CN1539971A - Method for preparing human interferon omega by using means of gene engineering - Google Patents
Method for preparing human interferon omega by using means of gene engineering Download PDFInfo
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Abstract
A process for preparing human interferon omega is disclosed. The Pichia yeast expression system and improved secretion-type expression carrier, pMEX9K are used for efficient expression of human interferon omega. Its advantage is high output.
Description
Technical field
The invention belongs to the genetically engineered field, relate to a kind of method of utilizing genetic engineering means to prepare human interferon omega.
Background technology
Human interferon omega is found in 1985, be with the interferon-alpha function is close but an interferoid molecule (Hauptmann R that antigenicity is different, Swetly P.A novel class of human type Iinterferons[J] .Nucleic Acids Res, 1985,13 (13): 4739-4749.).Utilize the standard antivirus test, the specific activity that has determined human interferon omega is 2.7～4 * 108U/mg.Induce by Sendai virus, successfully utilized human peripheral leucocytes to produce and be purified into human interferon omega (Adolf G R, Maurer-Fogy I, Kalsner I et al.Purification and characterization of natural humaninterferon omega 1.Two alternative cleavage sites for the signal peptidase[J] .JBiol Chem, 1990,265 (16): 9290-9295.).In addition, utilize different exogenous protein expression systems, in intestinal bacteria, insect cell and Chinese hamster ovary celI, expressed the human interferon omega of reorganization respectively.These make becomes possibility to this proteic further investigation.In the mouse model of several people's tumours, comprise ovarian cancer, melanoma and skin carcinoma etc., confirmed that human interferon omega has intravital anti-tumor function (Horton H M, Hemandez P, Parker S E, et al.Antitumor effects of interferon-omega:in vivotherapy of human tumor xenografts in nude mice[J] .Cancer Res, 1999,59 (16): 4064-4068.).Utilize people's hepatoma cell line, (Hagelstein J such as Hagelstein J, Kist A, Stremmel W, et al.Antiviral potential of interferon-omega on hepatitis B virusreplication in human hepatoma cells[J] .Arzneimittelforschung, 1998,48 (3): 343-347.) compared the restraining effect of various Interferon, rabbit to hepatitis B virus duplication, found that human interferon omega has the similar inhibition virus replication effect of same interferon-alpha, IFN-and tumor necrosis factor alpha.In external inhibition experiment, find that human interferon omega has the inhibition viral protein synthetic effect stronger than interferon-alpha to HIV virus.Because human interferon omega has stronger antiviral duplicating, effects such as inhibition of cell proliferation and immunomodulatory, this cytokine is expected to be used for the treatment of kinds of tumors, virus disease and to the drug-fast patient of interferon-alpha.
Pichia yeast expression system (Pichia pastoris) is a kind of eukaryotic expression system (the Cereghino J L that set up in recent years, Cregg J M.Heterologous protein expression in themethylotrophic yeast Pichia pastoris[J] .FEMS Microbiol Rev, 2000,24 (1): 45-66.), be to be used to one of the most successful expression system of expressing foreign protein.Because it had both had prokaryotic expression system, as escherichia expression system commonly used, breeding is rapid, and the cheap and convenience operation of expense has eukaryotic expression system again and can correctly process expressed protein, fold and appropriate glycosylated advantage.Simultaneously, recombinant protein is easy to purifying.Therefore be used for proteinic great expression just more and more widely.At present existing 200 multiple protein are expressed (Cregg JM in this expression system, Cereghino JL, Shi JY, et al.Recombinant protein expression in Pichia pastoris[J] .Mol Biotechnol, 2000,16 (1): 23-52.).
Pichia yeast expression system comprises in the born of the same parents expresses and the secreting, expressing dual mode, and wherein secreting, expressing is owing to its expression amount height, and glycosylation is complete, and disulfide linkage and higher structure form correctly, and especially the later separation purifying is simple and convenient and enjoy favor.
Pichia spp is a kind of methyl alcohol nutritional type yeast, its growth rapidly, culture condition is simple, can the high-density cultured continuously; Its genetic manipulation is similar to yeast saccharomyces cerevisiae, and technology is quite ripe; The promotor (pAOX1) of the alcohol oxidase gene that generally uses in the expression vector is inducible promoter, suppressed by glucose, and just transcriptional start, translation is very suitable for expression of exogenous gene behind the methanol induction.
