shaking flask is expressed the expression amount of each transformant, embodiment is that recombinant conversion of picking multi-copy integration is inoculated in 10mL BMGY substratum, under 28～30 ℃, 250r/min condition shaking culture to bacterium liquid OD600 be 2～6 o'clock, the centrifugal supernatant that goes replaces with the BMMY substratum and continues to cultivate, and every 24h adds methyl alcohol to final concentration 0.5%, after continuous induction 72h, collect bacterium liquid.The centrifugal rear collection supernatant of bacterium liquid is got the part supernatant after trichoroacetic acid(TCA) is concentrated, verifies that in the 15%SDS-PAGE electrophoresis relative molecular weight of rhIL-21 is 16kD.Be Western-blot with the IL-21 polyclonal antibody, rhIL-21 reaction is positive, and therefrom to obtain the transformant of high expression level be original engineering bacteria in screening, sees Fig. 2.
Result shows, after described expression cassette being incorporated on the karyomit(e) of pichia spp, can screen the engineering bacteria of many Expression of Plant Heights Huamn Interleukin 21 gene, wherein in figure No. 6 be the highest transformant of expression amount, therefore preferentially select as engineering bacteria.
2. a large amount of abduction deliverings of shaking flask
The original engineering bacteria of picking is inoculated in 100mL BMGY substratum, under 28～30 ℃, 250r/min condition shaking culture to bacterium liquid OD600 be 2～6 o'clock, the centrifugal supernatant that goes, replacing with the BMMY substratum continues to cultivate, added methyl alcohol to final concentration 0.5% in every 24 hours, respectively at 0,1,2,3,4,5,6,7 day set time collection bacterium liquid.The centrifugal rear collection supernatant of bacterium liquid is got the part supernatant after trichoroacetic acid(TCA) is concentrated, carries out SDS-PAGE and ELISA quantitatively (reference reagent box specification sheets carries out), and result shows that the 6th day expression amount is the highest, sees Fig. 3.The ELISA quantitative result shows that the expressing quantity of the 6th day is up to being about 230mg/mL.
Wherein substratum is semisynthetic medium, fills a prescription as follows:
The YPD nutrient solution: peptone 10g, yeast extract 5g is dissolved in distilled water, is settled to 960ml, 121 ℃ of autoclavings, 20min.After being chilled to room temperature, add 40mL 50% glucose, 4 ℃ of placements.If make flat board, add the 15g agar powder, add glucose after sterilization.
The BMGY/BMMY substratum: peptone 20g, yeast extract 10g is dissolved in distilled water, is settled to 700mL, 121 ℃ of autoclavings, 20min.After being chilled to room temperature, add successively 1M potassium phosphate buffer (pH6.0) 100mL, the 10X yeast is without nitrogenous source substratum (YNB) 100mL, 500X vitamin H 2mL, 10X glycerine 100mL is if the BMMY nutrient solution replaces glycerine with methyl alcohol.Use or 4 ℃ of preservations after mixing.
The detection of hIL-21
Utilize fast protein liquid chromatography system (
FPLC), use cation exchange medium SPSepharose Fast Flow to carry out purifying to the albumen of expressing in supernatant.Use ABIPROCISE
TM492cLC (GC320078) instrument carries out front 15 the amino acid whose detections of N end of the rhIL-21 of purifying, detection method is according to N-terminal sequencing standard method (SCI-S-006), front 15 aminoacid sequences of rhIL-21N end are: tyrosine-α-amino-isovaleric acid-L-glutamic acid-leucine-glutamine-aspartic acid-arginine-Histidine-methionine(Met)-Isoleucine-arginine-methionine(Met)-arginine-glutamine-leucine (YVELQDRHMI RMRQL), further having proved conclusively expression product is rhIL-21.
In addition, the result of biological activity test shows, described expression product can stimulate the propagation of human lymphocyte, and the stimulation degree is identical with commercially available IL-21 standard substance.
Comparative Examples 1
As a result, screening acquisition pichia spp transformant can not the secreting, expressing Huamn Interleukin 21 gene.
Comparative Examples 2
As a result, can express Huamn Interleukin 21 gene although screening obtains the pichia spp transformant, almost can't detect expressed Huamn Interleukin 21 gene in substratum, this points out this unit construction can not effectively realize the secreting, expressing of Huamn Interleukin 21 gene.
