CN1869228A - Production method of recombination human interferon gamma - Google Patents
Production method of recombination human interferon gamma Download PDFInfo
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- CN1869228A CN1869228A CN 200510026132 CN200510026132A CN1869228A CN 1869228 A CN1869228 A CN 1869228A CN 200510026132 CN200510026132 CN 200510026132 CN 200510026132 A CN200510026132 A CN 200510026132A CN 1869228 A CN1869228 A CN 1869228A
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Abstract
The invention relates to a reconstruction interferon gamma nucleotide sequence and the manufacture method to produce reconstruction interferon gamma, and the relative expression carrier and engineer cell construction, expression and purification technology. The method improves the expression quantity and the purification yield. It has the advantage of high expression, good stability, and simple technology. It could gain reconstruction human interferon gamma high efficiently, conveniently and in low cost.
Description
Technical field
The present invention relates to the genetically engineered field.More specifically, the invention provides a kind of method of High-efficient Production recombinant human interferon gamma, and the expression and the purifying process of the structure of related expression carriers and engineering cell, recombinant human interferon gamma.
Background technology
Interferon, rabbit is the soluble proteins with antiviral widely, antitumor and immunoregulation effect that is produced by various kinds of cell.Interferon, rabbit is not the molecule of homogeneous on the whole, and can be divided into 3 types according to producing cell: what white corpuscle produced is the α type; What inoblast produced is the β type; What the T cell produced is the γ type.Composite factors such as generation cell, acceptor and activity according to Interferon, rabbit are divided into 2 types with it: I type and II type.
I type Interferon, rabbit is called antiviral Interferon, rabbit again, and its biological activity is based on antiviral.
II type Interferon, rabbit claims type II interferon or IFN-γ again, and a kind of cytokine is mainly produced by the T cell; Main activity is to participate in immunomodulatory, is important immune-regulating factor in the body.IFN-γ has only a kind of protein of activity form, carries out in various degree glycosylation modified forming by the molecular weight polypeptide chain that is 18kD; People's interferon-gamma gene has only one, is positioned on human No. 12 karyomit(e), and the mature peptide of its coding is made up of 143 amino acid, and molecular weight 16KD has glycosyl, no intramolecular disulfide bond, and glycoprotein exists with the homodimer form.The acceptor of the acceptor of IFN-γ and I type Interferon, rabbit is irrelevant, and its gene is positioned on No. 6 karyomit(e), but is expressed in most karyocytes surface too.IFN-γ is unstable to acid, very easily destroys when pH2.0, utilizes this characteristic at an easy rate itself and I type Interferon, rabbit to be made a distinction.
Do not contain Interferon, rabbit in normal circumstances undertissue or the serum, only can lure just under the effect of some specific factor that cell produces Interferon, rabbit into.IFN-γ is mainly produced by CD8+T cell and some CD4+T cell (particularly TH1 cell), and the NK cell also can synthesize a spot of IFN-γ; These cells could secretion of gamma-IFN only be subjected to the activation of antigen or mitogen in immunne response after.
The main biological action of interferon-gamma:
1. antitumor action
In 3 types Interferon, rabbit, α, interferon-are effective not as IFN-to acute tumor treatment.IFN-has direct anti-proliferative effect to tumour cell, can be by prolonging growth and the breeding of cell cycle to delay tumour cell; Also can stop or the conversion process of the tumour cell that slows down by the expression that suppresses oncogene; Energy activating macrophage, NK cell etc. are with the direct killing cancer cells or suppress oncogene indirectly; Also but induced tumor necrosin and promote the expression of cancer cells to TNF acceptor, MHC II class antigen etc. makes it easily be discerned and be killed and wounded by cytotoxic T lymphocyte.People are transferred to tumour cell with the IFN-gene by retroviral vector, make tumour cell secrete IFN-voluntarily, and the knurl effect is killed in performance.This result of study shows that to the tumour cell of IFN-gene transfection, the kill capability of body obviously strengthens.
2. immunoregulation effect
The main activity of interferon-gamma is exactly to participate in immunomodulatory, plays dual regulation in vivo.On the one hand, IFN-can activate cells such as NK, strengthens antiviral, antineoplastic function; On the other hand, it can suppress B emiocytosis IgE, thereby avoids because of the allergy of the too high I of the generation type of IgE level, and it can also recover the function of suppressor T cell, reduces the local deposits of immunocomplex, suppresses the generation of III type allergy.