PPIC9K is the multiple copied secreted expression carrier that Invitrogen company releases.Size is 9.3kb.This carrier contains intestinal bacteria replication orgin Col E1 and penbritin and two resistant genes of kantlex, and wherein kalamycin resistance gene can also make pichia spp that G418 is produced resistance, can be used for the screening that multiple copied inserts.PPIC9K utilizes the secretion signal of alpha factor that goal gene is secreted to born of the same parents.Since pichia spp itself only secrete account for total amount 5% albumen to born of the same parents, therefore it is very convenient the albumen of secreting, expressing to be carried out separation and purification.
At present, still do not utilize Pichia pastoris yeast expression system to prepare the report of human interferon omega.
Summary of the invention
The present invention utilizes Pichia pastoris yeast expression system to prepare human interferon omega first.
The preparation method may further comprise the steps:
Make up secretor type Yeast expression carrier pMEX9K.The foreign protein that utilizes secreted expression carrier pPIC9K to express exists incorrect N end, these amino-acid residues that have more can influence and change expressed proteic 26S Proteasome Structure and Function (Goda S, Takano K, Yamagata Y.et al.Effect of extraN-terminal residues on the stability and folding of human lysozyme expressed inPichia pastoris[J] .Protein Eng, 2000,13 (4): 299-307.).In order to correct this defective, the contriver has carried out changing structure to secreted expression carrier pPIC9K, has been built into new expression vector pMEX9K.(Qi Lianquan, Chen Wei is in kept burning day and night etc. referring to article for the construction process of secretor type Yeast expression carrier pMEX9K.The structure of two novel multiple copied yeast expression vectors [J]. institute of Military Medical Science Institute periodical, 2002,26 (4): 264-266.).
Make up recombinant vectors pMEX9K ω.Construction process is seen accompanying drawing 1.With the method amplification human interferon omega gene of PCR, enzyme is cut carrier pMEX9K and PCR product and is reclaimed enzyme and cut product respectively, connects and transformed into escherichia coli.The enzyme of transformant cut identify and sequencing shows that construction of recombinant vector successfully.
Recombinant vectors pMEX9K ω is electric transformation receptor bacterium GS115 after linearizing, expresses.
The separation and purification of target protein.The fermentation supernatant is crossed the macrobead cation-exchange chromatography post of pre-equilibration, with sodium-chlor wash-out target protein peak, the target protein peak can directly be splined on the hydrophobic chromatography post, be splined on short grained cationic exchange coloum behind the wash-out, pass through further gel chromatography again, target protein has obtained good separation.
The determination of activity of target protein.According to document (the Chinese biological standard of articles council compiles. Chinese biological goods rules [M]. Beijing: Chemical Industry Press, 2000,373-375.) carry out.
The contriver has also explored the abduction delivering condition and the purifying process of high expression level bacterial strain.Discovery is in the yeast culture supernatant liquor, and the content of target protein accounts for 20%～30% of total protein in the culture supernatant inducing back 48～60h to reach the highest.After through four step chromatography purifications, the purity of target protein reaches more than 95%, and the rate of recovery is more than 30%.
The preparation method of human interferon omega provided by the invention has the output height, and expression and purifying are simple, and expense is cheap, is convenient to enlarge characteristics such as production, has vast market prospect.
Description of drawings
Fig. 1 is the building process synoptic diagram of human interferon omega expression vector.Wherein Kan+ is a kalamycin resistance gene; IFNW is that human interferon omega gene SS is α-factor signal peptide
Fig. 2 is that the SDS-PAGE of pichia spp transformant expression product analyzes collection of illustrative plates.
A.b.c. induce the expression supernatant of back transformant
D. the expression supernatant of transformant before inducing
E. target protein
M. molecular weight of albumen standard
Fig. 3 is that the SDS-PAGE after the separation and purification of human interferon omega analyzes
A. purified target protein;
B. after the digestion of PNGase F Glycosylase;
C.PNGase F Glycosylase;
M. molecular weight of albumen standard
Embodiment
The structure of embodiment one human interferon omega recombinant expression vector
1 material and method
1.1 plasmid, bacterial classification, virus and cell strain
Bacillus coli DH 5 alpha, pichia spp Pichia pastoris is available from magnificent company.