Comparative Examples 3
As a result, can express Huamn Interleukin 21 gene although screening obtains the pichia spp transformant, the Huamn Interleukin 21 gene comparatively small amt detected in substratum, this points out this unit construction is not preferred combination, is unfavorable for the secreting, expressing of Huamn Interleukin 21 gene.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Sequence table
＜110〉Shanghai Genon Bioengineering Co., Ltd
Shanghai Research Center of Genetically Modified
＜120〉method of pichia spp Restruction Huamn Interleukin 21 gene
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Tyr Val Glu Leu Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile
1 5 10 15
Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu
20 25 30
Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala
35 40 45
Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn
50 55 60
Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro
65 70 75 80
Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro
85 90 95
Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg
100 105 110
Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg
115 120 125
Thr His GlySer Glu Asp Ser
130 135
Claims (8)
1. the expression cassette of a Huamn Interleukin 21 gene, is characterized in that, described expression cassette from 5 ' to 3 ' comprises following element successively:
(1) the controlling gene pichia pastoris AOX 1 promoter of transcribing;
(2) control the signal peptide sequence of foreign protein secreting, expressing in pichia spp, described signal peptide sequence is yeast alpha factor signal peptide sequence;
(3) encoding sequence of Huamn Interleukin 21 gene;
(4) terminator codon;
(5) the AOX1 transcription terminator in terminator codon downstream;
Wherein said AOX1 promotor, yeast alpha factor signal peptide and AOX1 transcription terminator are respective element in the pPIC9K plasmid, and the encoding sequence of described Huamn Interleukin 21 gene is the nucleotide sequence of albumen shown in coding SEQ ID NO:4.
2. expression cassette as claimed in claim 1, is characterized in that, the encoding sequence of described Huamn Interleukin 21 gene is as shown in 268-672 of SEQ ID NO:1.
3. expression cassette as claimed in claim 1, is characterized in that, the encoding sequence of described signal peptide sequence is in SEQ ID NO:1 shown in the 1-267 position.
4. an expression vector, is characterized in that, described expression vector carries or contain the expression cassette of Huamn Interleukin 21 gene claimed in claim 1.
5. a Pichia yeast engineering, is characterized in that, is integrated with the expression cassette of Huamn Interleukin 21 gene claimed in claim 1 in the karyomit(e) of described pichia spp, and the secreting, expressing Huamn Interleukin 21 gene.
6. a method for preparing Huamn Interleukin 21 gene, is characterized in that, comprises step:
(a) under the condition that is fit to fermentation, cultivate Pichia yeast engineering claimed in claim 5, and induce pichia pastoris AOX 1 promoter to make engineering bacteria secreting, expressing Huamn Interleukin 21 gene;
(b) isolate the Huamn Interleukin 21 gene of secreting, expressing from fermented liquid.
7. method as claimed in claim 6, is characterized in that, step (a) is divided into thalli growth stage and abduction delivering stage.
8. method according to claim 7, is characterized in that, pichia pastoris AOX 1 promoter inductor used is methyl alcohol, and concentration is 0.5 ~ 1.0% (v/v).
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| CN103966253A (en) * | 2014-05-30 | 2014-08-06 | 中国科学技术大学 | Method for efficiently preparing recombinant human interluekin-33 protein |
| CN108728477B (en) * | 2017-04-24 | 2022-02-22 | 华东理工大学 | Efficient transposition mutation system and construction method |
| CN109593773B (en) * | 2018-11-22 | 2021-07-30 | 北京利德曼生化股份有限公司 | Method for expressing soluble growth stimulation expression gene 2 protein by using yeast expression system |
| CN111334546A (en) * | 2020-03-25 | 2020-06-26 | 南京工业大学 | Recombinant expression of human interleukin 2-red fluorescent protein in pichia pastoris and application of recombinant expression in vitro to slow release and promotion of T cell proliferation |
| CN113528566B (en) * | 2021-06-03 | 2022-12-20 | 自然资源部第三海洋研究所 | Yeast recombinant expression vector and construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1506463A (en) * | 2002-12-07 | 2004-06-23 | 中国人民解放军第三军医大学 | Method for yeast cell to express human interleukin 10 |
| CN1712536A (en) * | 2005-04-28 | 2005-12-28 | 中国人民解放军第三军医大学 | Expression of interleukin 24 from yeast cell |
| CN1827639A (en) * | 1999-03-09 | 2006-09-06 | 津莫吉尼蒂克斯公司 | Novel cytokine zalpha11 ligand |
| CN1869230A (en) * | 2006-05-31 | 2006-11-29 | 中国药科大学 | Nucleotid sequence of human interleukin 21 and method and application of producing matured human interleukin 21 |
-
2009
- 2009-11-20 CN CN 200910199126 patent/CN102021196B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1827639A (en) * | 1999-03-09 | 2006-09-06 | 津莫吉尼蒂克斯公司 | Novel cytokine zalpha11 ligand |
| CN1506463A (en) * | 2002-12-07 | 2004-06-23 | 中国人民解放军第三军医大学 | Method for yeast cell to express human interleukin 10 |
| CN1712536A (en) * | 2005-04-28 | 2005-12-28 | 中国人民解放军第三军医大学 | Expression of interleukin 24 from yeast cell |
| CN1869230A (en) * | 2006-05-31 | 2006-11-29 | 中国药科大学 | Nucleotid sequence of human interleukin 21 and method and application of producing matured human interleukin 21 |
Non-Patent Citations (3)
| Title |
|---|
| Ryutaro Asano et al.antitumor activity of interleukin-21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli.《FEBS Letters》.2002,第528卷全文. * |
| 李栋等.白细胞介素21的生物学功能及其抗肿瘤效应.《生命科学》.2009,第21卷(第4期),全文. * |
| 聂东宋等.外源蛋白在巴氏毕赤酵母中高效表达的策略.《吉首大学学报》.2001,第22卷(第3期),全文. * |
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