Adopt escherichia expression system expressing human interferon-gamma (as Chinese patent 941120910) now both at home and abroad mostly, interferon-gamma is that inclusion body is expressed, relate to albumen and become the renaturation difficulty, problems such as complex process, also have in the yeast of the employing born of the same parents and express (seeing Chinese patent 021368538), there is the thalline ultrasonication equally, purifying process complexity, problem such as the interferon activity of acquisition is on the low side.
System of the present invention provides a kind of method of High-efficient Production recombinant human interferon gamma.By optimizing the nucleotide sequence of coding recombinant human interferon gamma, adopt the transformed novel Pichia anomala expression system secreting, expressing human interferon gamma of yeast glycosylation, improve zymotechnique by optimizing, improve the expression level of recombinant human interferon gamma, easy purifying process simultaneously obtains high expression level, high yield, high reactivity, has the recombinant human interferon gamma of industrialization value.
Summary of the invention
Purpose of the present invention just provides a kind of method of production recombinant human interferon gamma of high-efficient simple.
Expression vector and engineering cell that another object of the present invention just provides the proteic encoding sequence of recombinant human interferon gamma and is used for this method.
In a first aspect of the present invention, just provided a kind of proteic nucleotide sequence of coding recombinant human interferon gamma of optimization, it is characterized in that, aminoacid sequence shown in the described nucleotide sequence coded SEQ ID NO:2, and nucleotides sequence is shown 95% above homogeny shown in described nucleotide sequence coded district and the SEQ ID NO:1.
In another preference, described nucleotide sequence contains the nucleotide sequence shown in the SEQ ID NO:1.
In a second aspect of the present invention, a kind of expression vector is provided, contain the described nucleotide sequence of claim 1.
In another preference, described expression vector is pPIC9K/rIFN-γ.
In a third aspect of the present invention, a kind of engineering cell is provided, it is characterized in that, be integrated with the described expression vector of claim 3.
In another preference, described engineering cell is through the transformed pichia spp of yeast glycosylation (Pichia Pastoris).
In another preference of the present invention, be integrated with the human interferon gamma gene coded sequence of 2-30 copy in the genome of described pichia spp host cell.
In another preference of the present invention, described pichia spp engineering cell comprises and utilizes methanol type fast and utilize methanol type at a slow speed.
In a fourth aspect of the present invention, provide a kind of production recombinant human interferon gamma proteic method, the method comprising the steps of:
A) under the expression condition that is fit to, cultivate host cell as claimed in claim 3, thereby secreting, expressing goes out human interferon gamma albumen; Preferably, described host cell is a pichia spp.
B) separation and purification goes out the human interferon gamma albumen of secreting, expressing.
In another preference of the present invention, the culture condition of described step (a) comprising:
Cultivation is divided into cultivation stage and induction period, it is 40-200 that cultivation stage cultivation bacterial concentration reaches OD600, induction time is 12-100hr, fermentation and inducing temperature remain on 25-32 ℃, the amount of trace element is 0.1-2ml/L, the pH value of inductive phase is 3-10, and inductive phase, methanol concentration was controlled at 0.1-5%.
In another preference of the present invention, the process of inducing can also be added casein hydrolysate (CA), peptone (peptone), Tryptones (Tryptone), arginine etc. as protective material.
In another preference of the present invention, described step (b) comprises step:
(i) fermented sample is removed precipitation by centrifugal and/or filter type, obtain fermented supernatant fluid;
(ii) fermented supernatant fluid is carried out ultrafiltration and concentration, exchange buffering liquid;
(iii) chromatography purification, described chromatography is selected from: cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, hydrophobic chromatography, affinity chromatography and combination thereof.
In another preference of the present invention, sample after DEAE Sepharose ion exchange column and Sepharose 200 gel-filtration columns, obtain purity greater than 95%, yield is at the pure product more than 50%.
Description of drawings
Fig. 1 is the structure route map of pPIC9k/IFN-γ expression plasmid.
Embodiment
The inventor is extensive studies by going deep into, optimization design by gene coded sequence, human interferon gamma encoding sequence (SEQ ID NO:1) after optimizing changed over to through the transformed methyl alcohol of yeast glycosylation utilize type pichia spp (P.pastoris), thereby realized the human interferon gamma efficient secretory expression, and optimized fermentation and purifying process.Finished the present invention on this basis.
The present invention is according to the natural mature peptide sequence of human interferon gamma, press codon-bias, under the condition that does not change aminoacid sequence, the complete proteic encoding sequence of gene synthesizing recombined human interferon-gamma, with this gene clone in the pUC19 after the sequence verification, with the ordinary method of molecular cloning, be cloned into expression vector pPIC9K, transform, be integrated into the P.pastoris host cell chromosome, by applying the transformant that antibiotic-screening goes out multi-copy integration, and then filter out the high expression level engineering cell.Shake flask test shows, induce 24 hours after, the culture supernatant target protein accounts for total protein more than 50%, more than the expression level 300mg/L.According to its speed that consumes methyl alcohol, judge that this engineering cell strain is Mut+.