1.2 enzyme, carrier, primer and main agents
Restriction enzyme, T4DNA ligase enzyme, plasmid extraction and recovery test kit and pGEMT carrier are all available from Promega company.Primer is given birth to worker company by Shanghai and is synthesized.FICOLL, TRIZOL, agarose is available from magnificent company.YNB is a Difico company product.The lower molecular weight standard protein is an AmershamPharmacia company product.Glycosylase PNGase F is available from New England Biolabs company.MEM substratum and new-born calf serum are available from Hyclone company.Other all chemical reagent are import or homemade analytical reagent.
1.3 substratum
1.3.1 intestinal bacteria substratum LB substratum: 1% peptone, 1% sodium-chlor, 0.5% yeast extract powder; The LBAK substratum: adding penbritin and kantlex to final concentration in the LB substratum respectively is 100 μ g/mL.
1.3.2 yeast culture base YPD substratum: 1% yeast extract powder, 1% peptone, 2% glucose.BMGY substratum: 1% yeast extract powder, 1% peptone, 1.34%YNB, 1% glycerine, the phosphate buffered saline buffer of 100mmol/LpH6.0,4 * 10
-5The % vitamin H.BMMY substratum: 1% yeast extract powder, 1% peptone, 1.34%YNB, 1% methyl alcohol, the phosphate buffered saline buffer of 100mmol/L pH6.0,4 * 10
-5The % vitamin H.
1.3.3 cell culture medium: adopt the MEM substratum, add 3%～10% new-born calf serum.
1.4 method
1.4.1 extraction, the enzyme of related plasmid DNA are cut, reclaim, are connected and conversion etc. all by reference (Sambrook J, Fritsch EF, Maniatis T.Molecular cloning:a laboratory manual[M] .New York:Cold Spring Harbor Laboratory Press, 1989,16-56) operation.
1.4.2 adopt FICOLL reagent, from human blood, separate white corpuscle, extract total RNA with TRIZOL, adopt RT-PCR method amplification human interferon omega gene, connect the pGEMT carrier, make up pGEMT ω plasmid.
1.4.3 the method with PCR amplifies this gene from the plasmid pGEMT ω that carries the human interferon omega gene, the dna sequence dna of human interferon omega gene is the nucleotide sequence of sequence 1 in the sequence table.Sequence 3 and sequence 4 are respectively the 5 ' end and 3 ' the end primer of amplification human interferon omega in the sequence table.Add corresponding restriction enzyme site simultaneously: the upstream adds Xho I point of contact, and the downstream adds Eco R I point of contact, and 5 ' first CTG codon is first codon of coding human interferon omega in the sequence 3.Cut carrier pMEX9K and PCR product with Xho I and Eco R I enzyme respectively and reclaim enzyme and cut product, connect and transformed into escherichia coli.The enzyme of transformant cut identify and sequencing shows that construction of recombinant vector successfully.The building process of recombinant vectors such as accompanying drawing 1.
1.4.4 cutting, the enzyme of recombinant plasmid pMEX9K ω DNA identifies that recombinant plasmid pMEX9K ω DNA cuts evaluation with Xho I and Eco R I enzyme, cuts separation and purification through Sal I enzyme then.
Efficiently expressing of embodiment two reorganization human interferon omegas
One, the same embodiment of material
Two, method
The recombinant plasmid pMEX9K ω that cuts through enzyme among the top embodiment is reclaimed, transform pichia spp GS115 with electrotransformation.
The screening of recombinant clone: the GS115 after the conversion obtains his through the MD plate screening
+Recombinant clone in MD, MM flat board, is cultivated 2～3d with each clone's dibbling simultaneously for 30 ℃, determines the Mut phenotype of each bacterial strain.
The abduction delivering of recombinant human interferon omega: the recombinant clone that filters out is inoculated among the 5ml BMGY, and 30 ℃ of 300r/min are cultured to OD
600Be 2.0～6.0; Room temperature 6, the centrifugal 4min of 000r/min.The thalline of collecting suspends with BMMY and is diluted to OD
600After being 1.0,30 ℃ of 300r/min induce.Every 12h adds methyl alcohol to 0.5%.Collect culture supernatant after inducing 48h, SDS-PAGE detects protein expression and measures its biological activity.