After obtaining engineering cell, just can be under the condition that is fit to the culturing engineering cell.In the present invention, the recombinant human interferon gamma fermentation condition of engineering bacterium expression is not particularly limited.Can adopt the fermentation condition of this area routine.For example, suitable medium includes, but is not limited to following composition:
(iv) nitrogenous source contains organic nitrogenous source and inorganic nitrogen-sourced, wherein organic nitrogen source such as peptone, yeast powder, or the mixture of the two, total concn 0.1-3%; Inorganic nitrogen-sourced as ammoniacal liquor or NH
4Cl, (NH
4)
2SO
4Deng ammonium salt, concentration 0-1.5%.
(v) inorganic salt comprise phosphoric acid salt, Chinese holly hydrochloride, Mg
2+Salt etc.Preferably, phosphate buffer concentration 20-200mM, pH5-10; Mg
2+Salt 0-5mM.
(vi) in substratum, add VITMAIN B1 VITAMIN as a supplement, concentration 1～1000PPM.
(vii) according to M
9Trace element formula in the substratum, trace element can add 0.1-2ml/L training liquid.
(viii) carbon source comprises glycerine, glucose, lactose etc.Can be single carbon source, also can be mixed carbon source.Preferably, carbon source is selected from down group: glycerol concentration is 0.1-3%; Lactose concn is 0.1-3%; Glucose concn is 0.1-1.5%.
For the extensive recombinant human interferon gamma that obtains, need in fermentor tank, express cultivation.The present invention has studied pilot scale fermentation technology, and the expression level after the optimization reaches 400mg/L:
Behind fermentation expression, the recombinant human interferon gamma albumen of expressing is separated.Usually, fermented sample earlier obtains fermented liquid supernatant in modes such as centrifugal, filtrations.Carry out preliminary purification and chromatography then and comprise cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, affinity chromatography etc.
Through different chromatography processes relatively, the purification process of optimization comprises:
1. fermented sample is obtained fermented supernatant fluid by centrifugal and/or filter type;
2. by saltouing and/or after ultrafiltration carries out preliminary purification, or, reach 95% pure product thereby obtain purity directly by ion-exchange, hydrophobic chromatography method.
Pure product purity is more than 95%, the about 200mg/L fermented liquid of yield.
The activity of pure product is measured by cytopathic-effect inhibition assay, and violet staining is measured D with microplate reader
570Value is at last with mark Huaihe River, world product proofreading activity unit.The ratio work of the pure product of determination of activity is about 5 * 10
7IU/mg.
Recombinant human interferon gamma can be made various formulations with routine techniques behind the purifying.
In an example of the present invention, made up the P.pastoris yeast expression engineering cell of producing recombinant human interferon gamma, methanol induction, high-level secretory expression need not become renaturation.
In another example of the present invention, the optimization of technology has by fermentation further improved the expression amount of recombinant human interferon gamma.
In another example of the present invention, because the protein excretion expression, but fermentation supernatant direct purification, purifying process is simple, can obtain pure product 200mg/L behind the purifying.
Stoste adds suitable auxiliary material behind the purifying, makes the powder ampoule agent for injection of recombinant human interferon gamma.The preliminarily stabilised test shows that this powder injection is stable.
Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low, and every liter of fermented liquid can obtain the pure product 200mg of recombinant human interferon gamma, than living about 5 * 10
7IU/mg.Be fit to industrialization production.
The invention has the advantages that:
What (1) select for use is through yeast glycosylation transformed pichia spp host cell, can carry out suitable glycosylation modified to it.
(2) the recombinant human interferon gamma protein gene after the optimization is highly suitable in the pichia spp and expresses, and has the characteristics of high expression level, high stable.
(3) by the crucial technological condition for fermentation of control, make expression level reach 400mg/L.
(4) purifying process is easy, rate of recovery height.Because secretory protein is not with the inclusion body formal representation, therefore simplified purification procedures, the purifying rate of recovery is improved greatly, make the scale operation recombinant human interferon gamma become possibility.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described 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, people such as Sambrook for example, 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 embodiment 1 expression plasmid and the acquisition of high expression engineering strain
According to the human interferon gamma natural acid sequence, press codon-bias, under the condition that does not change aminoacid sequence, the target gene sequences of full gene composite coding human interferon gamma albumen mature peptide is cloned among the pUC19 this gene order and sequence verification.