Three, result
To screen through the pichia spp GS115 that electrotransformation transforms, screen three transformants that expression amount is higher, carry out fermentation culture respectively and induce centrifugal and collection supernatant.Determination of activity is the result show, three positive colony supernatant activity are respectively 3.2 * 10
6, 1.6 * 10
6With 3.0 * 10
6U/mL; Utilize SDS-PAGE to detect protein expression, and compare result such as Fig. 2 with the low molecular weight protein (LMWP) standard.Protein band at 22,000 places among the figure is a target protein, and this molecular weight of albumen is greater than theoretical molecular, and supposition may be (in the aminoacid sequence of target protein, a potential glycosylation site Asn80-Met81-Thr82 being arranged) due to the glycosylation.Utilize Glycosylase PNGase F the digestion of target protein to be confirmed this supposition correct (Fig. 3).Measure its relative molecular mass and theoretical value basically identical (it is respectively 22,166.10 and 20,340.71 that MALDI-TOF measures the molecular weight of target protein before and after PNGase F digestion) through the postdigestive target protein of PNGase F through MALDI-TOF.
The separation and purification of embodiment three reorganization human interferon omegas
One, material: with embodiment one.
Two, method
Will induce the bacterium liquid of back 48h, 10, behind the centrifugal 10min of 000r/min, get supernatant with 0.45 μ m membrane filtration, be splined on the macrobead positively charged ion chromatography post of sodium acetate pre-equilibration, usefulness 0.5M sodium-chlor wash-out target protein peak; The target protein peak is splined on the hydrophobic chromatography post, and elution peak is splined on the small-particle ion exchange column, 500mol/L sodium-chlor wash-out target protein peak; The target protein peak is splined on Superdex 75 gel-filtration columns, and uses 20mmol/L PBS, 150mol/L sodium-chlor, pH7.2 wash-out target protein peak.
Three, result
Target protein has obtained good separation, finds that through thin layer scanning target protein purity is greater than 95%, as Fig. 3.
The evaluation of embodiment quadruple group human interferon omega
One, material: see embodiment one.
Two, methods and results:
The N terminal amino acid sequence of human interferon omega is analyzed.Utilize 491 sequential analysis of protein instrument of PE company, adopt the automatic edman degradation method, the contriver has measured N-terminal 15 amino-acid residues of target protein, and it is in proper order: LGCDLPQNHGLLSRN.Illustration purpose albumen has the aminoterminal aminoacid sequence with natural albumen unanimity.
The determination of activity of human interferon omega according to document (the Chinese biological standard of articles council compiles. Chinese biological goods rules [M]. Beijing: Chemical Industry Press, 2000,373-375.) carry out.Determination of activity is the result show, behind three step chromatography purifications, the rate of recovery of target protein is more than 35%, and the proteic specific activity of final purpose has reached 2.2 * 10
8U/mL, same document (Horton H M, Hernandez P.Parker S E, et al.Antitumor effects of interferon-omega:in vivo therapy of human tumorxenografts in nude mice[J] .Cancer Res, 1999,59 (16): 4064-4068.) the report result is similar.
The physics and chemistry of human interferon omega is identified.Recombinant protein behind the purifying is determined its relative molecular mass with (MALDI-TOF) mass-spectrometric technique of substance assistant laser desorpted flight time, carried out N terminal amino acid sequence mensuration and Glycosylase PNGase F digestion.The results are shown in embodiment two.