The expression vector establishment method is seen Fig. 1.Obtain purpose human interferon gamma gene by pcr amplification, with XhoI+EcoRI double digestion (MBI, 2*Tango
TM, 37 ℃) and PCR product and pPIC9 plasmid (available from Invitrogen company).Two fragments are connected with the T4 dna ligase, connect the product conversion and enter bacillus coli DH 5 alpha (available from Promega company), containing selected clone on the LB flat board of penbritin, preparing plasmid in a small amount, going out positive colony by double digestion/PCR evaluation and screening.The pPIC9/IFN-γ plasmid of a large amount of amplification conclusive evidences with BamH I+EcoR I double digestion, reclaims small segment; Plasmid pPIC9K (available from Invitrogen company) handles with identical enzyme, reclaims big fragment.Two fragments are connected with the T4 dna ligase, connect the product conversion and enter bacillus coli DH 5 alpha, select positive colony on the LB flat board of penbritin and carry out plasmid enzyme restriction and identify containing, prepare plasmid in a small amount, go out positive colony by double digestion/PCR evaluation and screening.Acquisition contains the recombinant plasmid pPIC9K/IFN-γ (interferon-gamma) of interferon-gamma.
Expression plasmid pPIC9/IFN-γ is after sequence verification, and with Sal I linearization for enzyme restriction, electroporation transforms and enters pichia spp SMD1168 (available from Promega company), is applied to be cultured to transformant in 30 ℃ on the RDB flat board and to grow screening high expression level bacterial strain.PPIC9K/IFN-γ plasmid (first sequence verification) is transformed the high expression level bacterial strain that has screened, screening G418 resistant strain.Identify the G418 resistance of yeast transformant with the dibbling method.G418 concentration is 1,2,3, the high resistance transformant of YPD plate screening of 4mg/mL with containing successively, and carries out little megger and reach the engineering yeast strain that screening obtains IFN-γ high expression level.According to its speed that consumes methyl alcohol, judge that this engineering cell strain is Mut+.
Embodiment 2 different pH are to the influence of expression level
Get mono-clonal, be inoculated in the BMGY primary seed solution, cultivate 17-20hr; In 1: 10 ratio two-stage inoculation (each condition has looped pipeline) in the 500ml of 50ml substratum Erlenmeyer flask, to cultivate about 24hr, 1% methanol induction, the pH of induction period need be regulated (directly regulating pH with phosphate buffer in the BMMY substratum).Induce the 24hr sampling, measure OD and pH simultaneously, add 1% methyl alcohol again and continue to induce.Coinduction 48hr.Induce preceding, induce different time sample detection SDS-PAGE.
Experimental result shows: shaking bottle optimal pH of horizontal expression IFN-γ between 5～9, best pH5-7.
Get mono-clonal, be inoculated in the BMGY primary seed solution, cultivate 17-20hr; , in the 1L of 250ml BMGY Erlenmeyer flask, cultivate about 4 ~ 8hr in 1: 10 ratio two-stage inoculation, last jar of fermentation, pH 5.0,20 ℃ of temperature, D0＞35% are treated to add 50% glycerine with 10 ~ 15 commentaries on classics degree streams after dissolved oxygen rises.Treat that glycerine exhausts, begin to induce with rotating speed 1 with 100% methyl alcohol after dissolved oxygen rises once more, gradually speed-raising.Induction period is that 10% CA, Peptone and each 300ml stream of Tryptone add jar (a 5L fermentor tank, a fermentation supernatant 3L) with concentration, induces 48hr to finish.Sample detection SDS-PAGE and protein content are to determine the optimum protein protective material of induction period.
Embodiment 4 recombinant human interferon gamma purifying
48h is cultivated and induced to high expression level bacterial strain extended volume, and centrifugal 10 minutes of 4 ℃ of following 5000rpm obtain fermented supernatant fluid, are splined on filling SP Sepharose FF ion exchange column, with the TrisHCl of the pH8.0 that contains 0.02m/L, 0-1mol/L NaCl wash-out; Sephadex G-25 post layer folding on the elutriant, the TrisHCl of 0.02m/L, pH8.0 solution equilibria wash-out desalination is collected protein peak.Go up sample SephacrylS-200HR again,, collect main peak with the PBS solution equilibria wash-out of pH7.4.
Through above purifying process, can get the pure product 200mg/L of albumen at last.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.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 institute restricted portion equally.
Sequence table
＜110〉Shanghai Xinshengyuan Medicine Research Co., Ltd.