Sequence table
＜110〉Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C
＜120〉a kind of method of utilizing genetic engineering means to prepare human interferon omega
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caccaaatga?ggagaatctc?ccctttcttg?tgtctcaagg?acagaagaga?cttcaggttc 120
ccccaggaga?tggtaaaagg?gagccagttg?cagaaggccc?atgtcatgtc?tgtcctccat 180
gagatgctgc?agcagatctt?cagcctcttc?cacacagagc?gctcctctgc?tgcctggaac 240
atgaccctcc?tagaccaact?ccacactgga?cttcatcagc?aactgcaaca?cctggagacc 300
tgcttgctgc?aggtagtggg?agaaggagaa?tctgctgggg?caattagcag?ccctgcactg 360
accttgagga?ggtacttcca?gggaatccgt?gtctacctga?aagagaagaa?atacagcgac 420
tgtgcctggg?aagttgtcag?aatggaaatc?atgaaatcct?tgttcttatc?aacaaacatg 480
caagaaagac?tgagaagtaa?agatagagac?ctgggctcat?cttga 525
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<211>174
<212>PRT
<213>
<400>2
Leu?Gly?Cys?Asp?Leu?Pro?Gln?Asn?His?Gly?Leu?Leu?Ser?Arg?Asn?Thr
1 5 10 15
Leu?Val?Leu?Leu?His?Gln?Met?Arg?Arg?Ile?Ser?Pro?Phe?Leu?Cys?Leu
20 25 30
Lys?Asp?Arg?Arg?Asp?Phe?Arg?Phe?Pro?Gln?Glu?Met?Val?Lys?Gly?Ser
35 40 45
Gln?Leu?Gln?Lys?Ala?His?Val?Met?Ser?Val?Leu?His?Glu?Met?Leu?Gln
50 55 60
Gln?Ile?Phe?Ser?Leu?Phe?His?Thr?Glu?Arg?Ser?Ser?Ala?Ala?Trp?Asn
65 70 75 80
Met?Thr?Leu?Leu?Asp?Gln?Leu?His?Thr?Gly?Leu?His?Gln?Gln?Leu?Gln
85 90 95
His?Leu?Glu?Thr?Cys?Leu?Leu?Gln?Val?Val?Gly?Glu?Gly?Glu?Ser?Ala
100 105 110
Gly?Ala?Ile?Ser?Ser?Pro?Ala?Leu?Thr?Leu?Arg?Arg?Tyr?Phe?Gln?Gly
115 120 125
Ile?Arg?Val?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Asp?Cys?Ala?Trp?Glu
130 135 140
Val?Val?Arg?Met?Glu?Ile?Met?Lys?Ser?Leu?Phe?Leu?Ser?Thr?Asn?Met
145 150 155 160
Gln?Glu?Arg?Leu?Arg?Ser?Lys?Asp?Arg?Asp?Leu?Gly?Ser?Ser
165 170
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gcctcgagaa?aagactgggc?tgtgatctg 29
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gggaattctt?atcaagatg 19
Claims (6)
1, a kind of method of utilizing genetic engineering means to prepare human interferon omega may further comprise the steps:
(1) makes up Yeast expression carrier;
(2) recombinant yeast expression vector of structure human interferon omega;
(3) expression of recombinant human interferon omega;
(4) separation and purification of recombinant human interferon omega.
2, method according to claim 1 is characterized in that described human interferon omega has the aminoacid sequence of sequence 2 in the sequence table or the nucleotide sequence that its gene has sequence 1 in the sequence table.
3, method according to claim 1, wherein said Yeast expression carrier have the Yeast expression carrier of correct aminoacid sequence for the target protein matter that can make expression.
4, method according to claim 1, wherein said Yeast expression carrier are pMEX9K.
5, method according to claim 1, wherein said human interferon omega recombinant yeast expression vector is the carrier that utilizes the described vector construction of claim 4.
6, method according to claim 5, wherein said human interferon omega recombinant yeast expression vector is pMEX9K ω.
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| CNA03123299XA CN1539971A (en) | 2003-04-25 | 2003-04-25 | Method for preparing human interferon omega by using means of gene engineering |
| PCT/CN2004/000374 WO2004096852A1 (en) | 2003-04-25 | 2004-04-19 | A RECOMBINANT HUMAN INTERFERON ϖ, THE METHOD FOR EXPRESSING IT AND THE USES OF IT |
| CN200480011049.1A CN1777620A (en) | 2003-04-25 | 2004-04-19 | Recombinant human omega interferon and its expressing method and use |
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| CN200480011049.1A Pending CN1777620A (en) | 2003-04-25 | 2004-04-19 | Recombinant human omega interferon and its expressing method and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1313491C (en) * | 2005-08-09 | 2007-05-02 | 中国科学院微生物研究所 | Cat omega interferon and its coding gene and uses |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1313491C (en) * | 2005-08-09 | 2007-05-02 | 中国科学院微生物研究所 | Cat omega interferon and its coding gene and uses |
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