＜120〉a kind of production method of recombinant human interferon gamma
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<210>1
<211>429
<212>DNA
＜213〉artificial sequence
<221>misc_feature
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＜223〉the recombinant human interferon gamma gene of You Huaing
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tttttcaata?gcaacaaaaa?gaaacgtgat?gacttcgaaa?agctgactaa?ttattcggta 300
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＜213〉homo sapiens
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Gln?Asp?Pro?Tyr?Val?Lys?Glu?Ala?Glu?Asn?Leu?Lys?Lys?Tyr?Phe?Asn?Ala?Gly
1 5 10 15
His?Ser?Asp?Val?Ala?Asp?Asn?Gly?Thr?Leu?Phe?Leu?Gly?Ile?Leu?Lys?Asn?Trp
20 25 30 35
Lys?Glu?Glu?Ser?Asp?Arg?Lys?Ile?Met?Gln?Ser?Gln?Ile?Val?Ser?Phe?Tyr?Phe?Lys
40 45 50 55
Leu?Phe?Lys?Asn?Phe?Lys?Asp?Asp?Gln?Ser?Ile?Gln?Lys?Ser?Val?Glu?Thr?Ile
60 65 70
Lys?Glu?Asp?Met?Asn?Val?Lys?Phe?Phe?Asn?Ser?Asn?Lys?Lys?Lys?Arg?Asp?Asp
75 80 85 90
Phe?Glu?Lys?Leu?Thr?Asn?Tyr?Ser?Val?Thr?Asp?Leu?Asn?Val?Gln?Arg?Lys?Ala
95 100 105
Ile?His?Glu?Leu?Ile?Gln?Val?Met?Ala?Glu?Leu?Ser?Pro?Ala?Ala?Lys?Thr?Gly?Lys
110 115 120 125
Arg?Lys?Arg?Ser?Gln?Met?Leu?Phe?Arg?Gly?Arg?Arg?Ala?Ser?Gln
130 135 140 143
Claims (7)
1. nucleotide sequence of human interferon gamma fusion rotein of encoding, it is characterized in that, aminoacid sequence shown in the described nucleotide sequence coded SEQ ID NO:2, and nucleotides sequence is shown 95% above homogeny shown in described nucleotide sequence coded district and the SEQ ID NO:1.
2. an expression vector is characterized in that, described expression vector contains the described nucleotide sequence of claim 1.
3. an engineering cell is characterized in that, it is integrated with the described expression vector of claim 2.
4. engineering cell as claimed in claim 3 is characterized in that it is a pichia spp, is the transformed pichia spp of a kind of yeast glycosylation.
5. method of producing human interferon gamma is characterized in that the method comprising the steps of:
(a) under the expression condition that is fit to, cultivate engineering cell as claimed in claim 3, thereby secreting, expressing goes out human interferon gamma albumen;
(b) separation and purification goes out the human interferon gamma albumen of expression.
6. method as claimed in claim 5 is characterized in that, the expression condition shown in the step (a) is: induction time is 12-120 hour, and preferable is 24-100 hour; Inducing pH is 3-10, and that preferable is 5-7.
7. method as claimed in claim 5 is characterized in that, the expression condition shown in the step (b) is:
(1). fermented liquid obtains to contain the supernatant liquor of target protein by simple centrifugal or ultrafiltration;
(2). by easy steps such as simple ion exchange chromatography, hydrophobic chromatographies, can obtain purity at the pure product more than 95%.
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|---|---|---|---|
| CN 200510026132 CN1869228A (en) | 2005-05-24 | 2005-05-24 | Production method of recombination human interferon gamma |
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|---|---|---|---|
| CN 200510026132 CN1869228A (en) | 2005-05-24 | 2005-05-24 | Production method of recombination human interferon gamma |
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|---|---|
| CN1869228A true CN1869228A (en) | 2006-11-29 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102030823A (en) * | 2010-06-21 | 2011-04-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Liver-targeted genetically engineered interferon and preparation method thereof |
| CN106349385A (en) * | 2016-09-30 | 2017-01-25 | 山东仙普爱瑞科技股份有限公司 | Process for extracting gama-interferon from pichia pastoris fermentation liquor |
-
2005
- 2005-05-24 CN CN 200510026132 patent/CN1869228A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102030823A (en) * | 2010-06-21 | 2011-04-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Liver-targeted genetically engineered interferon and preparation method thereof |
| CN106349385A (en) * | 2016-09-30 | 2017-01-25 | 山东仙普爱瑞科技股份有限公司 | Process for extracting gama-interferon from pichia pastoris fermentation liquor |
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